Retinal Degenerative Diseases XIX

John Ash
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Advances in Experimental Medicine and Biology 1415

John D. Ash · Eric Pierce · Robert E. Anderson ·

Catherine Bowes Rickman · Joe G. Hollyfield ·
Christian Grimm Editors

Retinal
Degenerative
Diseases XIX
Mechanisms and Experimental Therapy
Advances in Experimental Medicine
and Biology

Volume 1415

Series Editors
Wim E. Crusio, Institut de Neurosciences Cognitives et Intégratives
d’Aquitaine, CNRS and University of Bordeaux, Pessac Cedex, France
Haidong Dong, Departments of Urology and Immunology, Mayo Clinic
Rochester, MN, USA
Heinfried H. Radeke, Institute of Pharmacology and Toxicology, Clinic of
the Goethe University Frankfurt Main, Frankfurt am Main, Hessen,
Germany
Nima Rezaei, Research Center for Immunodeficiencies, Children’s Medical
Center, Tehran University of Medical Sciences, Tehran, Iran
Ortrud Steinlein, Institute of Human Genetics, LMU University Hospital
Munich, Germany
Junjie Xiao, Cardiac Regeneration and Ageing Lab, Institute of
Cardiovascular Sciences, School of Life Science, Shanghai University
Shanghai, China
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John D. Ash • Eric Pierce
Robert E. Anderson
Catherine Bowes Rickman
Joe G. Hollyfield • Christian Grimm
Editors

Retinal Degenerative
Diseases XIX
Mechanisms and Experimental
Therapy
Editors
John D. Ash Eric Pierce
Department of Ophthalmology Ocular Genomics Institute
University of Pittsburgh School Department of Ophthalmology
of Medicine Massachusetts Eye and Ear Infirmary
Pittsburgh, PA, USA Harvard Medical School
Boston, MA, USA
Robert E. Anderson
Health Sciences Center Catherine Bowes Rickman
University of Oklahoma Health Department of Ophthalmology
Sciences Center Duke Medical Center
Oklahoma City, OK, USA Durham, NC, USA

Joe G. Hollyfield Christian Grimm

Department of Ophthalmology Laboratory for Retinal Cell Biology
Cleveland Clinic Lerner College Department of Ophthalmology
of Medicine University Hospital Zurich
Cleveland, OH, USA University of Zurich
Schlieren, Switzerland

ISSN 0065-2598     ISSN 2214-8019 (electronic)

Advances in Experimental Medicine and Biology
ISBN 978-3-031-27680-4    ISBN 978-3-031-27681-1 (eBook)
https://doi.org/10.1007/978-3-031-27681-1

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The editors are pleased dedicate this publication to the memory
of our long-time friend and colleague, Alan M. Laties. Except
for the most recent years, Alan attended each of these biennial
retinal degeneration meetings since they began in 1984. Early
on Alan recognized the importance of our attempt to provide a
continuing international platform for discussions and scientific
exchange to take place among investigators focused on retinal
degeneration research. Through his scientific leadership at the
Foundation Fighting Blindness (formerly the Retinitis
Pigmentosa Foundation), we received the first meeting grant to
partially cover some of the expenses of the RD meeting held in
San Francisco in 1988. The Foundation has provided
continuing support for each of the subsequent meetings in the
form of travel grant support for young investigators.
Born in Beverly, Massachusetts, the son of Russian immigrants,
he attended Harvard College (BA, 1954) and completed
medical school at Baylor College of Medicine (MD, 1959),
followed by a residency in ophthalmology in the Hospital of the
University of Pennsylvania (1961–63). A United States Public
Health Service Special Research Fellowship supported his
vi 

research training in the Institute of Neurological Sciences at

the University of Pennsylvania (1963–64). He joined the faculty
at the University of Pennsylvania in 1965 where he moved
through the academic ranks until retiring as Emeritus
Professor of Ophthalmology at the Perelman School of
Medicine in 2020. He held joint appointments in
Ophthalmology and Neurology where he was the Irene Heinz
Given and John LaPorte Given Research Professor and the
Harold G. Scheie Research Professor in Ophthalmology. He
served as neuro-ophthalmologist at the Hospital of the
University of Pennsylvania while pursuing basic research on
the autonomic innervation of the eye, eye growth, and
therapeutic approaches to eye diseases. He has published 140
original research papers, 30 review articles, and presented
numerous invited lectures at major university medical centers
around the world on a variety of topics critical to the treatment
of diseases of the eye. He was an inventor holding multiple
patents in the area of ophthalmology.
In the early 1970s, Alan was approached by the Retinitis
Pigmentosa Foundation to help them develop a scientific plan
to support targeted research that would lead to an
understanding of the causes of retinitis pigmentosa. At the time,
it was recognized that these diseases were inherited, but only in
a very limited way (autosomal dominant, recessive or
X-linked). At the time, no mutations causing RP had been
identified and the Human Genome Project would not be
initiated for another 20 years. Alan agreed and organized the
first Scientific Advisory Board for this Foundation and served
as Chairman. In this leadership role, Alan helped identify and
direct funding to the first laboratory focused on degenerative
retinal disease research, the Berman-Gund Laboratory at the
Massachusetts Eye and Ear Infirmary, Harvard University.
Research Centers focused on retinal degeneration would later
be expanded to many medical centers in North America,
England, and Europe. Alan recognized the importance and
need for animal models with these inherited retinal diseases
and directed funds from the Foundation to support the
development of the dog models with RP identified by Dr.
Gustavo Aguirre at the College of Veterinary Medicine. In the
early 1980s, Alan initiated a scientific plan for the Foundation
to identify the major genes responsible for RP. This led in 1989
to the discovery of a mutation in the rhodopsin gene
 vii

responsible for an autosomal dominant form of retinitis

pigmentosa. Discovery of mutations in other genes causing
retinitis pigmentosa quickly followed. With the discovery of
RP-65, a gene that causes a recessive form of RP, gene therapy
in a dog model with this recessive disorder could be quickly
initiated because of Dr. Laties’ early support from the
Foundation of these dog model lines. Dr. Laties’ early
leadership was hugely important to gene therapy clinical trials
and a number of other therapies related to these inherited
retinal diseases. To honor Dr. Laties, the Foundation Fighting
Blindness named their physicians’ and physician-scientists’
career development award the Alan Laties Career Development
Program and honored him with the inaugural Llura Liggett
Gund Lifetime Achievement Award.
Dr. Laties was a gifted scientist, outstanding leader, and
compassionate human who enriched the lives of his
contemporaries. He played a key role in nurturing and
expanding research in inherited retinal diseases. He is survived
by his wife Deena Gu, a distinguished artist, daughter Jane
Laties, sons Alex P. Laties and Nicholas P. Robinson, and a
brother, David.
Preface

The XIX International Symposium on Retinal Degeneration was held from

September 26 to October 2, 2021. The symposium was initially planned for
October of 2020 in Mendoza, Argentina. However, the global pandemic made
this meeting impossible. With the availability of vaccines, we decided in
March of 2021 that it would be possible to organize the meeting for late
September of 2021. From the beginning, we planned the meeting as an in-­
person meeting with the capability of switching to a hybrid or fully online
meeting depending on the state of the pandemic, and we moved the in-person
meeting to the United States to reduce travel complications for most attend-
ees. As the delta variant began to surge in the weeks leading up to the meet-
ing, we had to activate the hybrid meeting. The meeting platform we
established allowed both in-person and virtual platform talks as well as both
in-person and virtual attendance. The platform was organized so that all pre-
sentations were live and all participants were able to ask questions. All pre-
sentations, including posters, were recorded and made available 4 months
after the meeting. The in-person sessions were held in the Sonesta Nashville
Airport Hotel in Nashville, TN. Because of COVID concerns, the in-person
attendance was small (118 scientists) compared to previous meetings (~250
scientists), but the overall attendance increased to 344 attendees. The virtual
option was the main driver for the increase in attendance. The meeting pro-
gram included four outstanding keynote presentations from Michael Chiang,
Director of the National Eye Institute on Artificial intelligence for clinical
care and research; Douglas Wallace, National academy of Science member
and Professor at the University of Pennsylvania on Mitochondria and the eti-
ology of disease; David Gamm, Professor at the University of Wisconsin-­
Madison on Ultrathin micromolded 3D scaffolds for outer retina
reconstruction; Valeria Canto-Soler, Professor at the University of Colorado
on Human iPSC-derived 3D retinal tissue for stem cell-based therapies for
retinal degenerative diseases. Drs Chiang and Wallace presented via the vir-
tual platform, while Drs Gamm and Canto-Soler presented from the podium.
The program also included 41 platform talks, with 28 presented in person
from the podium and another 13 presented virtually. In addition, 143 posters
were presented as short talks on the virtual platform. Seventy-three of the
posters were also presented in person during two well-attended poster ses-
sions. New and important data were presented at the meeting, and we were
mentioned in a written article published on NPR, and several attendees were
interviewed by reporters from Science and other journals.

ix
x Preface

The RD2021 Travel award competition was highly successful at attracting

qualified applicants. We received a 35% increase in TA applications for a total
of 196. The applications were reviewed by a panel of 14 expert reviewers,
including 6 women, 8 men, and sceintists from a recognized underrepre-
sented minority (URM). Since funding from European sources is dedicated to
European early career scientists, we included three reviewers from Europe.
Many of the panel members have been prior travel awardees. Each applica-
tion was assigned four reviewers, and reviewers independently scored appli-
cations on a 1–9 scale. Based on scores, the applications are ranked and
slotted into funding sources based on funding agency criteria. We were able
to support full travel awards for 60 in-person early career scientists and
another 41 virtual early-career scientists. This is the largest pool of awardees
at an RD meeting. The awards were balanced between men and women. In
addition, we implemented a new diversity and inclusion policy and dedicated
a minimum of six awards to underrepresented minorities (URM). In the end,
we were able to fund 11 URMs to attend the RD meeting.
Although the pandemic made the RD2021 meeting more complex and
more challenging to organize, the RD2021 meeting was, by all accounts, a
terrific success.

Pittsburgh, PA, USA John D. Ash

Boston, MA, USA Eric Pierce
Oklahoma City, OK, USA Robert E. Anderson
Durham, NC, USA Catherine Bowes Rickman
Cleveland, OH, USA Joe G. Hollyfield
Schlieren, Switzerland Christian Grimm
Contents

Part I Age-related Macular Degeneration

High-Resolution Imaging Mass Spectrometry of Human

Donor Eye: Photoreceptors Cells and Basal Laminar
Deposit of Age-­Related Macular Degeneration������������������������������������   3
David M. G. Anderson, Ankita Kotnala, Jeffrey D. Messinger,
Nathan Heath Patterson, Jeffrey M. Spraggins, Christine A. Curcio,
Richard M. Caprioli, and Kevin L. Schey
The Noncanonical Role of Complement Factor H
in Retinal Pigment Epithelium (RPE) Cells and Implications
for Age-Related Macular Degeneration (AMD)������������������������������������   9
Angela Armento, David Adrian Merle, and Marius Ueffing
 acular Pigment Carotenoids and Bisretinoid A2E���������������������������� 15
M
Ranganathan Arunkumar and Paul S. Bernstein
Disturbed Matrix Metalloproteinases Activity in Age-Related
Macular Degeneration ���������������������������������������������������������������������������� 21
Beatriz Martins and Rosa Fernandes
Current Views on Chr10q26 Contribution to Age-Related
Macular Degeneration ���������������������������������������������������������������������������� 27
Navdeep Gogna, Lillian F. Hyde, Gayle B. Collin, Lisa Stone,
Jurgen K. Naggert, and Patsy M. Nishina
Untargeted Lipidomic Profiling of Aged Human Retina
With and Without Age-Related Macular Degeneration (AMD)���������� 37
Ankita Kotnala, David M. G. Anderson, Jeffrey D. Messinger,
Christine A. Curcio, and Kevin L. Schey
Decoding Race and Age-Related Macular Degeneration:
GPR 143 Activity Is the Key�������������������������������������������������������������������� 43
Dorothy Tung and Brian S. McKay
 eroxisome Proliferator-Activated Receptor Gamma
P
Coactivator-­1Alpha (PGC-1α): A Transcriptional Regulator at
the Interface of Aging and Age-Related Macular Degeneration? ������ 49
Freya M. Mowat

xi
xii Contents

Regulation of ABCA1 by miR-33 and miR-34a

in the Aging Eye �������������������������������������������������������������������������������������� 55
Florian Peters and Christian Grimm
 he Role of Gene Expression Regulation on Genetic Risk
T
of Age-­Related Macular Degeneration�������������������������������������������������� 61
Rinki Ratnapriya
Elastin Layer in Bruch’s Membrane as a Target
for Immunization or Tolerization to Modulate Pathology
in the Mouse Model of Smoke-Induced Ocular Injury������������������������ 67
Bärbel Rohrer, Nathaniel Parsons, Balasubramaniam Annamalai,
Crystal Nicholson, Elisabeth Obert, Bryan Jones,
and Andrew D. Dick
Repurposing Drugs for Treatment of Age-Related
Macular Degeneration ���������������������������������������������������������������������������� 73
Sarah G. Francisco and Sheldon Rowan

Part II Extracellular Vesicles

Extracellular Vesicle RNA Contents as Biomarkers

for Ocular Diseases���������������������������������������������������������������������������������� 81
Heran Getachew and Eric Pierce
Proteomics of Retinal Extracellular Vesicles: A Review
into an Unexplored Mechanism in Retinal Health
and AMD Pathogenesis���������������������������������������������������������������������������� 87
Adrian V. Cioanca, Riccardo Natoli, and Yvette Wooff

Part III Gene Editing

Prime Editing Strategy to Install the PRPH2

c.828+1G>A Mutation ���������������������������������������������������������������������������� 97
Salvatore Marco Caruso, Yi-Ting Tsai, Bruna Lopes da Costa,
Masha Kolesnikova, Laura A. Jenny, Stephen H. Tsang,
and Peter M. J. Quinn
Analysis of CRB1 Pathogenic Variants Correctable
with CRISPR Base and Prime Editing�������������������������������������������������� 103
Bruna Lopes da Costa, Laura A. Jenny, Irene H. Maumenee,
Stephen H. Tsang, and Peter M. J. Quinn
Generation of an Avian Myeloblastosis Virus (AMV)
Reverse Transcriptase Prime Editor������������������������������������������������������ 109
Yi-Ting Tsai, Bruna Lopes da Costa, Salvatore Marco Caruso,
Nicolas D. Nolan, Sarah R. Levi, Stephen H. Tsang,
and Peter M. J. Quinn
Contents xiii

Part IV Gene Therapy

Preexisting Neutralizing Antibodies against Different

Adeno-Associated Virus Serotypes in Humans
and Large Animal Models for Gene Therapy���������������������������������������� 117
Divya Ail and Deniz Dalkara
Optimization of Capillary-Based Western Blotting
for MYO7A ���������������������������������������������������������������������������������������������� 125
Kaitlyn R. Calabro, Sanford L. Boye, and Shannon E. Boye
AAV Serotypes and Their Suitability for Retinal
Gene Therapy ������������������������������������������������������������������������������������������ 131
Lynn J. A. Ebner and Christian Grimm
Gene Augmentation for Autosomal Dominant CRX-Associated
Retinopathies�������������������������������������������������������������������������������������������� 135
Chi Sun and Shiming Chen
 xnip Gene Therapy of Retinitis Pigmentosa Improves
T
Cone Health���������������������������������������������������������������������������������������������� 143
Yunlu Xue

Part V Human Retinal Degeneration

Factors Affecting Readthrough of Natural Versus

Premature Termination Codons ������������������������������������������������������������ 149
Avigail Beryozkin, Kerstin Nagel-Wolfum, Eyal Banin,
and Dror Sharon
Integrating Computational Approaches to Predict
the Effect of Genetic Variants on Protein Stability
in Retinal Degenerative Disease�������������������������������������������������������������� 157
Michelle Grunin, Ellen Palmer, Sarah de Jong, Bowen Jin,
David Rinker, Christopher Moth, John A. Capra,
Jonathan L. Haines, William S. Bush, and Anneke I. den Hollander
 etwork Biology and Medicine to Rescue: Applications
N
for Retinal Disease Mechanisms and Therapy�������������������������������������� 165
Anupam K. Mondal and Anand Swaroop
Non-syndromic Retinal Degeneration Caused by Pathogenic
Variants in Joubert Syndrome Genes���������������������������������������������������� 173
Riccardo Sangermano, Egle Galdikaité-Braziené,
and Kinga M. Bujakowska
Exonic Variants that Affect Splicing – An Opportunity
for “Hidden” Mutations Causing Inherited Retinal Diseases�������������� 183
Yogapriya Sundaresan, Eyal Banin, and Dror Sharon
 nhanced S-cone Syndrome, a Mini-review������������������������������������������ 189
E
Yiyi Wang, Jessica Wong, Jacque L. Duncan, Austin Roorda,
and William S. Tuten
xiv Contents

Part VI Inflammation

 he Role of Microglia in Inherited Retinal Diseases���������������������������� 197

T
Asha Kumari and Shyamanga Borooah
CD68: Potential Contributor to Inflammation and RPE
Cell Dystrophy������������������������������������������������������������������������������������������ 207
Mayur Choudhary and Goldis Malek
 ene Expression of Clusterin, Tissue Inhibitor
G
of Metalloproteinase-1, and Their Receptors in Retinal
Pigment Epithelial Cells and Müller Glial Cells Is
Modulated by Inflammatory Stresses���������������������������������������������������� 215
Mengmei Zheng, Eun-Jin Lee, Shinwu Jeong,
and Cheryl Mae Craft

Part VII Mechanisms of Degeneration

 xonal Transport Defects in Retinal Ganglion Cell Diseases�������������� 223

A
Iskalen Cansu Topcu Okan, Fatma Ozdemir, and Cavit Agca
 onnexins Biology in the Pathophysiology of Retinal Diseases���������� 229
C
Alejandro Ponce-Mora, Andrea Yuste, Giuliana Perini-Villanueva,
María Miranda, and Eloy Bejarano
 ole of Nuclear NAD+ in Retinal Homeostasis�������������������������������������� 235
R
Emily E. Brown, Michael J. Scandura, and Eric Pierce
Retinal Pigmented Epithelium-­Derived Ectopic Norrin Does
Not Promote Intraretinal Angiogenesis in Transgenic Mice���������������� 241
Andrea E. Dillinger and Ernst R. Tamm
Caveolin-1 in Müller Glia Exists as Heat-Resistant, High
Molecular Weight Complexes ���������������������������������������������������������������� 249
Eric N. Enyong, Jami Gurley, Virginie Sjoelung,
and Michael H. Elliott
 ole of VLC-PUFAs in Retinal and Macular Degeneration���������������� 257
R
Aruna Gorusupudi, Uzoamaka Nwagbo, and Paul S. Bernstein
Ocular Amyloid, Condensates, and Aggregates – Higher-Order
Protein Assemblies Participate in Both Retinal Degeneration
and Function�������������������������������������������������������������������������������������������� 263
Michael H. Hayes, DaNae R. Woodard, and John D. Hulleman
 hotoreceptor Ion Channels in Signaling and Disease������������������������ 269
P
Shivangi M. Inamdar, Colten K. Lankford, and Sheila A. Baker
The Role of Peripherin-2/ROM1 Complexes in Photoreceptor
Outer Segment Disc Morphogenesis������������������������������������������������������ 277
Tylor R. Lewis, Muayyad R. Al-Ubaidi, Muna I. Naash,
and Vadim Y. Arshavsky
Contents xv

Human Mutations in Arl3, a Small GTPase Involved

in Lipidated Cargo Delivery to the Cilia, Cause
Retinal Dystrophy������������������������������������������������������������������������������������ 283
Amanda M. Travis and Jillian N. Pearring
Genotype–Phenotype Association in ABCA4-Associated
Retinopathy���������������������������������������������������������������������������������������������� 289
Maximilian Pfau, Wadih M. Zein, Laryssa A. Huryn,
Catherine A. Cukras, Brett G. Jeffrey, Robert B. Hufnagel,
and Brian P. Brooks
Retinal Pathoconnectomics: A Window into
Neurodegeneration���������������������������������������������������������������������������������� 297
Rebecca L. Pfeiffer and Bryan W. Jones
The Role of Ceramide in Inherited Retinal
Disease Pathology������������������������������������������������������������������������������������ 303
Xinye Qian, Tanmay Srinivasan, Jessica He, and Rui Chen
 xtracellular Matrix: The Unexplored Aspects
E
of Retinal Pathologies and Regeneration���������������������������������������������� 309
Dmitri Serjanov and David R. Hyde
Role of TFEB in Diseases Associated with
Lysosomal Dysfunction���������������������������������������������������������������������������� 319
Hsuan-Yeh Pan and Mallika Valapala
Retinoic Acid Receptor-Related Orphan Receptors
(RORs) in Eye Development and Disease���������������������������������������������� 327
Felix Yemanyi, Kiran Bora, Alexandra K. Blomfield,
and Jing Chen

Part VIII Mechanisms of Degeneration – Animal Models

A Novel Mouse Model for Late-­Onset Retinal Degeneration

(L-ORD) Develops RPE Abnormalities Due
to the Loss of C1qtnf5/Ctrp5�������������������������������������������������������������������� 335
Shyamanga Borooah, Anil Chekuri, Shikha Pachauri,
Bhubananda Sahu, Marina Vorochikhina, John J. Suk,
Dirk-­Uwe Bartsch, Venkata R. M. Chavali, Monica M. Jablonski,
and Radha Ayyagari
 omparison of Mouse Models of Autosomal Dominant Retinitis
C
Pigmentosa Due to the P23H Mutation of Rhodopsin�������������������������� 341
Shannon R. Barwick and Sylvia B. Smith
Compensatory Cone-Mediated Mechanisms in Inherited
Retinal Degeneration Mouse Models: A Functional
and Gene Expression Analysis���������������������������������������������������������������� 347
Alicia A. Brunet, David M. Hunt, Carla Mellough,
Alan R. Harvey, and Livia S. Carvalho
xvi Contents

Inhibition of Ryanodine Receptor 1 Reduces Endoplasmic

Reticulum (ER) Stress and Promotes ER Protein
Degradation in Cyclic Nucleotide-Gated
Channel Deficiency���������������������������������������������������������������������������������� 353
Fan Yang, Hongwei Ma, Rekha Garg, Alfred Lewin,
and Xi-Qin Ding
 ouse Choroid Proteome Revisited: Focus on Aging�������������������������� 359
M
Donita Garland, James Harnly, and Radha Ayyagari
Morphological and Functional Comparison
of Mice Models for Retinitis Pigmentosa ���������������������������������������������� 365
Prakadeeswari Gopalakrishnan, Avigail Beryozkin,
Eyal Banin, and Dror Sharon
 urrent Advancements in Mouse Models of Retinal Disease�������������� 371
C
T. J. Hollingsworth, Xiangdi Wang, Raven N. Simpson,
William A. White, Robert W. Williams,
and Monica M. Jablonski
Single-Cell Transcriptomic Profiling of Müller
Glia in the rd10 Retina���������������������������������������������������������������������������� 377
Duygu Sigurdsson and Christian Grimm

Methods for In Vivo Characterization of Proteostasis
in the Mouse Retina �������������������������������������������������������������������������������� 383
Yixiao Wang and Ekaterina S. Lobanova
Absence of PRCD Leads to Dysregulation in Lipid
Homeostasis Resulting in Disorganization
of Photoreceptor Outer Segment Structure������������������������������������������ 389
Sree I. Motipally and Saravanan Kolandaivelu

Expansion Microscopy of Mouse Photoreceptor Cilia ������������������������ 395
Abigail R. Moyel, Michael A. Robichaux, and Theodore Wensel
Rod Photoreceptor-Specific Ablation of Metformin Target,
AMPK, in a Preclinical Model of Autosomal
Recessive Retinitis Pigmentosa �������������������������������������������������������������� 403
Nicholas D. Nolan, Laura A. Jenny, Stephen H. Tsang,
and Xuan Cui
TLR2 Is Highly Overexpressed in Retinal Myeloid
Cells in the rd10 Mouse Model of Retinitis Pigmentosa ���������������������� 409
Alonso Sánchez-Cruz, Enrique J. de la Rosa,
and Catalina Hernández-Sánchez
Environmental Light Has an Essential Effect
on the Disease Expression in a Dominant RPE65 Mutation���������������� 415
Wenjing Wu, Yusuke Takahashi, Xiang Ma, Gennadiy Moiseyev,
and Jian-Xing Ma
Contents xvii

Microglia Preserve Visual Function in a Mouse Model

of Retinitis Pigmentosa with Rhodopsin-P23H Mutant ���������������������� 421
Chen Yu and Daniel R. Saban

Part IX Mechanisms of Degeneration – Metabolism

Measuring the Release of Lactate from Wild-Type

and rd1 Mouse Retina������������������������������������������������������������������������������ 429
Yiyi Chen, Laimdota Zizmare, Christoph Trautwein,
and François Paquet-Durand
Aerobic Glycolysis in Photoreceptors Supports Energy
Demand in the Absence of Mitochondrial Coupling���������������������������� 435
Daniel T. Hass, Celia M. Bisbach, Martin Sadilek,
Ian R. Sweet, and James B. Hurley

Redox Status in Retinitis Pigmentosa���������������������������������������������������� 443
L. Olivares-González, S. Velasco, I. Campillo, J. M. Millán,
and R. Rodrigo
Perspectives on Retinal Dolichol Metabolism,
and Visual Deficits in Dolichol Metabolism-Associated
Inherited Disorders���������������������������������������������������������������������������������� 449
Sriganesh Ramachandra Rao, Steven J. Pittler,
and Steven J. Fliesler
Retinal Metabolic Profile on IMPG2 Deficiency Mice
with Subretinal Lesions �������������������������������������������������������������������������� 457
Rong Xu, Yekai Wang, Jianhai Du, and Ezequiel M. Salido

Part X Neuroprotection

Glutathione Coating of Liposomes Enhances the Delivery

of Hydrophilic Cargo to the Inner Nuclear Layer
in Retinal Cultures ���������������������������������������������������������������������������������� 467
Gustav Christensen and François Paquet-Durand
Modification of Müller Glial Cell Fate and Proliferation
with the Use of Small Molecules ������������������������������������������������������������ 473
Marcus J. Hooper
A Potential Neuroprotective Role for Pyruvate Kinase
2 in Retinal Degeneration������������������������������������������������������������������������ 479
Jiaming Zhou, Michel Rasmussen, and Per Ekström

Part XI Photoreceptors

Critical Role of VEGF as a Direct Regulator

of Photoreceptor Function���������������������������������������������������������������������� 487
Jianyan Hu, Meili Zhu, Dai Li, Qiang Wu, and Yun-Zheng Le
xviii Contents


Lysine Ubiquitylation Drives Rhodopsin Protein Turnover���������������� 493
Allen P. F. Chen, Leon Chea, Eun-Jin Lee,
and Jonathan H. Lin
 Silico Prediction of MYO1C-­Rhodopsin Interactions
In
and Its Significance in Protein Localization
and Visual Function �������������������������������������������������������������������������������� 499
Glenn P. Lobo, Rakesh Radhakrishnan, Matthias Leung,
Andrew Gruesen, Hans-­Joachim Knölker, Frederik J. van Kuijk,
and Sandra R. Montezuma
A Ciliary Branched Actin Network Drives
Photoreceptor Disc Morphogenesis�������������������������������������������������������� 507
William J. Spencer and Vadim Y. Arshavsky

Part XII RPE


Revisiting the Daily Timing of POS Phagocytosis�������������������������������� 515
Antonio E. Paniagua, Harjas S. Sabharwal, Kausalya Kethu,
Andrew W. Chang, and David S. Williams
Inhibition of Bacterial Peptidoglycan Cytopathy
by Retina Pigment Epithelial PGRP2 Amidase������������������������������������ 521
Marlyn P. Langford, Laura A. Perilloux-Lyons,
and A. Scott Kavanaugh
Understanding Ischemic Retinopathies: The Role
of Succinate and Its Receptor in Retinal
Pigment Epithelium �������������������������������������������������������������������������������� 527
Bilge Esin Ozturk

The Amphipathic Helix in Visual Cycle Proteins: A Review���������������� 533
Sheetal Uppal, Eugenia Poliakov, Susan Gentleman,
and T. Michael Redmond

The Retinal Pigment Epithelium: Cells That Know the Beat!������������ 539
Elora M. Vanoni and Emeline F. Nandrot

Part XIII Stem Cell Models and Therapies

Retinal Organoids: A Human Model System

for Development, Diseases, and Therapies�������������������������������������������� 549
Sangeetha Kandoi and Deepak A. Lamba

Modeling Retinitis Pigmentosa with Patient-Derived iPSCs �������������� 555
Yeh Chwan Leong and Jane C. Sowden

Primary Retinal Cell Cultures as a Model to Study
Retina Biology������������������������������������������������������������������������������������������ 565
Germán A. Michelis, Luis E. Politi, and S. Patricia Becerra
Contents xix

Generation of CRB1 RP Patient-­Derived iPSCs

and a CRISPR/Cas9-­Mediated Homology-Directed
Repair Strategy for the CRB1 c.2480G>T Mutation���������������������������� 571
Bruna Lopes da Costa, Yao Li, Sarah R. Levi, Stephen H. Tsang,
and Peter M. J. Quinn
Inducing Neural Regeneration from Glia Using Proneural
bHLH Transcription Factors������������������������������������������������������������������ 577
Levi Todd

Index�������������������������������������������������������������������������������������������������������� 583
 Part I
Age-related Macular Degeneration
 High-Resolution Imaging Mass
Spectrometry of Human Donor Eye:
Photoreceptors Cells and Basal
Laminar Deposit of Age-­Related
Macular Degeneration

David M. G. Anderson, Ankita Kotnala,

Jeffrey D. Messinger, Nathan Heath Patterson,
Jeffrey M. Spraggins, Christine A. Curcio,
Richard M. Caprioli, and Kevin L. Schey

Abstract nohistochemistry. Although both techniques

Pathologies of the retina are clinically visual-
ized in vivo with OCT and ex vivo with immu-
D. M. G. Anderson · N. H. Patterson · R. M. Caprioli
· K. L. Schey (*)
Department of Biochemistry, Vanderbilt University,
Nashville, TN, USA
Mass Spectrometry Research Center, Vanderbilt
University School of Medicine, Nashville, TN, USA
e-mail: k.schey@vanderbilt.edu
A. Kotnala
Department of Biochemistry, Vanderbilt University,
Nashville, TN, USA
Department of Ophthalmology and Visual Sciences,
University of Alabama at Birmingham,
Birmingham, AL, USA
J. D. Messinger · C. A. Curcio
Department of Ophthalmology and Visual Sciences,
University of Alabama at Birmingham,
Birmingham, AL, USA
J. M. Spraggins
Mass Spectrometry Research Center, Vanderbilt
University School of Medicine, Nashville, TN, USA
Department of Ophthalmology and Visual Sciences,
University of Alabama at Birmingham,
Birmingham, AL, USA
Department of Cell and Developmental Biology,
Vanderbilt University School of Medicine,
Nashville, TN, USA
provide valuable information on prognosis
and disease state, a comprehensive method for
fully elucidating molecular constituents pres-
ent in locations of interest is desirable. The
purpose of this work was to use multimodal
imaging technologies to localize the vast num-
ber of molecular species observed with
matrix-assisted laser desorption ionization
imaging mass spectrometry (MALDI IMS) in
aged and diseased retinal tissues. Herein,
MALDI IMS was utilized to observe molecu-
lar species that reside in photoreceptor cells
and also a basal laminar deposit from two
human donor eyes. The molecular species
observed to accumulate in these discrete
regions can be further identified and studied to
attempt to gain a greater understanding of bio-
logical processes occurring in debilitating eye
diseases such as age-related macular degener-
ation (AMD).
Keywords
Age-related macular degeneration · Macula ·
Retinal pigment epithelium · Photoreceptors ·
Basal lamina deposit · MALDI IMS

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 3

J. D. Ash et al. (eds.), Retinal Degenerative Diseases XIX, Advances in Experimental Medicine
and Biology 1415, https://doi.org/10.1007/978-3-031-27681-1_1
4 D. M. G. Anderson et al.
1 Introduction
Matrix-assisted laser desorption ionization
imaging mass spectrometry (MALDI IMS) can
localize and display the tissue distributions of
hundreds to thousands of molecules, at cellular
resolution, without the need for antibodies or
radioisotopes [1]. With effective co-registration
to multimodal optical imaging and optical
coherence topography (OCT) microscopy, these
distributions can be accurately correlated to
very small histological features of the neural
retina and retinal pigment epithelium (RPE).
MALDI IMS methods have been used to exam-
ine eye tissues including the retina [2–4], optic
nerve [4–6], lens [7, 8], and cornea [9]. Distinct
cell and synaptic layers of the retina have unique
layer-specific lipid and metabolite signatures
distinguished by IMS [4, 10, 11]. By applying
multimodal optical imaging technologies with
accurate registration and incorporating data-rich
IMS images [12], cellular and subcellular local-
ization of specific molecules informative to cell-
specific biochemistry can be observed. Human
retinal lipid composition studies have been per-
formed in the past. The results, while valuable,
provide limited information on cellular origin,
as experiments require dissections followed by
solvent extractions. MALDI IMS offers a
“molecular microscope” that localizes tissue
components in situ by molecular weights [11],
simultaneously providing hundreds to thou-
sands of spatially resolved signals. In this study,
we used a newly developed method of high-
accuracy registration [12] to co-­ register high
spatial resolution IMS images with OCT auto-
fluorescence and histological images of the
same tissue to examine subcellular localizations
and molecular features of photoreceptors and
AMD pathology.
2 Methods
This section has been summarized from Anderson
et al. [3]; for detailed explanations, please see
this reference.
2.1 Tissue Acquisition
and Characterization
Whole eyes were obtained from deceased human
donors via Advancing Sight Network (formerly
the Alabama Eye Bank) by the UAB authors.
2.2 Tissue Handling and Ex Vivo
Imaging
Methods were optimized for multimodal ex vivo
clinical imaging of the ocular fundus [13]. Globes
with lens and iris in place were immersed in buff-
ered 4% paraformaldehyde overnight. Iris and
lens were removed before imaging.
For imaging with OCT and scanning laser
ophthalmoscopy, globes were immersed in buffer
facing frontward within a custom-built chamber
with a 60-diopter lens [13]. Spectral domain
OCT images were captured with a Spectralis
(HRA&OCT, HRA2; Heidelberg Engineering).
Tissues were embedded in 2.5% carboxy-
methyl cellulose (Sigma C9481), and serial
10 μm cryosections were collected on Superfrost
glass slides and on large, 45 × 45 mm in-house,
polylysine-coated indium-tin-oxide (ITO) slides
(Delta Technologies Loveland, CO, USA).
2.3 MALDI IMS Analysis
The matrices 2,5-dihydroxyacetophenone (DHA)
and 1,5-diaminonaphthalene (DAN) (Sigma
Aldrich, St. Louis, MO, USA) were applied to
tissue sections by sublimation [14]. MALDI IMS
data were acquired with a laser spot size of
10–15 μm in full scan mode using a Bruker
SolariX 9.4T FTICR mass spectrometer (Bruker
Daltonics Billerica, MA, USA). Following data
acquisition, an advanced image registration
workflow [12] was performed. More detailed
information of the image registration process can
be found in publications by Patterson et al. [12]
and Anderson et al. [3]. Molecular identifications
were made using LC-MS of chloroform-­methanol
extracts from adjacent tissue sections.
High-Resolution Imaging Mass Spectrometry of Human Donor Eye: Photoreceptors Cells and Basal…
3 Results
3.1 Signals Specific
to Photoreceptors and RPE
Figure 1a shows MALDI IMS and optical micros-
copy focusing on photoreceptors and their
­support cells. The RPE sends delicate processes
in the apical direction to contact photoreceptor
outer segments, near the RPE cell body for rods
and 10–15 μm above the cell body, to contact
cone outer segments, which are shorter. Figure 1a
is color-coded depiction of photoreceptor and
RPE compartments associated with IMS signals
in Fig. 1b (blue ONL, red inner segments, yellow
outer segments, green RPE).
Figure 1b shows MALDI IMS images over-
laid with H&E images from this donor. The pho-
toreceptors on the left side of the image are
attached to the RPE and detached from the RPE
on the right side, a common artifact which can
occur during sample preparation. In Fig. 1a, the
signal at m/z 818.575 was observed with high
abundance in the ONL and was identified as
PE(20:0_22:6) (blue). This region is comprised
of the photoreceptor cell bodies and processes of
Müller (radial) glia. A highly localized signal can
be observed with high abundance along a narrow
band aligned with photoreceptor inner segments
at m/z 1426.0 (red). This signal was identified as
Fig. 1 MALDI IMS signals consistent with localization
to photoreceptor and RPE compartments. (a) Schematic
diagram of outer retina, excerpted from Fig. 1a. Blue,
pink, yellow, and green bands indicate layers formed by
highly compartmentalized and vertically aligned photore-
ceptors and RPE cells in panels b, c. Layers: OPL outer
plexiform layer, ONL outer nuclear layer, ELM external
limiting membrane, RPE retinal pigment epithelium, BrM
Bruch’s membrane, R rod, C cone photoreceptors. (b–f)
5
a cardiolipin CL(70:5). Cardiolipins are highly
abundant in mitochondria, which are abundant in
the ellipsoid portion of photoreceptor inner seg-
ments. At the distal part of the photoreceptor
cells, outer segments are highly interleaved with
apical processes of RPE cells. A DHA-containing
PE(18:0_22:6) is observed at m/z 790.539 (yel-
low) in panel D which can be observed with high
abundance in the outer segments, while a signal
observed at m/z 728.596 (green) is localized
above and within the RPE.
3.2 Signals Specific to Basal
Laminar Deposit
Figure 2 shows multimodal imaging of a 93-year-­
old donor tissue with the imaging modalities
separated into panels. Figure 2a displays ex vivo
OCT hyperreflective foci (yellow arrowhead) and
an RPE elevation (green arrowhead) near the
fovea. Figure 2b shows that retinal layers are vis-
ible in H&E-stained sections after IMS data
acquisition. The inset magnifies BLamD (PASH
staining of an alternate section), clearly indicat-
ing thickened extracellular matrix between the
RPE plasma membrane and its native basal lam-
ina. Figure 2c shows autofluorescence of the ele-
vated RPE layer and anteriorly migrated RPE
cells, which account for high-risk-indicating
MALDI IMS images and H&E-stained tissue images
overlaid in perifoveal retina displaying signals from mul-
tiple lipid classes that localize to subcellular compart-
ments of the photoreceptor cells. (b) Overlay showing
four separate signals. (c) Localized to ONL. (d) Localized
to photoreceptor inner and outer segments. (e) Localized
to mitochondria-rich photoreceptor inner segments. (f)
Localized to RPE apical processes
6 D. M. G. Anderson et al.
Fig. 2 Imaging mass spectrometry (IMS) for molecularly
informed optical coherence tomography (OCT) and
tissue-­level target discovery. Asterisk, foveal pit; RPE,
retinal pigment epithelium. Color-coded arrowheads rep-
resent corresponding structures across modalities, in a
93-year-old human donor eye. (a) OCT B-scan shows
subretinal hyperreflective material (yellow) and an RPE
elevation (green). (b) H&E stained cryosection shows
hyperreflective foci of clinical OCT. Figure 2d
shows that a sphingomyelin-related lipid
(PE-Cer-NMe2(42:1)) at m/z 799.671 [15] is
highly abundant and localizes to BLamD and
RPE, building on previous histochemical and
chromatography findings of lipids in this deposit
[16, 17].
pigmented debris (yellow) and dysmorphic RPE overly-
ing BLamD. Insert, BLamD with basal mound (arrow).
Basal mounds contain soft druse material. Layers: GCL
ganglion cell, INL inner nuclear, HFL Henle fiber, ONL
outer nuclear. (c) Autofluorescent, pigmented debris (yel-
low) and dysmorphic RPE (green). (d) IMS reveals an m/z
signal at 799.673, restricted to BLamD and not detected in
the RPE
4 Conclusions
MALDI IMS combined with multimodal imag-
ing methods and ex vivo OCT provides a power-
ful tool to elucidate the molecular composition
and localization of molecular species in key
regions and pathology associated with ocular dis-
High-Resolution Imaging Mass Spectrometry of Human Donor Eye: Photoreceptors Cells and Basal…
ease. Understanding molecular processes occur-
ring in BLamD in early AMD is important as
they are early-identified histologic risk factors
for AMD progression [18] and are just now being
recognized clinically [19, 20].
Acknowledgments This project was supported by the
National Institutes of Health P41 GM103391 (R.M.C.)
and R01 EY027948 (C.A.C.). Support was also received
by a Research to Prevent Blindness Catalyst Award for
Innovative Research Approaches for Age-Related Macular
Degeneration to K.L.S.
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 The Noncanonical Role
of Complement Factor H in Retinal
Pigment Epithelium (RPE) Cells
and Implications for Age-Related
Macular Degeneration (AMD)

Angela Armento, David Adrian Merle,

and Marius Ueffing

Abstract pathway of the complement system, and the

Age-related macular degeneration (AMD) is a
complex degenerative disease of the retina.
Dysfunction of the retinal pigment epithelium
(RPE) occurs in early stages of AMD, and
progressive RPE atrophy leads to photorecep-
tor death and visual impairments that ulti-
mately manifest as geographic atrophy (GA),
one of the late-stage forms of AMD. AMD is
caused by a combination of risk factors includ-
ing aging, lifestyle choices, and genetic pre-
disposition. A gene variant in the complement
factor H gene (CFH) that leads to the Y402H
polymorphism in the factor H protein (FH)
confers the second highest risk for the devel-
opment and progression of AMD. FH is a
major negative regulator of the alternative
FH Y402H variant leads to increased comple-
ment activation, which is detectable in AMD
patients. For this reason, various therapeutic
approaches targeting the complement system
have been developed, however, so far with
very limited or no efficacy. Interestingly,
recent studies suggest roles for FH beyond
complement regulation. Here, we will discuss
the noncanonical functions of FH in RPE cells
and highlight the potential implications of
those functions for future therapeutic
approaches.
Keywords
Age-related macular degeneration (AMD) ·
Retinal pigment epithelium (RPE) cells ·
Intracellular complement factor H (CFH)

A. Armento (*) · M. Ueffing

Institute for Ophthalmic Research, Department for
Ophthalmology, Eberhard Karls University of
Tübingen, Tübingen, Germany
e-mail: angela.armento@uni-tuebingen.de;
marius.ueffing@uni-tuebingen.de
D. A. Merle
Institute for Ophthalmic Research, Department for
Ophthalmology, Eberhard Karls University of
Tübingen, Tübingen, Germany
Department of Ophthalmology, Medical University of
Graz, Graz, Austria
e-mail: david.merle@medunigraz.at
1 Introduction
Age-related macular degeneration (AMD) is a
complex and progressive disease of the macula,
which affects mainly the elderly population,
especially in the developed countries [1, 2]. Due
to demographic changes in western societies,
AMD incidence is constantly rising, and the con-
comitant visual impairment threatens not only

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 9

J. D. Ash et al. (eds.), Retinal Degenerative Diseases XIX, Advances in Experimental Medicine
and Biology 1415, https://doi.org/10.1007/978-3-031-27681-1_2
10
the individual’s quality of life but also poses a
significant burden on healthcare systems world-
wide [3]. The early stages of the disease are char-
acterized by the presence of enlarging drusen in
the retina. With disease progression, early AMD
advance to late stages: wet or dry AMD. Wet
AMD is defined by the presence of neovascular-
ization in the retina, while dry AMD is limited to
progressive RPE and retinal atrophy, called geo-
graphic atrophy (GA), without newly formed
vessels. Around 10% of the cases constitute wet
AMD, and intravitreal anti-VEGF is effective in
treating most cases. However, only recently,
one approved treatment has become available for
dry AMD in the US [2, 8].
The main pathogenetic driver for AMD is
advanced age. Specific genetic factors can pre-
dispose for AMD or protect from it. Unhealthy
lifestyle habits, like smoking or malnutrition,
can further increase the risk for AMD. The com-
bination of those risk factors leads to pathologi-
cal changes in the retina, involving a plethora of
cellular mechanisms promoting disease pro-
gression [4].
2 Complement System in AMD
Among the processes involved in AMD, the com-
plement system is associated to AMD at different
levels. First of all, many single nucleotide poly-
morphisms (SNPs) associated with AMD cluster
into genes of the alternative pathway of the com-
plement system with the Complement Factor H
(CFH) gene carrying the second highest risk for
AMD [5]. The most common risk haplotype
causes an amino acid substitution from tyrosine
to histidine at position 402 (called Y402H) in
proteins coded by CFH, which are full length FH
and a truncated version, FHL-1 [6, 7]. The com-
plement system is a part of the innate immune
system designed to recognize and mediate the
removal of pathogens or waste material [8]. The
eye is an immune-privileged organ, meaning that
inflammation needs to be strictly limited, and
therefore uncontrolled complement activation is
deleterious [8]. FH is a major negative regulator
of the complement system, and the AMD variant
A. Armento et al.
402H of FH causes uncontrolled complement
activation in vitro and in vivo [9]. Moreover,
complement factors accumulate in drusen [10],
and increased complement system activation has
been detected in AMD patients, independent of
their genotype [11].
Due to the strong implications of complement
system activation in AMD pathology, various
complement inhibitors have been tested in clini-
cal trials. However, most agents that entered
phase II were discontinued due to a reported lack
of efficacy. Interestingly, in some clinical trials, a
group of subjects showed improvements, while
others did not [12]. Apparently, one of the sus-
pected reasons for the observed lack of efficacy is
a lack of patient’s stratification and identification
of those patients that are likely to benefit from
complement inhibition. Moreover, several stud-
ies point to a lack of understanding of the role of
FH in AMD pathology, especially in RPE cells,
which are degenerating in AMD.
3 Noncanonical Role
of Complement Factor H
in RPE Cells
RPE cells exert several functions vital for retinal
homeostasis, which are impaired in AMD [13].
First of all, RPE cells are responsible for phago-
cytosis of shed photoreceptor outer segments
(POS) and recycling of visual pigments, needed
for a proper visual cycle. This process is energy
intensive, requiring constant mitochondrial res-
piration and glycolysis within RPE cells. Due to
a lack of the inner retina vasculature in the mac-
ula, RPE cells are vital to ascertain nutrient sup-
ply from the choriocapillaris. Any pathological
disruption or perturbance of metabolic activity
of RPE cells would affect the amount of nutri-
ents that can reach the neuroretina. Indeed, the
so-­called bioenergetics crisis of RPE cells has
been described for AMD pathology [14].
Moreover, RPE cells quench short UV light as
well as the continuous influx of peroxidized lip-
ids from phagocytosed photoreceptor outer seg-
ments and thus need a high degree of antioxidant
capacity as well as perfect functioning of their
The Noncanonical Role of Complement Factor H in Retinal Pigment Epithelium (RPE) Cells and…
mitochondria to protect the retina from exces-
sive oxidative stress.
Our recent work demonstrates that FH is
endogenously produced by RPE cells. Within
RPE cells, FH is acting locally supporting retinal
homeostasis [15, 16]. This function of FH is con-
text dependent, and we suggest that locally pro-
duced FH exerts specific functions at the
RPE/retina interface. Most of these newly dis-
covered functions (summarized in Fig. 1) affect a
balanced regulation of immune competence and
surveillance as well as regulating oxidative stress
response and energy metabolism, mechanisms
which are part of AMD pathology [8].
FH plays a protective role against oxidative
stress in RPE cells. Exogenously applied recom-
binant FH protects RPE cells from damage
induced by the exposure to lipid oxidation prod-
ucts [17, 18]. In parallel, loss of endogenous FH
in RPE cells, via CFH silencing, also led to
increased vulnerability to oxidative stress,
increased levels of oxidized lipids, and reduced
viability [15]. Interestingly, addition of recombi-
nant FH in CFH-silenced RPE cells did not cause
any rescuing effects [15]. This finding indicates
that, although exogenous FH is protective, a
proper intracellular FH function must be present
to ascertain physiological balance and resilience
to stress. This phenomenon may be explained by
impaired mitochondrial function seen in iPSC-­
RPE cells carrying the CFH Y402H polymor-
phism [19] and CFH-silenced hTERT-RPE1 cells
[15]. In both models, major processes involved in
energy production, glycolysis, and mitochondrial
respiration are impaired. In addition, a misbal-
ance in the degree of mitochondria turnover
seems to be involved, with an exaggerated
increase in mitophagy [15, 19]. Moreover, it has
been shown in iPSC-RPE carrying CFH 402H
variant that mitochondria are enlarged and accu-
mulating [20]. Mitochondria are essential for a
correct response to oxidative stress; therefore,
damage or dysfunction of these organelles is del-
eterious in such an oxidative milieu like the ret-
ina. Accumulation of oxidized lipids has been
reported in RPE cells under FH dysregulation
and induced oxidative stress, when FH function
was impaired as a consequence of CFH silencing
11
[15] or in the presence of the CFH 402H variant
[20]. Moreover, accumulation of lipids was
shown also in cfh Y402H mouse models of AMD,
especially when subjected to high cholesterol
diet [21].
The mechanism of action of intracellular FH
is not fully elucidated. Based on the data gener-
ated in iPSC-RPE cells carrying the CFH 402H
variant, it has been speculated that more than one
signaling pathway mediates the effects of FH
[19]. Specifically, it has been postulated that the
NF-kB pathway and mTOR pathway are likely
involved, in concomitance with autophagy and
proteasome activity dysregulation, which ulti-
mately lead to mitochondrial damage [19].
In our model of CFH-silenced hTERT-RPE1
cells, we could verify these hypotheses. Indeed,
we found that in absence of endogenous FH, both
the NF-kB and mTOR pathway are upregulated,
with NF-kB triggering pro-inflammatory cyto-
kine expression [22] and the mTOR pathway
modulating mitochondrial respiration, but not
glycolysis [23]. The mTOR pathway is a major
regulator of autophagy [24]. Dysfunction in the
autophagy-lysosomal axis in iPSC-RPE cells
with CFH high risk was reported but is not regu-
lated by this pathway. Its dysfunction rather
depends on the accumulation of complement
activation products [25]. Based on our analyses
of the intracellular FH protein interactions that
form a functional interactome in RPE cells, we
suggest a regulatory role of FH in interaction
with the ubiquitin proteasome system (UPS) and
RB1/E2F signaling, adding a new level of com-
plexity in the understanding of FH mechanism of
action [23]. Interestingly, and based on studies in
other cell types than RPE, it has already been
suggested that FH has a noncanonical intracellu-
lar function, independent from complement acti-
vation. In clear renal cell carcinoma cells and
kidney endothelial cells, FH knockdown impairs
cellular homeostasis, at least partially via NF-kB
activation [26, 27].
The synopsis of these findings indicates that
understanding the function of FH in a specific
cell and tissue context is key. In the context of
AMD, it is important to advance our understand-
ing on how RPE cells interact with the neuroret-
12
Fig. 1 Schematic representation of the noncanonical functions of FH in RPE cells
ina once intracellular FH dysregulation or a CFH
402H high risk variant perturbs intracellular
homeostasis within these cells. In this regard, we
have recently established a novel coculture model
of RPE and neuroretina. Using this system, we
show that CFH-silenced RPE cells promote reti-
nal degeneration, which could not be rescued by
the addition of exogenous FH. Moreover, we
showed that the damage occurred at the level of
mitochondrial activity and lipid oxidation in the
photoreceptors, rather than a canonical inflam-
mation or complement activation [16].
4 Future Perspective
Recent works of several groups, including ours,
emphasize a noncanonical and intracellular role
of FH in RPE cells. Besides their reduced capac-
ity to dampen alternative complement activity,
CFH risk polymorphisms such as Y402H impair
RPE homeostasis and are likely contributing to
promote AMD. In consequence, a simple comple-
A. Armento et al.
ment pathway inhibitor strategy to treat AMD
progression may not suffice. Rational re-­balancing
strategies in a form of combinatorial therapies
may be needed to target several drivers of AMD
molecular pathology in a systems-­oriented fash-
ion simultaneously. More specifically, the increase
of alternative complement pathway activity at the
Bruch’s membrane/RPE interface may have to be
considered concomitantly with pro-inflammatory,
oxidative stress-­ related perturbation and meta-
bolic disbalance of RPE and neuroretina to
achieve effective future therapeutic regimes for
dry AMD. Last but not least, a very thorough
stratification of patient groups may be needed to
move to an individualized and rational therapy for
dry AMD of the future.
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 Macular Pigment Carotenoids
and Bisretinoid A2E

Ranganathan Arunkumar and Paul S. Bernstein

Abstract Keywords

Lutein (L), zeaxanthin (Z), and meso-­ Abca4 · A2E · Antioxidant · Bisretinoids ·
zeaxanthin (MZ) are the three macular pig- Carotenoids · Lipofuscin · Macular pigments
ments (MP) carotenoids that uniquely · Photo-oxidation · Retina · Retinal pigment
accumulate in the macula lutea region of the epithelium
human retina. L and Z are obtained by humans
through dietary intake. The third MP, MZ, is
rarely present in diet, and its abundance in the 1 Introduction
human fovea is due to the metabolic conver-
sion of dietary L by the retinal pigment epithe- Carotenoids are natural pigments synthesized by
lium’s RPE65 enzyme. The major functions of photosynthetic bacteria, algae, yeast, and plants.
MP in ocular health are to filter high-intensity, L, Z, and MZ are oxygenated carotenoids (xan-
phototoxic blue light and to act as effective thophylls) obtained by humans through dietary
antioxidants for scavenging free radicals. The intake of leafy greens, fruits, and vegetables.
pyridinium bisretinoid, N-retinylidene-N-­ Among the 15 major dietary carotenoids detected
retinylethanolamine (A2E), contributes to in human serum, these three xanthophyll carot-
drusen formation in dry age-related macular enoids selectively accumulate with a unique dis-
degeneration (AMD) and to the autofluores- tribution in the macular region of human retina
cent flecks in autosomal recessive Stargardt [1]. The MP’s total concentration is about 1 mM
disease (STGD1). Retinal carotenoids attenu- at the fovea, and its concentration declines to less
ate A2E formation and can directly and indi- than 10 μM in the peripheral retina [2]. The ratio
rectly alleviate A2E-mediated oxidative of L:Z:MZ in the peripheral retina is about 3:1:0,
damage. In this chapter, we review these more and the concentration of total carotenoids rises
recently recognized interconnections between 100-fold in the macula lutea with a change in the
MP carotenoids and A2E bisretinoids. ratio of L:Z:MZ of about 1:1:1, as measured by
high-performance liquid chromatography
(HPLC) [3]. More recently, high-resolution con-
R. Arunkumar · P. S. Bernstein (*) focal resonance Raman microscopy imaging of
Department of Ophthalmology and Visual Sciences, the human retina from our laboratory has showed
John A. Moran Eye Center, University of Utah that the ratio of Z + MZ:L can be greater than 9:1
School of Medicine, Salt Lake City, UT, USA at the foveal center, while L is more diffusely dis-
e-mail: paul.bernstein@hsc.utah.edu

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 15

J. D. Ash et al. (eds.), Retinal Degenerative Diseases XIX, Advances in Experimental Medicine
and Biology 1415, https://doi.org/10.1007/978-3-031-27681-1_3
16 R. Arunkumar and P. S. Bernstein

tributed across the macula at a relatively lower aided by conjugated double-bond structure with
concentration [4]. Retinal carotenoids mainly the hydrophilic group on the ionone ring [2]. The
localize to the outer plexiform (Henle fiber) layer absorption maximum of L is around 445 nm, and
and the inner plexiform layer, with axial exten- Z is around 450 nm, corresponding with the haz-
sion from the inner limiting membrane to the ardous blue light range of 430–500 nm.
outer limiting membrane. The specificity in the Depending on the retinal carotenoid concentra-
distribution of retinal carotenoids in the human tion, they are estimated to absorb 40–90% of
retina may be due to the selective uptake of reti- incident short-wavelength, high-energy photo-
nal carotenoids by carotenoid binding proteins toxic blue light [8].
StARD3 (L binding protein) and GSTP1 (Z and Retinal carotenoids with conjugated double
MZ binding protein). The other major factors that bond (C=C) structures are associated with greater
influence the MP carotenoid distributions and singlet oxygen quenching. Z has 11 conjugated
concentrations between individuals are the double bonds and is 50% better at quenching sin-
amount of oral intake of carotenoids and their glet oxygen relative to L with its 10-conjugated
bioavailability [5]. L is present in higher concen- double bonds [9]. MZ, a stereoisomer of Z, with
trations in the diet, but it is converted to MZ by an identical 11-conjugated double-bond structure
the RPE65 enzyme, and the preferential accumu- would be expected to possess the same antioxi-
lation of Z and MZ at the foveal center may be dant properties as Z, but it exhibited better singlet
due to their higher antioxidant potential relative oxygen quenching properties relative to L and Z
to L. The fovea is prone to photo-damage caused [9]. Furthermore, the antioxidant activity of all
by light-induced oxidative stress from reactive three retinal carotenoids as a mixture showed bet-
oxygen species, and singlet oxygen can be gener- ter singlet oxygen quenching ability than any of
ated by photo-oxidation of A2E and other bisreti- the individual MP carotenoids. Retinal carot-
noid components of lipofuscin [6]. A2E acts as a enoids not only quench singlet oxygen, but they
photosensitizer in the presence of short-­ also quench superoxide anion radicals and
wavelength blue light and oxygen that generate hydroxyl radicals, the major causative agents of
reactive oxygen caused by photo-oxidation and lipid peroxidation and light-induced damage [10,
photo-degradation, resulting in retinal degenera- 11]. Generally, retinal carotenoids are better
tion and apoptosis of photoreceptor and RPE hydroxyl radical scavengers than superoxide
cells [7]. Supplementation with retinal carot- anion scavengers, and Z exhibits better hydroxyl
enoids potentially attenuates harmful blue light radical scavenging activity than lutein [10]. MP
and effectively scavenges singlet oxygen and carotenoids protect the retina, which is vulnera-
reactive free radicals in ocular tissues and in bio- ble to oxidative damage caused by exposure to
chemical assays. light, high concentration of oxygen, abundant
photosensitizers, and high concentrations of
polyunsaturated fatty acids (PUFAs).
2 Structure and Function
of the Retinal Carotenoids
3 A2E Bisretinoids
Retinal carotenoids are characterized by the pres-
ence of hydroxyl (O-H) functional groups In the visual cycle, the activation of rhodopsin by
attached at the 3 and 3′ positions of terminal ion- a photon of light during phototransduction results
one rings connected by an isoprenoid backbone in the isomerization of 11-cis-retinal to all-trans-­
structure (Fig. 1). The presence of hydroxyl retinal in photoreceptor outer segments and the
groups and the conjugated double bonds struc- release of all-trans-retinal, which is subsequently
ture determine the light-absorbing properties and reduced to all-trans-retinol and transported to the
antioxidant activities of retinal carotenoids. The RPE cells. In the RPE, the all-trans-retinol is
light filtering function of retinal carotenoids is either stored as a fatty acid ester or is converted
Macular Pigment Carotenoids and Bisretinoid A2E
HO
Lutein
HO
Zeaxanthin
HO
Meso-Zeaxanthin
Fig. 1 Structures of the macular carotenoids: lutein (L), zeaxanthin (Z), meso-zeaxanthin (MZ), and N-retinylidene-N-­
retinylethanolamine (A2E)
back to an 11-cis-retinoid that is transported back
to the photoreceptor outer segment, which can
bind to opsin to produce a regenerated visual pig-
ment. Some all-trans-retinal in the photoreceptor
outer segments reacts with phosphatidylethanol-
amine (PE) to form N-retinylidene-PE (NRPE),
which is transported or “flipped” by the ATP-­
binding cassette, subfamily A, member 4
(ABCA4) protein present in photoreceptor outer
segments. ABCA4 translocates NRPE across the
lipid bilayer from the luminal side to the cyto-
plasmic side of the disc membrane. NRPE
released on the cytoplasmic side is hydrolyzed
into all-trans-retinal and reduced to all-trans-­
retinol by retinol dehydrogenases (RDHs) [12]. If
ABCA4 does not effectively translocate NRPE, it
reacts with one more molecule of all-trans-­retinal
to form a toxic bisretinoid, dihydropyridinium-­
A2PE. Dihydropyridinium-A2PE is further
oxidized either to A2-dihydropyridine-PE
­ loss of membrane integrity and perturbs mem-
(A2-DHP-PE) by eliminating one hydrogen or to
phosphatidylethanolamine-pyridinium bisreti-
noid (A2PE) by eliminating two hydrogens in the
acidic and oxidizing environment (Fig. 2). During
phagocytosis of photoreceptor outer segments by
17
OH
OH
OH
N
OH
A2E
RPE, A2PE is further hydrolyzed to A2E by
phospholipase D inside RPE phagosomes.
Mutation or dysfunction in the ABCA4 gene
leads to excessive accumulation of A2E and other
bisretinoids in RPE lipofuscin, resulting in reti-
nal cell death and vision loss. A2E is reported to
accumulate with aging and dry AMD and in
STGD1 [13].
A2E (C42H58ON; molecular weight, 592) is a
well-characterized component of lipofuscin. It
exhibits absorbance in both the UV and visible
regions of the spectrum [A2E: absorbance max-
ima (λmax) are 440 and 340 nm, and iso-A2E’s
λmax are 430 and 340 nm]. A2E’s hydrophobic
side arms and positively charged hydrophilic
head group confer an amphiphilic structure
(Fig. 1). A2E has a detergent-like structure that
may influence membrane properties and inhibit
the lysosomal degradation of lipids. A2E induces
brane stability, and it inhibits cytochrome C oxy-
genase and the ATP-driven proton pump. A2E
photo-oxidation products can activate the com-
plement system and cause inflammation and
DNA damage [12, 13].
18
Fig. 2 The visual cycle and bisretinoid formation in pho-
toreceptor outer segments and RPE. When a photon of
light is captured by the visual chromophore, its 11-cis-­
retinal (11-cis-RAL) Schiff base chromophore photoi-
somerizes to all-trans-retinal (all-trans-RAL). The
released all-trans-RAL from opsin is reduced to all-trans-­
retinol (all-trans-ROL) by retinol dehydrogenases
(RDHs). Alternatively, some all-trans-RAL reacts with
phosphatidylethanolamine (PE) in the photoreceptor outer
segments to form N-retinylidene-PE (NRPE), which is
transported by ABCA4. The bisretinoid synthesis path-
way (red) is initiated when NRPE, rather than hydrolyzing
to all-trans-ROL and PE, reacts with a second molecule of
retinaldehyde. A multistep pathway leads to the formation
of the intermediate dihydropyridinium-A2PE. Auto-­
oxidation of dihydropyridinium-A2PE with loss of two
4 Retinal Carotenoids
Attenuate Bisretinoids
in Various Systems
4.1 In Vitro
A2E and its precursor A2PE can be irradiated
with 430 nm blue light and analyzed by fast atom
bombardment mass spectroscopy (FAB-MS).
The FAB-MS spectra after irradiation not only
showed a molecular ion peak at m/z 592 and 1223
attributable to A2E and A2PE, but they also
R. Arunkumar and P. S. Bernstein
hydrogens generates A2PE, and loss of one hydrogen gen-
erates A2-DHP-PE; phosphate hydrolysis of the latter
produces A2-DHP-E. A2E, lysoA2PE, and A2-GPE are
produced from A2PE. Reaction of the all-trans-RAL
dimer with PE with the formation of a Schiff base linkage
generates all-trans-retinal dimer-PE (atRALdi-PE), and
phosphate hydrolysis of the latter yields all-trans-retinal
dimer-ethanolamine (atRALdi-E). All-trans-ROL is
transported into RPE cells where all-trans-ROL is esteri-
fied to fatty acids to make all-trans retinyl esters (all-­
trans-­RE) by the enzyme lecithin retinol acyl transferase
(LRAT). All-trans-RE is converted to 11-cis retinol
(11-cis-ROL) by the RPE65 isomerohydrolase. 11-cis
retinol dehydrogenase (11cRDH) oxidizes 11-cis-ROL to
11-cis-RAL which enters the photoreceptor outer seg-
ments for the regeneration of opsin
showed additional peaks at m/z 608, 624, 640,
656, 672, and 688, as well as 1239, 1255, 1271,
and 1286. The additional higher m/z peaks are
photo-oxidation products of A2E and A2PE
formed by the insertion of oxygen atoms at the
C=C bonds along the side arms of A2E and
A2PE. When A2E and A2PE are irradiated at
430 nm in the presence of L and Z, the additional
photo-oxidized peaks are absent [14]. Singlet
oxygen quenching abilities of retinal carotenoids
were studied in the presence of endoperoxide
1,4-dimethylnapthalene (1 mM), which releases
Macular Pigment Carotenoids and Bisretinoid A2E 19

singlet oxygen with a half-life time of 5 h. @ 3.1-fold in the zeaxanthin-supplemented

25 °C. The endoperoxide was incubated with
A2E (500 μM) with and without retinal carot-
enoids (500 μM to 4 mM). A2E oxidation was
measured using HPLC, and it was found that reti-
nal carotenoids effectively quench singlet oxygen
products in a concentration-dependent manner.
Furthermore, Z was observed to be a better sin-
glet oxygen quencher than L [14].
group. A2E levels increased sixfold in the
unsupplemented control group, whereas there
was minimal increase in A2E in the MP carot-
enoid supplemented group, and their A2E lev-
els were significantly lower than the control
group [16].
4.5 Rodent Model

4.2 Cell Culture Until recently, the protective effects of macular

carotenoids against bisretinoids had not been
The protective effects of retinal carotenoids on studied in small laboratory mammals because
photo-oxidation of A2E were studied in ARPE-­ non-primates do not accumulate substantial
19 cells grown in Dulbecco’s Modified Eagle amounts of carotenoids in ocular tissues due to
Medium (DMEM) medium with 10 μM A2E for the presence of very active carotenoid cleavage
10 days. The cells accumulated A2E and were enzymes (Bco1 and Bco2). This led us to cross
incubated with L or Z (10 μM), and they were our Bco2−/− “macular pigment mice” that accu-
then exposed to blue light (430 nm). Cells incu- mulate dietary MP carotenoids in their retinas
bated with retinal carotenoids completely attenu- with a mouse model of STGD that accumulates
ated A2E photo-oxidation and inhibited A2E with increasing age. Abca4−/−/Bco2−/− mice
proteasome inactivation [15]. fed with L or Z have lower levels of A2E and iso-­
A2E compared to the control group with no
carotenoid supplementation, and there was a sta-
4.3 Human Donor Eyes tistically significant inverse correlation between
retinal carotenoids and A2E and iso-A2E levels
A2E concentrations in human cadaver eyes in RPE/choroid (Fig. 3). Furthermore, L and Z
increase with age in both the macula and in the supplementation improved visual performance in
peripheral retina, and A2E levels are threefold Abca4−/−/Bco2−/− mice compared to the control
lower in the macula region compared to the group [13].
peripheral retina despite excess light exposure
and high metabolic activity. Moreover, A2E con-
centrations are inversely related to retinal carot- 5 Summary
enoid concentrations [16]. Lipofuscin and A2E
levels are significantly lower in the fovea region The macular pigment carotenoids are distinct
where retinal carotenoids are present in abun- from other nutrients due to their selective accu-
dance [17]. mulation and unique spatial distribution in the
fovea, the region most vulnerable to excess light
and high metabolic oxygen. While it is widely
4.4 Avian Model appreciated that the beneficial properties of MP
carotenoids are in part mediated by their blue
The association between retinal carotenoids light-absorbing and antioxidant properties, it is
and A2E was studied in Japanese quail, whose now becoming increasingly understood that
retinal carotenoids are present as fatty acid attenuation of A2E formation and oxidation by L,
esters in distinct photoreceptor oil drops. After Z, and MZ are important protective effects as
16 weeks of feeding, total carotenoids rose well, further emphasizing their valuable role as
1.6-fold in the lutein-supplemented group and safe and cost-effective nutritional interventions
20
Fig. 3 Distribution of RPE/choroid A2E + iso-A2E levels
in relation to retinal carotenoids in Abca4−/−/Bco2−/− mice.
There was a statistically significant inverse correlation
between retinal carotenoids and A2E and iso-A2E levels
in RPE/choroid (p values: *p < 0.05). Red line refers to L
with R2 = 0.815, blue line refers to Z with R2 = 0.825, and
the orange dotted line refers to combined regression plot
of L, Z, and placebo group with R2 = 0.904. [13]
against visual loss from age-related and inherited
macular diseases.
Conflict of Interest No authors have any conflicts of
interest.
Grant Support EY-11600 and EY-14800; Research to
Prevent Blindness.
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 Disturbed Matrix
Metalloproteinases Activity
in Age-Related Macular
Degeneration

Beatriz Martins and Rosa Fernandes

Abstract ulation and TIMPs’ significant regulatory

Matrix metalloproteinases (MMPs) are a
tightly regulated family of proteolytic
enzymes that break down extracellular matrix
(ECM) and basement membrane components.
Because it is associated with development,
morphogenesis, tissue remodeling, and repair,
ECM remodeling is an important mechanism.
MMPs are thought to act as a double-edged
sword, as they contribute to maintaining pho-
toreceptors/retinal pigment epithelium (RPE)/
Bruch’s membrane (BM)/choroid complex
homeostasis and also contribute to the onset
and progression of age-related macular degen-
eration (AMD). Polymorphisms and/or altered
expression in MMPs and their tissue inhibitors
(TIMPs) are associated with age-related mac-
ular degeneration (AMD). Here, we review
the evidence for MMPs’ role in the onset and
progression of AMD via addressing their reg-
functions.
Keywords
Age-related macular degeneration · Matrix
metalloproteinases · Extracellular matrix ·
Bruch’s membrane · Geographic atrophy ·
Choroidal neovascularization
1 Introduction
Matrix metalloproteinases (MMPs), also known
as matrixins, belong to an important class of
zinc-dependent endopeptidases, the metzincins
superfamily [1]. This superfamily includes 21
human disintegrins and metalloproteinase
domain (ADAMs) and 19 secreted disintegrin
and metalloproteinase thrombospondin domain
(ADAMTSs), in addition to 23 MMPs expressed

B. Martins R. Fernandes (*)

Faculty of Medicine, Coimbra Institute for Clinical
and Biomedical Research (iCBR), University of
Coimbra, Coimbra, Portugal
Institute of Pharmacology and Experimental
Therapeutics, Faculty of Medicine, University of
Coimbra, Coimbra, Portugal
Faculty of Medicine, Coimbra Institute for Clinical
and Biomedical Research (iCBR), University of
Coimbra, Coimbra, Portugal
Institute of Pharmacology and Experimental
Therapeutics, Faculty of Medicine, University of
Coimbra, Coimbra, Portugal
Association for Innovation and Biomedical Research
on Light and Image (AIBILI), Coimbra, Portugal
e-mail: rcfernandes@fmed.uc.pt

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 21

J. D. Ash et al. (eds.), Retinal Degenerative Diseases XIX, Advances in Experimental Medicine
and Biology 1415, https://doi.org/10.1007/978-3-031-27681-1_4
22
in human tissue [2]. MMPs typically have an
80-amino acid propeptide, a 170-amino acid cat-
alytic metalloproteinase domain, a variable-­
length linker peptide (hinge region), and a
200-amino acid hemopexin (Hpx) domain. A
detailed description of MMPs structure and an
overview of their substrate preferences and their
association with extracellular matrix (ECM) com-
ponents and inhibitors can be found in other
excellent reports [2–4].
The MMPs are usually secreted as zymogens
by a variety of cells into the extracellular space
[3] or stay tethered to their plasma membrane [5].
In humans, there are six membrane-type (MT)-
MMPs [5]. In the generation of active MMPs,
enzymatic cleavages of the zymogens are needed.
Active MMP-2, for example, is made from pro-­
MMP-­2 by MMP-14 cleavage, while pro-MMP-9
is cleaved by MMP-3 [5, 6].
MMPs are mainly classified based on their
substrate preference and domain organization,
being grouped into gelatinases (MMP-2, MMP-­
9), collagenases (MMP-1, MMP-8, MMP-13),
stromelysins (MMP-3, MMP-10), MT-MMPs
(MMP-14, MMP-15, MMP-16, MMP-17), and
others [7]. Under normal conditions, both MMPs
and the tissue inhibitors of metalloproteinases
(TIMPs) have an important role in the regulation
of the turnover of ECM [2, 8]. A dysregulation of
these components can both contribute to patho-
logical ECM remodeling and serve as a nexus for
a variety of pathways involved in age-related
macular degeneration (AMD) pathogenesis.
2 MMPs Are Implicated in AMD
AMD is a retinal degenerative disease that is a
leading cause of central vision loss in the elderly.
This disease, in its early stages, is characterized
by the accumulation of drusen that causes pro-
gressive degeneration of the photoreceptors and
retinal pigment epithelium (RPE). The disease
can progress to geographic atrophy (GA), an
advanced form of dry AMD, and/or choroidal
neovascularization (CNV), also known as wet
AMD [9, 10].
B. Martins and R. Fernandes
The specific interplay of pathophysiological
events that converge to AMD and related degen-
eration mechanisms remain to be fully elucidated
[11]. Nevertheless, several studies have been
highlighting the alterations in the regulation of
ECM, by dysregulation of the activity of MMPs
and TIMPs, as one of the main mechanisms
involved in the development of AMD [12]. This
modulation of ECM turnover is associated with
the other molecular mechanisms involved in the
pathophysiology of the disease since some stud-
ies have shown that both oxidative stress and acti-
vation of the complement system can modulate
the activity of ECM components [13, 14].
Additionally, the formation of drusen, the hall-
mark of AMD, is also believed to be associated
with a dysregulation of the ECM components,
especially in the RPE and Bruch’s membrane
(BM) [15]. These alterations in ECM modulation
caused by dysregulation of MMP/TIMP complex
can also be influenced not only by environmental
but also by genetic factors. For example, poly-
morphisms in MMP-2 (rs243865) and in MMP-9
(rs3918424 and rs3918241) are associated with
increased risk of AMD, mainly in younger
patients (<65 years) [16–18]. Other MMP-2
polymorphisms (rs243865, rs243866, and
rs2287074) are associated with a lower likeli-
hood of AMD development [19, 20]. There are
also some TIMPs polymorphisms associated
with AMD. Despite some polymorphisms in
TIMP-2 (rs817909) and in TIMP-3 (rs5754227)
appear to have a protective effect, other polymor-
phisms in TIMP-3 (rs713685 and rs743751) are
more frequent in AMD patients [18, 21, 22]. As a
result, these findings highlight evidence that
changes in ECM turnover are essential mecha-
nisms implicated in the development of AMD
and may help explain the susceptibility to the dis-
ease’s late stages.
2.1 MMPs in Dry AMD
BM is a five-layered ECM located at the inter-
face of the retina and choriocapillaris. BM plays
an important role in cell-cell communication and
Disturbed Matrix Metalloproteinases Activity in Age-Related Macular Degeneration
regulates the exchange of oxygen and nutrients
between the choroid and the outer retina [23].
However, BM undergoes a number of age-related
alterations that may have a role in AMD patho-
genesis. Especially noteworthy, the accumulation
of cellular debris and ECM proteins leads BM to
thicken and to the formation of drusen [24].
Several studies have been demonstrated that
aging causes structural and functional changes in
the BM due to a decrease in the activity of the
gelatinase components of the MMP system. In
this case, increased levels of high molecular
weight components of the MMPs pathway
(HMW1 and HMW2) reduce the pool of the acti-
vated gelatinases MMP-2 and MMP-9 and con-
tribute to reduced matrix degradation and
thickness of BM [25–27]. Another study revealed
that higher TIMP-3 concentrations in the drusen
block MMPs activity, lowering proteolytic activ-
ity within the drusen [28]. Oxidative stress
reduces MMP-2 activity and leads to the forma-
tion of sub-RPE deposits that can be involved in
the formation of drusen during AMD develop-
ment [29].
Nevertheless, several other studies also
detected increased levels of other MMPs in
patients with GA. MMP-7 levels were found to
be lower in these patients, but MMP-9 and
TIMP-1 levels were higher [30, 31]. In vitro and
in vivo models of dry AMD led to increased
expression, secretion, and activation of MMP-1,
MMP-3, and MMP-9 and are associated with
decreased RPE cell viability [32–34]. Moreover,
MMP-9’s role in the breakdown of the RPE bar-
rier has been highlighted, as silencing of this
MMP can improve the barrier integrity [35, 36].
Regarding genetic alterations, only a few stud-
ies have related polymorphisms specifically with
dry AMD. Liutkeviciene et al. detected that a
polymorphism in the MMP-2 gene (rd243865) is
associated with the development of hard drusen
in AMD patients [37].
2.2 MMPs in Wet AMD
The choroid is a network of blood vessels, located
below the BM that is critical for nutrients and
23
oxygen supply to the outer retina. The oxidative
and inflammatory processes that occur in late
stages of AMD might promote a pro-angiogenic
environment, resulting in CNV, which is the hall-
mark of wet AMD. Neovessels from choroid,
also referred as CNV, develop beneath the RPE
or penetrate into the subretinal space in patients
with wet AMD [38, 39].
Similar to GA and BM thickness, in this case
also dysregulation of ECM turnover appears to
have a close relation with CNV. Increased activ-
ity of MMPs can degrade some components of
BM, essential to the growth of new vessels [40,
41]. Several studies have shown that patients with
wet AMD present increased levels of MMP-9 in
aqueous humor, vitreous, and plasma [42–44].
This increase in the levels of MMP-9 contributes
to CNV since this MMP can increase VEGF lev-
els (pro-angiogenic factor) by decreasing PEDF
levels (anti-angiogenic factor), both secreted by
the RPE [45]. MMP-2, MMP-7, and MMP-13
have likewise shown a rise in their expression and
levels in patients with CNV lesions [18, 46, 47].
Moreover, cellular and animal models of CNV
lesions reported increased expression of MMP-2
and MMP-9, which were involved in the forma-
tion of experimental CNV. Additionally, the inhi-
bition of these two gelatinases appears to be a
good strategy to treat CNV, since their elimina-
tion is associated with decreased CNV lesion size
[48–50]. Some studies have also been underlin-
ing the alterations in TIMPs activity as an impor-
tant mechanism underlying CNV. On both wet
AMD patients and animal models, decreased lev-
els of TIMPs (TIMP-2 and TIMP-3) contribute to
CNV susceptibility [18, 51].
Regarding genetic alterations, several studies
identified various polymorphisms on both MMPs
and TIMPs that are related to CNV and wet
AMD. Microsatellites in the promoter of MMP-­
9, along with other polymorphisms on the
MMP-9 gene (rs142450006, rs3918424), have
been associated with increased risk of CNV pro-
gression and wet AMD development [17, 52, 53].
Also polymorphisms on MMP-1 (rs1799750),
MMP-2 (rs243865), MMP-7 (rs11568818), and
MMP-20 (rs10895322) are associated with
increased CNV lesion size and development of
24
wet AMD [54–56]. Finally, a polymorphism in
the TIMP-3 gene (rs9621532) has a protective
effect and is associated with decreased risk of
wet AMD [57].
3 Concluding Remarks
Due to their ability to break down ECM constitu-
ents, MMPs are relevant components in many
pathological processes of AMD. Specifically,
they play an important role in the accumulation
of drusen and degeneration of photoreceptors/RPE
in dry AMD and pathological vessel growth in
wet AMD. TIMPs control changes in their
expression and activity. To better understand the
role of each MMP in AMD pathogenesis, further
research is needed.
Acknowledgments Funding provided by the Global
Ophthalmology Awards Program (GOAP), a Bayer spon-
sored initiative committed to supporting ophthalmic
research across the world. Thanks are also due to FCT
(Portugal) and Strategic Projects UIDB/04539/2020 and
UIDP/04539/2020 (CIBB), COMPETE-FEDER (POCI-
01-0145-FEDER-007440), and Centro 2020 Regional
Operational Program: BRAINHEALTH 2020
(CENTRO-01-0145- FEDER-­000008). The authors also
acknowledge FCT for the doctoral research grant
2020.04811.BD (to B.M.).
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 Current Views on Chr10q26
Contribution to Age-Related
Macular Degeneration
Navdeep Gogna, Lillian F. Hyde, Gayle B. Collin,
Lisa Stone, Jurgen K. Naggert,
and Patsy M. Nishina
Abstract
Age-related macular degeneration (AMD) is
the leading cause of blindness in the global
aging population. Familial aggregation and
genome-wide association (GWA) studies have
identified gene variants associated with AMD,
implying a strong genetic contribution to
AMD development. Two loci, on human Chr
1q31 and 10q26, respectively, represent the
most influential of all genetic factors. While
the role of CFH at Chr 1q31 is well estab-
lished, uncertainty remains about the genes
ARMS2 and HTRA1, at the Chr 10q26 locus.
Since both genes are in strong linkage disequi-
librium, assigning individual gene effects is
difficult. In this chapter, we review current lit-
erature about ARMS2 and HTRA1 and their
relevance to AMD risk. Future studies will be
necessary to unravel the mechanisms by which
they contribute to AMD.
Keywords
AMD · ARMS2 · HTRA1 · Linkage disequi-
librium · Genetics
N. Gogna (*) · L. F. Hyde · G. B. Collin · L. Stone ·
J. K. Naggert · P. M. Nishina
The Jackson Laboratory, Bar Harbor, ME, USA
e-mail: navdeep.gogna@jax.org
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023
J. D. Ash et al. (eds.), Retinal Degenerative Diseases XIX, Advances in Experimental Medicine
and Biology 1415, https://doi.org/10.1007/978-3-031-27681-1_5
1 Introduction
Age-related macular degeneration (AMD) is the
leading cause of irreversible central vision loss in
aging populations [1, 2]. By 2050, an estimated
22 million people in the USA [3], and, by 2040,
288 million people worldwide [4], will be
affected with AMD. Early stages of AMD are
characterized by the presence of few (<20)
medium-sized drusen and/or retinal pigment epi-
thelium (RPE) abnormalities [5–7]. Drusen,
lipid-rich, protein-containing deposits that accu-
mulate between the RPE and Bruch’s membrane,
are considered a “hallmark” of AMD [8].
Intermediate AMD exhibits at least one large
druse, multiple medium-size drusen, or geo-
graphic atrophy (GA) that does not extend to the
center of the macula [5]. Advanced or late AMD
is either non-neovascular (dry, atrophic, or non-
exudative) or neovascular (wet or exudative) [9].
Dry AMD, characterized by drusen and GA,
extends to macula’s center and affects the chorio-
capillaris, RPE cells, and photoreceptors (PRs)
without leakage of blood or serum into the retina
and macula [10]. In contrast, wet AMD is associ-
ated with RPE detachment and choroidal neovas-
cularization, leading to leakage and fibrovascular
scarring [11].
AMD is a complex, multifactorial disease
[12–24]. Risk factors identified from case-­
control, cross-sectional, and prospective cohort
studies [10, 25–34] include age [35], family his-
27
28
tory [28, 36], dietary choices [30, 37–40], race
and ethnicity [41–43], gender [21], and smoking
[25, 44–46]. In addition, genetic factors explain
46–71% of the variation in overall disease sever-
ity [47]. To date, a total of 52 independently asso-
ciated common and rare gene variants
(p < 5 × 10−8), distributed across 34 loci, have
been associated with AMD risk [48–51].
Genome-wide association (GWA) and family-­
based linkage studies identified genetic variants
in the complement factor H (CFH) gene on
Chr1q32 and two tightly linked genes—ARMS2
and HTRA1—on Chr10q26 as major contributors
to AMD risk [52–65]. Together, these increase
AMD predisposition by more than 40-fold [66].
While significant progress has been made toward
understanding the role of CFH in AMD risk [67–
70], uncertainty remains regarding the gene(s)
responsible at the Chr10q26 locus. Strong link-
age disequilibrium between ARMS2 and HTRA1
genes has made it difficult to distinguish individ-
ual gene effects by statistical methods [71].
Moreover, PLEKHA1 is also found in linkage
disequilibrium with ARMS2 [55]. Several studies
suggest that ARMS2/HTRA1 genetic variations
are more strongly associated with AMD risk than
PLEKHA1 [56, 72, 73]. In this chapter, we review
existing information about ARMS2 and HTRA1
and their association with AMD.
2 
ARMS2
The ARMS2 gene, age-related maculopathy sus-
ceptibility 2, is only present in higher primates
[74]. ARMS2 encodes an 11-kDa protein [75];
however the function, localization, and native
structure of the protein have not been firmly
established. An in silico approach to predict the
structure of ARMS2 concluded that it has 15
putative phosphorylation sites, four putative gly-
cation sites of ε-amino groups of lysine, two
putative O-linked glycation sites, and three
potential binding sites [76]. The alanine at posi-
tion 69 is predicted to be a functional residue for
protein binding.
N. Gogna et al.
2.1 Reported Tissue, Cellular
and Subcellular Expression
of ARMS2/ARMS2
The expression of ARMS2 was detected in pla-
cental tissue [55], at low levels in retinal samples
[56, 73] and later, ubiquitously, in human tissues
[77]. The Bgee gene expression database (v14.2)
currently lists 62 distinct human tissues with
varying ARMS2 mRNA levels [78].
Kanda et al. expressed human retinal ARMS2
mRNA in COS-1, ARPE-19, and JEG cells.
Subcellular fractionation, followed by co-­staining
with MitoTracker and cytochrome C oxidase
subunit IV, located ARMS2 to the mitochondrial
(Mt) outer membrane [73]. Subsequently, another
group co-stained ARMS2 with retinoschisin (an
extracellular protein that stains PR inner seg-
ments) and Mt cytochrome c oxidase subunit II in
human retinal sections. ARMS2 localized in the
Mt-enriched ellipsoid region of the inner seg-
ments of both rod and cone PR cells [79].
However, conflicting results were presented by
Wang et al. [80]. Using immunofluorescence
(IF), verified by immunoblotting, endogenous
ARMS2 in ARPE-19 cells and overexpressed
GFP-tagged and HIS-tagged exogenous ARMS2
in COS7 cells were found to localize in the cyto-
sol and not in Mt or any other organelle [80]. The
group further performed in silico analyses to
identify the protein features required for Mt tar-
geting and concluded that ARMS2 lacked canon-
ical mitochondrial targeting sequences [80].
Kortvely et al. transfected HEK293 cells with
plasmid constructs coding for normal or risk vari-
ant ARMS2. Immunostaining findings suggested
that ARMS2 is a secreted protein and a compo-
nent of the extracellular matrix (ECM), found
mainly in choroid pillars of human eye [81].
Using a yeast two-hybrid system, followed by
coprecipitation, ARMS2 was found to bind sev-
eral ECM proteins [81]. Further, ARMS2 colo-
calized with the endoplasmic reticulum (ER) in
transfected ARPE-19 cells expressing ARMS2.
Adding a small N-terminal tag to ARMS2 did not
have any effect, whereas the same tag at the
Current Views on Chr10q26 Contribution to Age-Related Macular Degeneration
C-terminus abolished ER colocalization, indicat-
ing that the C-terminus is integral to proper
­targeting [81]. A follow-up study concluded that
ARMS2 is secreted via an unconventional, Golgi-­
independent pathway. Blocking the canonical ER
to Golgi transport did not affect ARMS2 secre-
tion. Instead, ARMS2 was shown to colocalize
with calnexin-positive and protein disulfide
isomerase-negative vesicle-like structures and,
with GRASP65, a marker for unconventional
protein secretion. Exon 2 of ARMS2 codes for
eight amino acids (-SIIHTAAR) at the
C-terminus. By generating a set of mutants, the
two isoleucines were found to be indispensable
for correct targeting. The intracellular distribu-
tion of the A69S risk allele was similar to the
non-risk allele, with both protein variants secreted
into the culture medium [82].
Micklisch et al. found endogenous ARMS2
gene expression in human blood-derived mono-
cytes and in human-induced pluripotent stem
cell-derived microglia (iPSdM) by PCR and laser
scanning microscopy. Contrary to the study by
Kortvely et al. which showed translation of the
ARMS2 A69S variant, this study reported that
ARMS2 protein was absent in patients homozy-
gous for the ARMS2 AMD risk variant A69S or
the non-risk variant R38X [75].
2.2 
ARMS2 Risk Variants and Their
Putative Role in AMD
The missense variant, rs10490924 (G>T:A69S)
[73, 80, 82], encoding an alanine-to-serine sub-
stitution at position 69 (A69S) is the most fre-
quently observed ARMS2 risk allele in the human
population. Little is known about the effect of
this mutation on ARMS2 protein structure and
function. One study reports that the A69S varia-
tion in ARMS2 creates a new putative phosphory-
lation site that breaks a predicted α-helix [73]. It
has been suggested that the A69S change affects
ARMS2’s function in either Mt [73, 79] or its
role in the cytoskeleton in COS7cells [80]. A
recent study implicated ARMS2 as a surface com-
plement regulator [75]. Recombinant ARMS2,
expressed in Pichia pastoris, was shown to bind
29
to human apoptotic and necrotic cells and initiate
properdin-mediated complement activation and
C3b surface opsonization for phagocytosis [75].
Two additional variants of ARMS2 have been
reported: an insertion-deletion (indel) polymor-
phism (NM_001099667.1:c.*372_815del443
ins54) in the 3′untranslated region (3′-UTR) and
a coding nonsense polymorphism (R38X) [79].
The ARMS2 indel lies within the HTRA1 cis-­
regulatory region, upstream of its transcription
start site [83, 84], and is suggested to regulate
HTRA1 expression. In heterozygous retinal and
placental samples from human donors, Fritsche
et al. showed that this indel mutation destabilizes
the ARMS2 transcript by removing the polyade-
nylation signal and inserting a 54-bp element
known to mediate rapid mRNA turnover [79].
The group validated their findings by developing
recombinant ARMS2 isoforms, quantifying heter-
ologous ARMS2 expression in vitro and conclud-
ing that the indel polymorphism in ARMS2 leads
to reduced mRNA levels [83].
The ARMS2 variant identified by Fritsche
et al., rs2736911(C>T:R38X), is predicted to
result in a premature stop codon (R38X) and a
truncated protein [79]. Yang et al. observed a
50% decrease in mRNA in patients heterozygous
for R38X [85], presumably due to nonsense-­
mediated mRNA decay. They reasoned that the
loss of ARMS2 is insufficient to explain AMD
susceptibility as both the indel and R38X variants
result in a decrease in ARMS2, but only the for-
mer confers risk to AMD, while the latter is
mildly protective [85, 86]. This led to a hypothe-
sis of dual causality, in which concomitant down-
regulation of ARMS2 and upregulation of HTRA1
explains the disease [85]. Similar results were
obtained in another study that showed reduced
ARMS2 expression in human postmortem retinal
and RPE samples, heterozygous for R38X [83].
However, a subsequent investigation failed to
detect a rs2736911-dependent alteration in
ARMS2 expression [87].
Since ARMS2 is not found in mice, to gain a
better understanding of ARMS2 function, two
transgenic mouse models expressing human
ARMS2 and ARMS2 A69S were developed. The
constructs used a ubiquitous cytomegalovirus
Another random document with
no related content on Scribd:
The Project Gutenberg eBook of Stay off the
Moon!
This ebook is for the use of anyone anywhere in the United States
and most other parts of the world at no cost and with almost no
restrictions whatsoever. You may copy it, give it away or re-use it
under the terms of the Project Gutenberg License included with this
ebook or online at www.gutenberg.org. If you are not located in the
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you are located before using this eBook.

Title: Stay off the Moon!

Author: Raymond F. Jones

Illustrator: Virgil Finlay

Release date: December 5, 2023 [eBook #72337]

Language: English

Original publication: New York, NY: Ziff-Davis Publishing Company,

1962

Credits: Greg Weeks, Mary Meehan and the Online Distributed

Proofreading Team at http://www.pgdp.net

*** START OF THE PROJECT GUTENBERG EBOOK STAY OFF

THE MOON! ***
 Stay off the MOON!

By RAYMOND F. JONES

Illustrated by FINLAY

How do you fill a pipette and measure out

a half c.c. of hydrochloric acid into a test
tube—from a quarter million miles away?

[Transcriber's Note: This etext was produced from

Amazing Stories December 1962.
Extensive research did not uncover any evidence that
the U.S. copyright on this publication was renewed.]
The real problem, of course, is not quite that simple. You don't literally
fill a pipette or use a test tube; you activate metering circuits that
force tiny, ground-glass plungers a measured distance into reagent
pumps. You send signals that close some valves and open others,
and apply heat and adjust temperatures, and filter solutions, and
send the product to a spectrometer that determines what you've got
and how much.
Then you have to code it and get the information from the moon to
earth.
James Cochran had seen the equipment work through hundreds of
checkout analyses. But he didn't understand it. He was a chemist,
and he had drawn up the specifications for the chemical analyzers of
the Prospector, but it had been the electronic boys who dreamed up
the remote mechanization and the telemetry equipment that would
allow him to sit before a complex panel at the Center and direct his
chemical laboratory on the moon to learn what the moon was made
of. Some of the light-headed technicians who worked on the project
had dubbed it Operation Green Cheese, but Cochran had more
respect for the complexity of the effort.
It was Sunday midnight. The beginning of countdown was forty hours
away. Cochran's crew had finished the chemical checkout, but in the
assembly hangar technicians still swarmed about the Prospector,
giving final-check to the power and telemetry components.
Jim Cochran signed off the last of the check reports and dropped
them in the slot for delivery to the Project Director. He turned off the
lights over his own desk and went out to the hangar. Under the blaze
of fluorescent lights the device looked like some monstrous insect.
The differential housings over the worm-screw drives gleamed like a
red, segmented carapace. The blue appendages of the solar cell
boxes were extended as if in some frantic appeal. The radar dish and
the helical antenna extended mutely upward. And, like a furious
proboscis, the exploratory drill, which would pierce the moon's skin to
a depth of five hundred feet, seemed to gnaw at the concrete floor of
the hangar.
Sam Jarvis, supervisor of electronic checkout, saw Jim Cochran enter
and came over to him with a broad, weary grin. "AOK, so far! This
package is going to be perfect. If only the rocket boys will set up a
bird that will take it to the moon—"
"They'd better," said Jim. "I don't think I could ever go through this
again if they dump the Prospector in the drink."

Sam turned back to look at the robot machine and the swarming
technicians. "Yes, you could," he said. "All of us have gone through
heartbreak time and again the past five years, watching them blow
up, or fall back and burn in the atmosphere because the motors didn't
ignite. Or seeing them get all the way to the moon and have some
five-dollar transistor conk out. But we always have at it again. You
will, too. You're new, but you're one of us now. You never back out
when you've come this far."
Watching the Prospector, Jim knew Sam was right. It had taken some
persuasion to bring him to this point, however. Until a couple of years
ago he had believed he would be content with ivory-tower plastics
research for the rest of his life. The persuasion had been applied
when Mary's brother, Allan Wright, had made the astronaut team.
Allan and Jim had grown up together. There was no other person Jim
felt closer to except Mary and their two children. Allan had dreamed
of space when they were kids, and when he was fifteen, he said, "I'm
going out there. I don't know how. But, somehow, I'm going out there."
Now, he had been selected to captain the first Apollo voyage. He had
been born for that purpose, he said.
But while he was still in the general pool of astronauts he had opened
his campaign to get Jim into the space program. "They need the best
brains they can get," Allan said. "You haven't got any right to sit in a
musty old plastics lab while guys with half your ability try to get us into
space. NASA will take you tomorrow!"
Jim didn't try to tell him that his plastics lab wasn't exactly musty, or
that he didn't think of himself as one of the best brains in the country.
But Mary sided with Allan; she was almost as excited about space as
he was. In the end, Jim went to NASA. Within days, he had been
assigned to head the development of the Prospector chemical
mechanization.
It had been something of a jolt to pull up all the roots he had so
carefully put down for him and his family, and move to the hectic,
bustling, space-frontier community of the Center. But he wasn't sorry.
It put something new in the blood, something men had never known
before.
Space!
The great Saturn lifted slowly, on a vast blossom of fire, with snowy
lox streaming down its sides. Then it was gone, a twinkle of fire high
above, among the stars. That was all.
Mary and Jim Cochran continued to stare at the fading twinkle, and
finally they turned away. Allan had obtained permission to be in the
blockhouse during the firing. It hadn't been necessary for Jim to be
there. He didn't want to know the instant-by-instant telemetry reports
which told whether or not the flight was successful. Sam or Allan
would call him when they knew. That would be soon enough for him.
"Let's drive down to the beach and watch the moon from there," said
Mary. "We can't just turn around and go home, like—like nothing had
happened."
Jim smiled in the darkness. Mary was as eager as he was for the
success of the flight. And she didn't have his fear of failure, that kept
him from wanting to know the maybe-yes, maybe-no indications that
the telemetry would first show.
"Sure," he said, "that's a good place to watch it."
The moon.

They watched its reflection thrashing on the water as the breakers

rolled across, under, and around it. It was the same image that men
had watched and wondered about and feared—for a half million
years. The first creatures that had any semblance of manhood had
sat on their haunches on this same shore and watched the same
moon and the same water.
And felt the same fears, Jim thought.
He didn't know whether it was fear or not, but there was some sense
of awesome mystery that filled him when he looked at the moon. It
had been that way all his life. He remembered how it was when he
was a boy and he walked through the fields at night on his way home.
He had to pass Cramer's Pond, and when the moon was up its light
from the sky and its reflection from the pond seemed to fill the whole
earth with a cold, silver light. He always hurried past the pond on
such nights.
Mary felt it, too. "I wonder why the moon makes people feel the way
they do."
"How does it make people feel?"
"Oh, kind of—kind of—you know!"
Jim laughed aloud. This was a typical Mary Cochran explanation. But
it told him all he needed to know. What she said was quite true. He
did know.
The baying of dogs on a wintery, moonlit night.
The madness called lunacy.
Seeds must be planted just so, in relation to the moon, or the crop will
fail.
Men had always felt strange things about the moon. Would a Saturn
missile and a mechanical monster in its nose be able to destroy all
that?
Jim started the car. "Let's get back. I've got to know how the flight's
going!"

They were still in the blockhouse, but the tension was relaxed. They
were talking and watching the meters and cathode ray tubes without
the strain and fear of failure. Jim knew the answer even before Sam
and Allan walked up to him and slapped him on the back.
"Where the devil did you go?" said Sam. "I thought you were going to
be right behind me when we fired, and you weren't here at all!"
"It's like your first baby, you want to be there, and you don't. Was it a
good shot?"
"Was it a good shot?" Allan's face became ecstatic. "We've never had
a better one. On course all the way!"
The Project Director, Emil Hennesey was behind them. His face was
bleak. "I expected you to be here for the firing, Cochran," he said. "It
seems to display little interest in your end of the project that you didn't
feel it necessary to show up."
Jim looked at him steadily and shrugged without answer. Hennesey
was one guy whose presence on the space team Jim couldn't figure
out. He was an ex-Major, and he had no capacity for dreaming. Men,
machines, transistors, rockets—they were all the same thing, merely
objects to be made to obey.
"You are aware of your next sequence of duties, I trust?" said
Hennesey.
Jim nodded curtly. "I'll be ready."

Sixty-six hours to the moon. That's what it takes with marginal escape
velocity and free-fall conditions. But it was really five hundred
thousand years and sixty-six hours, Jim thought. Surely there hadn't
been a single hour in all that time when someone, somewhere on
earth had not felt the longing to solve the secret mystery of the moon.
Now they were about to find the answer. But what would they have
when they found it? They would know that the surface dust of the
moon consisted of certain percentages of silicates and oxides. They
would know that the under layers were composed of rocks, maybe of
granite or limestone or basalt. They would determine how much of
each.
And then it would be over. The quest of the ages would be answered
with a few simple statements that could be obtained in any high-
school chemistry lab—if the lab were on the moon.
Jim Cochran felt there had to be more to it than that.
Why do dogs howl at the moon on winter nights?
Why do men say that madness of the mind is lunacy?
Why must planting be done in the right phase of the moon?
Little sleep was had by any of the crew during the next two nights,
even though the instrument stage of the ship was now completely
inert except for occasional telemetry signals that were fed to the
computers for course checking and correction. The ship was simply
falling on its own momentum.
Six hours before moonfall, activities in the tracking center accelerated
and the tension increased. There was no question of hitting the
moon; the landing had to be made safe for the cargo of instruments.
Jim Cochran watched the operators during this period. He told
himself he didn't understand it, but he had actually learned a great
deal of electronics during the past two years. He had had to in order
to design and operate a chemical laboratory 240,000 miles away.
The television screens came on, showing the pock-marked surface of
the moon as the ship orbited. The thrill and the fear of the great
unknown began to rise in Jim's throat. By the silence in the room, he
was sure the others sensed it, too.
Abruptly, the braking command was given and the ship began to fall
out of orbit towards the planned landing in the Sea of Rains. On the
screens, the images swelled as the ship plummeted faster. In one
corner could be seen the spring-loaded extension legs, like those of
some great spider. It seemed impossible that these could cushion the
violent shock of landing.
The sudden surge of a retro rocket and the blast of moondust blinded
the television eye, but there was a sense of crazy, rocking, rolling
motion. Then the eye went dead.
Jim almost cried out. The ship couldn't have crashed.

An operator quickly switched controls and the screens came alive

again. He turned a dial slowly. The camera eye moved. It swept the
craggy horizon and the nearby floor of the Sea of Rains. Others had
seen this before, but it was the first time for Jim. He found himself
pushing forward, drinking in the sight eagerly.
"The moon—the moon—" he said softly to himself. But the others
heard it and they understood.
Signals were sent across space to collapse the landing legs and
unfold the sides of the instrument cone like the leaves of a flower.
The Prospector lay exposed to the environment for which it was built.
Slowly, in response to other signals, the worm-screw drives, which
had been retracted against the body of the vehicle, turned through an
arc and lowered to the surface. Locked in position, the drive screws
began turning slowly. The vehicle moved off the now-useless landing
support and became an entity of its own.
The ungainly arms of the solar cells automatically oriented toward the
sun; the antennas pointed toward earth. The scanning cameras in the
turret of the Prospector took control of the video circuits and the turret
slowly turned as the vehicle moved across the face of the moon. The
landing support remained behind and slowly dwindled like some
useless wreckage.
There was sudden pandemonium in the tracking center control room.
The operators laid down their headsets and began pounding each
other on the back, while ear-splitting Indian yells filled the air. Jim and
Sam found themselves beating each other on the arms and yelling
senselessly.
"We made it!" Sam cried. "We made it! We got your little old
laboratory up there for you!"

There were hours of testing and calibration yet to be done before the
Prospector could be used for its primary mission. Hundreds of
electronic circuits had to be checked to see that they survived the
takeoff and landing without becoming distorted or inoperative.
Jim went home for the rest of the night. When he returned the next
morning Sam reported that all circuits were go, and the Prospector
was his.
He had operated the laboratory in the Prospector many times, either
on a mock-up or from this control panel while the Prospector was in
the hangar. But he couldn't keep the faint tremor from his hand as he
reached for the first control that would manipulate the machine on the
moon.
The drill had been extended to operating position, but the head had
not yet been energized. Jim touched it to the fine dust of the floor of
the Sea of Rains. The drill went quickly to a depth of eighteen inches
in the dust before it struck something firmer.
"That kills the theory about eighty feet of that stuff, anyway," said Sam
as he read the instruments.
Jim was not interested in depth at this time. He fed some of the
surface material into the laboratory and set the controls to run the
preprogrammed analysis. They waited minutes; then the analysis
began to appear in cryptic symbols on a paper tape.
Jim glanced at it and frowned.
"What's the matter? Isn't it working right?" Sam asked anxiously.
Jim hesitated. "It indicates the presence of several silicates, some
carbonates, and a high percentage of oxides. These are mostly of
sodium, calcium, and iron, as you might expect. But there's
something wrong with your calibration. The atomic and molecular
characteristics aren't coming through right."
"The boys ran checks on the standard samples aboard the
Prospector last night," said Sam. "The results tallied exactly. I'll show
you the tapes."

Jim waited, puzzled, while Sam brought up the check tapes. When he
saw them, he shook his head. "There's a standard calcium carbonate
sample carried aboard the Prospector. Here's a calcium carbonate
picked off the surface. You can see the difference yourself. The
nominal analysis is the same, but the atomic weights and the energy
levels are just slightly different. That doesn't make sense unless your
circuits are out of calibration."
"Let's run another standard sample," said Sam.
Within a few minutes the calibration check had been repeated. Jim
held up the tape. Sam peered over his shoulder. "Just like the first
one," said Sam. "Nothing's wrong with the circuits. Maybe you've got
some new stuff there, that's never been identified before."
"That's hardly possible," said Jim. "There aren't any new elements in
the places where sodium and calcium and silica are supposed to be.
Yet, I don't understand how this can be. If the atomic weights are
different, and the energy levels are different, they have to be different
elements. It doesn't make sense."
"Well, why don't we push on," said Sam, "that is, if you've completed
the surface sampling in this spot. Some samples at lower depths may
give other indications."
Jim agreed. He drove the drill deeper into the face of the moon. At
ten-foot intervals he removed samples and ran them through the
analyzer. The results were the same down to the hundred-foot level.
All results showed common chemical elements with slightly variant
atomic characteristics.
Which made them different chemical elements!
After six hours, Jim stood up from the console and shook his head
wearily. "It's no good, Sam. There's something wrong that I can't put
my finger on. If it isn't in the circuits, I don't know where it is. But
these readings just aren't right. There's no use going deeper until we
find out where the error is."
Sam's face was somber. "There just isn't any error. There can't be.
Unless it was made by whoever put the moon together—"
"Please make a complete check of every analyzer and telemetry
circuit tonight, and we'll try again tomorrow. I want to think about this."

He thought about it, and he dreamed about it. And along about three
o'clock in the morning he sat bolt upright in bed and stared at the dim
moonlight on the opposite wall of the bedroom.
It wasn't possible, he told himself audibly. It just wasn't possible!
Mary stirred and leaned on one elbow beside him. "What's the matter.
Are you having nightmares?"
"Yeah—yeah, I guess I am. I'll be back in a minute, honey." He got up
and padded to the door. "I've got to make a phone call."
"At this time of night?"
But Jim was gone. He turned on the hall light and dialed Sam's
number. After a long time Sam answered sleepily.
"Wake up!" said Jim. "I've just figured it out!"
"Who the devil—? Oh, it's you, Jim. Figured out what? Do you know
what time it is."
"Do you know the results of your calibration re-check?"
"How would I know that? I've got the night crew on it, but I didn't ask
them to report to me in the middle of the night. Go back to bed, and
let's talk about it in the morning."
"They're not going to find anything wrong, Sam."
"I could have told you that."
"But the elements of the moon are different—and there's only one
explanation."
"What?"
"Think about it a minute, Sam. We take a spectrograph of the sun,
and we find the same elements that are here on earth. We turn it on
Alpha Centauri and find the same thing. We turn it to the farthest
stars we can find that give enough light to record by. Always the
same. Calcium is calcium, whether it's on the earth or on a star a half
billion light years away."
"So?" Sam's voice was tired, and he sounded as if he was listening
only because Jim was too good a friend to tell to go to hell for calling
in the middle of the night.
"So? So what?" Sam repeated.
"So we go to the moon," said Jim, "and all of a sudden calcium isn't
calcium, and the sodium on the moon isn't the same as the sodium
on earth and on the sun and on Alpha Centauri and the stars a half
billion light years away. Don't you see what that means!"
"No, I guess not," said Sam dully. "Maybe in the morning—"
"It means the moon just doesn't belong, Sam! It means the moon is
completely foreign to anything in the Solar System, in the whole
galaxy—in any galaxy we have been able to analyze. It means the
moon has come from somewhere else, from a region of space where
atoms and electrons are not even the same as atoms and electrons
here. It must be a somewhere that's so far away it's beyond the edge
of space as we know it!"
"I'll get dressed and come over," said Sam.

Mary made chocolate and toast, and they sat around the kitchen
table thinking and talking of the awesome implications of Jim's theory.
"If what you say is true," said Sam, "it might be that the slightest
contact with any substance of the moon would be sheer poison to a
human being. A returning vessel could never be permitted to enter
the earth's atmosphere, and decontamination would become one of
the major branches of science."
"That's entirely possible. It would complicate enormously the
problems of establishing a moon-base. A speck of moondust inside
the base might be as lethal as an unshielded reactor."
Mary was looking out the kitchen window toward the thin crescent of
moon that was setting over the city. She thought of Allan, who would
soon be voyaging to that alien world. "It's like a trap up there in the
sky. We should never have tried to reach it."
"No—it's not like that at all," said Jim vigorously. "We'll solve whatever
problems we find there. But think of it! We don't have to build a ship
capable of crossing billions of light years of space to see what's out
there. Something from out there has come to us and parked right in
our own front yard. We have a thousand times more reason to go
now!"
Sam toyed with his toast and dunked it in the chocolate. "I think you
ought to keep it quiet—until we really know, don't you."
"Why? I'll run some more tests, sure. I'll plug every loophole there can
possibly be. But unless I find something new I'm going to announce it.
Why shouldn't I?"
"I don't think Hennesey will like it, for one thing. Too sensational.
Even when we actually land there and confirm your analyses by on-
the-spot checks—it's still only a theory that the moon doesn't belong
to this galaxy. You'll never be able to prove that."
"What we've found already is proof enough!"
"Not for Hennesey. He'll ask to see the shipping manifest by which
the moon was transferred here. You know Hennesey."
"Sure, I know Hennesey," said Jim bitterly. "And he doesn't count. Not
in something like this. This is big, and important, and I'm going to
announce it. I want the credit for discovering it. It'll be called
Cochran's Theory, and some University will offer me at least an
honorary Ph.D. Is that bad?"
Sam shook his head. "Of course it's not bad. But I wonder what the
public reaction will be like, and how about Congress—especially if
this business about possible poisoning from moondust turns out to be
correct? There might be a lot of pressure to cut funds and maybe
cancel the whole moon project."
"If moondust is lethal, there's no better time to be warned against it
than right now!"

He spent the following week, eighteen hours a day, at the chemical

analysis panel in the tracking station. As long as the moon was above
the horizon, day or night, he kept experiments going—checking, re-
checking, calibrating, searching for loopholes.
There were no loopholes. There was no malfunction of equipment.
The atoms of the moon's elements were not the same as those of the
rest of the galaxy.
When Allan next came to visit on leave from his own station, Jim told
him what had been found. Allan's face paled a little as he listened in
awe to the story. He stood up and paced across the room as Jim
finished.
"I always knew there would be something—something extra in this,"
he said. "You look up at night and you know the moon can't be just
another piece of earth. Now—you've found out what it is!
"How far could it have come? How old can it be? Imagine stepping
out onto a world so old, from so far away!"
"You haven't heard it all," said Mary tensely.
"There's more?"
"They think perhaps the slightest touch of moon substance may be
lethal."
"We don't know," said Jim hastily. "It's a thought that Sam advanced.
But I think it's quite possible. We won't know until we can run animal
tests. But special precautions will have to be taken to decontaminate
the Apollo after you re-enter with your space-suits."
"This will mean engineering changes. Are they under way?"
"I haven't announced my theory yet. But the necessary changes to
provide for decontamination will have to be made."
"I wanted this as much as you," said Mary. "But now it's gone all
wrong. We should have gone on dreaming about the moon and let it
be a dream that never came true. I'm afraid of it now. No man should
set foot on something so alien and so different."
Allan put an arm around her shoulders and shook her gently. "Sis!
What kind of talk is that? You're talking to the guy who's going to be
the first one to put his foot on the moon."

From the mountain of data accumulated in his experiments, Jim wrote

his paper. Hennesey could control the publication of any material