Neurobiology of Disease 148 (2021) 105219

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Neurobiology of Disease
journal homepage: www.elsevier.com/locate/ynbdi

Leptin enhances adult neurogenesis and reduces pathological features in a

transgenic mouse model of Alzheimer’s disease
Michele Longoni Calió a, b, *, Amanda Cristina Mosini b, Darci Souza Marinho c,
Geisa Nogueira Salles d, Fernando Henrique Massinhani a, Gui Mi Ko e,
Marimélia Aparecida Porcionatto a, *
a
Department of Biochemistry, Laboratory of Neurobiology, Universidade Federal de São Paulo, São Paulo, SP 04039-032, Brazil
b
Department of Physiology, Laboratory of Neurobiology, Universidade Federal de São Paulo, São Paulo, SP 04039-032, Brazil
c
Department of Ginecology, Laboratory of Genetic Control, Universidade Federal de São Paulo, São Paulo, SP 04023-062, Brazil
d
Institute of Research and Development, Laboratory of Biomedical Nanotechnology, Universidade do Vale do Paraíba, São José dos Campos, SP, 12244-000, Brazil
e
Institute of Pharmacology and Molecular Biology INFAR, Universidade Federal de São Paulo, São Paulo, SP, 04044-020, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Alzheimer’s disease (AD) is the most common dementia worldwide and is characterized by the presence of senile
Alzheimer’s disease plaques by amyloid-beta (Aβ) and neurofibrillary tangles of hyperphosphorylated Tau protein. These changes
amyloid-beta lead to progressive neuronal degeneration and dysfunction, resulting in severe brain atrophy and cognitive
leptin
deficits. With the discovery that neurogenesis persists in the adult mammalian brain, including brain regions
neurogenesis
neural stem cells
affected by AD, studies of the use of neural stem cells (NSCs) for the treatment of neurodegenerative diseases to
hippocampus repair or prevent neuronal cell loss have increased. Here we demonstrate that leptin administration increases the
neurogenic process in the dentate gyrus of the hippocampus as well as in the subventricular zone of lateral
ventricles of adult and aged mice. Chronic treatment with leptin increased NSCs proliferation with significant
effects on proliferation and differentiation of newborn cells. The expression of the long form of the leptin re­
ceptor, LepRb, was detected in the neurogenic niches by reverse qPCR and immunohistochemistry. Moreover,
leptin modulated astrogliosis, microglial cell number and the formation of senile plaques. Additionally, leptin led
to attenuation of Aβ-induced neurodegeneration and superoxide anion production as revealed by Fluoro-Jade B
and dihydroethidium staining. Our study contributes to the understanding of the effects of leptin in the brain that
may lead to the development of new therapies to treat Alzheimer’s disease.

1. Introduction
Described over a century ago, Alzheimer’s disease (AD) is the most
common progressive dementia associated with aging (Gallaway et al.,
2017; Dai et al., 2018). However, the precise mechanisms of AD still are
not fully understood. The pathology of AD is mainly characterized by
multiple etiological and biochemical aberrations including the forma­
tion of senile plaques by aggregates of amyloid-beta (Aβ) peptides,
neurofibrillary tangles (NFTs) of hyperphosphorylated Tau protein,
microglial activation and oxidative stress, that are linked to abnormal
neuronal function, progressive degeneration and neuronal injury which
cause memory loss and cognitive dysfunction (Armstrong, 2011; Laur­
itzen et al., 2012; Zhang et al., 2011; Reitz and Mayeux, 2014; Heneka
et al., 2014; Jeong, 2017; Sanabria-Castro et al., 2017; Feitosa and da
Silva Oliveira, 2018).
Neurogenesis is the process of generating new neurons by neural
stem cells (NSCs) that occurs mainly in two brain regions, named
neurogenic niches – the subventricular zone (SVZ) of the lateral

Abbreviation: 2xTgAD, double transgenic AD model; AD, Alzheimer’s disease; Aβ, amyloid-beta; BDNF, brain-derived neurotrophic factor; BrdU, 5-Bromo-
2’deoxyuridine; DCX, doublecortin; DG, dentate gyrus; DPX, Dibutylphthalate Polystyrene Xylene; DHE, dihydroethidium; FJB, fluorojade B; GFAP, glia fibrillary
acid protein; GPx1, glutathione peroxidase 1; LepRb, Leptin receptor b; Map2, microtubule-associated protein 2; NeuN, neuronal specific nuclear protein; NFT,
neurofibrillary tangles; NSC, neural stem cells; PBS, phosphate-buffered solution; PCNA, proliferating cell nuclear antigen; ROS, reactive oxygen species; SGZ,
subgranular zone; Sod2, superoxide dismutase 2; SVZ, subventricular zone; WT, wild type.
* Corresponding author at.: Rua Pedro de Toledo, 669 – 3◦ andar, CEP 04049-032, Vila Clementino, São Paulo, SP, Brazil
E-mail addresses: michele.longoni@unifesp.br (M.L. Calió), marimelia.pocionatto@unifesp.br (M.A. Porcionatto).

https://doi.org/10.1016/j.nbd.2020.105219
Received 22 April 2020; Received in revised form 18 November 2020; Accepted 3 December 2020
Available online 8 December 2020
0969-9961/© 2020 Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
M.L. Calió et al.
ventricles (LVs) and the subgranular zone (SGZ) in the dentate gyrus
(DG) of the hippocampus (Alvarez-Buylla and Lim, 2004; Fuentealba
et al., 2012) but also disseminated throughout the central nervous sys­
tem (CNS) (Ming and Song, 2011; Lin and Iacovitti, 2015).
Although the adult brain of mammals, including humans, generates
new neurons throughout life (Altman and Das, 1965; Eriksson et al.,
1998; Spalding et al., 2013; Ernst et al., 2014), the proliferative activity
of NSCs and the generation of new neurons markedly declines during
aging and is deregulated in neurodegenerative diseases such as AD,
which may contribute to a decline in memory function and some degree
of cognitive impairment (Clelland et al., 2009; Lazarov et al., 2010).
Moreover, the differences observed in the neurogenic process of
neurodegenerative diseases may reflect different stages of the pathology
(Jin et al., 2004a; Rodriguez et al., 2008; Rodriguez et al., 2011). The
discovery that quiescent precursor cells can be activated even in the
aged or damaged brain has contributed to the emergence of the hy­
pothesis that neurodegenerative diseases could be treated by stimulating
the adult neurogenesis. In that case, NSCs may be an endogenous source
of healthy cells where the derived progeny might be re-directed to areas
of degeneration, where they would be involved in cell replacement,
providing a promising therapeutic approach (Gage, 2000; Lie et al.,
2004; Hormoz, 2013; Zhang et al., 2017; Matarredona et al., 2018).
Consequently, several studies have been conducted to investigate how
the promotion of the neurogenic potential of these cells in AD improves
the symptoms of the disease like spatial memory in experimental animal
models (Becker et al., 2007; Taniuchi et al., 2007; Naumann et al., 2009;
Markiewicz et al., 2011; Sahay et al., 2011; Kanemoto et al., 2013;
Hamilton et al., 2015; Winner and Winkler, 2015; Zhang et al., 2017; Tai
et al., 2017).
Clinical evidence indicates that diet and lifestyle are major risk
factors for developing AD and disturbances in metabolism are also
linked to the development of the pathology (Dosunmu et al., 2007; Yin
et al., 2016). The hormone leptin is known to regulate food intake and
energy balance, being also involved in many other processes such as
immune response, stress, and neurodevelopment (Folch et al., 2012;
Meyer et al., 2014; Bellefontaine et al., 2014). Indeed, lower leptin levels
are associated with an elevated risk of AD, as well as aberrant leptin
function (Lieb et al., 2009; Baranowska-Bik et al., 2015). Within the past
decade, researchers showed a role for leptin in neuroprotection and
hippocampal neurogenesis. In animal models, leptin administration fa­
cilitates learning and alters synaptic plasticity (Moult et al., 2010; Luo
et al., 2015; Ghasemi et al., 2016). Additionally, this adipokine atten­
uates Tau hyperphosphorylation in neuronal cells and protects against
Aβ-induced neurotoxicity in cellular models (Garza et al., 2008; Doherty
et al., 2012; Liu et al., 2015a, 2015b). Thus, there is growing evidence
that leptin-based therapies may be beneficial in AD.
Although the pathway linking neurogenesis and AD is still under
debate, mainly due to the different animal models used, it is important
that further studies be done to determine the role of leptin in neuro­
genesis and whether it can be considered as a therapeutic strategy in AD.
We used the double transgenic APPswe/PS1dE9 (2xTgAD) mouse
model of different ages for investigating the temporal changes of neu­
rogenesis and the action of leptin during the disease progress in the two
main neurogenic niches. The 2xTgAD mouse lineage reproduces a subset
of neuropathological, biochemical and behavioral changes identified in
patients with AD, such as massive Aβ accumulation and plaque forma­
tion, vascular deficits and memory impairment (Holcomb et al., 1998;
Jankowsky et al., 2002).
Our findings demonstrate that leptin plays a role in extra-
hippocampal neurogenesis, in regions involved in neural cell replace­
ment, regardless of age or disease stage. Collectively, we show that
leptin participates in several events that reduce the pathology and exert
multiple functions besides neurogenesis, contributing to the Aβ clear­
ance and decreasing oxidative stress. We hypothesized that strategies
targeted at stimulating this process could have significant therapeutic
value, suggesting that leptin may be used as an approach for the
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Neurobiology of Disease 148 (2021) 105219
treatment of AD and probably other neurodegenerative diseases.
2. Material and methods
Animals - Three-month-old (Young) and twelve-month-old (Aged)
male heterozygous double transgenic APP/PS1 mice (2xTgAD) - over­
expressing a chimeric mouse/human mutant APP (amyloid precursor
protein) (Mo/HuAPP695swe) and a mutant human presenilin 1 (PS1-
dE9), - colony B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo/J (from Jackson
Laboratory, Bar Harbor, ME) and nontransgenic littermates C57BL/6J
mice (wild type – WT) were used. The animals, weighing 25–30g, were
purchased from the Center of Development of Experimental Models for
Biology (CEDEME–UNIFESP), at the Universidade Federal de São Paulo,
São Paulo, Brazil. Mice were genotyped to confirm the presence or
absence of the transgenes. Animals were housed and maintained on a
12-h light/dark cycle (lights on at 06:00 a.m.) in a humidity and
temperature-controlled environment with ad libitum access to food and
water. All procedures were carried out in accordance with the internal
Ethical Committee on Animal Research of the Universidade Federal de
São Paulo UNIFESP/EPM (CEP 6882070814). All efforts were made to
minimize animal suffering and to reduce the number of animals used.
Drugs - Recombinant mouse leptin (R&D Systems) was resuspended
in sterile saline solution (0.9 % NaCl).
Leptin treatment – To study the effect of leptin on cell proliferation in
the neurogenic niches, a dose of 1mg/kg was used. This dose was chosen
based on its efficacy in inducing neurogenesis in mice (Garza et al.,
2008). Animals of both ages and lineages (n = 10 per group) received
intraperitoneal (i.p.) injection of saline or leptin (1mg/kg), once a day at
the end of the light cycle for 7 consecutive days. Bodyweight was
assessed daily during the treatment. In the last two days of treatment,
half of the mice (n = 5 per group) were injected with 5-Bromo-2-deox­
yuridine (BrdU; Sigma, St. Louis, MO), an analog of the nitrogen base
thymidine that is incorporated into the DNA of the proliferative cells.
Animals received four intraperitoneal injections of BrdU (50mg/kg, in
0.1M sodium phosphate buffer [PBS], pH7.4) within 48 h, at 8-h in­
tervals between each dose, corresponding with the S-phase of cell di­
vision in mammals. These mice were transcardially perfused 8h after the
last BrdU injection. The brains of these animals were used for immu­
nohistochemical evaluation of BrdU-labeled cells.
Biochemical measurements of leptin in serum - Serum samples were
obtained from blood collected from all groups of aged animals and
stored at -80◦ C for correlating the decrease in plasma leptin levels and
the onset of AD. Briefly, the blood was obtained at the time of euthanasia
of the animals and a small sample was placed in tubes at ambient tem­
perature for 1-2 hours in an upright position. The tubes were then
centrifuged at 4 ◦ C for 10 minutes at 3000g and the clear serum was
removed and stored. The concentrations of leptin levels from non­
transgenic WT and 2xTgAD mice were measured in serum by an enzyme-
linked immunosorbent assay (ELISA) kit Quantikine Mouse Leptin
Immunoassay (R&D Systems) according to the manufacturer’s recom­
mendations. Leptin levels were normalized to total protein levels and
expressed as picograms of leptin content per milliliters of total proteins
(pg/mL). Values were determined using a four-parameter nonlinear
regression equation.
Tissue Preparation – The animals that received BrdU injections (n = 5
per group) were terminally anesthetized with a ketamine cocktail
(43mg/kg ketamine, 10mg/kg xylazine, and 1.4mg/kg acepromazine)
and transcardially perfused through the ascending aorta using 0.1M PBS
(pH 7.4) and by 4% paraformaldehyde (PFA, pH 7.4) in 0.1M PBS. The
brain was removed from the skull and post-fixed overnight, also in 4%
PFA, before being transferred to a 30% (w/v) sucrose solution in 0.1M
PBS for 48h. Brains were embedded in O.C.T. frozen tissue embedding
media (HistoPrep™, Fisher Scientific) and coronally cryosectioned at
30μm thick sections on a cryostat (Leica CM 1850). The sections were
ordered in a systematic and uniformly-random manner (SURS) (Gun­
dersen, 2002) and stored free-floating in anti-freezing solution (30%
M.L. Calió et al.
sucrose, 30% ethylene glycol, 0.1M PBS) in a multi-well plate until
processing for immunofluorescence. For the molecular biology experi­
ment, hippocampi of the remaining animals (n = 5 per group) were
frozen on dry ice, processed for real-time polymerase chain reaction
(PCR), being stored at -80◦ C for later analysis.
BrdU Immunofluorescence staining - Once BrdU is incorporated into
the DNA, the material was pretreated with a nucleic acid denaturant to
make it accessible to the BrdU antibody. Brain sections were briefly
washed in 0.1M PBS, incubated in 1% hydrogen peroxide for 10 min,
and treated with 2N HCl solution in 0.1% Triton X-100 for 30 min
succeed by incubation with 0.1M borate buffer (pH 8.5) for 10 min.
Next, the slices were immersed in a blocking solution with 0.1% Triton
X-100 and 10% normal goat serum in PBS for 1h and then incubated
with rat monoclonal anti-BrdU (1:300, Molecular Probes) in the block­
ing solution overnight at 4◦ C. After washing in PBS, the fragments were
then incubated with goat anti-rat Alexa Fluor 647nm (1:300, Molecular
Probes) in blocking solution for 1h at room temperature. A negative
sample test was performed using serum rather than the primary anti­
body to establish the specificity of the immunostaining. The slides were
examined under a laser scanning confocal microscope (Zeiss LSM 810
Axiovert; Carl Zeiss GmbH, Germany).
Fluorescence Immunolabeling - To determine the phenotypes of BrdU-
labeled (BrdU+) cells, brain sections were processed for doublecortin
(DCX) and glial fibrillary acidic protein (GFAP) fluorescent double la­
beling. The Leptin-receptor (LepRb) antibody was used to check whether
these cells were responding to leptin treatment and expressing its re­
ceptor. Briefly, free-floating SVZ and hippocampus sections were
collected and first pretreated as described above, and then incubated
with rat anti-BrdU antibody (1:300, Molecular Probes) and guinea pig
anti-DCX (1:500, Merk/Millipore) or chicken anti-GFAP (1:500, Merk/
Millipore). To detect the leptin receptor, Aβ deposits and microglia,
hippocampus sections from mice were immunostained overnight
without DNA denaturation step and the following primary antibodies
were used: rabbit anti-LepRb (1:1000, Abcam) or mouse anti-βAmyloid
6E10 (1:1000, Covance/Biologend) and goat anti-Iba1 (ionized calcium-
binding adaptor molecule, 1:1000, Wako). After washing in 0.1 M PBS
three times, sections were incubated for 1h with fluorescent secondary
antibodies conjugated with Alexa Fluor 647nm goat anti-rat IgG to
reveal immunoreactivity of BrdU, Alexa Fluor 488nm goat anti-guinea
pig IgG to reveal DCX or Alexa Fluor 594nm goat anti-chicken IgG to
reveal immunoreactivity of GFAP, Alexa Fluor 594nm goat anti-rabbit
IgG to reveal LepRb, Alexa Fluor 488nm goat anti-mouse IgG to reveal
Aβ plaques or Alexa Fluor 594nm rabbit anti-goat IgG to reveal Iba1,
respectively (1:300 for all secondary antibodies, Molecular Probes) and
counterstained with the nuclear tracer 4’6’-diamidino-2-phenylindole
(DAPI, 5mM; 1:10000, Sigma). Then, the sections were rinsed in PBS,
ordered and mounted onto coated glass slides, and coverslipped with
Fluoromount G™ (Thermo Fisher Scientific). The slides were analyzed
in a blind manner using a computer-based digitizing image system (Zeiss
Axiovert 100M; Carl Zeiss, Germany) connected to an LSM 810 Confocal
Laser Scanning System. The laser and detector were maintained at
constant settings when imaging each staining set.
Fluoro-Jade B labeling – FJB is a fluorochrome derived from fluores­
cein and is commonly used in neuroscience to label degenerating neu­
rons in brain tissue. Fluoro-Jade B (Histo-Chem Inc) staining was carried
out as described by Schmued and Hopkins (2000). Briefly, PFA-fixed
brain slices were mounted on 1.5% gelatin-coated slides, dehydrated
overnight at room temperature and kept into a heater for 30 min at 40◦ C
before staining. The slices were dipped for 5 min in a solution of 1%
sodium hydroxide in 80% alcohol before placed for 2 min in 70%
ethanol, and lastly for 1 min in distilled water. Sections were then
oxidized for 10 min by immersion in 0.06% KMnO4, under slow shaking.
After numerous rinses in distilled water, slices were incubated for 30
min in 0.004% FJB dye in 0.1% acetic acid, washed thoroughly in
distilled water, and placed into the heater set to 40◦ C until the material
was entirely dry. Lastly, the tissues were cleared in xylene and
3
Neurobiology of Disease 148 (2021) 105219
coverslipped using DPX (Dibutylphthalate Polystyrene Xylene)
mounting medium (Sigma).
Superoxide anion detection - Superoxide was detected with the
oxidative fluorescent probe DHE (dihydroethidium; Molecular Probes),
which reacts with O.2, resulting in ethidine fluorescence. To remove the
differences in cellular densities, DAPI was used in all of the frozen slices.
The frozen brain sections were incubated in a light-protected humidified
chamber at room temperature with DHE (5mM) for 5 min and then
counterstained with the nuclear tracer DAPI (5mM; Sigma). The slides
were immediately analyzed with a computer-based scanning image
system (Zeiss Axiovert 100M; Carl Zeiss, Germany) connected to an LSM
810 Confocal Laser Scanning System. Fluorescence was detected with
510–560nm excitation and 590nm emission filters. The results are
expressed as the DHE/DAPI ratio according to Oudot et al. (2006).
Cell Quantification and Analyses - The SVZ and hippocampus coronal
sections were mounted on glass slides and were coded. The cell counts
were conducted by personnel blinded to the experimental condition of
each sample. The brain was sectioned at 30μm, and every eight sections
were spaced 240μm apart throughout the entire SVZ and the hippo­
campus extent was used to assess the number of labeled cells. This en­
sures that the same cell will not be counted twice on consecutive sections
and guarantees that the area counted for each brain is constant. A
sequence of eight slices per animal was obtained using the Smooth
Fractionator method (Gundersen, 2002), representing the entire volume
of the dorsal hippocampus or anterolateral SVZ. Cell counting was
performed at 40x magnification. In the SVZ, the cells of the anterolateral
region were considered while in the hippocampus cells of the SGZ and
the gyrus were counted. The three planes (X, Y and Z) of z-stack images
(1.5μm/z-stack) were visualized to confirm the extent of cell colocali­
zation using the EzColocalization plugin for ImageJ analysis software.
Cells single labeled for BrdU, Iba1 and FJB or double-labeled for BrdU/
DCX, BrdU/GFAP or DCX/LepRb were counted. Cell body was defined as
the counting unit. The Cell Counter tool of ImageJ software (National
Institute of Health - NIH Image, https://imagej.nih.gov/ij/34) was used
to estimate the number of BrdU double stained cells. The percentage of
BrdU cells double-labeled was calculated by dividing the number of
double-labeled cells by the total number of BrdU cells and multiplying
100. As mentioned, the morphometric analysis was performed using
ImageJ software.
Microglia Density and morphological Analysis - The number of Iba1
microglial cells has been reported as the number of somas per acquired
area (300 mm2). To quantify microglia density, the images were
analyzed by Optical Fractionator tool of Stereo Investigator software. A
z-axis projection based on the maximal intensity signal throughout the
thickness of the section was obtained and after threshold setting, the
number of cells was recorded. Data are expressed as the density of
positive fluorescent cells versus total area (Grimaldi et al., 2019).
Quantification of the morphological features of the microglia was
carried out by taking z-stacks of 10 ROI per slice which were then
converted to maximum intensity projection tiff files. Microglia
morphology was analyzed to identify changes in the resting or activated
state, such as alterations on cell body and cell ramification between
groups. Photomicrographs of microglia at 40x magnification from all
groups were used. We used ImageJ software and appropriate plugins
(AnalyzeSkeleton v.3.2.2 and FracLac v.2.5) to convert all photomi­
crographs to binary, skeletonized and outline images to analyze cell
morphology. In addition, cell somas were manually counted in each
photomicrograph. We summarized the number of process endpoints and
length from the Analyze Skeleton plugin (http://imagej.net/AnalyzeSke
leton31) data output and normalized all data by the number of microglia
cell somas in each image to calculate the number of microglia end­
points/cell and microglia process length/cell. To compare microglia
complexity, we used FracLac plugin for ImageJ (https://imagej.nih.gov/
ij/plugins/fraclac/FLHelp/Introduction.htm), which quantifies each
cell’s contour bounded by the endpoints and process lengths, calculating
the fractal dimension. In addition, we evaluated the span ratios as
M.L. Calió et al.
indicators of cell shape, and density as an indicator of cell size. In
Table S1, we summarize skeleton (endpoints and process length) and
fractal analysis measures (density, fractal dimension and span ratio) in
terms of measure, unit, sampling and interpretation (Supplemental Data
Table S1).
Estimations of Amyloid Plaque Density and Volume - Quantitative
image analysis of Aβ plaques was performed as previously described (Liu
et al., 2015a, 2015b; Furcila et al., 2019). The number of Aβ plaques per
field (density), diameter and volume of plaque occupied area were
estimated with the aid of Stereo Investigator software (Stereo Investi­
gator 11.0, MicroBright Field Inc., MBF Bioscience; Williston, USA),
using its Optical and Area Fraction Fractionator tool in the entire hip­
pocampal area of each slice. To estimate the Aβ plaque diameter, the
edges of the plaque were delineated with the Nucleator tool of Stereo
Investigator software. This tool provides the volume of each Aβ plaque
analyzed, as well as the relative area occupied by them in each examined
hippocampal subfield to provide the percentage (%) of area coverage by
Aβ plaques. All Aβ plaques with a diameter between 5 -100μm (this cut-
off ensured analysis of discrete plaques - Gowrishankar et al., 2015)
were examined.
A minimum of six coronal sections were selected for each animal, at
equal 240-μm intervals with 10x and 40x magnification. Field selection
was performed by choosing 3 evenly-spaced random fields encompass­
ing all of the hippocampi of each section. These regions of interest were
delineated using 10x objective lenses and plaque counting was per­
formed with 40x objective lenses. Images were captured using a digital
camera mounted to a Nikon ECLIPSE 80i inverted microscope (Tokyo,
Japan) with a motorized stage connected to a computer. All analyses
used z projections of 3 optical slices at the center of the plaque. For each
mouse, quantitative analysis was performed on an area of about 3 x 3
mm for each image at 40x magnification (Yuan and Grutzendler, 2016).
Real-time quantitative PCR - To determine the action of leptin on gene
expression, quantitative reverse transcription PCR (qPCR) was per­
formed. The hippocampi of five animals of each group were collected,
and total RNA was extracted using Tryzol® (Invitrogen/Thermo Fisher
Scientific) according to the manufacturer’s instructions. The comple­
mentary DNA (cDNA) was generated from the extracted RNA using the
SuperScript™First-Strand Synthesis System (Thermo Fisher Scientific)
for RT-PCR. Relative quantitation was performed using 25 ng of reverse-
transcribed mRNA (cDNA samples) that were amplified with TaqMan
PCR Master Mix and TaqMan probes. The reactions were run on a 7500
Real-Time PCR System (Applied Biosystems) according to the manu­
facturer’s instructions. After amplification, the PCR products were
analyzed with the ABI PRISM sequence detection software (Applied
Biosystems). In addition, melting curve analysis was performed to
confirm the authenticity of the PCR products. Quantitative gene
expression was analyzed by the comparative CT method (2-∆∆CT) ac­
cording to Livak and Schmittgen (2001). TaqMan probes to caspase-3,
NeuN, Map2a, DCX, GFAP, βIII Tubulin, Ki67, PCNA, Nestin or
Custom TaqMan Array Fast Plates to Oxidative stress were used (all
probes and mixes were purchased from Applied Biosystems). The
expression of each gene was normalized by the mean of the five
endogenous controls (β-actin, 18S, Gusb, Gapdh and Hprt).
In the analysis Custom TaqMan Array Fast Plates to Oxidative stress,
the Student t-test was used for statistical analysis, and the comparison
between the groups was made as follows: the groups treated with leptin
were compared with their respective untreated groups, of the same
lineage and age, considering the treatment factor (untreated vs. treated).
In another analysis, the untreated groups of aged animals were
compared with groups of young animals of the same genotype, taking
into account the age factor (young vs aged). Finally, the remaining
group of young untreated 2xTgAD mice was correlated with the group of
untreated age-matched littermate wild-type animals, where the geno­
type factor was considered for comparison purposes (WT vs 2xTgAD).
Statistical Analysis – Multiple comparisons of treatment vs. age or
treatment vs. genotype was performed by one or two-way analysis of
4
Neurobiology of Disease 148 (2021) 105219
variance (ANOVA) with repeated measures followed by Tukey’s
correction post hoc. When pertinent, a nonparametric Student t-test was
applied. The data are expressed as mean ± standard error of the mean
(SEM). Significance was set at *p < 0.05, **p < 0.01, ***p < 0.001.
Statistical analyzes were performed using the GraphPad Prism software
version 5.0 (GraphPad Software).
3. Results
3.1. Leptin Changes Gene Expression of Proliferation and Immature
Neural Cells Markers in the Hippocampus
We treated WT and 2xTgAD mice with leptin for 7 consecutive days
(See Supplemental Data Fig. S1A). Bodyweight of the mice was
measured before (Supplemental Data Fig. S1B) and throughout the 7
days of leptin treatment. The percentage of body weight loss are shown
in Fig. S1D. The treatment with leptin did not affect the weight loss of
animals.
We observed that there is a tendency in the decrease of serum leptin
concentration in 2xTgAD animals, although there was no significant
difference. Even after treatment, we did not detect a significant
enhancement in leptin levels in serum samples in 2xTgAD mice (See
Supplemental Data Fig. S1C).
After this, we investigated the expression profile of genes involved in
neurogenesis in the hippocampus: glial fibrillary acid protein (GFAP -
neural stem cell and astrocyte marker); Nestin (amplifying and neural
stem cell marker); proliferating cell nuclear antigen (PCNA) and Ki67
(proliferation and cell-cycle markers); doublecortin (DCX - neuronal
progenitor marker); βIII-tubulin, microtubule-associated protein2a
(Map2a) and neuronal-specific nuclear protein (NeuN - mature neuron
markers).
We observed that treatment with leptin increased the expression of
Nestin by young WT, and young and aged 2xTgAD mice (Fig. 1B). The
expression of DCX was increased in all treated groups (Fig. 1E) and
decreased with aging in both genotypes, whereas the upregulation of
PCNA was observed only in young animals of both strains (Fig. 1C).
GFAP expression was downregulated in leptin-treated aged 2xTgAD
animals, but not in the other groups (Fig. 1A). Finally, no difference was
observed in the expression of tubulin βIII, Map2a and NeuN with the use
of leptin, but a decrease in the expression of Map2 and NeuN genes was
found in aged 2xTgAD mice in comparison to the WT or young animals
(Fig. 1F-H).
3.2. Age and genotype-dependent reduced proliferation of SVZ and SGZ
cells is increased after leptin treatment
In the present study, we asked whether leptin regulates adult neu­
rogenesis in the SVZ and the hippocampus. To address this question, we
labeled newborn cells by administrating BrdU in the last two days of
treatment with leptin and quantified the number of BrdU+ cells in the
SGZ and the SVZ of treated and untreated, young and aged, WT and
2xTgAD mice. In the hippocampus, BrdU+ cells usually are present in the
internal layer of the granular cell layer of the DG, while in SVZ, neural
stem cells can be found in several areas, although we have focused on
the anterolateral region (Fig. 2A). We found a significantly higher
number of BrdU+ cells in the SVZ of all leptin-treated groups (Fig. 2B).
Analysis of the SGZ revealed that all groups, except the young WT mice,
showed an increased number of BrdU+ cells following treatment with
leptin (Fig. 2C). In addition, in SVZ, both young and aged 2xTgAD an­
imals demonstrated a lower number of proliferative cells compared to
WT animals of both ages and a decrease in the number of these cells was
also observed in aged WT animals when compared to young WT animals
(Fig. 2B). In SGZ, the comparison of both ages or genotypes showed a
decrease in aged 2xTgAD animals when compared with aged WT ani­
mals or young double transgenic animals (Fig. 2C).
M.L. Calió et al. Neurobiology of Disease 148 (2021) 105219

Fig. 1. Gene expression analysis by qPCR of proliferative and neural markers in the hippocampus of mice treated or not with leptin in different ages. The animals
were injected i.p. with vehicle (PBS) or leptin (1mg/kg) daily for 7 days. Leptin treatment increased the expression of most genes analyzed. Notice the difference in
the expression of GFAP (A), Ki67 (D) and DCX (E) between young and aged mice or treated mice. The data were normalized using four endogenous controls and are
relative to the PBS control group; n = 5 per group. One-way ANOVA, data are shown as mean ± SEM, represented by error bars;*p < 0.05, **p < 0.01 ***p < 0.001.

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M.L. Calió et al. Neurobiology of Disease 148 (2021) 105219

Fig. 2. In vivo leptin administration increases proliferation in the SVZ and SGZ neurogenic niches. BrdU labeling was used to evaluate proliferation in both
neurogenic niches. The animals were injected i.p. with vehicle (PBS) or leptin (1mg/kg) daily for 7 days. BrdU+ cells were counted in the SVZ of the lateral ventricle
and in the SGZ of the dentate gyrus of the hippocampus. (A) Representative images showing BrdU+ cells in the SVZ and SGZ from control (PBS) and treated (leptin)
WT and 2xTgAD mice of different ages. Regions used for the analysis and quantification of BrdU+ cells showing increased proliferation in the treated groups from
both genotypes in the SVZ (B) and SGZ (C). Scale bar = 50μm. Data are expressed as mean ± SEM, n = 5 animals per group. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 3. Leptin administration decreases astrogliosis in the hippocampus. GFAP labeling was used to evaluate the proliferation of neural stem cells and astrocytes or
astrogliosis in both neurogenic niches. Double positive BrdU+/GFAP+ cells were counted in the SVZ of the lateral ventricle and in the hippocampus. (A) Repre­
sentative images showing BrdU+ (white) and GFAP+ (red) cells in SVZ and SGZ from control (PBS) and treated (leptin) WT and 2xTgAD mice of different ages. (B)
Quantification of BrdU+/GFAP+ cells shows no difference in the proliferation of neural stem cells in the SVZ when mice received leptin. (C) Quantification of BrdU+/
GFAP+ cells shows decreased proliferation of astrocyte in the hippocampus of the leptin treated group. Scale bar = 50μm. Data are expressed as mean ± SEM, n = 5
animals per group. *p < 0.05; **p < 0.01; ***p < 0.001.

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M.L. Calió et al. Neurobiology of Disease 148 (2021) 105219

3.3. Leptin improves temporal changes during aging and AD by increasing
neurogenesis in the SVZ and SGZ and decreasing astrogliosis in the
hippocampus
To explore how leptin affected neurogenesis in AD, we analyzed the
expression of DCX (a marker for immature progenitor neurons), GFAP (a
marker for astrocytes), and BrdU (a marker for proliferation) in the SVZ
and DG of mice of different ages and treatment conditions.
Aβ can trigger the neuroinflammatory process to produce a diversity
of inflammatory cytokines, causing neurotoxic effects in the brain. GFAP
expression was therefore examined as an indicator of astrogliosis. In
SGZ, the presence of proliferative GFAP+ cells was higher in the AD
model even at 3 months of age and this difference became even more
significant at 12 months of age, compared to wild-type animals. In
addition, an increase in these cells was also observed in aged 2xTgAD
animals compared to young animals (Fig. 3C). We also observed that
leptin treatment led to a decrease in the number of GFAP+ cells in the
hippocampus of aged 2xTgAD animals (Fig. 3A,C), suggesting that leptin
may reduce astrogliosis. The number of GFAP-labeled cells in the SVZ
was not significantly altered by leptin treatment in WT and transgenic
mice (Fig. 3A,B).
Representative confocal images also showed that after 7 days of
leptin treatment there was a significant increase in the number of DCX
coexpressed with BrdU cells in all treated groups of animals (Fig. 4).
Higher numbers of DCX+ cells were observed in both regions of interest
including the anterolateral area in SVZ and SGZ in the hippocampal
dentate gyrus (Fig. 4B,C). Regarding age, in SGZ, both genotypes
showed a decrease in the number of proliferative neuroblasts in the
groups of aged animals. However, aged 2xTgAD animals showed an even
lower number of cells compared to WT animals (Fig. 4C). Curiously, the
presence of neuroblasts is more evident in the SVZ, in all groups
analyzed, when compared to the hippocampus, where the action of
leptin was more significant in the aged 2xTgAD (Fig. 4C). Thus, the
increments in the number of neuroblasts were approximately 1.5 fold in
the SVZ and 30 fold in the hippocampus of aged 2xTgAD group.
3.4. Leptin treatment upregulates the expression of LepRb in adult neural
stem/progenitor cells
We next sought to elucidate the mechanisms involved in transducing
the effects of leptin in neurogenesis and differentiation. It has been
described in the literature that LepRb is expressed in the dentate gyrus
(Scott et al., 2009), and to determine if leptin treatment would affect
LepRb expression, we used qPCR and immunocytochemistry. qPCR
analysis using RNA isolated from hippocampal tissue of untreated and
treated groups of both ages and genotypes showed that the expression
level of LepRb was significantly higher in all treated groups compared to
control (Fig. 5A). Interestingly, there was no significant change in LepRb
expression as a function of age in either WT or 2xTgAD group (Fig. 5A).
To investigate which type of neural progenitor was expressing leptin
receptor, the presence of LepRb on the hippocampus and SVZ was
further confirmed by immunohistochemical staining with antibodies
against LepRb and DCX of treated animals of both age and genotype
(Fig. 5B). The immunoreactivity for LepRb was observed in a large
number of DCX+ cells. As negative control, the primary antibody was
omitted from the reaction and no immunostaining was seen in this case.
3.5. Leptin restores Impaired Sod2 and GPx1 expression and leads to
decreased superoxide production in 2xTgAD mice brains
We used Custom TaqMan Array Fast Plates to Oxidative stress and
Antioxidant defense (See Supplemental Data Table S2 and S3) to

Fig. 4. Leptin administration increases the generation of neuroblasts in the SVZ and hippocampal SGZ. DCX labeling was used to evaluate the production of
neuroblasts by neural stem cells in the two neurogenic niches. Double positive BrdU+/DCX+ cells were counted in the SVZ of the lateral ventricle and in the SGZ of
the dentate gyrus of the hippocampus. (A) Representative images showing BrdU+ (white) and DCX+ (green) cells in the SVZ and SGZ from control (PBS) and treated
(leptin) WT and 2xTgAD mice of different ages. (B) Quantification of BrdU+/DCX+ cells shows an increased generation of neuroblasts in the treated groups in the
SVZ. (C) Quantification of BrdU+/DCX+ cells shows an increased generation of neuroblasts in the treated groups in SGZ. Scale bar = 50μm. Data are expressed as
mean ± SEM, n = 5 animals per group. *p < 0.05; **p < 0.01; ***p < 0.001.

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M.L. Calió et al. Neurobiology of Disease 148 (2021) 105219

Fig. 5. Neural stem cells and neuroblasts express LepRb. (A) Gene expression analysis by qPCR for LepR in the hippocampus. Leptin treatment increased the
expression of the receptor on all treated groups. (B) Representative images show double staining LepR+/DCX+ cells in SVZ and SGZ of treated animals. Scale bar
= 50μm.

investigate whether neurodegeneration and leptin treatment could
affect the gene expression pattern of antioxidant enzymes. The number
of upregulated or downregulated genes for each group are listed in
Table 1. Most genes related to antioxidant enzymes that could have their
relative expression (RE) quantified, did not show a significant change in
the treated groups (Table 2), except for superoxide dismutase-2 (Sod2)
and Glutathione peroxidase-1 (GPx1) in 2xTgAD treated animals
(Fig. 6). Interestingly, even in the younger DA models, the treatment
with leptin-induced an increase in gene expression of these two genes.
No significant variation in the level of mRNA was detected in the hip­
pocampus considering age. Regarding the WT animals, treatment with
leptin was effective only in young animals, increasing the SOD2 gene
expression.
Since our results showed significant differences in Sod2 expression in
leptin-treated AD animals, we focused on analyzing whether the anti­
oxidant activity of leptin could decrease the level of superoxide pro­
duced and attenuate the oxidative stress in AD animals. For that, we
used dihydroethidium (DHE), which is a detector of intracellular su­
peroxide. Leptin treatment significantly reduced cellular superoxide
levels in the hippocampus (Fig. 7C, D) of 2xTgAD animals. The indicator
of the presence of superoxide production was four times greater in the
dentate gyrus of untreated aged 2xTgAD than in treated animals, which
exhibited a significant decrease in the DHE/DAPI ratio, confirming the
protective role of this hormone. Curiously in SVZ, leptin decreased su­
peroxide production also in younger animals.
3.6. Leptin reduces Aβ plaque accumulation and neurodegeneration
To describe the capacity of leptin to reduce brain Aβ levels, we used
transgenic mice at the age of 12 months, treated and untreated with
leptin. Our data show that Aβ staining in the hippocampus of aged
2xTgAD mice was significantly reduced after treatment with leptin
(Fig. 8B). The hippocampus of aged 2xTgAD mice treated with leptin
showed a lower frequency of plaques per occupied area (Fig. 8C) and
also demonstrated smaller proportions of plaques of larger sizes,
although the number of plaques of small diameter was relatively higher
than in untreated animals of the same age and genotype (Fig. 8E).
Taking into account the age and genotype of animals, we observed
that the number of FJB+ cells increases in the hippocampus. An example
of the contrasting findings between control and leptin groups can be
visualized in Fig. 8D. In fact, we observed that FJB labeling in the hip­
pocampus of 2xTgAD mice decreased significantly after treatment with
leptin as compared with the control group and was similar to WT mice
(See Supplemental Data Fig. S2). In addition, the hippocampus of aged
transgenic mice contains more neurodegenerating cells when compared
to young mice and transgenic mice treated with leptin. We also observed
FJB staining in other areas, such as cortical regions and thalamus (data
not shown). Due to the association between brain-derived neurotrophic
factor (BDNF) levels and the onset of AD, we investigated BDNF mRNA
expression in the animals. BDNF was detected in all groups, with sig­
nificant increments in aged 2xTgAD leptin-treated group while lower
levels of BDNF expression were associated with the progression of pa­
thology (Fig. 8F).

Table 1
Number of genes related to oxidative stress in the hippocampus that were regulated positively by leptin above a 2.0 ratio (Upregulated genes) or were negatively
regulated by leptin below a 0.5 ratio (Downregulated genes). It was considered that genes that showed value between 0.5 and 2.0 had no difference in genic expression
after treatment with leptin (No difference genes).
Number of genes

Upregulated* Downregulated* No difference*a Undeterminedb Total of genes analyzed #

Young WT + Leptin 4 26 6 55 91
Aged WT + PBS 8 23 5 55 91
Aged WT + Leptin 5 27 5 54 91
Young 2xTgAD + PBS 4 17 7 63 91
Young 2xTgAD + Leptin 13 21 1 56 91
Aged 2xTgAD + PBS 4 24 6 57 91
Aged 2xTgAD + Leptin 4 27 5 55 91
*
Compared to the untreated control group of each age and genotype
a
Genes with values of expression between 0.5 and 2; compared to untreated control across all 3 replicates
b
Genes that did not show any CT value across all 3 replicates
#
Without the five endogenous controls used

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M.L. Calió et al. Neurobiology of Disease 148 (2021) 105219

Table 2
Fold increase of the relative expression (RE) of genes related to antioxidant enzymes that were upregulated by leptin
Relative Expression

Gene Young WT + Aged WT + Aged WT + Young 2xTgAD + Young 2xTgAD + Aged 2xTgAD + Aged 2xTgAD +
symbol Leptin* PBS# Leptin* PBSα Leptin* PBS# Leptin*

Apc 0,001 0,008 0,002 0,004 0,070 0,004 0,002

Ccs 0,062 0,063 0,032 0,064 0,130 0,064 0,032
Gpx1 0,980 2,047 2,547 2,007 7,943 1,002 3,602
Gpx2 0,002 0,002 0,002 0,004 0,004 0,008 0,002
Gpx3 0,016 0,062 0,004 0,008 0,067 0,008 0,004
Gpx4 0,250 0,516 0,126 0,504 2,075 0,501 0,126
Gpx7 0,001 0,002 0,002 0,004 0,004 0,004 0,002
Gpx8 0,124 0,127 0,126 0,251 0,256 0,248 0,126
Prdx3 0,004 0,008 0,004 0,008 0,033 0,004 0,004
Prdx4 0,246 0,500 0,122 0,499 2,014 0,254 0,122
Ptgs1 0,987 2,045 0,992 0,993 2,049 1,005 0,992
Sod2 2,775 1,962 2,460 0,996 8,165 0,993 2,826
*
Relative to control group: PBS of same age and genotype
#
Relative to control group: Young PBS, of the same genotype
α
Relative to control group: Young WT PBS

Fig. 6. Leptin increases Sod2 and GPx1 and reduces oxidative stress in the hippocampus of 2xTgAD mice. In vivo leptin administration for 7 days upregulated the
expression of Sod2 (A) and GPx1 gene expression (B) in the hippocampus of 2xTgAD but not in wild type. The data were normalized using four endogenous controls
and are relative to the young WT PBS control group. Data are expressed as mean ± SEM, n = 5 animals per group. *p < 0.05; **p < 0.01.

These results suggest a novel mechanism linking leptin action to
BDNF expression in AD, while it also implies the neurogenesis in this
pathology.
3.7. Leptin treatment attenuates the increased number and alters the
morphology of microglial cells resulting from AD in the hippocampus of
2xTgAD elderly animals
To verify neuroinflammation in the hippocampus, microglia were
immunolabeled with anti-Iba1 and counted (Fig. 9A). For this purpose,
we chose 12-months-old mice, because, at this stage, the 2xTgAD ani­
mals already have massive amyloid-beta plaque formation in the hip­
pocampus and cortex, high levels of microgliosis, and microglial
activation as well as alterations in microglia cytokine production
(Babcock et al., 2015; Manocha et al., 2016).
Leptin treatment largely depleted the DG of Iba1+ cells in both ge­
notypes (Fig. 8A and B). After 7 days of treatment with leptin, a few
randomly distributed Iba1+ cells were observed in WT animals while in
2xTgAD treated mice, the remaining Iba1+ cells were observed gathered
mainly around the sites of amyloid-beta plaques (Fig. 8C). Interestingly,
2xTgAD animals started out having higher numbers of Iba1+ cells
compared to WT animals (Fig. 8B), and the Iba1 cell reduction in
2xTgAd animals treated with leptin was more efficient compared to WT
treated animals. While only 22.84% of Iba1 cells were diminished in the
WT hippocampus, 52.2% of the cells were diminished in the 2xTgAD
hippocampus (Fig. 9B).
The morphology of Iba1 immunoreactive microglia was estimated in
the hippocampus of 12-months-old mice. Several features were analyzed
as endpoints, process length, cell size, cell complexity and cell shape.
In all the groups examined, microglia exhibited the typical branched
morphology with small cell bodies, multiple long proximal branches
extending in all directions and extensive ramification of the distal pro­
cesses. In several areas, the microglia of AD animals appeared less
branched, with an atrophied appearance (Fig. 10 A). The analysis of
microglial cells in the data set showed significant differences between
the groups treated with leptin and the untreated groups, in both geno­
types in the analysis of process length and density (Fig. 10 C and F). The
branching index (endpoints) increased only in the treated WT animals.
The evaluation of this characteristic also showed that the microglia is
more branched in the 2xTgAD animals compared to the WT strain,
whereas in the 2xTgAD mice treated with leptin, the microglial cells
exhibited a lower number of branches when compared with the WT
animals that received leptin (Fig. 10 B).
No difference was observed after statistical comparisons of the data
regarding the geometric structure (fractal dimension) (Fig. 10 D) while
only animals 2xTgAD that received leptin showed an increase in the

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Fig. 7. Leptin reduces superoxide production in the hippocampus of 2xTgAD mice. (A) Representative confocal images of superoxide staining in the hippocampus of
WT and 2xTgAD animals treated and not treated with leptin for 7 days. Dihydroethidium staining (DHE - red). The nuclei were stained with DAPI (blue). The ratio
intensity of DHE/DAPI fluorescence was used to calculate the oxidative stress rate. Intense levels of DHE indicate the greater occurrence of superoxide anion for­
mation. (B) Quantification of superoxide anion by the analysis of pixilation in SVZ. A decrease in leptin-treated 2xTgAD animals of both ages was observed when
compared to untreated animals. (C) Quantification of superoxide anion by the analysis of pixilation in the hippocampus. A decrease in leptin-treated aged 2xTgAD
animals was observed when compared to untreated animals. Scale bar = 50μm. Data are expressed as mean ± SEM represented by error bars, n = 5 animals per
group. ***p < 0.001.

span ratio when compared to untreated animals (Fig. 10 E).
4. Discussion
Leptin is known to repress appetite and reduce body weight gain
(Zhang et al., 2007b) and has been shown to enhance hippocampal
neurogenesis (Garza et al., 2008; Perez-Gonzalez et al., 2011). Leptin is a
pleiotropic hormone and as an adipokine, it plays multiple functions in
reproduction, glucose homeostasis, bone formation, tissue remodeling,
inflammation, as well as in other elements of the endocrine and immune
systems (Paz-Filho et al., 2010). Interestingly, growing evidence shows
that leptin, a peptide hormone classically associated with energy bal­
ance and metabolic control, is also importantly implicated in learning
and memory (Morrison, 2009) performing a direct and indirect role as a
cognitive enhancer, especially in the context of metabolic disorders and
dementia.
Since dietary limitation and physical activity have been shown to
improve hippocampal neurogenesis, it is possible that the effects of
leptin on neurogenesis could be mediated by its action in food intake and
metabolism regulation (Trejo et al., 2001; Lee et al., 2002). However, in
our study, the treatment with leptin did not affect the weight of treated
animals. Garza et al. (2008) reported that bodyweight gain decreased
without a significant effect on hippocampal neurogenesis after acute
leptin injection. Furthermore, it was found that chronic leptin admin­
istration did not alter locomotor activity (Overton et al., 2001). These
data demonstrate that the action of leptin on neurogenesis maybe is
dissociated from its effect on energy homeostasis.
Although there is evidence that leptin could be synthesized in the
brain, it is known that most of the leptin found in the CNS is derived
from peripheral white adipose tissue (WAT) (Campfield et al., 1996).
Changes in leptin levels found in age-related diseases are considered
important factors for the origin of cognitive deficits. Several clinical and
epidemiological studies have addressed the association between low
leptin levels and the risk of AD, supporting the concept that a disrupted
leptin metabolism and/or signaling contributes to the pathogenesis of
AD. In this regard, Power et al. (2001) found lower levels of serum leptin
in patients with AD underweight than in controls, being the first study
correlating Alzheimer’s disease with low leptin. Studies have shown a
strong inverse association of dementia and AD with leptin levels both in
plasma and in cerebrospinal fluid (CFS) (Lieb et al., 2009; Theodor­
opoulou et al., 2012; Bonda et al., 2014). Another study showed lower
levels of circulating leptin in AD subjects as well as its strong positive
correlation with the severity of dementia (Khemka et al., 2014). Simi­
larly, several other case-control studies have confirmed that lower
serum leptin is associated with AD (Bigalke et al., 2011; Karaoulanis
et al., 2012). Contradicting this, another study has shown no difference
in serum leptin levels in AD patients compared to controls (Theodor­
opoulou et al., 2012). Similarly, distinct research including AD patients
and normal controls has failed to find any significant alteration in the
leptin levels between these groups (Warren et al., 2012). A negative
relationship between leptin levels and severity of dementia has also been
observed by Baranowska-Bik et al. (2015). The results of these studies
show that individuals with low leptin levels had a greater decline in their
cognitive ability than those with normal levels (Holden et al., 2009).
We observed that there is a tendency in the decrease of serum leptin
concentration in 2xTgAD animals, although there was no significant
difference. Even after treatment, we did not detect a significant
enhancement in leptin levels in serum samples in mice. Importantly, our
results seem contradictory to the finding of some of these clinical studies
showing that higher leptin levels are related to reduced risk of AD and

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M.L. Calió et al. Neurobiology of Disease 148 (2021) 105219

Fig. 8. Leptin decreases the accumulation of Aβ in the hippocampus and increases BDNF expression. Leptin-treated 2xTgAD mice exhibit reduced levels of Aβ
deposits (green) compared with control (PBS) 2xTgAD mice. (A) Representative images of Aβ staining in the hippocampus of aged WT and 2xTgAD mice treated with
PBS or leptin. The three planes (X, Y and Z) of Z-stack images (1.5μm/z-stack) were visualized to confirm the extent of cell colocalization or the diameter and area
occupied by the amyloid plaque. Scale bar = 100μm. (B) Quantitative analysis of Aβ shows a lower number of plaques in the brain of 2xTgAd treated mice. The
arrows point at the senile plaques, which are magnified in the inset. Data are expressed as mean ± SEM, n = 5 animals per group. **p < 0.01. (C) Quantification of
areas (percentage) of Aβ plaques in the hippocampus of all groups studied. (D) Quantitative analysis of FJB staining revealed a significant reduction in the number of
labeled cells after leptin treatment, indicating less neurodegeneration. (E) Quantitative analysis of Aβ sizes shows a lower number of larger plaques in the brain of
2xTgAd treated mice. (F) Aged 2xTgAD animals show downregulation of BDNF when compared with WT animals, and leptin administration restores the levels of
BDNF. One-way ANOVA, data are expressed as mean ± SEM, n = 5 animals per group. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 9. Leptin attenuates microglial cell number in the hippocampus of WT and 2xTgAD mice. (A) Representative images of Iba-1+ cells, in the hippocampus sections
of aged WT and 2xTgAD animals (B) Quantitative analysis of Iba-1+ stained cells. (C) Higher magnification of amyloid plaques seen in 2xTgAD - PBS animal. One-way
ANOVA, data are expressed as mean ± SEM, n = 5 mice per group, 4-8 sections per animal; ** p < 0.01; *** p < 0.001. Scale bar = 100μm in (A) and 20μm in (C).

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M.L. Calió et al. Neurobiology of Disease 148 (2021) 105219

Fig. 10. Microglia morphology, evidenced by the density and the presence and the length of secondary/tertiary branches in the hippocampus. (A) Pre-processing of
cell digital image. After a random selection of cells from the tissue picture, the noise was removed by filtering the overall background to get a shape extraction. Next,
the image was changed to grayscale and then transformed into a binary image using the same threshold for all pictures. The binary image was edited to clear the
background and to join all branches so that the cell image would be formed by a continuous set of pixels. Finally, a skeletonized shape and its pairwise outline shape
were used for morphological parameters measures shown (B-F). One-way ANOVA, data are expressed as mean ± SEM, n = 5 mice per group, 4-8 sections per animal;
** p < 0.01; *** p < 0.001. Scale bar = 50μm.

low leptin is a risk factor for AD (Power et al., 2001; Lieb et al., 2009;
Bigalke et al., 2011; Rizoulis et al., 2005; Khemka et al., 2014) and re­
sults from animal studies that suggest that lower plasma leptin levels are
related to the role of leptin in memory decline in transgenic mice models
overexpressing APP (Greco et al., 2010; Ishii et al., 2014). Indeed
transgenic models that have the same mutations linked to familial AD,
also exhibit reduced serum leptin levels (Fewlass et al., 2004).
Regarding the data observed in our study, both results may be due to
leptin half-life in plasma, which is about 40 min. Also considering that
the levels of circulating leptin correlate directly with the amount of
adipose tissue (Bhatia et al., 2017) and that the weight of the 2xTgAD
animals was greater than that of the WT animals, we suggest that the
discrepancy in terms of circulating leptin observed in this study may
have been influenced by the supposed greater amount of fat present in
AD animals. Similarly, Peng et al. (2014) also observed a trend toward
decreased levels of leptin levels in transgenic AD mice compared with
the control.
It is known that circulating leptin levels are modulated by fasting or
cold exposure (Hardie et al., 1996). As leptin is secreted in a circadian
manner, the amount of circulating leptin is dynamic and changes
throughout the day according to food intake or energy expenditure
(Ahima et al., 1998; Ramos-Lobo et al., 2017). Since in this study the
animals had ad libitum access to water and food, this may have influ­
enced the leptin levels observed here. Moreover, as a trend of decreasing
leptin levels was observed in 2xTgAD animals compared to age-matched
controls, we believe that by increasing the n we would obtain similar

12
M.L. Calió et al.
results to those shown in previous studies.
Although our findings appear to be contradictory to most previous
studies, a few studies also demonstrate that no difference in leptin levels
was observed between the different genotypes (Peng et al., 2014; Lee
et al., 2018; Yeh et al., 2020) and some clinical studies have found no
apparent link between plasma leptin levels and AD (McGregor and
Harvey, 2018). In fact, Maioli et al. (2015) demonstrated alterations in
brain leptin signaling despite unchanged CSF leptin levels in AD. Lee
et al. (2018) observed that the level of leptin was comparable between
WT and the same AD model used in our study. Another recent study also
showed that the fasting levels of leptin were not different among WT and
the transgenic AD model (Yeh et al., 2020). One possible reason for this
divergence is that the plasma levels of leptin may not be a good indicator
of leptin levels in the CNS once altered blood-brain transport of leptin
has been documented in AD (Dietrich et al., 2008). Besides serum leptin
levels may not reflect the intracerebral concentration of that peptide.
The determination of the levels of these peptides in the CSF requires an
invasive procedure and for this reason it was not evaluated in our
investigation. Despite this, new studies would be helpful in identifying
whether changes in leptin levels correlate and track with disease pro­
gression and worsening AD pathology. While there is potential for leptin
to serve as both a novel therapeutic intervention and diagnostic target,
particularly early in AD (Tezapsidis et al., 2009), additional studies are
clearly needed to test the clinical utility of leptin in AD.
In the adult brain, neurogenesis occurs in the SGZ of the hippo­
campal dentate gyrus and the SVZ of the lateral ventricles. While
migration of the newly generated cells in the SGZ is restricted to the
hippocampus, where they participate in learning and memory forma­
tion, cells from the SVZ are destined to renew interneurons of the ol­
factory bulb (Kriegstein and Alvarez-Buylla, 2009). SVZ-migrating NSCs
are thought to participate in the attempt to regenerate an injured area
(Conover and Notti, 2007; Clelland et al., 2009; Deng et al., 2009;
Garthe et al., 2009; Mundim et al., 2018) including neurological diseases
such as AD (Chen et al., 2012; Fainstein et al., 2018).
Previous studies have shown that neurogenesis happens at a dimin­
ished rate with the advancing of age, impacting its physiological role in
neuronal replacement and consequently its ability to participate in the
repair mechanisms triggered by injury or disease (Enwere et al., 2004;
Katsimpardi and Lledo, 2018; Kuhn et al., 2018). Despite this, the aged
brain partially retains its neurogenic capacity through different mech­
anisms (Kempermann et al., 2002; Moreno-Jimenez et al., 2019). In
conditions like AD, which occurs at an increasing frequency with
advancing age, the ability of the brain to mobilize new neurons opens
novel possibilities for cell-replacement and provides a promising ther­
apeutic methodology. Numerous studies have demonstrated that
increasing neurogenesis might promote functional recovery in the
damaged area in the brain (Jin et al., 2002; Jin et al., 2004a; Kern et al.,
2011; Diaz-Moreno et al., 2013).
It is known that the neurogenic process can be triggered by physio­
logical factors, such as growth factors and environmental enrichment,
and by pathological processes, including trauma and neurodegeneration
(Perez-Gonzalez et al., 2011; Mundim et al., 2018). In vivo and in vitro
studies have shown that leptin stimulates neurogenesis in the hippo­
campal dentate gyrus by increasing cell proliferation (Garza et al., 2008)
and neuroprotection (Weng et al., 2007; Zhang et al., 2007b). Adult
hippocampal neurogenesis is a highly plastic process that responds
swiftly to neuronal activity. Adult hippocampal neurogenesis can be
regulated at the level of neural stem cell recruitment and activation,
progenitor proliferation, as well as newborn cell survival and differen­
tiation (Pineda and Encinas, 2016).
A dysfunction in hippocampal neurogenesis has been observed in
multiple models of AD and human AD brain tissue and may be related to
the cognitive and memory dysfunction (Yamashita et al., 2006; Demars
et al., 2010; Winner et al., 2011; Perry et al., 2012; Moreno-Jimenez
et al., 2019), whereas the increase of endogenous neurogenesis can
improve the spatial memory in experimental models (Mirochnic et al.,
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Neurobiology of Disease 148 (2021) 105219
2009; Sahay et al., 2011). Despite this, the increase in neurogenesis may
not be sufficient to compensate the damages that occur in this pathology
(Jin et al., 2004a).
Since the hippocampus is one of the first areas to be affected in AD,
most studies about neuroregeneration examine the involvement of SGZ-
born neuroblasts in brain repair, and neurogenesis at the SVZ has not
been widely investigated so far mainly in terms of possible regulation of
adult neurogenesis by leptin. Therefore, in the present study, one of our
questions was whether the neurogenic action of leptin seen in SGZ could
occur similarly in SVZ, which could help replace damaged cells and/or
prevent neurodegeneration. If the progression of the disease is related to
the loss of the functional capacity of the NSCs, since neuroregeneration
could be affected in several regions of the brain and not only in the
hippocampus, the discovery of therapeutic strategies that increase
neurogenesis or prevent the decrease of the proliferative process in these
areas could be a new treatment option for neurodegenerative diseases.
In addition, SVZ NSCs are better characterized than SGZ in terms of
functional responses such as proliferation and migration, both in phys­
iological and pathological conditions (Fainstein et al., 2018). Besides,
the neurodegeneration in AD affects the entire neocortex and several
areas of the brain, rather than just the hippocampus, highlighting the
need to study NSCs populations from different niches, including the SVZ,
SGZ, and others.
For this reason, we analyzed two neurogenic niches and evaluated
whether leptin would be able to regulate adult neurogenesis both in the
SVZ and the SGZ. Since neurogenesis is tightly related to AD, under­
standing the mechanisms of alterations in this process will contribute to
understanding AD pathophysiology.
Here, we have demonstrated that the administration of leptin in­
creases the proliferation of neural progenitors in the main neurogenic
niches and reduces neurodegeneration, even in the transgenic AD mouse
model of 12 months of age, thereby suggesting by this mechanism that
dead or damaged neurons could be replaced.
We studied gene expression of some proliferation and differentiation
markers expressed in the brain, asking whether their expressions were
modified in the transgenic mice and if leptin could alter this pattern.
Leptin induced an increase in gene expression of DCX, a highly
expressed gene in neuroblasts, which was found at low levels in all
groups of 2xTgAD animals prior to treatment, although no difference
was observed between the genotypes. Increased PCNA expression was
only observed in WT animals of both ages, whereas increased Ki67 and
nestin expression was also be observed in transgenic animals. Interest­
ingly, some genes related to proliferation showed a lessened response to
leptin-evoked expression. This data suggests a possible interaction be­
tween age and genotype factors. We hypothesize that certain prolifera­
tive genes are more activated in the presence of Aβ plaques, which are
only found in aged AD models. In this sense, it is known that PS1 reg­
ulates NSCs differentiation both in vitro and in vivo (Demars et al., 2011)
while sAPPα acts as a proliferation factor on NSCs in the adult brain
(Demars et al., 2013; Baratchi et al., 2012).
Unlike expected by the effect of leptin on cell proliferation, this
hormone decreased the gene expression of GFAP, which was twice as
high in the aged 2xTgAD animals compared to WT and treated animals.
This fact suggests that the decrease in GFAP is linked to the reduction of
astrogliosis observed in aged transgenic AD animals.
The decreased levels of neurotrophins have been found in AD in prior
studies (Stopa et al., 1990; Hock et al., 2000). This environment
impaired the maturation of NSCs into differentiated neurons. Our results
of gene expression contrast with prior findings from Jin et al. (2004b),
from the hippocampus of AD patients, showed increased, rather than
impaired, expression of the immature neuronal marker. These dissimi­
larities raise the possibility that different forms of AD may diverge
concerning their association with neurogenesis, particularly considering
the sporadic form of AD.
However, it is not known whether the newly generated neurons can
differentiate into mature neurons. Furthermore, using qPCR, no
M.L. Calió et al.
differences were observed in the gene expression of the mature neuronal
markers (βIII-tubulin, Map2 and NeuN) when leptin was used, and lower
expression was observed in the aged 2xTgAD animals compared to
young 2xTgAD and aged WT mice. These findings are consistent with
previous report that showed a decrease in MAP2 expression in the
dentate gyrus in AD (Li et al., 2008).
This is likely to be due to the fact that the one-week period used for
treatment is not sufficient for the complete neuronal maturation process.
Thus, although we have observed an increase in neuroproliferation, we
cannot affirm that the newly generated neurons have differentiated into
mature neurons. In this sense, previous report showed that the decrease
in MAP2 expression in the AD dentate gyrus is mainly attributable to an
interruption in the maturation of developing immature granular cells as
a reaction to changes in the AD microenvironment (Li et al., 2008).
Most studies of the pathogenesis of AD have focused on the degen­
eration of existing synapses and neuronal cells and only a few studies
explore the possibility of a defect or decreased capacity in the produc­
tion of new neural cells from NSCs.
Neurogenesis in the hippocampus plays an important role in the
maintenance and improvement of memory and learning (Shors et al.,
2001; Snyder et al., 2005). Thus, disruption of neurogenesis in the
dentate gyrus can be involved in the age-associated decline of hippo­
campal learning and memory, also observed in AD. The balance between
neurogenesis and neurodegeneration is thought to be critical for the
maintenance of these functions (Gould et al., 1999). However, these
cognitive abilities require not only the birth of new neurons to replace
the lost cells but also the differentiation of these newborn cells. The
present study demonstrates for the first time the differences between WT
and 2xTgAD animals on neurogenesis in different niches that are
compromised in AD. Besides this, we also evaluated the effect of leptin,
addressing the genotype effects.
Using immunohistochemistry, we found that the proliferation of cells
is dramatically decreased in both SVZ and SGZ in AD. Our results indi­
cate that NSCs from Alzheimer’s disease mice exhibit decreased func­
tional properties in terms of their ability to proliferate, even in the early-
onset of disease in SVZ, when compared to WT animals.
This loss of proliferative capacity is accentuated with advancing age
in both genotypes and not only in a late-onset neurodegenerative dis­
ease, as would be expected. In 2xTgAD animals, NSCs proliferative
function has been shown to decrease at the age of 3 months (young
animals), a time point before Aβ accumulation in the brain. At the age of
12 months, a time point with advanced Alzheimer’s pathology, the
proliferation of NSCs proves to be even more diminished and dysfunc­
tional. Interestingly, leptin is able to increase cell proliferation in SVZ in
both genotypes, regardless of the age of the animals.
Concerning the SGZ, dramatically reduced proliferation in the den­
tate gyrus has been observed in aged transgenic mice for the mutated
form of APP, although a reduction in proliferation has also been noticed
in aged WT animals when compared to young WT animals but less
markedly.
Here we show that overexpression of mutant APP and PS1 in the
transgenic mice reduces the proliferation of neural progenitors in the
hippocampus of aged animals. However, we observed that leptin in­
creases the number of proliferative cells both in the aged WT and young
and aged double transgenic animals. Thus, collectively, the fact that we
observed a decrease in the proliferation of NSCs even before the onset of
AD suggests that mutations linked to AD may have deleterious proper­
ties in neurogenesis, regardless of the effect of Aβ deposition. These data
suggest that in addition to advancing age, both the expression of
transgenes and the brain environment of AD can impact the proliferation
of NSCs. Indeed, the neurogenic process in the adult brain could also be
viewed as one form of neural plasticity, being triggered by many factors
that are not only due to injury or situations that require cell replace­
ment. In fact, several studies summarized the idea that the pathology of
AD may reflect the fundamental failure of neural plasticity (Mesulam,
1999; Haughey et al., 2002; Arendt, 2005; Kizil and Bhattarai, 2018).
14
Neurobiology of Disease 148 (2021) 105219
To determine whether the genotype and leptin treatment could also
affect differentiation of neural stem cells in neuroblasts or astrocytes, or
even affect the proliferation of neural progenitors, cell differentiation
was assessed using DCX and GFAP labeling.
Our results regarding the coexpression of BrdU with DCX in the SGZ
indicated that the number of these coexpressing cells decreased with age
in both genotypes. At the age of 12 months, the number of BrdU/DCX+
cells decreased in the DG of the AD mice model when compared to the
WT animals. These results show that there is a decreased generation of
DG neuroblasts during the progression of AD. Namely, AD temporally
declines neurogenesis. On the other hand, leptin treatment has been
shown to increase the number of BrdU/DCX coexpressing cells in both
genotypes and ages.
Concerning SVZ, the quantity of BrdU/DCX+ cells did not vary ac­
cording to age or between genotypes. Also, our data showed increased
expression of the immature neuronal marker DCX in the SVZ of treated
mice from both ages and genotypes.
Whether enhanced neurogenesis would be beneficial or not fully
understood. It is important to know what occurs when neuronal activity
is increased leading to abnormal hyperexcitatory levels. Pineda and
Encinas (2016) discuss the effects of neuronal hyperexcitation on hip­
pocampal neurogenesis. In this sense, the contribution of aberrant
neurogenesis is seen to be linked to the generation of neurons with
ectopic location and mossy fiber sprouting (Parent et al., 1997), which
are features evidenced by epilepsy and show little effect in AD. Several
studies have shown that increased neurogenesis, especially in the hip­
pocampus, is useful in AD cases, improving cognition and memory since
adult neurogenesis is a form of hippocampal plasticity with NSCs
bearing little resemblance to their mature counterparts (Deng et al.,
2009; Greco et al., 2010; Kim et al., 2010; Maioli et al., 2012; Perez-
Gonzalez et al., 2014; Akers et al., 2014; Ghasemi et al., 2016). More­
over, there is significant cross-talk between many of the important
proteins that regulate the progression of familial AD and adult neuro­
genesis (Lazarov and Marr, 2010, 2013). PS1, for example, regulates
NSCs differentiation both in vitro and in vivo (Demars et al., 2011), while
sAPPα acts as a proliferation factor on NSCs in the adult brain (Demars
et al., 2013; Baratchi et al., 2012).
Chronic neuroinflammation is also common to nearly all neurode­
generative diseases, and it contributes to their pathophysiology (Heneka
et al., 2014). Astrocyte cells are capable of local proliferation under
certain conditions, with indications that their progeny may resume
normal physiological function over time (Bardehle et al., 2013; Buffo
et al., 2008). It is known that injury and degeneration can cause astro­
gliosis and subsequent astrocyte proliferation, although the exact mo­
lecular stimulus of proliferation is currently unknown (Doetsch, 2003).
Nevertheless, although anti-inflammatory and immunosuppressive
therapies have demonstrated some efficacy in neurodegenerative dis­
ease models, these treatments have largely failed in the clinic (Arvani­
takis et al., 2008; Wyss-Coray and Rogers, 2012).
Concerning SGZ, DCX expression gradually decreases with
advancing age, but GFAP expression increases mainly in 2xTgAD ani­
mals, and the two markers seem to have contrary expression patterns
with aging. Since GFAP is also an indicator of NSCs, it is uncertain
whether the BrdU/GFAP+ cells that we have identified are proliferative
NSCs or reactive astrocytes. Given the strong GFAP staining as well as
consistent results from previous studies (Schmitz et al., 2004; Lee et al.,
2014; Zeng et al., 2016), it is likely that they are the latter, but we cannot
conclusively classify them as reactive astrocytes without further anal­
ysis. Also, it is known that astrogliosis is found in AD and other neuro­
degenerative diseases (Wright et al., 2013; Hostenbach et al., 2014).
Reactive astrocytes play critical roles in modulating neuroinflammation
and oxidative stress, which are among the main aspects of the patho­
genesis of AD (Agostinho et al., 2010). Consequently, targeting neuro­
inflammation in the early stages is considered to be one of the
methodologies to treat or delay the progression of AD.
In this sense, our data show that leptin treatment may decrease
M.L. Calió et al.
astrogliosis in aged 2xTgAD animals as seen by the exacerbated prolif­
eration in the hippocampus of these animals which is a consequence of
the neurodegenerative lesions found in this pathology. Our findings are
consistent with the results of Garcia et al. (Garcia et al., 2014), which
observed a large number of GFAP+ cells in the hippocampus of this
animal model at the age of 9 and 12 months, compared to WT mice. In
addition, in both the 2xTgAD and WT animals, of both ages, there were
no significant changes in the number of BrdU/GFAP+ within the SVZ.
Leptin receptors are highly expressed in the CA1 and CA3 regions of
the hippocampus, as well as in the DG (Mercer et al., 1996; Shanley
et al., 2002). Leptin receptor-deficient animal models (db/db mice)
exhibit intense deficits in spatial memory, learning and insufficient short
term memory processing (Li et al., 2002; Farr et al., 2006; Oomura et al.,
2006)
To further characterize the association between leptin and neuro­
genesis in AD, we assessed the mRNA expression and the presence of the
long form of leptin receptor (LepRb) in 2xTgAD and wild-type (WT)
leptin-treated mice at different ages and analyzed the co-localization of
LepRb with the marker of neural differentiation in the neurogenic
niches. Gene expression analysis by qPCR for LepR in the hippocampus
showed no difference between groups, regardless of age or genotype.
Besides, leptin treatment increased the expression of its receptor on all
treated groups. The overexpression of LepRb suggests its involvement in
the observed improvement. Double staining with LepR/DCX showed
that some cells coexpressed these markers, in the two areas analyzed.
It is well accepted that the environment of the AD brain is toxic to
neural cells mainly due to the formation of Aβ oligomers, which can
trigger AD pathogenesis (Hardy and Selkoe, 2002; Rapoport et al., 2002)
and can be one of the reasons why there is a limitation on the ability to
repair neuronal death via neurogenesis in AD (Haughey et al., 2002;
Zhang et al., 2007a). 2xTgAD mice brains showed that the Aβ plaques
were localized in the hippocampus and entorhinal cortex (data not
shown). In addition, Caille et al. (2004) established another theory that
sAPP (soluble amyloid precursor protein) regulates the proliferation of
NSCs responsive to EGF in SVZ due to the lack of sAPP-binding sites in
the SGZ, thereby causing regional differences in adult neurogenesis.
Therefore, it might be probable that this regional difference in neuro­
genesis was reliant on the amount of Aβ deposition.
Leptin has been shown to reduce brain Aβ levels in 6-months old
CRND8 transgenic mice following 8 weeks of treatment (Greco et al.,
2010). Our findings presented here indicate that leptin administration
reduced the accumulation of Aβ deposits in 12 months-age 2xTgAD
mice, coinciding with previously published studies (Perez-Gonzalez
et al., 2011; Perez-Gonzalez et al., 2014). Recent findings suggest that
the inhibitory effect of this hormone concerning Aβ accumulation is
presumably attributed to its inhibitory action of APP processing through
the amyloidogenic pathway, regulating BACE expression, the most
important β-secretase involved in APP processing (Perez-Gonzalez et al.,
2014). In the present study, we found that AD model animals treated
with leptin had a greater number of smaller plaques. However, mice in
this group exhibited larger plaques at a lower frequency than untreated
animals. Our treatment with leptin, by inducing the modulation of the
immune response and decreasing the size of senile plaques, demon­
strated the capacity to inhibit Aβ accumulation. Additionally, our data
support the observations of Greco et al. (2010) who revealed that 8
weeks of leptin treatment significantly reduced the amyloid burden in
the hippocampus of 6-month old TgCRND8 mice, compared to age and
gender-matched saline-treated transgenic mice. This result was parallel
to a decrease in the average size of plaques also analyzed in this study.
Therapeutic explorations on AD have been constantly conducted on
the pathogenesis and subsequently tailored therapeutic intervention,
especially in the neurobiological field, and the discovery of neuro­
trophins (NTs) was a milestone, which provided a new perspective into
neurogenesis and neuron survival (Bothwell, 2014). Brain-derived
neurotrophic factor (BDNF) is the most widely distributed NT in the
adult brain and has key roles in neuronal survival, synaptic plasticity
15
Neurobiology of Disease 148 (2021) 105219
and memory storage in the hippocampus (Lu et al., 2013). More
recently, it has also been described as important in the central control of
energy homeostasis (Rios, 2013; Vanevski and Xu, 2013). BDNF is
widely distributed in the brain, being highly expressed in the hippo­
campus, cortex, and basal forebrain, where it is important for learning,
memory, and higher thinking (Huang and Reichardt, 2001; Yamada and
Nabeshima, 2003; Voineskos et al., 2011) and in several hypothalamic
nuclei, where it also regulates feeding and body weight (Rios, 2013).
Although BDNF is needed in the developmental stages, BDNF levels have
been shown to decrease in tissues with aging (Tapia-Arancibia et al.,
2008). In humans, decreased hippocampal volume is associated with
decreasing BDNF levels, suggesting there is a relationship that might
explain some of the cognitive declines that occur during aging (Erickson
et al., 2010; Buchman et al., 2016). Changes of BDNF level and mRNA
expression have been also reported in brain samples as well as blood of
AD patients, which also indicates a potential role of BDNF in the path­
ogenic process of AD (Huang and Reichardt, 2001; Peng et al., 2009;
Angelucci et al., 2010; Laske et al., 2011; Voineskos et al., 2011; Francis
et al., 2012a; Ventriglia et al., 2013; Nagata et al., 2014; Prakash and
Kumar, 2014; Song et al., 2015; Li et al., 2016; Marsh and Blurton-Jones,
2017). Correspondingly, higher blood BDNF levels seem to delay the
rate of cognitive decline in AD (Bollen et al., 2013). As early as 1991, it
was found that BDNF mRNA decreased in the hippocampus of in­
dividuals with AD, which suggested that BDNF may be related and
contribute to the progression of cell loss in AD (Phillips et al., 1991).
Compared with aged controls, BDNF protein expression, as well as
mRNA levels, were found decreased in hippocampus and frontal, pari­
etal, and temporal cortex of AD postmortem samples (Connor et al.,
1997; Hock et al., 2000; Holsinger et al., 2000; Michalski and Fahne­
stock, 2003; Peng et al., 2005), meanwhile, similar results are also
replicated in AD animal models (Francis et al., 2012b; Kim et al., 2010;
Li et al., 2009; Maioli et al., 2012; Meng et al., 2013; Naert and Rivest,
2012; Peng et al., 2009). In addition, another study demonstrated that
the induction of Alzheimer’s disease model leads to a decrease in BDNF
mRNA levels in the hippocampus (Smith et al., 1995).
BDNF functions depend not only on its site of action but also on a
complex array of metabolic and environmental factors that regulate its
expression in a brain region-specific manner. It is currently accepted
that peripheral metabolic cues such as leptin increases BDNF mRNA
expression in the nucleus of the hypothalamus. Leptin is an adipocyte-
derived hormone that acts as an anorexigenic stimulus and it is known
to acutely induce BDNF gene expression in the ventromedial nucleus of
the hypothalamus of fasted mice (Komori et al., 2006; Unger et al.,
2007) but is unclear whether leptin regulates mRNA BDNF in the hip­
pocampus, as a modulator of neural activation. In our experiments,
changes in hippocampal BDNF gene expression were present in the aged
treated group in which significantly higher levels were observed.
However, it is unlikely that leptin alone contributed to the increase in
BDNF expression observed in these animals.
In a previous study, Guo et al. (2008) demonstrated that chronic
intraventricular leptin infusion is capable of protecting the neurons in
the hippocampus from cell death induced by neuronal insults (Guo et al.,
2008). Besides the formation of Aβ plaques, we evaluated the neuro­
degeneration in leptin-treated and untreated mice. In this sense, our data
are contradictory to Garza et al. (2008) who observed that leptin did not
show effects on cell survival, but are consistent with the findings of
Perez-Gonzalez et al. (2014) who used lentivirus gene delivery to
overexpress leptin in the brains of AD animals. Curiously, they also
observed that leptin significantly reduced neuronal degeneration in the
hippocampus of the AD model, also suggesting that leptin has a neuro­
protective effect.
It is known that oxidative stress can initiate the process of apoptosis
and that abnormalities in the regulation and expression of antioxidant
enzymes may have a role in mechanisms of CNS aging and neuro­
degeneration. In fact, neurons are at particular risk from damage caused
by free radicals because of high rates of oxygen consumption and high
M.L. Calió et al.
contents of polyunsaturated fatty acids (Zemlan et al., 1989; Uysal et al.,
2012; Radi et al., 2014). Several lines of evidence point that the mo­
lecular mechanisms that link AB to the onset of neurotoxicity are the
production of reactive oxygen species induced by the presence of this
oligomer. Actually, the oxidative stress resulting from the production of
ROS is considered a common effector of the cascade of degenerative
events in many neurological disorders (Cimini et al., 2009).
In the present study, we also evaluated the action of leptin on gene
expression of antioxidant enzymes. Besides, we checked whether leptin
was able to decrease previously existing levels of superoxide in the
neurogenic niches of AD animals. Although the main cause of neuronal
death in AD is the formation of senile plaques, in vivo evidence showed
that mitochondrial dysfunction and redox imbalance triggers or worsens
AD-related neuronal deficits (Calon et al., 2004; Lustbader et al., 2004;
Beal, 2005).
Studies suggest that there are multiple mechanisms by which
oxidative stress may accumulate and create dysfunctional neuronal re­
sponses in AD and that the development of the AD phenotype requires
multiple insults (Zhu et al., 2004). Since our previous results demon­
strate the ability of leptin to decrease neurodegeneration in animal
models of AD, we decided to investigate whether leptin could improve
the existing oxidative stress profile in this pathology. In order to obtain a
better insight into the ROS scavenging defenses of the hippocampus of
Tg mice, we studied GPx1 and SOD2. We have demonstrated the exis­
tence of significantly abnormal increased superoxide generation and the
downregulation of Sod2 expression in the hippocampus of aged 2xTgAD,
suggesting a negative correlation between cellular superoxide produc­
tion and Sod2 decline in advanced AD.
The analysis of gene expression shows relevant differences between
treated and untreated Tg animals of both ages for the two genes
evaluated.
The literature shows that there is no difference in the expression of
some genes related to the oxidative response in the hippocampus of AD
models. Several studies have shown that there are different reactions in
response to the perturbation of the redox status, depending on the
specific brain area. In this sense, it is known that the cortex is more
reactive while the hippocampus is more prone and susceptible earlier to
the oxidative insult (Scheff et al., 2007; Cimini et al., 2009).
In this perspective, our results corroborate the findings of Cimini
et al. (2009) who observed that there is no difference in the levels of
mRNA of GPx1 or SOD2 in the hippocampus of WT animals and trans­
genic AD mice. The cortex is physiologically more exposed to ROS, and
therefore basally endowed with more effective antioxidant defenses. In
this perspective, the increases of SOD2 and GPX1 in the cortex of AD
animals observed by Cimini et al. (2009) may be interpreted as a
compensatory response to an early perturbation of the redox status. This
response likely consists of stabilization and/or activation of the enzy­
matic proteins, rather than induction, since the corresponding mRNA
levels were unchanged. Differential effects of oxidative stress on tran­
scriptional or post-transcriptional regulation of antioxidant enzymes
have been previously reported (Rohrdanz et al., 2001).
We also found that the fluorescence intensity of DHE staining was
increased in non-treated aged 2xTgAD in comparison to WT and that the
superoxide levels decreased after treatment with leptin in these animals,
in both niches analyzed. Besides, no difference was found in the WT
group. Surprisingly, in SVZ, the antioxidant action of leptin was detected
in younger animals, whereas in the hippocampus, the same result could
only be observed in the aged animals. Interestingly, the hippocampus
and the subventricular zone show a different behavior, making it
possible to hypothesize a higher susceptibility of the former area to the
oxidative insult in AD (Scheff et al., 2007) and also to leptin treatment.
We believe that leptin’s role in decreasing DHE is due to its immuno­
modulatory capacity through microglia, which is responsible for the
production of ROS in the hippocampus of aged 2xTgAD mice as well as
astrocytes (von Bernhardi et al., 2015). With aging and the deposition of
Aβ, the microglia’s response becomes exaggeratedly dysregulated and
16
Neurobiology of Disease 148 (2021) 105219
reactive, which could justify the fact that we observed a more evident
result to treatment with leptin in these animals.
Taken together, these results suggest that Sod2 protects the aging
brain against oxidative stress in AD by controlling superoxide over­
production and that leptin treatment can restore this antioxidant
pattern. Moreover, the significant decrease in superoxide anion (O-2)
production observed after treatment indicates that leptin also has anti­
oxidant action in AD.
Microglial activation has been considered a crucial player in the
pathological process of multiple human neurodegenerative diseases
(Heneka et al., 2015; Navarro et al., 2018). In this study, we briefly
highlight our data about neuroinflammation in the hippocampal area,
including microgliosis and astrogliosis. Recent studies mention that
Iba1is widely expressed in microglia of the hippocampus and cortex of
amyloidogenic transgenic models (Unger et al., 2018; Navarro et al.,
2018). In these mice, microglial cells clustered primarily at sites around
amyloid-beta plaques (Nimmerjahn et al., 2005; Unger et al., 2018)
supporting our interpretation of Iba-1 staining in the hippocampus of
aged 2xTgAD mice. We observed that the number of microglial cells was
higher in the 2xTgAD animals compared to WT mice. In this sense, leptin
was able to attenuate the number of Iba1+ cells, compared to the PBS
group, and that the number of cells labeled after treatment was similar
to the WT animals. On the other hand, in the WT-leptin group, the Iba-1
immunoreactive microglia was similar to the PBS group. Total microglia
number in both Alzheimer’s patients and animal models is increased and
correlates with disease severity and progression (Olmos-Alonso et al.,
2016). This suggests that disease pathology promotes microglial
proliferation.
This corroborates a previous publication that attests that leptin de­
creases the level of activated microglia in the hippocampus 1 day after
transient ischemia (Yan et al., 2011), indicating that this hormone is also
effective in reducing this neuroinflammatory marker.
It is known that AD brains exhibit at least two different morpho­
logical microglial phenotypes. Morphologically, microglia are classified
as ramified (quiescent/resting), or ameboid/phagocytotic (activated),
(Tremblay et al., 2011). The cells associated with the amyloid plaques
display an activated amoeboid-like phenotype while those present in
other regions have a homeostatic-like morphology (Krabbe et al., 2013).
Microglia behavior in AD may also depend heavily upon the stage of the
disease (Clayton et al., 2017). In accordance with these studies, in
2xTgAD animals we observed that the microglia had heterogeneous
morphology, from the branched homeostatic state to atrophied and
amoeboid forms, depending on the observed region. Indeed, regional
heterogeneity of microglia could be also observed during homeostasis.
During pathology, disease-associated microglia emerge with distinct
transcriptional profiles that reflect specific activation states (Priller and
Prinz, 2019).
Microglial activation plays a neuroprotective role and could improve
AD pathology by reducing Aβ levels in APP-based models (Michaud
et al., 2013). Differently, chronic activation that occurs from excessive
neuronal damage in neurological diseases can lead to the constant
release of pro-inflammatory molecules and harmful production of ROS
which results in adverse effects. Moderate increases in these cytokines
are generally considered a normal part of aging (Clayton et al., 2017).
However, large increases, as observed in AD, lead to excessive neuro­
toxicity that drives the neurodegenerative process and, in consequence,
could be implicated in the pathology of AD (Spangenberg et al., 2016;
Clayton et al., 2017; Navarro et al., 2018).
According to Navarro et al. (2018), the high accumulation of Aβ
produces a fast microglial response. In this same study, it was observed
that in samples from patients in advanced stages of AD, there was a
degenerative process of microglial cells, which compromised the role of
the immune response to damage, mainly in the dentate gyrus (Navarro
et al., 2018). Interestingly, our data showed that in aged 2xTgAD mice,
leptin also induced some morphological changes in microglia.
Considering that microglial cells are implicated in many beneficial
M.L. Calió et al.
functions, such as Aβ phagocytosis, senile plaque compaction and limi­
tation of Aβ toxicity and elimination of damaged neurons (Yuan et al.,
2016; Ulland et al., 2017), an impaired microglial response could
exacerbate the progression of pathology. On the other hand, there is also
abundant evidence that microglia can be harmful to neurons by
secreting factors to injure these cells (Colonna and Butovsky, 2017;
Liddelow et al., 2017), suggesting a possible participation of this hor­
mone in the modulation of the immune response in AD. Promisingly,
microglia activation seems to be an important target reached by leptin
treatment.
Our results suggest that leptin has several important actions in the
brain: first, it has a stimulatory effect on the proliferation and differ­
entiation of neuronal progenitors and may also have an immunomod­
ulatory effect. Second, it has neuroprotective and antioxidant capacity
by reducing amyloid-beta plaques, neurodegeneration and oxidative
stress. These data demonstrate the utility of leptin as a potential thera­
peutic approach for AD, although essential features are needed to ensure
its translation to the clinic in the future.
5. Conclusions
In summary, our results suggest that leptin increases the production
of newborn cells in two neurogenic niches even in the aged AD brain.
These data sustain an unusual role of leptin in the course of adult neu­
rogenesis and neurodegeneration in other neurogenic niches than the
hippocampus, providing new understandings into the process of
neurogenic control in dementia. Future efforts are needed to clarify
which signaling mechanisms are involved in the neurogenic and neu­
roprotective function of this hormone. Our data also provide the basis
for further analysis of the role of leptin, as an alternative or adjuvant, to
slow down or delay the central pathology associated with AD.
Funding
This work was supported by Fundação de Amparo à Pesquisa do
Estado de São Paulo – FAPESP (grant numbers 2014/24341-9 and 2015/
19231-8), Conselho Nacional de Desenvolvimento Científico e Tec­
nológico – CNPq (grant number 404646/2012-3), and Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior – CAPES (Finance Code
001).
Credit And Author Contributions Statement
Michele L. Calió conceptualized the project, designed methodology,
carried out the immunohistochemistry, qPCR experiments, performed
all formal and statistical analysis, interpreted the data, wrote and edited
the manuscript and the fig.s. Darci S. Marinho performed DNA extrac­
tion and genotyped all the animals, separating them for the experiments.
Geisa N. Salles carried out serum extraction, ELISA experiments and
tissue preparations. Amanda F. Mosini contributed to RNA and cDNA for
molecular analysis and qPCR experiments. Fernando H. Massinhani
performed morphometrical data collection and analysis and assisted cell
quantification. Gui M. Ko selected and crossed the heterozygous animals
and maintained the colony of transgenic mice and assisted the treatment
of animals. Marimélia A. Porcionatto conceptualized the project,
designed experiments, interpreted the data, provided guidance and su­
pervision, was responsible for funding acquisition and reviewed the
manuscript. The work presented here was carried out in collaboration
between all authors. All authors revised and approved the final version
of the manuscript. Conflict of interest: The authors declare no conflict of
interest.
Acknowledgments
We would like to thank Professor Beatriz Monteiro (Universidade
Federal de São Paulo) for the 6E10 and Iba1 antibodies and Clivandir
17
Neurobiology of Disease 148 (2021) 105219
Severino (Universidade Federal de São Paulo) for technical assistance.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.nbd.2020.105219.
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