Characterization of Behavioural and Neuro-pathological Changes in a Translational Huntington’s

Disease Mouse Model for Potential Preventative Therapies

Jingyun WU 1, Luisa MÖHLE 1, Thomas BRÜNING 1, Iván EIRIZ 1, Jens PAHNKE 1,2,3,4

1 University of Oslo and University Hospital Oslo, Norway; 2 University Medical Center and Universiy of Lübeck, Germany;
3 University of Latvia, Department of Pharmacology, Riga, Latvia; 4 University of Tel Aviv, Israel
E-mail correspondence to Jens Pahnke (jens.pahnke@medisin.uio.no)

Abstract Motor Performance Tests: Rotarod, Wire Hang test Anxiety-like behaviour: OF
Huntington’s disease (HD) is an inherited and autosomal-dominant neurodegenerative disease and Pole Test. ♂
♂ ♂
characterized by the disordered control of voluntary movement, psychiatric disturbance, and A B
cognitive impairment. We generated the zQ175Δneo HD mice from zQ175 mice to perform a
temporal characterization of several behavioural and neuropathological features. The
zQ175Δneo mice exhibited motor coordination dysfunctions and body weight loss at an early age
of around 29 weeks. In addition, zQ175Δneo demonstrated muscular problems, anxiety-like
behaviours, striatal atrophy, testicular atrophy and increased astrogliosis after 36 weeks of age.
However, microglia activation and whole brain atrophy only manifested at a late symptomatic
stage of our experimental set-up. Overall, the zQ175Δneo is a reliable knock-in mouse model
that showed significant HD-like phenotypes at a heterozygous state, which is more relevant to
human HD. Removing the neo-cassette resulted in a more robust and long-term onset of HD
symptoms, which enabled the zQ175Δneo mice to show severe and progressive phenotypes and Figure 3: The motor Figure 4: Psychiatric features
♂
provide a great value for preclinical studies. C performance of wild type like anxiety-like behaviours
(empty circles) and zQ175Δneo was assessed at 7-week
mice (filled circles) was intervals by performing open
assessed at 7-week intervals field test and the time a
by performing (A) accelerating mouse spent in the center of
rotarod performance, (B) wire an open field was recorded.
hang, and (C) pole test N=4-6. Only male animals
protocols. N=4-6. Only male data are only shown.
animals data are only shown.

Brain atrophy Reactive Gliosis in Striatum

A B 57-week WT 57-week zQ175Δneo

A B

IBA-1
Figure 1: Graphic summary of the main findings of zQ175Δneo HD mouse model characterization.
C ♂ D ♂ C D

GFAP
Material and Methods
Animals Subjects were male and female heterozygous zQ175Δneo mice generated from the
original zQ175 mouse model and their corresponding wildtype (WT) littermates. Body weight and
food intake were monitored weekly during the course of the study. E ♂ F ♂
Behavioural Experiment Paradigm 7 timepoints were chosen to characterize the behavioural Figure 5: General brain atrophy and striatal
and neuropathological characteristics of zQ175Δneo mouse model at 7 different stages of the atrophy were determined in zQ175Δneo mice
mouse lifespan. To eliminate any possible confounding influence in mouse behaviour tests due compared to wild type littermates.
to repeated behavioural testing, seven distinct cohorts of animals were set up. Every cohort Representative images of haematoxylin-eosin-
consisted of sex-matched zQ175Δneo mice and their counterparts WT (N=6 per genotype per stained coronal brain sections from 57-week-
sex). All animals were submitted to the following sequence of behavioural tests: the open field old female wild type (left) and zQ175Δneo mice
test (OF), accelerating rotarod test (RR), pole test and wire hang test. Each cohort was sacrificed (right) at (A) +0.8 mm and (B) -1.80 mm
after the behaviour test battery, and brain and testis were collected. Figure 6: Representative images of the IBA1-
distance from bregma. Dashed outlines stained and GFAP-stained striatum area of
Immunohistochemistry (IHC) Formalin-fixed hemispheres were embedded in paraffin and cut indicate the measured area of (A) striatum and
in 4-μm-thick coronal sections. Sections (approximately Bregma −2.0 mm) were stained for 57-week-old (A, C) WT and (B,D) zQ175Δneo
(B) hemisphere. Scale bars indicate 1000 µm. mice. Scale bar indicates 50µm. IBA1+ (E)
microglia (IBA1, 1:1,000, FUJIFILM Wako Chemicals Europe GmbH, 019–19741), astrocytes (C) Quantification of striatal area and half
(GFAP, 1:500) using a BOND-III® automated immunostaining system (Leica Biosystems GmbH, and GFAP+ (F) cells in the striatal area were
hemisphere area was assessed at 7-week measured at 7-week intervals during the time
Germany) with a haematoxylin counterstain. Quantitative analysis was performed semi- intervals during the time course of the study.
automatically using deep-learning algorithms generated with DeePathology™ STUDIO course of the study. N=6.
N=6.
(DeePathology Ltd., Israel)
Western Blot Striatum and testis tissues protein were extracted obtained in the ice-cold RIPA muHTT Levels in Striatum
buffer, and then homogenized by electric homogenizer. The lysates were centrifuged at 20,000
Female striatum Male striatum
rpm at 4°C for 30 min, and the supernatants were collected for western-blots. The muHTT
(PolyQ) antibodies MW1(1:1000, Merck) was applied for characterizing the muHTT levels. Age Age
15 22 29 36 43 50 57 15 22 29 36 43 50 57
(weeks) (weeks)
MW1 tubulin

MW1 tubulin

Body Weight and Representative Images

250 kDa 250 kDa

A 50 kDa 50 kDa

Figure 7: MW1 detects the muHtt protein in the striatal extracts of zQ175Δneo mice throughout the time
course of the study

Results summary

 Body weight loss and motor coordination malfunction were observed at an early stage of the zQ175Δneo
mouse model.
 Muscle-related motor dysfunctions manifested while the disease progresses at around 36 weeks.
B  IHC staining showed an early increase of activated astrocytes In the striatum and cortex while
activated microglia only showed an robust increase at a late timepoint at 57 weeks.
 Overall, zQ175Δneo mouse model with a C57BL/6 background displayed an early and enhanced HD-like
neuropathological phenotypes. The time course of HD progression and the heterozygous state of this
model are more relatable to human situation and the age of HD-phonotype onset of zQ175Δneo mouse
model (10 months old) is corresponding to the human HD onset age (after 40 years old).
 To this end, the present study is highly clinically relevant as it provided an insight in the molecular and
cellular characterizations underlying the novel HD model. In addition, the results generated from this
study that zQ175Δneo mice warrant further study as a promising model for HD treatment exploration.

C D Figure 2: (A) Male and (B)
female zQ175Δneo mouse body
weight measurement during the
time course of 57 weeks. N=6.
Representative photos of (C)
15-week old and (D) 57-week
old female zQ175Δneo mice.
Funding Information
Deutsche Forschungsgemeinschaft/ Germany (DFG 263024513); Nasjonalforeningen (16154),
HelseSØ/ Norway (2019054, 2019055, 2022046); Barnekreftforeningen (19008); EEA grant/Norges
grants (TAČR TARIMAD TO100078); Norges forskningsrådet/ Norway (295910 NAPI, 327571
PETABC).
www.pahnkelab.eu