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Original research
Loss of Wtap results in cerebellar ataxia and degeneration of
Purkinje cells
Yeming Yang
a
,
b
,
1
, Guo Huang
a
,
b
,
1
, Xiaoyan Jiang
a
, Xiao Li
a
, Kuanxiang Sun
a
, Yi Shi
a
,
b
,
Zhenglin Yang
a
,
b
,
*
, Xianjun Zhu
a
,
b
,
c
,
*
a
The Sichuan Provincial Key Laboratory for Human Disease Gene Study and Department of Laboratory Medicine, Center for Medical Genetics, Sichuan
Provincial People’s Hospital, University of Electronic Science and Technology of China, Chengdu, Sichuan 610072, China
b
Research Unit for Blindness Prevention of Chinese Academy of Medical Sciences (2019RU026), Sichuan Academy of Medical Sciences and Sichuan
Provincial People’s Hospital, Chengdu, Sichuan 610072, China
c
Key Laboratory of Tibetan Medicine Research, Chinese Academy of Sciences and Qinghai Provincial Key Laboratory of Tibetan Medicine Research,
Northwest Institute of Plateau Biology, Xining, Qinghai 810008, China
ARTICLE INFO
Article history:
Received 6 December 2021
Received in revised form
3 March 2022
Accepted 4 March 2022
Available online 15 March 2022
Keywords:
N
6
-methyladenosine
Wtap
METTL3
METTL14
Purkinje cell
Ataxia
Cerebellum
ABSTRACT
N
6
-methyladenosine (m
6
A) modification, which is achieved by the METTL3/METTL14/WTAP methyl-
transferase complex, is the most abundant internal mRNA modification. Although recent evidence indicates
that m
6
A can regulate neurodevelopment as well as synaptic function, the roles of m
6
A modification in the
cerebellum and related synaptic connections are not well established. Here, we report that Purkinje cell
(PC)-specific WTAP knockout mice display early-onset ataxia concomitant with cerebellar atrophy due to
extensive PC degeneration and apoptotic cell death. Loss of Wtap also causes the aberrant degradation of
multiple PC synapses. WTAP depletion leads to decreased expression levels of METTL3/14 and reduced
m
6
A methylation in PCs. Moreover, the expression of GFAP and NF-L in the degenerating cerebellum is
increased, suggesting severe neuronal injuries. In conclusion, this study demonstrates the critical role of
WTAP-mediated m
6
A modification in cerebellar PCs, thus providing unique insights related to neurode-
generative disorders.
Copyright ©2022, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and
Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved.
Introduction
The cerebellum is responsible for smooth and purposeful
movements, postural adjustments to maintain balance, and learning
of new motor skills. Purkinje cells (PCs) are the sole efferent neurons
in the cerebellar cortex and are considered the most functionally
important neurons for the coordination of body movement. They are
arranged in a monolayer and possess extensive dendrites that
project into the molecular layer (ML), where they receive inputs from
the two major excitatory streams: climbing fibers (CFs), which
synapse directly on PCs, and mossy fibers, which synapse on
granule cells (GCs). The axons of the GCs extend into the ML, where
they give rise to parallel fibers (PFs) and transmit afferent informa-
tion from mossy fibers to PCs (Wang et al., 2014a). In addition,
basket and stellate cells, which serve as inhibitory interneurons in
the ML of the cerebellar cortex, play an essential role in cerebellar
physiology by providing feed-forward inhibition to efferent PCs. PC
axons make inhibitory synaptic contacts with neurons in deep
cerebellar nuclei and thus represent the sole output of the cerebellar
cortex, which in turn projects to the thalamus and many other brain
regions (Cerminara et al., 2015).
Given their central role in cerebellar function, PCs are the most
extensively studied type of cerebellar neuron and were investigated
in the present study. In humans, defects in PCs have been implicated
in a variety of hereditary ataxias (HAs), which comprise a heteroge-
neous group of neurological disorders characterized by typical
neuropathological features, such as cerebellar atrophy and pro-
gressive PC loss (Ashizawa et al., 2018). Most HAs are autosomal
dominant disorders, while a few of them are autosomal recessive or
X-linked. Traditionally, autosomal dominant HAs are associated with
coding CAG repeat expansions that lead to neuronal protein aggre-
gation and consequent cell death (Sch€
ols et al., 2004). However,
genetic research has identified multiple mutations in a large number
of genes, indicating that there are additional pathological
*Corresponding authors.
E-mail addresses: zliny@yahoo.com (Z. Yang), xjzhu@uestc.edu.cn (X. Zhu).
1
These authors contributed equally to this work.
Contents lists available at ScienceDirect
Journal of Genetics and Genomics
Journal homepage: www.journals.elsevier.com/journal-of-genetics-
and-genomics/
https://doi.org/10.1016/j.jgg.2022.03.001
1673-8527/Copyright ©2022, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and
Science Press. All rights reserved.
Journal of Genetics and Genomics 49 (2022) 847e858
mechanisms involving widely diverse pathways essential for the
structure, homeostasis, function and survival of PCs that can explain
the characteristic PC death, cerebellar atrophy and resulting ataxia in
HAs (Jayadev and Bird, 2013). Although an increasing number of
causative genes of HA have been identified, the precise molecular
mechanisms are still largely unknown.
N
6
-methyladenosine (m
6
A) modification is one of the most
common methylation modifications of eukaryotic mRNA (Meyer
et al., 2012;Wang et al., 2019) and influences fundamental as-
pects of RNA metabolism, including alternative splicing, stability,
nuclear export, degradation, and translation (Zhao et al., 2017).
m
6
A modification has been shown to be a dynamic and reversible
modification that is executed by a multicomponent complex
comprising methyltransferase-like (METTL)-3/14 and Wilms’tumor
1-associated protein (WTAP) as the key methyltransferases and
removed by the demethylases FTO and ALKBH5. During the
methylation process, METTL3 acts as the catalytic component,
while METTL14 is involved in structural stabilization and RNA
substrate recognition (Wang et al., 2016a,2016b). WTAP interacts
with METTL3 and METTL14 to facilitate the transport of the
METTL3-METTL14 heterodimer to nuclear speckles enriched with
pre-mRNA processing factors, which is essential for the catalytic
activity of m
6
A methyltransferase (Ping et al., 2014). m
6
A-modified
transcripts are then transported to the cytoplasm and can be
recognized by a series of m
6
A readers, such as YTHDF1/2/3,
IGF2BP1/2/3 and YTHDC1/2, which then exert regulatory effects
(Wang et al., 2014b;Hsu et al., 2017).
RNA m
6
A methylation has aroused extensive attention and has
been widely investigated. In the mammalian nervous system, m
6
A
regulation has been proven to play important roles in brain devel-
opment (Yoon et al., 2017;Wang et al., 2018), cognitive function
(Widagdo et al., 2016;Walters et al., 2017;Shi et al., 2018;Zhang
et al., 2018), axonal regeneration (Weng et al., 2018), and synaptic
function (Merkurjev et al., 2018). In particular, the cerebellum is
among the brain areas with the highest levels of m
6
A methylation
(Chang et al., 2017). Mettl3 depletion in the mouse nervous system
using Nestin-Cre causes severe developmental defects in the cere-
bellum by controlling the mRNA stability of genes involved in cere-
bellar development and apoptosis (Wang et al., 2018). Furthermore,
deficiency of another m
6
A eraser, ALKBH5, was reported to affect
the metabolism of RNA of a subset of cell fate determination genes
and thus impair postnatal cerebellar development (Ma et al., 2018).
Therefore, these data suggest that m
6
A modification plays a funda-
mental role in the regulation of cerebellar development and neuro-
genesis. Additionally, previous study showed spatiotemporal-
specific expression of METTL3, METTL14, and WTAP from birth to
adulthood in the mouse cerebellum, especially in PCs. Notably, a cell
type-specific regulation was presented, with a remarkable reduction
of their expression in GCs while a mild increase in PCs (Ma et al.,
2018), implying that WTAP plays an important role not only in the
early cerebellar development, but also in the neurological function
maintenance and survival of PCs during adulthood. Due to the lack of
specific spatiotemporal knockout animal models, the exact role of
WTAP and the specific mechanisms of m
6
A modification in the
cerebellar PCs are not known and warrant further investigation.
In the present study, we generate mice with cerebellar PC-
specific conditional knockout of Wtap, which encodes a compo-
nent of the m
6
A writer protein complex. Our data show that loss of
Wtap in PCs causes early-onset ataxia concomitant with cerebellar
atrophy due to extensive PC degeneration. Further investigations
demonstrate that deletion of Wtap causes the aberrant degradation
of multiple PC synapses. Wtap depletion lead to decreased protein
levels of METTL3/14, resulting in reduced m6A methylation levels in
PCs. Overall, our findings highlight WTAP as a regulator of cerebellar
PC survival.
Results
Generation of Wtap conditional knockout mice using Pcp2-Cre
line
As a key regulator of m
6
A methyltransferases, WTAP facilitates
the proper nuclear localization of the METTL3-METTL14 hetero-
dimer and maintains the catalytic activity of m
6
A methyltransferases.
To investigate the role of WTAP in the cerebellar cortex, we intro-
duced two loxP sites into the Wtap genomic region flanking exon 3
by CRISPR/Cas9 system-assisted homologous recombination
(Fig. 1A). To generate Wtap conditional knockout mice, the resulting
Wtap floxed mice were crossed with L7 promoter (Pcp2-Cre)
transgenic mice, which constitutively express Cre recombinase in
cerebellar PCs from approximately postnatal day (P)6 (Barski et al.,
2000)(Fig. 1A). PCR genotyping was performed to confirm the
specific deletion of Wtap exon 3 and distinguish Wtap
flox/flox
;Pcp2-
Cre mice (hereafter referred to as Wtap
PKO
mice), Wtap
flox/þ
;Pcp2-
Cre mice, and Wtap
flox/flox
mice (used as control, hereafter referred
to as Wtap
f/f
mice) (Fig. 1B). All experimental mice used and corre-
sponding examinations were shown as a flowchart (Fig. S1).
We introduced ROSA26-tdTomato reporter mice (Fig. S2A and
S2B) to visualize the specific expression of Pcp2-Cre in cerebellar
PCs. PCs were labeled with an antibody specific to Calbindin-D28K
(hereafter referred to as Calbindin), and Pcp2-Cre was distinctly
expressed in Calbindin-marked PCs (Fig. S2C), suggesting precise
excision. To evaluate the excision efficiency of WTAP in PCs, we
extracted RNA and protein from Wtap
f/f
and Wtap
PKO
mouse cere-
bellar cortex samples. The expression level of WTAP in the Wtap
PKO
mouse cerebellar cortex was reduced to ~59% and ~52%, respec-
tively, of that in the control mice, as measured by RT-qPCR (Fig. 1C)
and Western blotting (Fig. 1D and 1E). Considering that WTAP is also
expressed in other cell types in the cerebellar cortex, the excision
efficiency of WTAP in PCs was relatively satisfactory. The immuno-
fluorescent staining further verified the specific WTAP depletion
(Fig. 1F). The WTAP fluorescence signal was robust in Wtap
f/f
PCs,
and WTAP mainly expressed in the nucleus of all PCs (Fig. 1F, ar-
rows). In contrast, WTAP was obviously absent from PCs in Wtap
PKO
mice (Fig. 1F, arrows), suggesting that WTAP was specifically
deleted in PCs.
Specific ablation of Wtap in cerebellar PCs causes ataxia and
cerebellar atrophy in mice
Wtap
PKO
pups wereborn at Mendelian ratios, and no differencewas
observed inbirth rate between these mice andcontrols. In addition, the
Wtap
PKO
mice exhibited a body weight growth rate that was compa-
rable to thatof the controls beforethe age of 21 weeks, whilethe growth
rate was slightly decreased by the age of 27 weeks (Fig. 2A). By 9
weeks, the Wtap
PKO
mice were extremely ataxic and unable to walk
smoothly. When suspended by their tails, the mutant mice displayed
dramatic tremors with an abnormal hindlimb clasping reflex indicative
of a neurological deficit(Fi g. 2B).Footprint pattern analysisshowed that
the Wtap
PKO
mice had an abnormal gait characterized by a shortened
stride length (Fig. 2Cand2D), demonstrating typical features of cere-
bellar ataxia. Furthermore, the rotarod test revealed that the motor
coordination of the mutant mice was significantly impaired (Fig. 2E).
The morphology of the cerebellum, as the center of motor coor-
dination, was next examined in 4-month-old littermates. Macro-
scopic histological examination showed that the cerebella of
Wtap
PKO
mice was markedly smaller, exhibiting severe atrophy
compared to other brain regions (Fig. 2F). This observation was
confirmed by hematoxylin and eosin (H&E) staining of cerebellar
sections (Fig. 2G), which revealed that the cerebellar area was
Y. Yang, G. Huang, X. Jiang et al. Journal of Genetics and Genomics 49 (2022) 847e858
848
reduced by approximately 61.5% in the mutant animals but showed
no obvious alterations in other brain regions (Fig. 2H and 2I).
Extensive degeneration of PCs in Wtap
PKO
mice
To further confirm that PC degeneration occurs in the cerebella of
mutation mice, cerebellar sections obtained from mice of different
ages (from 3 to 12 weeks old) were immunostained with Calbindin to
label PCs. Surprisingly, PC loss was first detected in the Wtap
PKO
mouse cerebellum at 6 weeks of age, and PC degeneration became
more pronounced over time (Fig. 3A). Quantitative analysis revealed
24.3%, 62.8%, and 87.9% reductions in the number of Calbindin-
positive PCs in Wtap
PKO
mice compared with controls at 6, 9, and
12 weeks of age, respectively (Fig. 3B).
Pcp2-cre is initially expressed at P6 and is fully established by
approximately 2e3 weeks after birth (Barski et al., 2000). Wtap
PKO
mice at P1 showed a normal cerebellar morphology that was
indistinguishable from that of their Wtap
f/f
littermates (Fig. S3). We
immunostained cerebellar sections from P9 and P15 mice for
Calbindin to determine whether any of the structural changes could
be detected in the early stage of Wtap abaltion. However, no sig-
nificant difference in PC number/morphology or dendritic
morphological features was observed between Wtap
f/f
and
Wtap
PKO
mice at both stages (Figs. S4 and S5). At P21, shrunken
PC somas as well as mild decrease of ML thickness were
observed, whereas no apparent PC loss was observed at this time
point (Fig. S6).
We next used 9-week-old mice for detailed analysis of PC
degeneration. H&E staining of the cerebellar cortex samples
revealed neurons with dystrophic characteristics, including shrunken
somas and depauperate dendrites (Fig. 3C, upper panels). The
thickness of the ML, in which the dendrites of PCs resided, was
reduced by ~27.9% in the Wtap
PKO
mouse cerebellum at 9 weeks of
age (Fig. 3C and 3D), which was consistent with the increase in PC
loss and ataxic gait exhibited by Wtap
PKO
mice. Calbindin immuno-
labeling further revealed extensive PC degradation in Wtap
PKO
mice.
Fig. 1. WTAP was deleted in cerebellar PCs from Wtap
PKO
mice. A: Schematic showing the strategy used to generate Wtap
flox/flox
;Pcp2-Cre (Wtap
PKO
) mice. In the Wtap conditional
knockout allele, exon 3 is flanked by two loxP sites. When mice carrying the floxed allele are crossed with Pcp2-Cre-expressing mice, exon 3 is deleted, resulting in a frame-shift deletion
and disruption of Wtap gene expression in PCs. B: Genotyping of Wtap
PKO
mice. Genomic DNA from mouse tail lysates was amplified using the Wtap-loxP-F and Wtap-loxP-R primer
pair and the Pcp2-Cre-F and Pcp2-Cre-R primer pair. C: RT-qPCR analysis revealed reduced expression of Wtap in the cerebella of 1-month-old Wtap
PKO
mice (n¼5). D: Reduced
expression levels of WTAP in the cerebella of 1-month-old Wtap
PKO
mice, as revealed by Western blotting analysis of cerebellar lysates from Wta p
f/f
and Wtap
PKO
mice using a WTAP
antibody. E: Quantification of the relative protein levels of WTAP (n¼6). F: Immunofluorescence analysis of cerebellar cryosections using a WTAP antibody (green), Calbindin antibody
(red) and DAPI (blue). Yellow arrowheads indicate the nuclei of PCs. The right panel shows the percentage of WTAP positive PCs (n¼3). The dotted lines represent the dividing line
between the IGL and PCL. *,P<0.05; **,P<0.01; ***,P<0.001. All data are presented as the mean ±SEMs. GFAP, glial fibrillary acid protein; H&E, hematoxylin and eosin staining;
IGL, internal granular layer; ML, molecular layer; PCL, Purkinje cell layer; SEM, standard error of the mean. Scale bar, 25
m
m(F).
Y. Yang, G. Huang, X. Jiang et al. Journal of Genetics and Genomics 49 (2022) 847e858
849
DAB staining of paraffin sections of the Wtap
PKO
mouse cerebellum
showed the presence of extensive degenerative features, including a
decrease in soma size and a marked reduction in the region occupied
by dendrites, prior to cell body loss (Fig. 3C, middle panels). Like-
wise, immunofluorescence staining using antibodies against Cal-
bindin and the specific neuronal marker NeuN was performed to label
the nuclei of PCs and GCs, respectively (Fig. 3C, lower panels).
Quantification of the cell number indicated that the GC number was
slightly reduced (P¼0.136, no significance) in the Wtap
PKO
mouse
cerebellum, although dramatic PC degeneration occurred at this time
point (Fig. 3E), implying that ML and subsequent cerebellar atrophy
are mainly caused by PC degradation.
We next assessed the dendritic spine of PCs using 3-week-old
mice, which have not shown visible PC loss. Golgi staining was first
performed to investigate synaptic changes, and the results revealed
that PCs in the Wtap
PKO
mouse cerebellar cortex exhibited normal
overall morphology and dendritic branching patterns (Fig. 4A). How-
ever, the dendritic spine density was significantly decreased in
Wtap
PKO
mice (Fig. 4A and 4B), with the spines exhibiting a stubby
morphology instead of the filopodia-like morphology observed in
control mice (Fig. 4A).
Cerebellar PCs, the only output neurons in the cerebellar cortex,
receive excitatory inputs from PFs and CFs, as well as inhibitory
inputs from basket and stellate interneurons (Fig. 4C). The reduction
in spine density and abnormal dendritic spine morphology in PCs
prompted us to examine PF-PC synapses in Wtap
PKO
mice. Cere-
bellar cryosections from 3-week-old animals were prepared and
subjected to double immunofluorescence staining with antibodies
against vGLUT1, a PF-specific presynaptic marker, and the PC
marker Calbindin. Regrettably, we detected no difference in cere-
bellar vGLUT1 expression between littermate controls and Wtap
PKO
mice, suggesting that PF synapses were not altered (Fig. 4D). We
next stained cerebellar sections for vGLUT2 to assess CF synapses,
which make excitatory connections with PCs. Strikingly, an ~70%
decrease in the density of vGLUT2-positive puncta was observed in
sections from Wtap
PKO
mice, suggesting a decrease in the number of
CF synapses (Fig. 4E). The two types of excitatory inputs to PCs can
be distinguished anatomically, as CFs form synapses with primary
dendrites adjacent to the PC soma, while PFs form synapses with
secondary and tertiary dendrites. To obtain further evidence that the
CF synapse density but not the PF synapse density was decreased,
we stained sections from WTAP-deficient mice for HOMER1, a
marker of postsynaptic excitatory inputs to PCs, and found that the
HOMER1 puncta density was distinctly reduced in the primary den-
drites but not the distal tertiary dendrites of PCs (Fig. 4F), suggesting
decreased CF-PC synapses. Additionally, we analyzed inhibitory
Fig. 2. Wtap
PKO
mice exhibited ataxia phenotypes and cerebellar atrophy. A: Body weight changes of the Wtap
f/f
and Wtap
PKO
mice from 3 to 27 weeks of age. B: Hindlimb clasping
phenotype of 9-week-old Wtap
PKO
mice during tail suspension.The blue arrowheads indicate the clasped feet. C:Wtap
PKO
mice dragged their hind feet when walking, as evident by ink
traces (blue, front paws; red, rear paws). D: Statistical analysis of the stride length of Wtap
f/f
and Wtap
PKO
mice (n¼6). E: Differences in performance in the accelerating rotarod test
were observed between Wtap
f/f
(n¼9) and Wtap
PKO
mice (n¼8). F: Comparison of the micromorphology of the brains of 4-month-old Wtap
f/f
and Wtap
PKO
mice. Photographs of mid-
sagittal brain sections are shown in the lower panel. G:H&E-stained sagittal cerebellar sections from 4-month-old Wtap
f/f
and Wtap
PKO
mice. Hand I: Surface areas of the whole brains
(H) and cerebella (I)ofWtap
f/f
and Wtap
PKO
mice (n¼3). *,P<0.05; ***,P<0.001; #, no significance. All data are presented as the mean ±SEM. Scale bar, 250
m
m(G).
Y. Yang, G. Huang, X. Jiang et al. Journal of Genetics and Genomics 49 (2022) 847e858
850
inputs to PCs. Staining of the cerebellar cortex for vGAT, a marker of
GABAergic presynaptic terminals, revealed that the density of vGAT
puncta was unchanged (Fig. 4G), suggesting that WTAP ablation had
little impact on inhibitory inputs to PCs. In addition, we compared
synaptic protein levels of cerebellar lysates from Wtap
f/f
and Wtap
PKO
mice by Western blotting. Consistent with immunostaining results,
vGLUT2 protein level in Wtap
PKO
mice was remarkably decreased,
while no difference in other synaptic proteins was observed
compared to controls (Fig. 4H).
Immunostaining of cerebellum sections using vGLUT2 antibody
from P15 littermates revealed similar synaptic deficits with that in 3-
week-old mice, characterized by reduced vGLUT2 puncta in ML of
Wtap
PKO
cerebellum (Fig. S7). This data suggested that the synaptic
phenotype preceded PC degeneration. Moreover, the synaptic
conditions of PCs in 9-week-old littermates, which exhibited exten-
sive PC degeneration, were also examined. Both immunostaining
and Western blotting data demonstrated that all synaptic types
connected with PCs, including PF and inhibitory input synapses were
decreased to varying degrees (Fig. S8). To gain insight into internal
synaptic changes, we analyzed cerebellar synapses by transmission
electron microscope (TEM). Consistent with the light microscopy
results, the bouton size of CF-PC synapses was reduced in Wtap
PKO
mice (Fig. 4I and 4J), whereas the density and length of the post-
synaptic density (PSD) of PF-PC synapses appeared normal
(Fig. 4Ke4M). Overall, these results indicate that PC-specific deletion
of Wtap led to extensive PC degeneration.
WTAP regulates PC survival via controlling METTL3/14
expression and m6A modification
WTAP interacts with METTL3 and METTL14 and serves as a
regulatory subunit that is essential for m
6
A methyltransferase ac-
tivity (Ping et al., 2014). This prompted us to examine the expression
level and cellular distribution of METTL3/14 in WTAP-deficient
cerebellar PCs. Immunofluorescence staining of sagittal cerebellar
sections from 1-month-old mice indicated that the levels of both
METTL3 and METTL14 were decreased to varying degrees in
WTAP-deficient PCs (Fig. 5Aand5B). Specifically, METTL3 was
distinctly localized in the PC nuclei in Wtap
f/f
mice, while it showed a
markedly lower content in Wtap
PKO
mice than in control mice
(Fig. 5A, arrows); Likewise, METTL14 also showed a nuclear
localization, and the METTL14 fluorescence in WTAP-depleted PCs
was drastically decreased (Fig. 5B, arrows). Consistent with the
immunostaining data, despite unchanged Calbindin protein level,
Western blotting analysis further verified that WTAP depletion
significantly decreased the protein levels of METTL3 and METTL14
to 42% and 61%, respectively, of those in the control group (Fig. 5C
and 5D). These findings suggest that WTAP depletion leads to
reduced expression levels of the core subunits METTL3 and
METTL14 of m
6
A methyltransferase complex in cerebellar PCs. To
investigate whether the alteration in the protein level of methyl-
transferase complex components affects m
6
A methylation, the
global m
6
A methylation profile in the cerebellar cortices of 1-month-
Fig. 3. Degeneration of PCs in Wtap
PKO
mice. A: Immunofluorescence labeling of cerebellar cryosections from Wtap
f/f
and Wtap
PKO
littermates of different ages using a Calbindin
antibody (green) and DAPI (blue). The lobules are indicated by Roman numerals. Scale bar, 500
m
m. B: Quantification of Calbindin-labeled PCs at different ages (n¼3). C: The upper
panel shows histological abnormalities in the cerebellum in Wtap
PKO
mice, as visualized by H&E staining. The middle panel show immunostaining for Calbindin in paraffin sections, and
immunofluorescence labeling of cerebellar cryosections using Calbindin (green) and NeuN (red) antibodies is shown in the lower panel. Scale bar, 200
m
m. Higher-magnification images
of lobules VIII/IX are shown in the right panel of each image (scale bar, 25
m
m). The yellow arrowheads indicate PCs. The double sided arrows indicate the ML thickness. D: Quantitative
assessment of the thickness of the ML (n¼5). E: Quantitative analysis of the number of GCs and PCs (n¼5). **,P<0.01; ***,P<0.001; #, no significance. All data are presented as
the mean ±SEM. H&E, hematoxylin and eosin staining; IGL, internal granular layer; ML, molecular layer; PCL, Purkinje cell layer; NeuN, neuronal nuclei.
Y. Yang, G. Huang, X. Jiang et al. Journal of Genetics and Genomics 49 (2022) 847e858
851
old Wtap
f/f
and Wtap
PKO
were assessed by RNA dot blot analysis.
As expected, there was a nearly 30% reduction in the overall m
6
A
level in the cerebellar cortices of Wtap
PKO
mice compared to those
of controls (Fig. 5Eand5F).
Transcript levels of Mettl3 and Mettl14 were measured by RT-
qPCR, which showed a ~23% reduction in transcript levels of
Mettl3 in Wtap
PKO
mouse cerebellar cortex compared to control mice,
while a comparable level of that of Mettl14 (P¼0.124, no significance)
was detected between these two groups (Fig. 5G). Considering that
the protein levels of METTL3 and METTL14 were decreased to a
greater extent (42% and 61%) in Wtap
PKO
group, which could be
caused by the impaired stability of m
6
A methyltransferase complex.
Therefore, the interactions between WTAP with METTL3 and METT14
were confirmed by co-IP. The Flag-tagged WTAP was transfected
into HEK293T cells together with HA-tagged METTL3 or METTL14
(Fig. 5H and 5I). Immunoprecipitations of HA-METTL3 or HA-
METTL14 revealed that WTAP stably binds to METTL3 and
METTL14. In addition, we knocked down (KD) WTAP expression by
Fig. 4. WTAP deficiency in PCs decreases the number and size of CF synapses but has no effect on PF synapses or inhibitory synapses. A: Images of Golgi staining of tissues from 3-
week-old Wtap
f/f
and Wtap
PKO
mice showing a reduced PC spine number upon WTAP depletion. B: Quantification of the spine number per 10
m
m dendrite (n¼12). C: Schematic of the
cellular circuitry in the cerebellar cortex. PF (blue) and F (green) excitatory inputs onto PCs, basket cells and stellate inhibitory interneurons (brown) in the ML are shown. DeG:
Immunofluorescence labeling of cerebellar cryosections from 3-week-old Wtap
f/f
and Wtap
PKO
mice using a vGLUT1 (D), vGLUT2 (E), HOMER1 (F), and vGAT (G) antibody (green).
Calbindin (red) was used to label PCs. The PF synapse density and postsynaptic density of interneurons were quantified by measuring the vGLUT1 and HOMER1 staining intensity,
respectively (n¼6). The CF synapse density and stellate/basket-cell synapse density were quantified by measuring the density of vGLUT2 and vGAT puncta, respectively (n¼6). The
dotted white lines represent the dividing lines between PCL and ML. H: Representative immunoblots and quantification of synaptic protein levels in cerebellar cortex from 3-week-old
mice. Protein level data were normalized according to GAPDH (n¼6). Iand J: Electron microscopy images (I) and quantification (J) showing that WTAP depletion decreased CF synapse
size (n¼8). The dotted red lines show the boundary of indicated synapses. KeM: Electron microscopy images (K) and quantification of the PSD (L) and PSD length of PF synapses (M)
(n¼8). *,P<0.05; ***,P<0.001; #, no significance. All data are presented as the means ±SEM. The dotted red lines show the boundary of indicated synapses. CF, climbing fiber; PF,
parallel fiber; PC, Purkinje cell. Scale bars, 10
m
m(A,DeG), 0.2
m
m(Iand K).
Y. Yang, G. Huang, X. Jiang et al. Journal of Genetics and Genomics 49 (2022) 847e858
852
lentivirus-mediated WTAP shRNA in HEK293T cells, and next per-
formed rescue experiments for WTAP KD cells using constructed
shRNA-resistant cDNA for WTAP (resWTAP, see Materials and
methods for details). Western blotting revealed that while shRNA
successfully knocked down the WTAP expression and caused
reduced METTL3/14 contents, resWTAP-added KD cells re-
expressed WTAP and thus increased the protein levels of METTL3/
14 compared to those in empty vector added KD cells (Fig. 5J and
5K). Thus, our data demonstrate that WTAP maintains the normal
expression of m
6
A methyltransferase subunit METTL3 and METTL14
in cerebellar PCs.
Exacerbation of neuronal injury and apoptotic cell death in the
degenerating cerebellum
As radial astrocytes found in the cerebellum, Bergmann glia
display a unique interaction with PCs, as the fibers of these glia
extend into the ML and enwrap the synapses on PC dendrites. Under
pathological conditions, astrocytes are rapidly activated and exert
neuroprotective or toxic effects (Buffo et al., 2010). Changes in as-
trocytes were also assessed in the present study. Cerebellar cry-
osections obtained from 9-week-old mice were immunostained for
glial fibrillary acidic protein (GFAP), the most well-known marker of
Fig. 5. Loss of Wtap causes reduced METTL3/14 expression and decreased m
6
A methylation in Wtap
PKO
PCs. Aand B: Immunofluorescence labeling of cerebellar cryosections from 1-
month-old Wtap
f/f
and Wtap
PKO
mice using a METTL3 (A) or METTL14 (B) antibody (green). Calbindin (red) was used to label PCs. DAPI (blue) was used to counterstain the nuclei. Scale
bar, 25
m
m. Higher-magnification images of the area indicated by the white triangle are shown in the left bottom (scale bar, 10
m
m). The yellow arrowheads indicate normal expression of
METTL3/14 in the nuclei of PCs from Wtap
f/f
mice. C: Western blotting of cerebellar lysates from Wtap
f/f
and Wtap
PKO
mice using WTAP, Calbindin, METTL3 and METTL14 antibodies. D:
Quantification of relative protein levels in the cerebella of Wtap
f/f
and Wtap
PKO
mice (n¼6). E: mRNA was isolated from the cerebellar cortex, and then dot blot analysis with an m
6
A
antibody was performed. Methylene blue staining was used to control for differences in loading. F: Quantification of relative m
6
A levels in (E)(n¼6). G: RT-qPCR analysis revealed reduced
expression of Mettl3 and Mettl14 in the cerebellar cortex from 1-month-old Wtap
PKO
mice (n¼4). Hand I: Western blotting of coimmunoprecipitations of METTL3-HA with WTAP-Flag.
HEK293T cells were co-transfected with the indicated constructs. METTL3-HA (H) or METTL14-HA (I) was immunoprecipitated and the amount of co-precipitated WTAP-Flag was
determined by immunoblotting, shown are representative immunoblots. Jand K: Western blotting (J) and quantification (K) of WTAP, METTL3 and METTL14 protein levels. Lentivirus-
mediated WTAP-specific shRNA (shWTAP) was prepared, and shWTAP-resistant cDNA (resWTAP) was created by introducing silent mutations in Flag-tagged WTAP to render it resistant
to shRNA-mediated knockdownof WTAP protein levels. HEK293T cells were co-transfected with shWTAP, along with emptyvector (Vec) or resWTAP. Control shRNA was also transfected
as shControl (shCtrl). Flag was used to verify the resWTAP expression. Both METTL3 and METTL14 levels in resWTAP-added group were elevated compared with the levels in vector-
added groups (n¼3). *,P<0.05; **,P<0.01; ***,P<0.001. All data are presented as the mean ±SEM. IGL, internal granular layer; ML, molecular layer; PCL, Purkinje cell layer.
Y. Yang, G. Huang, X. Jiang et al. Journal of Genetics and Genomics 49 (2022) 847e858
853
reactive astrocytes and gliosis. In the control cerebellum, PFs with
low GFAP immunoreactivity crossed the ML (Fig. 6A, upper panel). In
contrast, in degenerated areas of the Wtap
PKO
mouse cerebellum,
Bergmann glial processes in the ML were thicker, were more disor-
ganized and showed more intense GFAP immunoreactivity (Fig. 6A,
lower panel), indicating that Bergmann glia exhibited astrogliosis-like
activation. To determine the impact of WTAP deletion in PCs on
Bergmann glia, Calbindin was co-labeled with brain lipid-binding
protein (BLBP), a marker for Bergmann glia, which confirmed that
the Bergmann glia alignment appeared normal in Wtap
PKO
cerebellar
cortex compare to that in controls (Fig. S9).
In addition, an abnormal immunostaining pattern was also
observed for neurofilament light chain (NF-L) in the Wtap
PKO
mouse
cerebellum; NF-L immunoreactivity was stronger and more disor-
ganized in these mice than in control mice. Indeed, elevation of the
expression of NF-L, a major constituent neurofilament, has often
been used as a potential biomarker of neuronal axons and death
(Yuan et al., 2017). The intense NF-L immunoreactivity in the Wtap
PKO
mouse cerebellum indicated severe cerebellar injury. Western blot-
ting analysis further confirmed these immunostaining results,
revealing that the protein levels of GFAP and NF-L were increased by
1.98- and 1.47-fold, respectively, in the Wtap
PKO
mouse cerebellar
cortex compared with the control mouse cerebellar cortex (Fig. 6C
and 6D).
To further determine whether PC loss was caused via apoptotic
signaling pathway, cerebellar cryosections obtained from 6-week-old
mice were immunostained for cleaved caspase-3 (casp-3), which is a
critical executioner of apoptosis. The expression level of cleaved casp-
3 was strikingly upregulated in massive Calbindin labeled PCs in
Wtap
PKO
cerebellum (Fig. 6E), suggesting that the caspase cascade
was triggered. The apoptosiswas further confirmedby TUNEL staining.
As shown in Fig.6Fand6G, the number of TUNEL-positivePCs (labeled
Fig. 6. Neuronal injury is exacerbated and apoptosis is increased in the degenerating cerebella of Wtap
PKO
mice. Aand B: Sections from of 9-week-old Wtap
f/f
and Wtap
PKO
mouse
cerebella were costained for the PC marker Calbindin (red) and the astrocyte marker GFAP (A) or NF-L (B) (green). DAPI (blue) was used to counterstain the nuclei. Scale bar, 100
m
m.
The lobules are indicated by Roman numerals. Higher magnification images of lobules VI/V are shown in the lower panel of each image (scale bar, 25
m
m). Cand D: Representative
immunoblots (C) and quantification (D) of WTAP, GFAP and NF-L expression in cerebellar lysates from 9-week-old Wtap
f/f
and Wtap
PKO
mice. GAPDH was used as the loading control
(n¼6) E: Immunofluorescence labeling of cerebellum cryosections from 6-week-old littermates with cleaved caspase-3 (green) and Calbindin (red). Scale bar, 100
m
m. Yellow ar-
rowheads represent cleaved caspase-3-positive cells. Inset images show a zoomed image cropped from areas indicated by white arrows (scale bar, 1
m
m). F: TUNEL staining (green) of
cerebellar cryosections from 6-week-old Wtap
f/f
and Wtap
PKO
littermates. Calbindin (red) was used to label PCs. The nuclei were counterstained with DAPI (blue). TUNEL-positive PCs
(green, yellow arrowheads) in the Wtap
PKO
mouse cerebellum were detected. Scale bar, 50
m
m. Inset images show a zoomed image cropped from areas indicated by white arrows (scale
bar, 1
m
m). G: Quantitative assessment of the number of TUNEL-positive cells in the PCL in whole sections (n¼6 from three samples). H: Representative TEM images of PC somas from
6-week-old Wtap
f/f
and Wtap
PKO
mice. Apoptotic PCs were observed in the Wtap
PKO
cerebellum. Scale bar, 2
m
m. *,P<0.05; **,P<0.01; ***,P<0.001. All data are presented as the
mean ±SEM. GFAP, glial fibrillary acidic protein; NF-L, neurofilament light chain; IGL, internal granular layer; ML, molecular layer; PCL, Purkinje cell layer.
Y. Yang, G. Huang, X. Jiang et al. Journal of Genetics and Genomics 49 (2022) 847e858
854
by Calbindin) in the Wtap
PKO
mouse cerebellar cortex was markedly
higher than that in control littermates, indicating ongoing apoptosis of
PCs in the Wtap
PKO
mouse cerebellar cortex.At the ultrastructurallevel,
dying PCs exhibited cytoplasmic condensation and cytosolic electron-
dense structures, autophagic-like vacuoles, and membrane whorls,
which are indicative of typical apoptotic cell death (Fig. 6H). Thus, PC
loss in Wtap
PKO
mice is caused by the apoptotic pathway.
Discussion
In the past several years, m
6
A modification has been recognized
as a pervasive internal mRNA modification that plays critical roles in
neural development.
In rodents, deletions of writers (METTL3, METTL14), erasers (FTO)
and readers (YTHDF2, FMR1) have profound effects on neural
developmental. In particular, newborn pups with conditional deletion
of METTL3 or METTL14 are smaller in size and die before P25 (Yoon
et al., 2017), and both acute knockdown and specific ablation of
METTL3/14 induce marked cortical and cerebellar defects, including a
reduction in the number of PCs and an increase in apoptosis of
cerebellar GCs (Ma et al., 2018;Wang et al., 2018). In addition, FTO-
deficient mice show a decrease in body weight and a dramatic
decrease in the size of the whole brin and distinct brain regions (Li
et al., 2017). Additionally, YTHDF2 knockout in mice causes lethality
at late embryonic developmental stages, delaying cortical neuro-
genesis (Li et al., 2018). In addition to its essential roles in controlling
embryonic neurodevelopment, m
6
A has also been proposed to
regulate adult neurogenesis. FTO inhibition decreases the prolifera-
tion and differentiation of adult neural stem cells (aNSCs) in the hip-
pocampus and consequently induces learning and memory
impairments (Cao et al., 2020). Another study provided further evi-
dence of the role of m
6
A in adult neurogenesis by demonstrating that
METTL3 deficiency significantly inhibits the proliferation of aNSCs and
affects the morphological maturation of newborn neurons in the adult
brain (Chen et al., 2019). Collectively, these findings emphasize the
central role of m
6
A in neuronal progenitor cells and adult stem cells
during neurogenesis in the brain. In this study, we generated Wtap
conditional knockout mice using L7 promoter (Pcp2-Cre) transgenic
mice to investigate its role in cerebellar PCs. Wtap
PKO
mice exhibited
no observable phenotypes until six weeks after birth (Fig. 3A). Addi-
tionally, a mild ataxic gait was first observed in Wtap
PKO
mice at
approximately six weeks of age, which is in accordance with the rapid
PC loss at this time point (Fig. 3A and 3B). Moreover, we examined
cerebellar sections from P1, P9, P15, and P21 PKO mice and found
that Wtap
PKO
mice showed a normal cerebellar morphology and PC
development that was indistinguishable from that of their Wtap
f/f
lit-
termates (Figs. S3eS6). Our findings suggest that loss of Wtap in PCs
led to early ataxia and PC degeneration.
PCs, the only output neurons in the cerebellar cortex, receive
excitatory inputs from PFs and CFs. Here, we find that WTAP ablation
could specifically affect synaptic contacts between CFs and PCs, as
indicated by reductions in the number and size of CF-PC synapses
(Fig. 4). Increasing studies in neurons have indicated that neuronal
synapses are enriched not only in m
6
A-modified mRNAs but also in
m
6
A effectors (Merkurjev et al., 2018;Yu et al., 2018;Zhuang et al.,
2019). Transcriptome-wide m
6
A profiling of the mouse cerebellum
and cerebral cortex revealed that m
6
A peaks are enriched in functions
pertaining to transcriptional regulation, axonal guidance, synapse
assembly and organization (Chang et al., 2017). In particular, m
6
A
regulatory proteins such as YTHDF1-3 and FTO are localized in den-
dritic regions in mouse brain slices as well as in hippocampal neuronal
cultures. Selective knockdown of Ythdf1 and Ythdf3 in hippocampal
neurons leads to synaptic dysfunction, including immature spine
morphology and blunted excitatory synaptic transmission (Merkurjev
et al., 2018), suggesting a vital role for m
6
A modification in neuronal
synapses. Golgi staining suggested a decrease in dendritic spine
density and aberrant spine morphology in Wtap
PKO
mice (Fig. 4A and
4B), which is an early feature of PC degeneration.
Strikingly, our Wtap
PKO
mice displayed an abnormal gait with
progressive motor and cerebellar nerve dysfunction that was highly
reminiscent of HA pathologies in humans. HAs are genetic disorders
that affect the cerebellum and its connections and can be inherited in
an autosomal dominant, autosomal recessive, or X-linked manner.
Progress in this field has been challenging owing to the large number
of genetic ataxic syndromes, many of which have overlapping clinical
features. In general, disease-causative mechanisms fall into two
major categories according to the associated genetic mutation:
those associated with microsatellite repeat expansions and those
related to point mutations. To date, dozens of disease-causative
genes have been reported to cause HAs in humans via different
modes of inheritance, including ATXN1/2/37/8/10,SPTBN2,CAC-
NA1A,TTBK2,FGF12,ANO10,KCNA1, and ATP2B3 (Shakkottai and
Paulson, 2019). Unfortunately, these known genes explain only some
HA cases, and the genetic cause of disease in the majority of HA
patients remains largely unclear. In addition to classical genetics,
multiple layers of epigenetic modifications, including DNA, chro-
matin, and histone modifications, have been shown to be required for
normal cerebellar function and survival and have been implicated in
various neuropathies (Yang et al., 2019), prompting wide interest in
elucidating how epigenetic regulators govern the development and
function of brain neural circuits, including in the cerebellum. Here, we
provide novel evidence that m
6
A methylation catalytic complex
subunit WTAP plays essential roles in cerebellar PC function, which
raises the possibility that epigenetic m
6
A modification may partici-
pate in the development of HA pathologies. Further investigation in
HA patient cases for genetic variants in genes encoding m6A
methylation writers, readers, and regulatory factors is warranted.
Materials and Methods
Mouse model
All animal experiment protocols were approved by the Institu-
tional Animal Care and Use Committee of Sichuan Provincial Peo-
ple’s Hospital (Chengdu, Sichuan, China). All animals were housed in
a temperature-controlled room (24
C) on a 12-h light/dark cycle. The
mice were maintained on the C57BL/6J background.
Mice with cerebellar PC-specific Wtap deletion were generated us-
ing the Cre-loxP system. Wtap
loxP/loxP
mice (C57BL/6-Wtap
em1(flox)Smoc
)
were purchased from Shanghai Model Organisms Center (Shanghai,
China). Briefly, Cas9 mRNA and gRNA were obtained by in vitro tran-
scription. The donor vector contained a 1.0 kb 5
0
arm, 0.5 kb flox region
and 1.0 kb 3
0
arm and subjectedto in-fusion cloning. Theprepared Cas9
mRNA,gRNA and donor vector weremicroinjectedinto the fertilized ova
of C57BL/6J mice to obtain F
0
generation mice. Then, the male F
0
chimeric mice were mated with female C57BL/6J mice to generate
heterozygous F
1
founders. Heterozygous F
1
mice were crossed to
generate Wtap
flox/flox
offspring.
To achieve Wtap depletion specifically in cerebellar PCs,
Wtap
flox/flox
mice were crossed with B6.129-Tg(Pcp2-cre)2Mpin/J
transgenic mice (Jackson Laboratory, stock number:
004146) (Barski et al., 2000) to yield progeny with the genotype
Wtap
flox/þ
;Pcp2-Cre. Then, the Wtap
flox/þ
;Pcp2-Cre mice were
crossed with Wtap
flox/flox
animals to generate Wtap
flox/flox
;Pcp2-Cre
mice (Wtap
PKO
). Wtap
flox/flox
littermates (Wtap
f/f
) were used as
controls. All experiments were performed using the male litter-
mates. In addition, to determine the specificity of Cre expression, a
tdTomato reporter gene was used (strain name: Cg-Gt(ROSA)
26Sortm14(CAG-tdTomato)Hze/J; Jackson Laboratory, stock
number: 007914). In the presence of the Cre enzyme, the stop
Y. Yang, G. Huang, X. Jiang et al. Journal of Genetics and Genomics 49 (2022) 847e858
855
codon before the tdTomato expression cassette was removed,
which permits the expression of tdTomato (red fluorescence) in
Cre-positive cells.
Genotyping by PCR
Genomic DNA samples obtained from mouse tails were geno-
typed using PCR. The floxed Wtap alleles and Pcp2-Cre were gen-
otyped using the corresponding primers (Table S1). The tdTomato
reporter mice were genotyped using primers provided by the JAX
mouse service (Table S1). All amplification reactions were performed
using a master mix (Invitrogen, CA, USA) according to the manu-
facturer’s instructions. PCR was performed under the following
conditions: 95
C for 5 min followed by 32 cycles of 95
C for 15 s,
60
C for 30 s and 72
C for 30 s. The PCR products were separated
by DNA electrophoresis on a 3% agarose gel.
Cell culture and transfection
HEK293T cells were purchased from National Infrastructure of cell
line Resource (Wuhan, China) and were recently authenticated by
STR profiling. They were cultured in DMEM with high glucose
(Hyclone, South Logan, UT, USA) supplemented with 10% fetal
bovine serum (Gibco, Grand Island, NY, USA) and 100 U/mL peni-
cillin/streptomycin (Invitrogen, Waltham, MA, USA) in an incubator
set to 37
C with 5% CO
2
. The recombinant expression plasmids,
pcDNA3.1-WTAP-Flag, pcDNA3.1-METTL3-HA tag, and pcDNA3.1-
METTL14-HA tag were purchased from Youbio Inc. (Youbio,
Changsha, China). For transfection, cells were seeded in plates
(Corning, NY, USA) and transiently transfected with Flag-tagged
WTAP or HA-tagged METTL3/METTL14 plasmid using Lipofect-
amine 3000 (Invitrogen, CA, USA) according to the manufacturer’s
instructions, and the cell lysis were harvested after 48 h.
RNA interference and rescue
Lentiviruses carrying shRNA targeting WTAP (5
0
-GTTATGGCAA-
GAGATGAGTTA -3
0
) and a negative control shRNA (5
0
-TTCTCC-
GAACGTGTCACGT-3
0
) were purchased (Genechem, Shanghai,
China). HEK293T cells were seeded in six-well plate and incubated
for 24 h before infection. WTAP lentiviral particles (1 10
8
TU/mL;
MOI ¼5) mixed in serum-free medium with transfection reagent were
transfected into cultured HEK293T cells. The medium was replaced
by culture medium after 12 h. Cells were harvested 72 h after
transfection. For rescue experiments, shWTAP-resistant cDNA
(resWTAP) was created by Youbio Inc. (Youbio, Changsha, China),
which was introduced three silent mutations (c.G63C, c.A66C, and
c.A69G) within the shWTAP target sequence (5
0
-GTTATGGCAAGA-
GATGAGTTA-3
0
). HEK293T cells were co-transfected by WTAP-
specific shRNA (MOI ¼5) together with shRNA-resistant WTAP
(1
m
g) to resistant to the shRNA-mediated knockdown. All experi-
mental procedures were performed strictly according to the pro-
tocols provided by the manufacturers.
Behavioral analysis
For the hindlimb clasping test (Wang et al., 2018), the mice were
suspended by their tails over a cage for up to 2 min to assess
abnormal hindlimb clasping. Clasping was considered crossing of
the hindlimbs for more than three seconds. For the rotarod test (Yang
et al., 2018), 9-week-old mice were placed on a rod that rotated at a
speed of 20 rpm. The latency to fall off the rod was measured. The
rotarod apparatus (UniBiolab, Shanghai, China) was used to evaluate
the animals’motor performance. Only male mice were used in the
behavioral experiments.
Histology and cell quantification
For H&E staining, deeply anesthetized animals were trans-
cardially perfused with PBS followed by 4% paraformaldehyde.
Isolated organs were fixed in 4% paraformaldehyde overnight at
4
C. Then, the fixed tissues were embedded in paraffin, cut into
5
m
m sections and stained using an H&E staining protocol. The
slices were scanned by Pannoramic DESK (3D HISTECH) and
visualized or measured using CaseViewer 2.4 software (3D
HISTECH). The thickness of the ML in midsagittal sections
obtained from lobules IV, V, and VI (midway down the adjoining
fissures) was measured as previously described (Yang et al., 2018).
The number of GCs in 1000
m
m
2
areas in lobule V was counted.
Measurements were obtained from at least three sections from three
mice of each genotype and averaged.
Immunohistochemistry
For immunohistochemistry, anesthetized mice were fixed by
transcardial perfusion with PBS followed by 4% paraformaldehyde in
100 mM phosphate buffer (pH 7.4). The animals’heads were post-
fixed in 4% paraformaldehyde in 100 mM phosphate buffer (pH 7.4)
overnight. The brains were dissected out, rinsed with PBS and cry-
oprotected in 30% sucrose until the tissues sunk to the bottom.
Cerebella were then cut sagittally down the middle, and were
embedded in optimal cutting temperature (OCT) compound. Tissue
was sectioned at 10e12
m
m thickness on a Leica Cyrostat
(CM3050S). The first four cerebellar slices were imaged per animal,
and three animals were included in the analysis. After blocking and
permeabilization with 10% normal donkey serum and 0.2% Triton X-
100 in phosphate buffer for 1 h, the sections were labeled with pri-
mary antibody overnight at 4
C. The primary antibodies used are
shown in Table S2. The sections were rinsed in PBS three times,
Alexa Fluor 594/488-conjugated goat anti-mouse/rabbit secondary
antibody (Cat# A11005 and A11008, Invitrogen, Waltham, MA, USA,
1:500 dilution) was applied, and the nuclei were counterstained with
DAPI (Cat# D8417, Sigma, St Louis, MO, USA) for 1 h at room tem-
perature. Images were captured with a Zeiss LSM 800 confocal
scanning microscope. Synapse density was determined by counting
the number of synapses per unit area or measuring the mean fluo-
rescence intensity of targeted area. For quantification of fluores-
cence intensity, target area was outlined, and the mean fluorescence
intensity of selected channel was measured using ZEN imaging
software 2.3 (Carl Zeiss). The fold change was calculated by com-
parison with fluorescence intensity of Wtap
f/f
controls. All Calbindin
labeled PCs in fields of midsagittal sections were counted, and the
results obtained from three sections were averaged.
Terminal deoxynucleotidyl transferase-mediated dUTP nick
end labeling (TUNEL) assay
Apoptotic cell death was assessed by TUNEL staining according
to the manufacturer’s protocol (Cat# 11684795910 Roche Di-
agnostics, Indianapolis, IN, USA) using prepared frozen sections.
Images were captured with a Zeiss LSM 800 confocal scanning
microscope.
Golgi staining
Golgi staining was performed as described previously (Liu et al.,
2017). Briefly, mouse brains were fixed in 10% formalin for 24 h
and then immersed in 3% potassium bichromate for 3 days in the
dark. The solution was changed each day. Then, the brains were
transferred to 2% silver nitrate solution and incubated for 24 h in the
dark. Sections were cut at a thickness of 60
m
m using a vibratome, air
Y. Yang, G. Huang, X. Jiang et al. Journal of Genetics and Genomics 49 (2022) 847e858
856
dried for 10 min, dehydrated in 95% and 100% ethanol, cleared in
xylene, and coverslipped. For measurement of spine density, only
spines that emerged perpendicular to the dendritic shaft were
counted.
RNA extraction and RT-qPCR
Total RNA was extracted using TRIzol reagent (Cat# T9424,
Sigma, Saint Louis, MO, USA) as recommended by the manufac-
turer. First-strand cDNA was synthesized using an iScript cDNA
Synthesis Kit (Cat# 170e8890, Bio-Rad, Hercules, California, USA).
Quantitative PCR was carried out using iTaq SYBRMix (Cat#
1725120 Bio-Rad, Hercules, California, USA) and a CFX384 Touch
Real-Time PCR Detection System (Cat# BJ005303, Bio-Rad, Her-
cules, California, USA). The primers, which were designed using
Primer3Plus, were showed in Table S1. The expression of each target
genes was normalized to the actin mRNA level, and the fold change
was calculated by delta-delta Ct analysis.
RNA m
6
A dot blot assays
mRNA was purified from total RNA using the PolyATtract®
mRNA Isolation System III (Cat#Z5300, Promega, Madison, WI,
USA) following the protocols supplied with the kit. The mRNA was
subjectedtodoublingdilutionwithDEPCwaterandspottedontoa
NC membrane (Cat# HATF00010, Millipore, Billerica, MA, USA).
The membranes were dried and then UV crosslinked at 1200 W,
blockedwith5%skimmilkandincubatedwithanm
6
Aantibody
and anti-mouse or anti-rabbit HRP-conjugated secondary anti-
body (1:5000; Bio-Rad, Hercules, CA, USA), and the signal was
developed using SuperSignal West Pico Chemiluminescent Sub-
strate according to the manufacturer’s instructions (Pierce,
Rockford, IL, USA). The same RNAs were spotted on the mem-
brane and stained with 0.02% methylene blue in 0.3 M sodium
acetate as a loading control.
Western blotting
Tissues were grinded and lysed in RIPA buffer (Cat# R0010,
Solarbio, Beijing, China) containing protease inhibitor cocktail (Cat#
11697498001, Roche, Redwood City, CA, USA) and phosphatase
inhibitor (Cat# 4906845001, Roche, Redwood City, CA, USA) for
20 min while rotating. After ultrasonication, adding 5Laemmli
sample buffer (Cat# P1040, Solarbio, Beijing, China) to the lysis and
then boiling for 5 min at 95
C. The protein concentration of the ly-
sates was determined using a DC Protein Assay according to the
manufacturer’s instructions (Cat# 500-0122, Bio-Rad, Hercules,
CA, USA). Equal amounts of protein were separated on SDS poly-
acrylamide gels and transferred onto NC membranes (Cat#
HATF00010, Millipore, Billerica, MA, USA). The blots were blocked
with 8% nonfat dry milk in TBS with Tween®20 detergent for 2 h at
room temperature. Then, the membranes were incubated with pri-
mary antibodies in blocking solution overnight at 4
C. The primary
antibodies used for Western blotting are shown in Table S2.The
primary antibodies were detected with anti-mouse or anti-rabbit
HRP-conjugated secondary antibodies (1:5000; Bio-Rad, Hercu-
les, CA, USA), and the signal was developed using SuperSignal™
West Pico PLUS Chemiluminescent Substrate (Cat# 34577,
Thermo, Waltham, USA). The relative intensity of the immunoreac-
tive bands was quantified using the gel analysis tool provided in
ImageJ software. The intensity of the proteins of interest was
normalized to that of GAPDH. At least three independent Western
blotting assays were conducted, and one typical blot is presented.
Coimmunoprecipitation (Co-IP)
Transfected HEK293T cells were harvested and lysed in lysis
buffer (100 mM NaCl, 20 mM Tris-HCl [pH 7.4], 0.5% NP-40, 1 mM
PMSF, 1 mM Na
3
VO
4
,1mM
b
-glycerophosphate, 1 mM NaF and
1Cocktail) and incubated on ice for 30 min with periodic agitation.
Lysates were cleared by centrifugation at 17,000 gfor 15 min at 4
C.
20
m
L of lysates should be taken out as input control, and the
remaining supernatant was incubated with HA antibody-conjugated
agarose beads (Sigma, St. Louis, MO, USA) at 4
C overnight while
rotating. The beads were washed four times with co-IP washing buffer
(50 mM Tris HCl, pH 7.4, 300 mM KCl, 0.1% NP-40, 1.5 mM MgCl
2
,
0.5 mM AEBSF, 1 mM DTT). Elution of the proteins was conducted by
adding 5Laemmli sample buffer (Cat# P1040, Solarbio, Beijing,
China) to the beads and boiling the IP setup for 5 min at 95
C. The
prepared samples were further analyzed by Western blotting.
Transmission electron microscopy (TEM)
Anesthetized mice were fixed by transcardial perfusion with PBS
followed by 2.5% glutaraldehyde in cacodylate buffer; the animals’
heads were postfixed in 2.5% glutaraldehyde in cacodylate buffer (pH
7.2) overnight at 4
C. The cerebella were dissected from the heads,
immersion-fixed for 8 h, and sectioned sagittally at 70-nm intervals
using a vibratome. The cerebellar sections were incubated in 1%
osmium tetroxide for 1 h, washed in 0.1 M phosphate buffer, dehy-
drated in an ascending series of ethanol and propylene oxide solu-
tions and embedded in Epon (25 g Epon 812, 13 g dodecenyl succinic
anhydride [DDSA], 12 g nadic methyl anhydride [NMA] and 1 mL
2,4,6-Tris [dimethylaminomethyl] phenol [DMP-30]; Electron Micro-
scopy Sciences). Ultrathin sections (70 nm) were cut and stained with
uranyl acetate and lead citrate. The sections were imaged with a
Philips CM120 scanning transmission electron microscope.
Statistical analysis
Statistical analysis was performed using GraphPad Prism 6
software. The normal distribution of the data sets was assessed
using the ShapiroeWilk test. For normally distributed data, statistical
significance was determined by Student’s t-test or ANOVA. If the
data were not normally distributed, a nonparametric statistic was
used. Pvalues were calculated by Student’s t-test or one-way
ANOVA followed by Tukey’s, Dunnett’s or Sidak’s multiple compar-
isons test as appropriate. P<0.05 was considered to indicate sta-
tistical significance.
CRediT authorship contribution statement
Yeming Yang: Investigation, Methodology, Validation, Writing -
Original draft. Guo Huang: Investigation, Validation. Xiaoyan Jiang:
Validation. Xiao Li: Resources. Kuanxiang Sun: Validation. Yi Shi:
Resource. Zhenglin Yang: Conceptualization, Methodology, Writing
- Review &Editing. Xianjun Zhu: Conceptualization, Methodology,
Writing - Review &Editing, Project administration.
Conflict of interest
All authors declare that there are no competing interests.
Acknowledgments
This study was supported by the National Natural Science
Foundation of China (82121003, 81970841, and 81790643), the
Department of Science and Technology of Sichuan Province
(2021YFS0386, 2021YFS0369, 20ZYD038, 20ZYD037,
Y. Yang, G. Huang, X. Jiang et al. Journal of Genetics and Genomics 49 (2022) 847e858
857
2020JDZH0026, 2021JDZH0022), the CAMS Innovation Fund for
Medical Sciences (2019e12M-5-032), Huanhua Distingished Scholar
grant, and the Department of Chengdu Science and Technology
(2021-YF05-01316-SN). The funders had no role in the study design,
data collection and analysis, or preparation of the manuscript. The
authors would like to thank Chengdu LiLai Biotechnology Co., Ltd.,
for technical assistance with histology analysis.
Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.jgg.2022.03.001.
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