Functional Cooperation of α-Synuclein and Tau
Is Essential for Proper Corticogenesis
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言語: English
出版者: Society for Neuroscience
公開日: 2024-05-20
キーワード (Ja):
キーワード (En): alpha-synculein, corticogenesis,
gliogenesis, neurogenesis, tau
作成者: Wang, Shengming, Fu, Yu, Miyata, Takaki,
Matsumoto, Sakiko, Shinoda, Tomoyasu, Itoh, Kyoko,
Harada, Akihiro, Hirotsune, Shinji, Jin, Mingyue
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http://hdl.handle.net/10466/0002000802
This work is licensed under a Creative Commons
Attribution 4.0 International License.

The Journal of Neuroscience, September 14, 2022 • 42(37):7031–7046 • 7031

Development/Plasticity/Repair

Functional Cooperation of a-Synuclein and Tau Is Essential
for Proper Corticogenesis
Shengming Wang,1 Yu Fu,1 Takaki Miyata,2 Sakiko Matsumoto,1 Tomoyasu Shinoda,2 Kyoko Itoh,3
Akihiro Harada,4 Shinji Hirotsune,1 and Mingyue Jin1,5
1

Department of Genetic Disease Research, Osaka Metropolitan University Graduate School of Medicine, Osaka 545-8585, Japan, 2Department of Anatomy andCell Biology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan, 3Department of Pathology and Applied Neurobiology, Graduate School of
Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan, 4Department of Cell Biology, Graduate School of Medicine, Osaka University,
Osaka 565-0871, Japan, and 5Guangxi Key Laboratory of Brain and Cognitive Neuroscience, Guilin Medical University, Guilin, Guangxi 541199, China

Alpha-synuclein (aSyn) and tau are abundant multifunctional neuronal proteins, and their intracellular deposits have been linked tomany neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease. Despite the disease relevance, their physiological roles remain elusive, as mice with knock-out of either of these genes do not exhibit overt phenotypes. To reveal functional cooperation, we generated aSyn2/2tau2/2 double-knock-out mice and characterized the functional cross talk between these proteinsduring brain development. Intriguingly, deletion of aSyn and tau reduced Notch signaling and accelerated interkinetic nuclear migration of G2 phase at early embryonic stage. This significantly altered the balance between the proliferative and neurogenic divisions ofprogenitor cells, resulting in an overproduction of early born neurons and enhanced neurogenesis, by which the brain size wasenlarged during the embryonic stage in both sexes. On the other hand, a reduction in the number of neural progenitor cells in the
middle stage of corticogenesis diminished subsequent gliogenesis in the aSyn2/2tau2/2 cortex. Additionally, the expansion and maturation of macroglial cells (astrocytes and oligodendrocytes) were suppressed in the aSyn2/2tau2/2 postnatal brain, which in turn
reduced the male aSyn2/2tau2/2 brain size and cortical thickness to less than the control values. Our study identifies importantfunctional cooperation of aSyn and tau during corticogenesis.
Key words: alpha-synculein; corticogenesis; gliogenesis; neurogenesis; tau
Significance Statement
Correct understanding of the physiological functions of aSyn and tau in CNS is critical to elucidate pathogenesis involved in the etiology of neurodegenerative diseases including Alzheimer’s disease and Parkinson’s disease. We show here that aSyn and tau arecooperatively involved in brain development via maintenance of progenitor cells. aSyn and tau double-knock-out mice exhibitedan overproduction of early born neurons and accelerated neurogenesis at early corticogenesis. Furthermore, loss of aSyn and tau
also perturbed gliogenesis at later embryonic stage, as well as the subsequent glial expansion and maturation at postnatal brain.
Our findings provide new mechanistic insights and extend therapeutic opportunities for neurodegenerative diseases caused by aberrant aSyn and tau.

Received Feb. 26, 2022; revised July 7, 2022; accepted July 21, 2022.
Author contributions: S.H., and M.J. designed research; S.W., Y.F., T.M., S.M., T.S., A.H., S.H., and M.J.
performed research; S.M. and K.I. contributed unpublished reagents/analytic tools; S.W., Y.F., S.M., and M.J.
analyzed data; and S.H. and M.J. wrote the paper.
This work was supported by Japan Society for the Promotion of Science Grants JP17H04047 to S.H., JP18K06936 andJP21K06821 to M.J., and JP19K16525 and JP22K06887 to S.M, and Japan Agency for Medical Research and Development
Grant JP18ek0109390 to S.H., M.J., and S.M. This work was also supported by the Natural Science Foundation ofGuangxi Province (No. 2022GXNSFAA035622 to M.J.). We thank Miyuki Kira (Osaka Metropolitan University) and Yoriko
Yabunaka (Osaka Metropolitan University) for technical support of DNA sequencing and HPLC analysis, Hiromichi
Nishimura (Osaka Metropolitan University) and Junko Hirohara (Osaka Metropolitan University) for mouse breeding, theOsaka Metropolitan University Graduate School of Medicine for results data that were partially obtained using the
Research Support Platform, Dr. Ayano Kawaguchi for data analysis and discussion (Nagoya University), and Namiko
Noguchi (Nagoya University) and Makoto Masaoka (Nagoya University) for in utero electroporation.
The authors declare no competing financial interests.
Correspondence should be addressed to Shinji Hirotsune at shinjih@omu.ac.jp or Mingyue Jin at
jinmingyue@omu.ac.jp.
https://doi.org/10.1523/JNEUROSCI.0396-22.2022
Copyright © 2022 the authors

Introduction

Alpha-synuclein (aSyn) and tau are typically characterized as
intrinsically disordered proteins (Weinreb et al., 1996; Chang etal., 2021) and are implicated in various neurodegenerative diseases, including Alzheimer’s disease (AD; Chang et al., 2021) andParkinson’s disease (PD; Polymeropoulos et al., 1997). Tau is amajor neuronal microtubule-associated protein (MAP) abundantly expressed in neuronal axons (Cleveland et al., 1977;
Drubin and Kirschner, 1986), whereas aSyn is a synaptic protein
highly concentrated in nerve terminals (Maroteaux et al., 1988).
Previous studies have also revealed that aSyn and tau promoteand stabilize microtubule (MT) polymerization (Alim et al.,
2004; Qiang et al., 2006; Cartelli et al., 2016; Toba et al., 2017).
Consistent with these effects, intracellular deposition of aSyn

7032 • J. Neurosci., September 14, 2022 • 42(37):7031–7046

and tau disrupts neuronal MTs (Chen et al., 2007) and impairsaxonal transport (Calogero et al., 2019), common features inmany neurodegenerative disorders in early onset individuals(Millecamps and Julien, 2013; Prots et al., 2018).
Neuronal MTs are essential for cell morphology, neurodevelopment, maintenance of physiological functions, and axonaltransport (Sleigh et al., 2019). Hence, mutations in tubulins or
MAPs have been linked to neurodevelopmental (Poirier et al.,2007; Jin et al., 2017) and neurodegenerative disorders (Calogero
et al., 2019; Shafiq et al., 2021). Unexpectedly, although aSyn
and tau are expressed throughout brain development (Hsu et al.,
1998; Fiock et al., 2020), neither aSyn-deficient (Abeliovich etal., 2000) nor tau-deficient (Harada et al., 1994; Ke et al., 2012)
mice showed clear phenotypes during brain development. Thesefindings indicate the possibility of some functional redundancy
among neuronal MAPs (Harada et al., 1994). Interestingly, aSyn
and tau are codeposited in multiple neurodegenerative diseases,
such as AD, dementia with Lewy bodies, and PD (Forman et al.,
2002; Ishizawa et al., 2003; Moussaud et al., 2014). These findings
strongly demonstrate that aSyn and tau play important roles inboth cognition and movement. To exclude functional redundancy and clarify the functional cross talk between aSyn and tau,
we crossed two single-knock-out (single-KO) strains to generate
aSyn / tau / double-KO mice. We hypothesized that simultaneous deletion of two neuronal MAPs, aSyn and tau, may affectembryonic brain development, probably because of disruption of
MT behaviors.
During mammalian corticogenesis, two kinds of neural progenitor cells (NPCs) radial glial cells (RGCs) and intermediate
progenitor cells (IPCs) are present in the ventricular zone (VZ)
and subventricular zone (SVZ), respectively (Greig et al., 2013).
These cells proliferate and give rise to postmitotic neurons. The
coordinated proliferation and differentiation of NPCs and neuronal migration play a pivotal role in creating a highly organized
brain structure and proper neuronal networks (Taverna etal., 2014). In this study, we investigated the cooperative functions of aSyn and tau during brain development by dissecting aSyn / tau / mice. Surprisingly, compared with thewild-type (WT) strain and the two single-KO strains, theaSyn / tau / mice had smaller brains, suggesting the presence of microcephaly in the adult mice. In contrast, NPCs
from aSyn / tau / mice exited the cell cycle prematurely atembryonic day (E)11 and displayed facilitated neurogenesis
from E12 that led to an enlarged brain during the embryonicstage. We also found that aSyn / tau / mice showeddiminished gliogenesis at the late embryonic stage and subsequent expansion and maturation in the postnatal brain.
Moreover, this downregulated gliogenesis, expansion, andmaturation led to the reduced brain size in aSyn / tau /mice compared with WT and single-KO mice. Together,
these findings indicate that functional cooperation between
aSyn and tau plays multiple roles in different developmental
processes.

Materials and Methods
All methods were performed in accordance with the approved guidelines. All experimental protocols were approved by the Osaka
Metropolitan University Graduate School of Medicine. Separate approval was provided for the recombinant DNA experiments (Osaka
Metropolitan University no. 708) and animal experiments (Osaka
Metropolitan University no. 21094).
Animal sources and maintenance . Both sexes of mouse embryos andpups were used at E11–E15 and postnatal day (P)0–P11, respectively;

Wang et al. · Functional Cross Talk between a-Synuclein and Tau during Brain Development

and adult male mice were used at 6 weeks of age, as indicated in themain text and figures. Homozygous a-synuclein (aSyn) KO mice
(B6;129  1-Sncatm1Rosl/J, strain #003692) were purchased from The
Jackson Laboratory, and heterozygous tau KO mice (B6;129-Mapt tm1(Cre))
were generated by Akihiro Harada (Department of Cell Biology, Graduate
School of Medicine, Osaka University) and housed in the Center for
Animal Resources and Development, Kumamoto University. In thisstudy, mice were maintained on a C57BL/6 background, WT C57BL/6
mice were used as controls, and homozygous aSyn / , tau / andaSyn / tau / mice were used as experimental mice. All mice werehoused in an environmentally controlled room in the Institute ofLaboratory Animals of the Graduate School of Medicine, Osaka
Metropolitan University. Each cage contained fewer than six mice, andmice were provided access to food and water ad libitum.
Generation of double-knock-out mice. aSyn and tau double knockout
mice were generated by crossing aSyn / and tau / mice. Genotyping
was performed by standard PCR, with denaturation at 94°C for 1 min
followed by 35 cycles of denaturation at 94°C for 20 s, annealing at 55°C
for 30 s, and extension at 72°C for 1 min. The following primers wereused: Tau forward (F), 59-TATGGCTGACCCTCGCCAGGAGTTT-39;
Tau reverse (R), 59-TCCACCCCACTGACCTTTTAAGCC-39; aSyn F,
59-ATGGATGTGTTCATGAAAGGACTTTCAA-39; and aSyn R, 59ACTTCCCAAATGCCACCAGGC-39.
Calculation of brain surface area. Mice at E14, P11, and 6 weeks
were chosen for calculation of the brain surface area. Samples were harvested in PBS at 4°C, and images were acquired using optical microscope(Olympus BX53) at 1.25 or 2 magnification. The surface areas were
circled in the images and calculated with Fiji ImageJ software.
Sectioning of brain samples. Embryos were sacrificed and dissected
in PBS and then fixed with periodate-lysine-paraformaldehyde (PLP) at
4°C for 2 h (E11–E13 embryos), 3 h (E14 embryos) and 5 h (E15
embryos). Pups and adult mice were perfused with PLP solution (P0, 5
ml/pup; P7 and P11, 15 ml/pup; 6 weeks old, 60 ml/mouse), and thebrains were fixed by continuous incubation in PLP overnight at 4°C. For
sectioning, samples were washed in PBS, cryoprotected in 20% sucrose/
PBS overnight at 4°C, embedded in optimal cutting temperature compound and sliced at a 16 mm thickness on a Leica CM 1950 cryostat.
Nissl staining of sections was performed with 0.25% thionin acetate salt
(catalog #861340, Sigma-Aldrich).
Immunohistochemistry. Brain sections were washed in PBS and subjected to antigen retrieval in 10 mM citrate buffer, pH 6.0, for 10–15 min
at ;80°C. After washing, the sections were blocked in 1% BSA supplemented with 0.1% Triton X-100, 0.01% NaN3, and primary antibodies
overnight at 4°C. The primary antibodies used in this study included
mouse anti-Pax6 (1:200; catalog #ab78545, Abcam), rabbit anti-Pax6(1:200; catalog #901301, BioLegend), rabbit anti-Tbr2 (1:500; catalog#ab23345, Abcam), rabbit anti-Tbr1 (1:500; catalog #ab31940, Abcam),rabbit anti-Ki67 (1:200; catalog #ab16667, Abcam), mouse anti-glial
fibrillary acidic protein (GFAP; 1:500; catalog #G3893, Sigma-Aldrich),mouse anti-Olig2 (1:500; catalog #MABN50, Millipore), rabbit anti-Cux1(1:500, catalog #sc-13024, Santa Cruz Biotechnology), rat anti-Ctip2(1:200; catalog #ab18465, Abcam), mouse anti-S100 b (1:500; catalog#AMAb91038, Sigma-Aldrich), mouse anti-neurogenin 2 (Ngn2; 1:200;
catalog #MAB3314, R&D Systems), mouse anti-Ascl1 (1:200; catalog#556604, BD Biosciences), rabbit anti-Notch1 (1:100; catalog #3608, Cell
Signaling Technology), rat anti-5-Bromo-29-deoxyuridine (BrdU; 1:100;
catalog #ab6326, Abcam), rabbit anti-cleaved caspase-3 (1:200; catalog#966, Cell Signaling Technology), and rabbit anti-Iba1 (1:1000; catalog#019-19741, Waco) antibodies. The secondary antibodies, including Alexa
Fluor 488-conjugated antibodies (goat anti-mouse IgG, catalog #A11001;
goat anti-rabbit IgG, catalog #A11008; goat anti-rat IgG, catalog #A11006,
Invitrogen) and Alexa Fluor 546-conjugated antibodies (goat anti-mouse
IgG, catalog #A11003; goat anti-rabbit IgG, catalog #A11010; goat anti-rat
IgG, catalog #A11081, Invitrogen) were diluted 1:500 in blocking buffer.
The brain sections were also stained with 49,69-diamidino-2-phenylindole(DAPI; 1:500; catalog #D523, Dojindo) for 2 h at room temperature (RT)in the dark and mounted with FluorSave Reagent (catalog #345789,
Millipore). Images were acquired using an LSM800 laser confocal microscope (Zeiss) with a 40 or water-immersion 63 objective lens.

Wang et al. · Functional Cross Talk between a-Synuclein and Tau during Brain Development

BrdU treatment. BrdU (catalog #B9285, Sigma-Aldrich) was injected
intraperitoneally into mice (50 mg/g mouse weight), and the mice werehoused in a temperature-controlled room with access to food and water
ad libitum until they were sacrificed. For BrdU staining, brain sections
were subjected to antigen retrieval with 2 N HCl for 10 min, washedwith PBS, and incubated with primary antibodies.
RNA preparation and qRT-PCR. Total RNA samples were extracted
from nine of E12 embryonic cortices/genotype using an RNeasy Plus
Mini Kit (QIAGEN) and reversely transcribed into cDNA with
SuperScript Reverse Transcriptase III (Invitrogen) and nucleotide oligodT primers. Quantitative PCR (qPCR) was performed on a QuantStudio
5 Real-Time PCR System (Applied Biosystems) using PowerUp SYBRGreen Master Mix (Applied Biosystems). The following primers wereused: Notch1 F, 59-GGTGAACAATGTGGATGCTG-39; Notch1 R, 59ACTTTGGCAGTCTCATAGCTG-39; b -actin F, 59-GCCTTCCTTCTTGGGTATG-39; and b -actin R, 59-ACCACCAGACAGCACTGTG-39.
Cell culture. Mouse embryonic fibroblasts (MEFs) were separated
from embryos (E12–E13) and cultured in high glucose D-MEM (catalog#043-30085, Wako), supplemented with 10% FBS (catalog #171012,
NICHIREI BIOSCIENCES INC), 1 L-glutamine (catalog #25030-081,
Invitrogen,) and 1 antibiotic mixture (penicillin-streptomycin solution, catalog #168-23191, Wako). To assess microtubule reorganization,
MEFs were cultured on poly-L-lysine (PLL)-precoated coverslips in sixwell plates and incubated with nocodazole (at concentrations of 0, 1, and2 mg/ml in D-MEM) for 1 h at RT. Then the cells were rinsed with PBS
for subsequent immunostaining. Lenti-X 293T cells were cultured inhigh glucose D-MEM, supplemented with 5% FBS, 1 L-glutamine, and1 antibiotic mixture for lentivirus preparation.
Western blot analysis. Brain neocortices dissected from E12 embryos
were lysed in lysis buffer (T-PER Tissue Protein Extraction Reagent, catalog #78510, Thermo Scientific; 1 Protease Inhibitor Cocktail, #78425,
Thermo Fisher Scientific; 5 mM EDTA; 1 mM DTT). For immunoblotting, proteins were separated on acrylamide gels under reducing conditions and were then electrophoretically transferred to PVDF
membranes. The membranes were incubated with blocking buffer(5% skim milk in TBS-T) and were then probed with antibodies against
Notch intracellular domain (NICD; 1:1000; catalog #ab52301, Abcam),Hes1 (1:1000; catalog #sc-166410, Santa Cruz Biotechnology), Hes5
(1:1000; catalog #ab25374, Abcam) and b -actin (1:1000; catalog #A2228,
Sigma-Aldrich), followed by incubation with a horseradish peroxidaseconjugated secondary antibody at a 1:1000 dilution [Donkey AntiMouse IgG (H1L), catalog #715-035-150, Jackson ImmunoResearch;
Donkey Anti-Rabbit IgG (H1L), catalog #711-035-152; Jackson
ImmunoResearch]. Blots were developed using ECL Prime detection reagent (Cytiva), and chemiluminescence was detected with a FUSION Solo S
imaging system (VILBER).
Immunocytochemistry. MEFs were fixed with prewarmed 4% paraformaldehyde (PFA; catalog #163-20145, Wako) for 15 min at 37°C,
treated with 0.1% Triton X-100 for 5 min, and then blocked in 4% BSA
before primary antibody treatment. The cells were incubated with rabbit
anti- b -tubulin antibody (1:300; catalog #ab6046, Abcam) in blocking
solution for 1 h at RT. An Alexa Fluor 546-conjugated secondary antibody (goat anti-rabbit IgG, 1:1000; catalog #A11010, Invitrogen) wasused, and the nuclei were stained with DAPI (1:500; catalog #D523,
Dojindo) for 1 h at RT in the dark. Images were acquired using an
LSM700 confocal microscope (Zeiss) with a 20 objective lens.
Lentivirus preparation. Lentiviruses were prepared as described previously (Toba et al., 2017). Briefly, the EB3 cDNA fragment fused tomNeonGreen (mNG) was subcloned into the self-inactivating lentiviral
vector pCSII-EF-MCS-IRES2, which was then cotransfected with the
packaging plasmids pCAG-HIVgp and the VSV-G and Rev-expressing
pCMV-VSV-G-RSV-Rev into Lenti-X 293T cells (5  106 n/p 10 dish)by using PEI MAX. After 48 h of culture, lentiviral particles were collected by ultracentrifugation at 17,000 rpm at 20°C for 2 h (CP80NX,
P28S rotor, Himac). The pellet was then resuspended in 600 ml of PBS,
aliquoted at 20 ml/tube, and stored at 80°C until use.
EB3 tracking analysis. MEF cells were cultured in PLL-precoatedglass bottom dishes (35 mm, catalog #3910-035, Iwaki) and induced tooverexpress EB3-mNG via lentivirus transduction (4 ml/dish, 48 h). Live

J. Neurosci., September 14, 2022 • 42(37):7031–7046 • 7033

cell images were recorded with an Olympus IX70 microscope coupled toa digital charge-coupled device (CCD) camera (EM-CCD C9100-13,
Hamamatsu Photonics) with an oil immersion objective lens (Plan Apo60, numerical aperture 1.40, Nikon Instech) and Aqua Cosmos 2.6
software (Hamamatsu Photonics). Moving EB3 particles were tracked
using Mark2 tracking software (provided by Ken’ya Furuta).
In utero electroporation and slice culture. In utero electroporation(IUE) was performed using pregnant WT and aSyn / tau / mice aspreviously described (Okamoto et al., 2013; Shinoda et al., 2018) withminor modifications. Approximately 1 ml of the plasmid mixture (pEF1aCre, pEF1a-LPL-Lyn-EGFP, and pBA-LPL-H2B-RFP with 0.02% Fast
Green) was injected into the lateral ventricle of each intrauterine E12
embryo. Then, the head of each embryo was placed between the disks of aforceps-type electrode (3 mm disk electrode, model CUY650P3, NEPA
GENE), and the genes were electroporated into the cerebral walls by electronic pulses (30–50 V, 50 ms, five times). At E13 (or E14), coronal cerebral wall slices prepared at a 200–250 mm thickness were embedded in
type I collagen gel in a 35 mm glass bottom dish (Iwaki), and live images
were recorded with a CellVoyager CV1000 microscope system (Yokogawa
Electric) at 10 min intervals.
Quantification and statistical analyses. All experimental data presented here were obtained from at least three independent replicates,
and analyses were performed with blinding to the genotype or condition.
Cells were counted in a 100 mm column for E11–E15 cortices and in a
300 mm column for P0, P7, P11, and 6-week-old cortices; neuronal celldensities were calculated in a 100  100 mm2 area for P7, P11, and 6week-old cortices with ZEN software (Zeiss; details provided in each figure legend). For comparisons between two or more groups, unpaired
two-tailed Student’s t tests or one-way ANOVA followed by Tukey’s
post hoc test, respectively, were used to calculate p values. Significancewas set at p , 0.05, and the data are presented as mean 6 SEM; *p ,0.05, **p , 0.01, ***p , 0.001.

Results

Generation of aSyn2/2tau2/2 double-knock-out mice
Although aSyn and tau are highly abundant MAPs expressed
throughout brain development (Hsu et al., 1998; Fiock et al.,2020), mice with single-KO of either gene do not display overt
phenotypes (Ke et al., 2012; Pathak et al., 2017), indicating
the existence of some degree of functional redundancy
among neuronal MAPs. To eliminate this functional redundancy and address the functional cross talk between aSyn
and tau, we generated aSyn / tau / double-KO mice bycrossing aSyn / mice (Abeliovich et al., 2000) with tau /mice (Muramatsu et al., 2008; Fig. 1A). aSyn / tau / mice wereviable and fertile, and survived to adulthood. Both male and female
aSyn / tau / mice had normal body sizes and weights relative tothose of WT and single-KO (either aSyn / or tau / ) littermatesand did not exhibit any obvious morphologic defects. We first examined the brain structure at 6 weeks after birth. We found clear reductions in brain size in 6-week-old aSyn / tau / mice (Fig. 1B,C).
In addition, the cortical layers and thickness were assessed after
immunolabeling with the markers Cux1 (layers II–IV) and Ctip2(layers V–VI; Nieto et al., 2004; Arlotta et al., 2005). Although,the thickness of layers II–VI was significantly decreased inaSyn / tau / mice (Fig. 1D,E), the numbers of neurons in eachlayer probed with Cux1 and Ctip2 were unaffected (Fig. 1D,F,G).
These results strongly suggest that aSyn and tau play essential roles
during brain development.
Deletion of aSyn and tau promotes neurogenesis
Corticogenesis depends on the highly regulated proliferation anddifferentiation of NPCs. To examine the effects of aSyn and tau
during brain development, we first assessed the behavior of cortical NPCs at E14, a middle stage of corticogenesis. In contrast toWang et al. · Functional Cross Talk between a-Synuclein and Tau during Brain Development

7034 • J. Neurosci., September 14, 2022 • 42(37):7031–7046

the small brains observed at 6 weeks, thebrains of aSyn / tau / mice at E14 weresignificantly larger than those of WT andsingle-KO mice (Fig. 2A). This result further
indicated that abnormalities occurred during the proliferation and differentiation of
RGCs in aSyn / tau / mice. Next, we
assessed proliferative RGCs using the Sphase marker BrdU and the cycling cellmarker Ki67 at E14. Two hours after peritoneal injection of BrdU, brain samples werecollected, and the sections were stained with
antibodies against BrdU and Ki67 (Fig. 2B,
C). Despite the brain enlargement observed
at E14, the number of BrdU1Ki671 cycling
NPCs was clearly reduced in aSyn / tau /mice (Fig. 2C). However, the numbers of
Pax61 RGCs did not differ among the WT
and KO mice (Fig. 2D,E). On the other
hand, the number of Tbr21 IPCs was significantly reduced in aSyn / tau / cortices (Fig. 2D,F,G). Interestingly, we also
found that the numbers of differentiatedneurons as identified by the DAPI1Pax6Tbr2- were remarkably increased in the VZ
and SVZ in aSyn / tau / mice (Fig. 2D,
H). These findings showed that although thenumber of IPCs was decreased, neurogenesis was remarkably enhanced by deletion ofaSyn and tau. Consistent with this finding,the number of Tbr11 postmitotic neurons
was remarkably increased in the aSyn /tau / cortical plate (CP), accompanied by
an apparent increase in CP thickness (Fig.
2I). These data indicated that the promotionof neurogenesis was concomitant withincreases in the cortical thickness and brain
size. We further examined the NPC pool at
E15 and found that the numbers of both
RGCs and IPCs were significantly decreased
in aSyn / tau / cortices (Fig. 2J–M), suggesting that the promotion of neurogenesis
led to excess consumption of RGCs andconsequently reduced the number of NPCs.

Figure 1. Loss of aSyn and tau reduced the brain size in adult mice. A, Generation of aSyn / tau / mice by crossing aSyn / mice with tau / mice. aSyn / tau / mice were confirmed by genotyping (left) and Western blotting(right). B, C, Comparison of cortical surface areas in 6-week-old (6w) WT and KO mice. Scale bar, 2 mm. D–G, Corticalarchitecture in 6-week-old mice, as visualized with antibodies against Cux1 (layers II–IV, red) and Ctip2 (layers V–VI,
green). The thickness of cortical layers II–VI (E) and the numbers of Cux11 upper layer neurons (F) and Ctip21 deep
layer neuronal cells (G) were quantified in WT and KO mice. In this study, cell nuclei were stained with DAPI (blue). Scalebar, 200 mm. The quantitative data are presented as mean 6 SEM, N = 3 mice per genotype from three separate litters.
The p values were calculated by one-way ANOVA with Tukey’s post hoc test; n.s., not significant; *p , 0.05, **p , 0.01.

Loss of aSyn and tau induces prematureneurogenesis
In E14 aSyn / tau / brains, despite the increased numbers of
postmitotic neurons and the thickened CP, differentiation to
IPCs was diminished probably because of the promotion of
direct neurogenesis from RGCs. To address the origin of the conflicting data at E14, we performed analyses at E12, an earlier
stage of neurogenesis, and the time at which the RGC-IPC-neuron differentiation system is initiated (Bond et al., 2020). As
expected, immunohistochemistry revealed that the number ofcycling RGCs (Pax61Ki671) was significantly increased in E12
aSyn / tau / brains (Fig. 3A,B). This increase may have contributed to the maintenance of the RGC pool until E14. To trace
differentiation, we labeled mitotic NPCs with BrdU at E12 andthe mice were sacrificed after 24 h (Fig. 3C). Brain sections were
stained with antibodies against BrdU, Ki67, and Tbr1. Comparedwith WT and both types of single-KO cortices, aSyn / tau /cortices displayed increases in the numbers of both mitotic

progenitors (BrdU1Ki671; Fig. 3D,E) and cell cycle-exiting
neurons (BrdU1Ki67-/BrdU1; Fig. 3D,F). Consistently, thenumber of postmitotic neurons identified by either Tbr11 or
BrdU1Tbr11/BrdU1 was clearly increased in the aSyn /tau / neocortex (Fig. 3G–I). Unexpectedly, we found that postmitotic neurons probed with antibody specific for the deep layer
marker Ctip2 were detected only in E11 aSyn / tau / mice,strongly indicating that loss of aSyn and tau functions resulted
in precocious neurogenesis from E11 (Fig. 3J,K)
Next, we investigated the differentiation of RGCs to IPCs at
E12. In contrast to the findings at E14, the number of Tbr21
IPCs was increased significantly in the aSyn / tau / group(Fig. 4A,B), suggesting that enhanced proliferation and differentiation of RGCs occurred from E12. Ngn2, another IPC marker,
is the main determinant of cortical neuronal fate (Miyata et al.,
2004; Kawaguchi et al., 2008; Han et al., 2018). Consistent withthe data shown in Figure 4, A and B, the number of Ngn21 IPCs
was also increased in the aSyn / tau / group (Fig. 4C,D). TheseWang et al. · Functional Cross Talk between a-Synuclein and Tau during Brain Development

J. Neurosci., September 14, 2022 • 42(37):7031–7046 • 7035

Figure 2. Loss of aSyn and tau promoted neurogenesis. A, Comparison of cortical surface areas at E14. Scale bar, 200 mm. B, Schematic illustration of the BrdU experiments and the image
acquisition area as indicated in the white rectangle. C, Analyses of cycling NPCs after BrdU injection at E14. Two hours after BrdU injection, brain sections were stained with anti-BrdU (S phasemarker, green) and anti-Ki67 (cycling cell marker, red) antibodies, and cycling NPCs in the VZ and SVZ were quantified. BrdU1Ki671 cells were counted in 100 mm columns in sections fromeach genotype. Scale bar, 50 mm. D–H, Quantification of the NPC pool and generated neurons at E14 by immunostaining for the RGC marker Pax6 (red) and the IPC marker Tbr2 (green).
Representative immunostaining images are shown in (D). NPCs were probed with Pax6 and/or Tbr2 markers (D–G), and the generated neurons identified by the DAPI1Pax6-Tbr2- (H) in the
VZ and SVZ were counted per 100 mm column for each genotype. Scale bar: (in D) D–H, 50 mm. I, Statistical analysis of postmitotic neurons in the neocortex after probing with Tbr1. The cells
were counted in a 100 mm column. J–M, Quantification of NPCs in E15 cortices. RGCs and IPCs at E15 were stained with anti-Pax6 (red) and anti-Tbr2 (green) antibodies, respectively, and representative images are shown in J. The quantitative RGCs and/or IPCs are shown in K–M. The quantified data are presented as mean 6 SEM, N = 3 embryos per genotype from three separate
litters. The p values were calculated by one-way ANOVA with Tukey’s post hoc test; n.s., not significant; **p , 0.01, ***p , 0.001.

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Wang et al. · Functional Cross Talk between a-Synuclein and Tau during Brain Development

Figure 3. Proliferation and differentiation of RGCs were facilitated in aSyn / tau / E12 cortices. A, B, Statistical analyses of cycling RGCs in E12 cortices. Cycling RGCs were probed with
antibodies against Pax6 (red) and Ki67 (green, A), and the numbers of Pax61 and Ki671 cells were determined and are shown in B. C–F, Cell cycle exit assay during E12–E13. Schematic
illustration of the BrdU experiments and the image acquisition area as indicated with the red rectangle (C). Representative images of E13 cortices after BrdU and Ki67 costaining are shown in
D. Statistical analyses of cycling cells (BrdU1Ki671, E) and cell cycle exiting cells (BrdU1Ki67-/BrdU1, F) are presented. G–I, Cycling cells were labeled with BrdU (green) at E12, and differentiated postmitotic neuronal cells were labeled with Tbr1 (red) at E13. Representative immunostaining images are shown in G, and the postmitotic neurons were counted in 100 mm columns
and are shown in H and I. J, K, Premature neurogenesis caused by deletion of aSyn and tau. Differentiated neurons were probed with an anti-Ctip2 antibody (green, J) and quantified (K). Cellcounting was performed in 100 mm columns for each genotype. The data are presented as mean 6 SEM, N = 3 embryos per genotype from three separate litters. The p values were calculated
by one-way ANOVA with Tukey’s post hoc test; *p , 0.05, ***p , 0.001. Scale bars: A (for A, B,) D (for C–F), G (for G–I), and J (for J, K), 50 mm.

IPCs are committed to differentiation into postmitotic neurons.
Together, these results suggest that enhanced neurogenesis isinduced by both RGCs and IPCs.
Considering the importance of the Notch signaling pathway
in the maintenance of RGCs and neuronal differentiation(Kopan and Ilagan, 2009), we investigated Notch1 expression at
E12. Notch1 was observed mainly in the VZ and SVZ in all tested
E12 cortices, but the intensity of Notch1 immunofluorescence
was apparently decreased in aSyn / tau / cortices (Fig. 4E,F),
indicating reduced expression and impaired activity of Notch1.
To confirm this, we also performed qPCR and found that themRNA level of Notch1 in E12 aSyn / tau / mice was clearlylower than that in WT and both types of single-KO mice (Fig.
4G). Activation of Notch1 triggers the cleavage of the NICD,
which then translocates to the nucleus to induce the expression of Hes proteins, ultimately inhibiting neuronal differentiation (Kopan and Ilagan, 2009). Western blot analysis
revealed that the expression levels of NICD and its downstream Hes1 and Hes5 were remarkably decreased in E12
aSyn / tau / cortices (Fig. 4H,I). The increased number of
Ngn21 IPCs in aSyn / tau / cortices also supported thisfinding. These results indicate that the acceleration of neurogenesis in aSyn / tau / mice is caused by downregulationof Notch signaling.

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Figure 4. A reduction in Notch signaling promotes neurogenesis in aSyn / tau / E12 cortices. A, B, Differentiation of IPCs was examined with Tbr2, an IPC marker. Tbr21 cells in the
VZ and SVZ were quantified in 100 mm columns. C, D, Differentiation of IPCs was examined with the proneuronal marker Ngn2. Ngn21 cells in the VZ and SVZ were quantified in 100 mm columns. E, F, Deletion of aSyn and tau led to a reduction in Notch1 expression in NPCs. Insets, Enlarged images from each boxed region. The mean Notch1 expression intensity in the VZ and
SVZ was quantified. G, Notch1 mRNA expression was quantified by qPCR. H, I, The expressions of NICD, Hes1, and Hes5 in E12 cortices were evaluated with Western blot analysis. The quantitative graphs are presented as means of triplicate values per genotype from three separate litters. J–L, The P0 cortical architecture was visualized by staining for the layer markers Cux1 (green)
and Ctip2 (red, J), and the Cux11 (K) and Ctip21 (L) neurons were quantified per 300 mm column. The quantitative data are presented as mean 6 SEM, N = 3 brains per genotype fromthree separate litters. Statistical analyses were performed with one-way ANOVA with Tukey’s post hoc test; *p , 0.05, **p , 0.01, ***p , 0.001. Scale bars: (in A) A, B, (in C) C, D, and (inE), E, F, 50 mm; (in J) J–L, 100 mm.

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Wang et al. · Functional Cross Talk between a-Synuclein and Tau during Brain Development

Figure 5. INM at the early embryonic stage was accelerated in aSyn / tau / mice. A–C, After nocodazole treatment, MEFs were stained with an anti-tubulin antibody (red,A), and the number of cells with straight MTs (B) and the mean MT length (C) were determined; N = 3 repeats of independent experiments, 30 cells per genotype. D, Quantified
mean velocity of EB3-mNG particles in MEFs; N = 3 repeats of independent experiments, 30 particles from different cells per genotype. E, F, Apical movement of nuclei/somata
during G2 phase in slice culture. After in utero electroporation of Lyn-EGFP (membrane, green) and H2B-RFP (nuclei, red) at E12, E13 cerebral wall slices were observed (E), andthe mean velocity of INM in G2 phase was quantified (F). At least 10 slices were observed, and 18 cells for the WT group and 33 cells for the aSyn / tau / group wereselected for INM quantification. G, H, After 16 h of observation of the cultured slices from E, 49.5 and 75.3% of electroporated WT and aSyn / tau / RGCs were found tohave escaped from the VZ, respectively. For quantification, five slices for the WT and eight slices for the aSyn / tau / were used. I, J, After IUE of Lyn-EGFP and H2B-RFP at
E12, slice culture was performed at E14. In electroporated cells, 66.5% of WT and 90% of aSyn / tau / RGCs escaped from the VZ. For quantification, five slices for the WT
and seven slices for the aSyn / tau / were used. The data are presented as mean 6 SEM, N = 3 brains from three different litters for each genotype. The p values were calculated by one-way ANOVA with Tukey’s post hoc test or Student’s t test; n.s., not significant; *p , 0.05, **p , 0.01, ***p , 0.001. Scale bars: (in A) A–C, 20 mm; (in E) E, F,(in G) G, H, and (in I) I, J, 50 mm.

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J. Neurosci., September 14, 2022 • 42(37):7031–7046 • 7039

Movie 1. EB3-mNG overexpression in WT MEFs induced by lentivirus infection. MT dynamics were visualized after overexpression of EB3-mNG in WT MEFs. Representative time lapse
movie was shown. Live cell imaging was collected every 200 ms for 1 min. Scale bar, 10mm. [View online]

Movie 3. EB3-mNG overexpression in tau-/- MEFs induced by lentivirus infection. MT dynamics were visualized after overexpression of EB3-mNG in tau-/- MEFs. Representative time
lapse movie was shown. Live cell imaging was collected every 200 ms for 1 min. Scale bar,
10 mm. [View online]

Movie 2. EB3-mNG overexpression in aSyn-/- MEFs induced by lentivirus infection. MT dynamics were visualized after overexpression of EB3-mNG in aSyn-/- MEFs. Representative
time lapse movie was shown. Live cell imaging was collected every 200 ms for 1 min. Scalebar, 10 mm. [View online]

Movie 4. EB3-mNG overexpression in aSyn-/-tau-/- MEFs induced by lentivirus infection.
MT dynamics were visualized after overexpression of EB3-mNG in aSyn-/-tau-/- MEFs.
Representative time lapse movie was shown. Live cell imaging was collected every 200 ms
for 1 min. Scale bar, 10 mm. [View online]

To examine whether the cortical architecture is affected inenlarged aSyn / tau / cortices, P0 neocortices were immunostained with the layer-specific markers Cux1 and Ctip2. We
observed that the numbers of deep layer Ctip21 neurons andupper layer Cux11 neurons were clearly increased in the aSyn /tau / CP (Fig. 4J–L), suggesting that cortical hypertrophy is associated with enhanced neurogenesis.

G2 phase interkinetic nuclear migration was accelerated inaSyn2/2tau2/2 mice
RGCs are apicobasally elongated, and their nuclei andsomata move vertically within the VZ in a cell cycle–dependent manner, a process called interkinetic nuclear migration(INM). Coupling of MT regulation with the cell cycle controls
the timing of INM. This nuclear/somal movement is intimately
linked to differentiation and neurogenesis (Miyata et al., 2014).
To determine the effects of impaired MT stability and dynamics
on neurogenesis, we traced cell cycle–dependent INM
in WT and aSyn / tau / cortical slices, through in vivo
experiments. After coexpression of Lyn-EGFP and H 2 BRFP by IUE at E12, embryonic cerebral walls were isolated
at E13 and subjected to slice culture and live imaging.
Compared with WT RGCs, G2 phase nuclear/somal movements toward the apical surface were faster in aSyn /tau / (Fig. 5E,F, Movies 5, 6). After 16 h of observation,
49.5% 6 3.87 and 75.3% 6 3.87 of the RGCs coelectroporated with Lyn-EGFP and H2B-RFP had escaped from the VZ
in WT and aSyn / tau / slices, respectively (Fig. 5G,H).
Additionally, 2 d after IUE at E12, almost all Lyn-EGFP and
H2B-RFP-coexpressing cells had reached the CP in theaSyn / tau / E14 slices (Fig. 5I,J), emphasizing that accelerated neurogenesis occurred during the E12–E14 stage in
the aSyn / tau / group as shown in Figure 2. Collectively,
these data indicate that the facilitated neurogenesis coupledwith the accelerated INM during G2 phase led to an overproduction of early born neurons in aSyn / tau / mice.

MT dynamics is altered in aSyn2/2tau2/2 MEFs
aSyn and tau share a common function as MT regulators(Moussaud et al., 2014). MTs play essential roles in the proliferation and differentiation of NPCs (Conde and Cáceres, 2009). To
analyze the effects of aSyn and tau on MT organization, we
assessed the MT network in fibroblasts isolated from WT and
KO embryos. MEFs did not display any morphologic abnormalities. However, after MT depolymerization via nocodazole treatment, we observed that newly extended MTs were shorter andless straight in aSyn / tau / MEFs (Fig. 5A–C). We theninvestigated the effects on MT dynamics after overexpression of
the plus end-binding protein EB3-mNG in the four genotypes of
MEFs by lentiviral transduction. Compared with WT, MEFs
derived from aSyn / or tau / showed a tendency of decreasein MT polymerization, whereas aSyn / tau / MEFs exhibited adrastic decrease in MT elongation (Fig. 5D, Movies 1, 2, 3, 4). These
results suggest that functional loss of aSyn and tau impairs MT
reorganization and dynamics. The altered MT reorganization anddynamics provide a molecular basis for the abnormal proliferationand differentiation of NPCs in aSyn / tau / mice.

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Wang et al. · Functional Cross Talk between a-Synuclein and Tau during Brain Development

Movie 6. G2 phase INM in aSyn-/-tau-/- cerebral slices. Lyn-EGFP (cell membrane) andH2B-RFP (cell nucleus) were electroporated into aSyn-/-tau-/- E12, and brain slice culture andapical INM during G2 phase were observed at E13. Scale bar, 20 mm. [View online]

Movie 5. G2 phase INM in WT cerebral slices. Lyn-EGFP (cell membrane) and H2B-RFP(cell nucleus) were electroporated into WT E12, and brain slice culture and apical INM during
G2 phase were observed at E13. Scale bar, 20 mm. [View online]

Loss of aSyn and tau diminishes gliogenesis in the later stageof corticogenesis
Our results indicated that aSyn and tau play an essential role inneurogenesis through proper maintenance of NPCs. Loss of bothaSyn and tau gave rise to premature neurogenesis at E11 andenhanced neurogenesis from E12, consequently resulting in adecrease in the number of RGCs at E15. In general, gliogenesis isfollowed by neurogenesis. Presumably, a reduction in NPCs canaffect subsequent gliogenesis in the later embryonic stage. To
confirm this hypothesis, we examined the behaviors of astrocytes
and oligodendrocytes at P11. Consistent with the observations in
the brains of 6-week-old mice, we observed a significant decreasein the cortical surface area at P11 aSyn / tau / mice (Fig. 6A,
B). The cortical thickness was also compared after Tbr1 immunostaining. The thickness of cortical layers II–VI was clearlydecreased in aSyn / tau / mice at P11 compared with that in
WT and both genotypes of single-KO mice (Fig. 6C,D). In contrast, the Tbr11 deep layer cell density in aSyn / tau / mice
was higher than that of WT, aSyn / , and tau / mice (Fig. 6C,
E). Neuronal death was also examined using an apoptosis
marker, cleaved caspase-3, and no clear apoptotic cells weredetected, even in aSyn / tau / mice (Fig. 6F,G). Consistent
with this observation, no differences were found in the numberof activated microglia (Iba11; Fig. 6H–J), which normally
respond to neuronal cell damage and remove the damaged cellsby phagocytosis in the CNS. These results corroborate our finding that the later stage of cortical development was affected inaSyn / tau / cortices without apparent neuronal cell death.
We next examined P11 brain sections by using S100 b , amarker of differentiated astrocytes, and Olig2, a marker of precursor and mature oligodendrocytes (Takebayashi et al., 2000).
Compared with WT and both genotypes of single-KO mice,aSyn / tau / exhibited markedly decreased numbers ofWang et al. · Functional Cross Talk between a-Synuclein and Tau during Brain Development

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Figure 6. Brain size was reduced in aSyn / tau / mice. A, B, Comparison of brain size in P11 mice. Scale bar, 1 mm. C–E, Quantification of the thickness of cortical layers II–VI (D) andthe deep layer neuron density (E) at P11 after immunostaining for Tbr1 (green, C). Scale bar, 200 mm. F–J, Neuronal death was investigated with cleaved caspase-3 (F, G) and Iba1 (H–J)
markers. Scale bar, 200 mm. The data are presented as mean 6 SEM, N = 3 brains from three different litters for each genotype. The p values were calculated by one-way ANOVA with
Tukey’s post hoc test; n.s., not significant; *p , 0.05, ***p , 0.001.

S100 b 1 astrocytes in the CP and white matter (WM; Fig. 7A–
E). Similarly, the numbers of Olig21 oligodendrocytes werereduced in the CP and WM of aSyn / tau / mice (Fig. 7F–J).
Furthermore, we examined the maturation of astrocytes and oligodendrocytes. The expression of GFAP, a mature and developing astrocyte marker (Yang and Wang, 2015), was examined byimmunohistochemistry. The intensity of GFAP staining was significantly decreased in both the pial and protoplasmic regions in
aSyn / tau / cortices (Fig. 8A–C). Mature oligodendrocytes
were probed with an anti-myelin basic protein (MBP) antibody.
Similar to findings in astrocytes, the density of MBP in oligodendrocytes was clearly decreased in the WM of aSyn / tau /mice at P11 (Fig. 8D,E).
To determine whether the diminished gliogenesis was attributable to a decrease in the number of IPCs, we immunostained E15
brain sections with an antibody against Ascl1, an IPC marker biased toward the oligodendrocyte lineage (Han et al., 2021). As we
expected, the number of Ascl11 IPCs was significantly reduced in
E15 aSyn / tau / cortices (Fig. 8F). Collectively, these results
indicate that the enhancement of early stage neurogenesis in
aSyn / tau / mice affects subsequent gliogenesis. Furthermore,
loss of aSyn and tau function impaired the expansion and maturation of astrocytes and oligodendrocytes in postnatal brains. This
impairment may have contributed to the reduction of brain size in
P11 and adult mice.

To determine the switch point of brain size, we first examined P7 cortices as expansion of astrocytes occurs during
the first postnatal week (Clavreul et al., 2019). P7 cortices
were immunostained with Cux1 and Ctip2 markers, and thecortical thickness was compared. The thickness of corticallayers II-VI in aSyn / tau / mice at P7 was comparable
to that in WT and two types of single-KO mice (Fig. 9A,B).
Conversely, aSyn / tau / mice displayed a significant increase in the number of upper layer Cux11 neurons, but not
deep layer Ctip21 neurons (Fig. 9A,C,D). Moreover, increased neuronal cell densities in the upper and deep layers
were confirmed in aSyn / tau / mice (Fig. 9A,E,F). These
results prompted us to further examine the behavior of neuronal cells in cortices from P11 and 6-week-old mice.
Similarly, we confirmed an increase in the number of upper
layer Cux11 neurons in aSyn / tau / mice at P11 (Fig.
9G–I), with higher neuronal cell densities in both the upperand deep layers (Fig. 9G,J,K). Consistent with the P7 and P11
cortices, increases in the neuronal cell densities in boththe upper and deep layers were detected in 6-week-old
aSyn / tau / mice (Figs. 1D, 9L,M). These results strongly
suggest that the reduction in brain size in adult aSyn / tau /mice is potentially because of the downregulation of gliogenesis
and expansion and maturation of microglial cells without neuronal loss.

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Wang et al. · Functional Cross Talk between a-Synuclein and Tau during Brain Development

Figure 7. Astrogenesis and oligodendrogenesis were diminished in aSyn / tau / mice. A–E, Statistical analyses of astrocytes in P11 cortices. P11 brain sections were stained for S100 b(green, A) and quantification of S100 b 1 astrocytes in the CP (C) and WM (E) was performed per 300 mm column. Each area surrounded by a yellow rectangle (CP) is enlarged and shown in
B, and the white boxed area (WM) is shown in D. Scale bar, 100 mm. F–J, Quantification of oligodendrocytes with the marker Olig2 at P11. P11 brain sections were immunostained with antiOlig2 antibody (green, F), and quantification of Olig21 oligodendrocytes in the CP (H) and WM (J) was performed per 300 mm column. Each area in the yellow box is shown in G, and thewhite boxed area is shown in I. Scale bar, 100 mm. The quantitative data are presented as the mean 6 SEM, N = 3 brains from three different litters for each genotype. The p values were calculated by one-way ANOVA with Tukey’s post hoc test; **p , 0.01, ***p , 0.001.

Discussion

aSyn and tau are abundant neuronal MAPs, and their abnormal intracellular aggregates have been linked to various neurodegenerative disorders (Vasili et al., 2019; Vacchi et al.,2020). Despite extensive endeavors, their physiological roles
remain elusive because of the subtle phenotypes in mice withknock-out of the individual genes (Ke et al., 2012; Pathak etal., 2017). In this study, we demonstrated that aSyn and tau
cooperatively play multiple developmental roles in a stagedependent manner.
Cortical neurons and macroglial cells arise from a common
proliferative RGCs (Rowitch and Kriegstein, 2010). Before theonset of neurogenesis, RGCs undergo symmetric cell division to
produce two daughter cells that adopt the progenitor fate (Rakic,
1982; Taverna et al., 2014). We show here that loss of aSyn andtau affected the balance between proliferative and neurogenic
divisions of RGCs, resulting in an overproduction of early bornneurons. This led to an enlarged brain in aSyn / tau / embryos.
The finding of facilitated neurogenesis was supported by the inutero electroporation experiments using E12–E14 cerebral slices,in which aSyn / tau / G2 phase nuclear/somal movements
became faster at E13, and almost all cells coelectroporated with
Lyn-EGFP and H2B-RFP had escaped from the VZ to the neocortex at E14. Consistent with this, an increase in the number of postmitotic neurons and an increased neocortical thickness wereobserved during the embryonic stage that was retained until theneonatal stage in aSyn / tau / mice. Notably, enhancement ofneurogenesis led to an increase in cortical thickness, without causing histologic layer defects, indicating that aSyn and tau are cooperatively involved in the maintenance of NPCs, but notpostmitotic neuronal migration during cortical lamination.
Differentiation of RGCs toward IPCs was also facilitatedin E12 embryos but decreased in E14 aSyn / tau / mice.
Moreover, a reduction in the RGC pool was found at E15.

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Figure 8. Downregulated maturation of astrocytes and oligodendrocytes was detected in aSyn / tau / mice. A–C, Astrocyte maturation was confirmed by GFAP immunostaining at
P11. The areas surrounded by a white rectangle are enlarged and shown in B and C. The GFAP density in both the pial and protoplasmic areas of the cortex were quantified. D, E,
Oligodendrocyte maturation was confirmed by MBP immunostaining at P11. The white boxed area is enlarged and shown in E. F, Differentiated IPCs at E15 analyzed by staining for Ascl1, a basal progenitor cell marker biased toward oligodendrogenesis. The cells were counted in a 100 mm column. Mean MBP densities in WM were measured in a 50  50 mm2 area. The quantitative data are presented as mean 6 SEM, N = 3 brains from three different litters for each genotype. The p values were calculated by one-way ANOVA with Tukey’s post hoc test; *p , 0.05,***p , 0.001. Scale bars: (in A) A–C, (in D) D, E, 100 mm; (in F), 50 mm.

Presumably, the premature and facilitated neurogenesis at theearly embryonic stage caused excess exhaustion of RGCs inaSyn / tau / mice. As we expected, the reductions in the RGC
and IPC pools in aSyn / tau / cortices also affected subsequentgliogenesis at later embryonic stage, and this effect was accompanied by impaired expansion and maturation of astrocytes andoligodendrocytes in the postnatal brain. Clavreul et al. (2019)
reported that expansion of astrocytes occurs during the first postnatal week, and P7–P21 is the maturation phase during which
each astrocyte increases the volume and complexity of their processes. Accordingly, at P11 and 6 weeks of age, aSyn / tau /mice displayed smaller brains than WT and two types of single-

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Wang et al. · Functional Cross Talk between a-Synuclein and Tau during Brain Development

Figure 9. The downregulated brain size in aSyn / tau / mice was caused by diminished gliogenesis. A–F, Quantification of neuronal cells in P7 cortices. Cortical neurons were probedwith Cux1 (red) and Ctip2 (green) markers (A), and the thickness of layers II–VI (B), the neuron numbers (C, D) and the cell densities (E, F) were quantified. The cells were counted in a
300 mm column, and the cell densities were calculated in a 100  100 mm2 area. Scale bar, 200 mm. G–K, Quantification of neuronal cells in P11 cortices. Cortical neurons were probed with
Cux1 (red) and Ctip2 (green) markers (G), and the neuron numbers (H, I) and cell densities (J, K) were quantified. The cells were counted in a 300 mm column. Scale bar, 200 mm. L, M, The
Cux11 neuron density in layers II–IV (L) and Ctip21 neuron density in layers V–VI (M) were quantified in the cortices from the 6-week-old mice (Fig. 1D). The quantitative data are presented
as mean 6 SEM, N = 3 brains from three separate litters for each genotype. The p values were calculated by one-way ANOVA with Tukey’s post hoc test; n.s., not significant; *p , 0.05,**p , 0.01, ***p , 0.001.

KO mice, accompanied by increased neuronal cell densities in theupper and deep layers, without neuronal cell death. Notably,
astrocytes interact with multiple types of cells, including
neurons, glial cells, and endothelial cells in blood vessels,
whereas mature oligodendrocytes generate a myelin sheaththat surrounds and insulates axons and facilitates the transmission of neural impulses in the CNS (Hartline andColman, 2007; Chang et al., 2016). Emerging evidence suggests that the loss of beneficial roles of glial cells can contribute to neurodegenerative conditions (Tremblay et al., 2019).
Dysregulation of gliogenesis and their expansion and maturation caused by deletion of aSyn and tau provide important

insights into elucidating pathogeneses of neurodegenerative
diseases including PD and multiple system atrophy.
MT organization plays pivotal roles during proliferation anddifferentiation of NPCs, including the cell cycle–dependent INM(Spear and Erickson, 2012; Cooper, 2013; Lasser et al., 2018).
aSyn and tau share a common feature as neuronal MAPs. We
found that absence of aSyn and tau diminished MT reorganization and dynamics. Conversely, G2 phase INM velocity was
clearly accelerated in aSyn / tau / brain slices, and neurogenesis was significantly enhanced from the early embryonicstage. Previous work revealed that mutation in a microtubule-motor-associated protein dynactin perturbs INM andWang et al. · Functional Cross Talk between a-Synuclein and Tau during Brain Development

accelerated retinal neurogenesis in the zebrafish (Del Bene etal., 2008). Furthermore, Notch signaling plays crucial rolesin the proliferation and differentiation of NPCs (Mase et al.,2021). In aSyn / tau / , we also established that Notch signaling was reduced during neurogenesis. Downregulation of
Notch target genes leads to upregulation of proneural genes
and neuronal differentiation (Kawaguchi et al., 2008). In linewith this, increased numbers of Ngn2-expressing IPCs weredetected in E12 aSyn / tau / cortices. Collectively, thedata indicate that the precocious and the facilitated neurogenesis at early embryonic stage in aSyn / tau / brains
is probably because of the reduction in Notch signaling
and accelerated INM. Both intracellular Notch signaling
conduction and INM depend on microtubule-associatedmotor proteins (Del Bene et al., 2008; Tsai et al., 2010; Hu
et al., 2013). Presumably, diminished MT reorganization
perturbs conduction of Notch signaling and cell cycle–dependent INM. More detailed studies are needed to better understandthe mechanisms underlying the facilitated proliferation and differentiation of NPCs in aSyn / tau / mice.
Intracellular codeposition of pathologic aSyn and tau has
been linked to many neurologic disorders, including AD and PD(Moussaud et al., 2014; Li et al., 2016; Henderson et al., 2019).
Furthermore, cross-seeding of aSyn and tau has been investigated in vitro and in vivo (Li et al., 2016; Lu et al., 2020). For thisreason, the interaction between aSyn and tau is considered todevelop neurodegenerative disorders. Here, we revealed previously unrecognized functional cross talk between aSyn and tau
during neurogenesis and gliogenesis. Our findings might provide
new mechanistic insights and expand the therapeutic opportunities for neurodegenerative diseases caused by aberrant aSyn and/or tau.

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