Histone bivalency regulates the timing of cerebellar granule cell development.

Developing neurons undergo a progression of morphological and gene expression changes as they transition from neuronal progenitors to mature neurons. Here we used RNA-seq and H3K4me3 and H3K27me3 ChIP-seq to analyze how chromatin modifications control gene expression in a specific type of CNS neuron: the mouse cerebellar granule cell (GC). We found that in proliferating GC progenitors (GCPs), H3K4me3/H3K27me3 bivalency is common at neuronal genes and undergoes dynamic changes that correlate with gene expression during migration and circuit formation. Expressing a fluorescent sensor for bivalent domains revealed subnuclear bivalent foci in proliferating GCPs. Inhibiting H3K27 methyltransferases EZH1 and EZH2 in vitro and in organotypic cerebellar slices dramatically altered the expression of bivalent genes, induced the down-regulation of migration-related genes and up-regulation of synaptic genes, inhibited glial-guided migration, and accelerated terminal differentiation. Thus, histone bivalency is required to regulate the timing of the progression from progenitor cells to mature neurons.

Novel genetic features of human and mouse Purkinje cell differentiation defined by comparative transcriptomics.

Comparative transcriptomics between differentiating human pluripotent stem cells (hPSCs) and developing mouse neurons offers a powerful approach to compare genetic and epigenetic pathways in human and mouse neurons. To analyze human Purkinje cell (PC) differentiation, we optimized a protocol to generate human pluripotent stem cell-derived Purkinje cells (hPSC-PCs) that formed synapses when cultured with mouse cerebellar glia and granule cells and fired large calcium currents, measured with the genetically encoded calcium indicator jRGECO1a. To directly compare global gene expression of hPSC-PCs with developing mouse PCs, we used translating ribosomal affinity purification (TRAP). As a first step, we used Tg(Pcp2-L10a-Egfp) TRAP mice to profile actively transcribed genes in developing postnatal mouse PCs and used metagene projection to identify the most salient patterns of PC gene expression over time. We then created a transgenic Pcp2-L10a-Egfp TRAP hPSC line to profile gene expression in differentiating hPSC-PCs, finding that the key gene expression pathways of differentiated hPSC-PCs most closely matched those of late juvenile mouse PCs (P21). Comparative bioinformatics identified classical PC gene signatures as well as novel mitochondrial and autophagy gene pathways during the differentiation of both mouse and human PCs. In addition, we identified genes expressed in hPSC-PCs but not mouse PCs and confirmed protein expression of a novel human PC gene, CD40LG, expressed in both hPSC-PCs and native human cerebellar tissue. This study therefore provides a direct comparison of hPSC-PC and mouse PC gene expression and a robust method for generating differentiated hPSC-PCs with human-specific gene expression for modeling developmental and degenerative cerebellar disorders.

The proteasome regulator PI31 is required for protein homeostasis, synapse maintenance, and neuronal survival in mice.

Proteasome-mediated degradation of intracellular proteins is essential for cell function and survival. The proteasome-binding protein PI31 (Proteasomal Inhibitor of 31kD) promotes 26S assembly and functions as an adapter for proteasome transport in axons. As localized protein synthesis and degradation is especially critical in neurons, we generated a conditional loss of PI31 in spinal motor neurons (MNs) and cerebellar Purkinje cells (PCs). A cKO of PI31 in these neurons caused axon degeneration, neuronal loss, and progressive spinal and cerebellar neurological dysfunction. For both MNs and PCs, markers of proteotoxic stress preceded axonal degeneration and motor dysfunction, indicating a critical role for PI31 in neuronal homeostasis. The time course of the loss of MN and PC function in developing mouse central nervous system suggests a key role for PI31 in human neurodegenerative diseases.

N-cadherin provides a cis and trans ligand for astrotactin that functions in glial-guided neuronal migration.

Prior studies demonstrate that astrotactin (ASTN1) provides a neuronal receptor for glial-guided CNS migration. Here we report that ASTN1 binds N-cadherin (CDH2) and that the ASTN1:CDH2 interaction supports cell-cell adhesion. To test the function of ASTN1:CDH2 binding in glial-guided neuronal migration, we generated a conditional loss of Cdh2 in cerebellar granule cells and in glia. Granule cell migration was slowed in cerebellar slice cultures after a conditional loss of neuronal Cdh2, and more severe migration defects occurred after a conditional loss of glial Cdh2 Expression in granule cells of a mutant form of ASTN1 that does not bind CDH2 also slowed migration. Moreover, in vitro chimeras of granule cells and glia showed impaired neuron-glia attachment in the absence of glial, but not neuronal, Cdh2 Thus, cis and trans bindings of ASTN1 to neuronal and glial CDH2 form an asymmetric neuron-glial bridge complex that promotes glial-guided neuronal migration.

ASTN2 modulates synaptic strength by trafficking and degradation of surface proteins.

Surface protein dynamics dictate synaptic connectivity and function in neuronal circuits. ASTN2, a gene disrupted by copy number variations (CNVs) in neurodevelopmental disorders, including autism spectrum, was previously shown to regulate the surface expression of ASTN1 in glial-guided neuronal migration. Here, we demonstrate that ASTN2 binds to and regulates the surface expression of multiple synaptic proteins in postmigratory neurons by endocytosis, resulting in modulation of synaptic activity. In cerebellar Purkinje cells (PCs), by immunogold electron microscopy, ASTN2 localizes primarily to endocytic and autophagocytic vesicles in the cell soma and in subsets of dendritic spines. Overexpression of ASTN2 in PCs, but not of ASTN2 lacking the FNIII domain, recurrently disrupted by CNVs in patients, including in a family presented here, increases inhibitory and excitatory postsynaptic activity and reduces levels of ASTN2 binding partners. Our data suggest a fundamental role for ASTN2 in dynamic regulation of surface proteins by endocytic trafficking and protein degradation.

Murine astrotactins 1 and 2 have a similar membrane topology and mature via endoproteolytic cleavage catalyzed by a signal peptidase.

Astrotactin 1 (Astn1) and Astn2 are membrane proteins that function in glial-guided migration, receptor trafficking, and synaptic plasticity in the brain as well as in planar polarity pathways in the skin. Here we used glycosylation mapping and protease protection approaches to map the topologies of mouse Astn1 and Astn2 in rough microsomal membranes and found that Astn2 has a cleaved N-terminal signal peptide, an N-terminal domain located in the lumen of the rough microsomal membranes (topologically equivalent to the extracellular surface in cells), two transmembrane helices, and a large C-terminal lumenal domain. We also found that Astn1 has the same topology as Astn2, but we did not observe any evidence of signal peptide cleavage in Astn1. Both Astn1 and Astn2 mature through endoproteolytic cleavage in the second transmembrane helix; importantly, we identified the endoprotease responsible for the maturation of Astn1 and Astn2 as the endoplasmic reticulum signal peptidase. Differences in the degree of Astn1 and Astn2 maturation possibly contribute to the higher levels of the C-terminal domain of Astn1 detected on neuronal membranes of the central nervous system. These differences may also explain the distinct cellular functions of Astn1 and Astn2, such as in membrane adhesion, receptor trafficking, and planar polarity signaling.

Purkinje cells derived from TSC patients display hypoexcitability and synaptic deficits associated with reduced FMRP levels and reversed by rapamycin.

Accumulating evidence suggests that cerebellar dysfunction early in life is associated with autism spectrum disorder (ASD), but the molecular mechanisms underlying the cerebellar deficits at the cellular level are unclear. Tuberous sclerosis complex (TSC) is a neurocutaneous disorder that often presents with ASD. Here, we developed a cerebellar Purkinje cell (PC) model of TSC with patient-derived human induced pluripotent stem cells (hiPSCs) to characterize the molecular mechanisms underlying cerebellar abnormalities in ASD and TSC. Our results show that hiPSC-derived PCs from patients with pathogenic TSC2 mutations displayed mTORC1 pathway hyperactivation, defects in neuronal differentiation and RNA regulation, hypoexcitability and reduced synaptic activity when compared with those derived from controls. Our gene expression analyses revealed downregulation of several components of fragile X mental retardation protein (FMRP) targets in TSC2-deficient hiPSC-PCs. We detected decreased expression of FMRP, glutamate receptor δ2 (GRID2), and pre- and post-synaptic markers such as synaptophysin and PSD95 in the TSC2-deficient hiPSC-PCs. The mTOR inhibitor rapamycin rescued the deficits in differentiation, synaptic dysfunction, and hypoexcitability of TSC2 mutant hiPSC-PCs in vitro. Our findings suggest that these gene expression changes and cellular abnormalities contribute to aberrant PC function during development in TSC affected individuals.

Consensus Paper: Cerebellar Development.

The development of the mammalian cerebellum is orchestrated by both cell-autonomous programs and inductive environmental influences. Here, we describe the main processes of cerebellar ontogenesis, highlighting the neurogenic strategies used by developing progenitors, the genetic programs involved in cell fate specification, the progressive changes of structural organization, and some of the better-known abnormalities associated with developmental disorders of the cerebellum.

Mice lacking Astn2 have ASD-like behaviors and altered cerebellar circuit properties.

Astrotactin 2 (ASTN2) is a transmembrane neuronal protein highly expressed in the cerebellum that functions in receptor trafficking and modulates cerebellar Purkinje cell (PC) synaptic activity. We recently reported a family with a paternally inherited intragenic ASTN2 duplication with a range of neurodevelopmental disorders, including autism spectrum disorder (ASD), learning difficulties, and speech and language delay. To provide a genetic model for the role of the cerebellum in ASD-related behaviors and study the role of ASTN2 in cerebellar circuit function, we generated global and PC-specific conditional Astn2 knockout (KO and cKO, respectively) mouse lines. Astn2 KO mice exhibit strong ASD-related behavioral phenotypes, including a marked decrease in separation-induced pup ultrasonic vocalization calls, hyperactivity and repetitive behaviors, altered social behaviors, and impaired cerebellar-dependent eyeblink conditioning. Hyperactivity and repetitive behaviors were also prominent in Astn2 cKO animals. By Golgi staining, Astn2 KO PCs have region-specific changes in dendritic spine density and filopodia numbers. Proteomic analysis of Astn2 KO cerebellum reveals a marked upregulation of ASTN2 family member, ASTN1, a neuron-glial adhesion protein. Immunohistochemistry and electron microscopy demonstrates a significant increase in Bergmann glia volume in the molecular layer of Astn2 KO animals. Electrophysiological experiments indicate a reduced frequency of spontaneous excitatory postsynaptic currents (EPSCs), as well as increased amplitudes of both spontaneous EPSCs and inhibitory postsynaptic currents (IPSCs) in the Astn2 KO animals, suggesting that pre- and postsynaptic components of synaptic transmission are altered. Thus, ASTN2 regulates ASD-like behaviors and cerebellar circuit properties.

Histone bivalency regulates the timing of cerebellar granule cell development.

Developing neurons undergo a progression of morphological and gene expression changes as they transition from neuronal progenitors to mature, multipolar neurons. Here we use RNA-seq and H3K4me3 and H3K27me3 ChIP-seq to analyze how chromatin modifications control gene expression in a specific type of CNS neuron, the mouse cerebellar granule cell (GC). We find that in proliferating GC progenitors (GCPs), H3K4me3/H3K27me3 bivalency is common at neuronal genes and undergoes dynamic changes that correlate with gene expression during migration and circuit formation. Expressing a fluorescent sensor for bivalent H3K4me3 and H3K27me3 domains revealed subnuclear bivalent foci in proliferating GCPs. Inhibiting H3K27 methyltransferases EZH1 and EZH2 in vitro and in organotypic cerebellar slices dramatically altered the expression of bivalent genes and induced the downregulation of migration-related genes and upregulation of synaptic genes, inhibited glial-guided migration, and accelerated terminal differentiation. Thus, histone bivalency is required to regulate the timing of the progression from progenitor cells to mature neurons.

Altered chromatin occupancy of patient-associated H4 mutants misregulate neuronal differentiation.

Chromatin is a crucial regulator of gene expression and tightly controls development across species. Mutations in only one copy of multiple histone genes were identified in children with developmental disorders characterized by microcephaly, but their mechanistic roles in development remain unclear. Here we focus on dominant mutations affecting histone H4 lysine 91. These H4K91 mutants form aberrant nuclear puncta at specific heterochromatin regions. Mechanistically, H4K91 mutants demonstrate enhanced binding to the histone variant H3.3, and ablation of H3.3 or the H3.3-specific chaperone DAXX diminishes the mutant localization to chromatin. Our functional studies demonstrate that H4K91 mutant expression increases chromatin accessibility, alters developmental gene expression through accelerating pro-neural differentiation, and causes reduced mouse brain size in vivo, reminiscent of the microcephaly phenotypes of patients. Together, our studies unveil a distinct molecular pathogenic mechanism from other known histone mutants, where H4K91 mutants misregulate cell fate during development through abnormal genomic localization.

A schizophrenia-associated mutation of DISC1 perturbs cerebral cortex development.

Disrupted-In-Schizophrenia-1 (DISC1), originally identified at the breakpoint of a chromosomal translocation that is linked to a rare familial schizophrenia, has been genetically implicated in schizophrenia in other populations. Schizophrenia involves subtle cytoarchitectural abnormalities that arise during neurodevelopment, but the underlying molecular mechanisms are unclear. Here, we demonstrate that DISC1 is a component of the microtubule-associated dynein motor complex and is essential for maintaining the complex at the centrosome, hence contributing to normal microtubular dynamics. Carboxy-terminal-truncated mutant DISC1 (mutDISC1), which results from a chromosomal translocation, functions in a dominant-negative manner by redistributing wild-type DISC1 through self-association and by dissociating the DISC1-dynein complex from the centrosome. Consequently, either depletion of endogenous DISC1 or expression of mutDISC1 impairs neurite outgrowth in vitro and proper development of the cerebral cortex in vivo. These results indicate that DISC1 is involved in cerebral cortex development, and suggest that loss of DISC1 function may underlie neurodevelopmental dysfunction in schizophrenia.

Lrp12/Mig13a reveals changing patterns of preplate neuronal polarity during corticogenesis that are absent in reeler mutant mice.

During corticogenesis, the earliest generated neurons form the preplate, which evolves into the marginal zone and subplate. Lrp12/Mig13a, a mammalian gene related to the Caenorhabditis elegans neuroblast migration gene mig-13, is expressed in a subpopulation of preplate neurons that undergo ventrally directed tangential migrations in the preplate layer and pioneer axon projections to the anterior commissure. As the preplate separates, Lrp12/Mig13a-positive neurons polarize in the radial plane and form a pseudocolumnar pattern, prior to moving to a deeper position within the emerging subplate layer. These changes in neuronal polarity do not occur in reeler mutant mice, revealing the earliest known defect in reeler cortical patterning and suggesting that the alignment of preplate neurons into a pseudolayer facilitates the movement of later-born radially migrating neurons into the emerging cortical plate.

Astn2, a novel member of the astrotactin gene family, regulates the trafficking of ASTN1 during glial-guided neuronal migration.

Glial-guided neuronal migration is a key step in the development of laminar architecture of cortical regions of the mammalian brain. We previously reported that neuronal protein astrotactin (ASTN1) functions as a neuron-glial ligand during CNS glial-guided migration. Here, we identify a new Astn family member, Astn2, that is expressed at high levels in migrating, cerebellar granule neurons, along with Astn1, at developmental stages when glial-guided migration is ongoing. Biochemical and flow cytometry experiments show that ASTN2 forms a complex with ASTN1 and regulates surface expression of ASTN1. Live imaging of Venus-tagged ASTN1 in migrating cerebellar granule cells reveals the intracellular trafficking of ASTN1-Venus, with ASTN1-Venus accumulating in the forward aspect of the leading process where new sites of adhesion will form. Treatment of migrating neurons with Dynasore, a soluble noncompetitive inhibitor of Dynamin, rapidly arrests the migration of immature granule cells in a reversible manner, suggesting the critical importance of receptor trafficking to neuronal locomotion along Bergmann glial fibers in the developing cerebellum. Together, these findings suggest that ASTN2 regulates the levels of ASTN1 in the plasma membrane and that the release of neuronal adhesions to the glial fiber during neuronal locomotion involves the intracellular trafficking of ASTN1.

PCSK9 reduces the protein levels of the LDL receptor in mouse brain during development and after ischemic stroke.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a major role in cholesterol homeostasis through enhanced degradation of the LDL receptor (LDLR) in liver. As novel inhibitors/silencers of PCSK9 are now being tested in clinical trials to treat hypercholesterolemia, it is crucial to define the physiological consequences of the lack of PCSK9 in various organs. LDLR regulation by PCSK9 has not been extensively described during mouse brain development and injury. Herein, we show that PCSK9 and LDLR are co-expressed in mouse brain during development and at adulthood. Although the protein levels of LDLR and apolipoprotein E (apoE) in the adult brain of Pcsk9(-/-) mice are similar to those of wild-type (WT) mice, LDLR levels increased and were accompanied by a reduction of apoE levels during development. This suggests that the upregulation of LDLR protein levels in Pcsk9(-/-) mice enhances apoE degradation. Upon ischemic stroke, PCSK9 was expressed in the dentate gyrus between 24 h and 72 h following brain reperfusion. Although mouse behavior and lesion volume were similar, LDLR protein levels dropped ∼2-fold less in the Pcsk9(-/-)-lesioned hippocampus, without affecting apoE levels and neurogenesis. Thus, PCSK9 downregulates LDLR levels during brain development and following transient ischemic stroke in adult mice.

Altered temporal sequence of transcriptional regulators in the generation of human cerebellar granule cells.

Brain development is regulated by conserved transcriptional programs across species, but little is known about the divergent mechanisms that create species-specific characteristics. Among brain regions, human cerebellar histogenesis differs in complexity compared with nonhuman primates and rodents, making it important to develop methods to generate human cerebellar neurons that closely resemble those in the developing human cerebellum. We report a rapid protocol for the derivation of the human ATOH1 lineage, the precursor of excitatory cerebellar neurons, from human pluripotent stem cells (hPSCs). Upon transplantation into juvenile mice, hPSC-derived cerebellar granule cells migrated along glial fibers and integrated into the cerebellar cortex. By Translational Ribosome Affinity Purification-seq, we identified an unexpected temporal shift in the expression of RBFOX3 (NeuN) and NEUROD1, which are classically associated with differentiated neurons, in the human outer external granule layer. This molecular divergence may enable the protracted development of the human cerebellum compared to mice.

LIS-less neurons don't even make it to the starting gate.

The manuscript by Tsai et al. (935-945) is a tour de force analysis of a controversial issue in developmental neurobiology, namely the molecular basis of the devastating human brain malformation, type I lissencephaly (Lis1) (Jellinger, K., and A. Rett. 1976. Neuropadiatrie. 7:66-91). For several decades, defects in neuronal migration have been assumed to underlie all defects in cortical histogenesis. In the paper by Tsai et al., the authors use a variety of elegant approaches, including the first real-time imaging of cortical neurons with reduced levels of LIS1, to demonstrate that LIS1 and dynactin act as regulators of dynein during cortical histogenesis. A loss of LIS1 results in both a failure to exit the cortical germinal zone and abnormal neuronal process formation. Thus, the primary action of the mutation is to disrupt the production of neurons in the developing brain as well as their migration.

A gene expression atlas of the central nervous system based on bacterial artificial chromosomes.

The mammalian central nervous system (CNS) contains a remarkable array of neural cells, each with a complex pattern of connections that together generate perceptions and higher brain functions. Here we describe a large-scale screen to create an atlas of CNS gene expression at the cellular level, and to provide a library of verified bacterial artificial chromosome (BAC) vectors and transgenic mouse lines that offer experimental access to CNS regions, cell classes and pathways. We illustrate the use of this atlas to derive novel insights into gene function in neural cells, and into principal steps of CNS development. The atlas, library of BAC vectors and BAC transgenic mice generated in this screen provide a rich resource that allows a broad array of investigations not previously available to the neuroscience community.

Gene expression profiling of preplate neurons destined for the subplate: genes involved in transcription, axon extension, neurotransmitter regulation, steroid hormone signaling, and neuronal survival.

During mammalian corticogenesis a series of transient cell layers establish laminar architectonics. The preplate, which forms from the earliest-generated neurons, separates into the marginal zone and subplate layer. To provide a systematic screen for genes involved in subplate development and function, we screened lines of transgenic mice, generated using bacterial artificial chromosome methodology (GENSAT Project), to identify transgenic lines of mice that express the enhanced green fluorescent protein (EGFP) reporter in preplate neurons destined for the subplate. Gene expression profiling of RNA purified from EGFP-positive neurons identified over 200 genes with enriched expression in future subplate neurons. Major classes of subplate-enriched genes included genes involved in transcriptional processes, cortical development, cell and axon motility, protein trafficking and steroid hormone signaling. Additionally, we identified 10 genes related to degenerative diseases of the cerebral and cerebellar cortex. Cre recombinase-based fate mapping of cells expressing Phosphodiesterase 1c (Pde1c) revealed beta-galactosidase positive cells in the ventricular zone, as well as the subplate, suggesting that subplate neurons and cortical projection neurons may be derived from common progenitors. These experiments therefore reveal genetic markers, which identify subplate neurons from the earliest stages of their development, and genes with enriched expression in subplate neurons during early stages of corticogenesis.

Tag-Team Genetics of Spinocerebellar Ataxia 6.

In this issue of Neuron, Du et al. (2019) demonstrate that the bicistronic CACNA1A gene encodes a transcription factor α1ACT, mutations in which are associated with SCA6, that controls expression of genes important for cerebellar Purkinje cell development and excitability. Reduction of α1ACT in the adult is well tolerated, suggesting a potential new therapy for SCA6.