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Cellular diversification is critical for specialized functions of the brain including learning and memory1. Single-cell RNA sequencing facilitates transcriptomic profiling of distinct major types of neuron2–4, but the divergence of transcriptomic profiles within a neuronal population and their link to function remain poorly understood. Here we isolate nuclei tagged5 in specific cell types followed by single-nucleus RNA sequencing to profile Purkinje neurons and map their responses to motor activity and learning. We find that two major subpopulations of Purkinje neurons, identified by expression of the genes Aldoc and Plcb4, bear distinct transcriptomic features. Plcb4+, but not Aldoc+, Purkinje neurons exhibit robust plasticity of gene expression in mice subjected to sensorimotor and learning experience. In vivo calcium imaging and optogenetic perturbation reveal that Plcb4+ Purkinje neurons have a crucial role in associative learning. Integrating single-nucleus RNA sequencing datasets with weighted gene co-expression network analysis uncovers a learning gene module that includes components of FGFR2 signalling in Plcb4+ Purkinje neurons. Knockout of Fgfr2 in Plcb4+ Purkinje neurons in mice using CRISPR disrupts motor learning. Our findings define how diversification of Purkinje neurons is linked to their responses in motor learning and provide a foundation for understanding their differential vulnerability to neurological disorders.Subpopulations of Purkinje neurons display distinct transcriptomic responses and functions in associative learning. [ABSTRACT FROM AUTHOR]

Regulation of chromatin plays fundamental roles in the development of the brain. Haploinsufficiency of the chromatin remodeling enzyme CHD7 causes CHARGE syndrome, a genetic disorder that affects the development of the cerebellum. However, how CHD7 controls chromatin states in the cerebellum remains incompletely understood. Using conditional knockout of CHD7 in granule cell precursors in the mouse cerebellum, we find that CHD7 robustly promotes chromatin accessibility, active histone modifications, and RNA polymerase recruitment at enhancers. In vivo profiling of genome architecture reveals that CHD7 concordantly regulates epigenomic modifications associated with enhancer activation and gene expression of topologically-interacting genes. Genome and gene ontology studies show that CHD7-regulated enhancers are associated with genes that control brain tissue morphogenesis. Accordingly, conditional knockout of CHD7 triggers a striking phenotype of cerebellar polymicrogyria, which we have also found in a case of CHARGE syndrome. Finally, we uncover a CHD7-dependent switch in the preferred orientation of granule cell precursor division in the developing cerebellum, providing a potential cellular basis for the cerebellar polymicrogyria phenotype upon loss of CHD7. Collectively, our findings define epigenomic regulation by CHD7 in granule cell precursors and identify abnormal cerebellar patterning upon CHD7 depletion, with potential implications for our understanding of CHARGE syndrome. CHARGE syndrome that affects cerebellar development can be caused by haploinsufficiency of the chromatin remodeling enzyme CHD7; however the precise role of CHD7 remains unknown. Here the authors show CHD7 promotes chromatin accessibility and enhancer activity in granule cell precursors and regulates morphogenesis of the cerebellar cortex, where loss of CHD7 triggers cerebellar polymicrogyria. [ABSTRACT FROM AUTHOR]

The development and function of the brain require tight control of gene expression. Genome architecture is thought to play a critical regulatory role in gene expression, but the mechanisms governing genome architecture in the brain in vivo remain poorly understood. Here, we report that conditional knockout of the chromatin remodeling enzyme Chd4 in granule neurons of the mouse cerebellum increases accessibility of gene regulatory sites genome-wide in vivo. Conditional knockout of Chd4 promotes recruitment of the architectural protein complex cohesin preferentially to gene enhancers in granule neurons in vivo. Importantly, in vivo profiling of genome architecture reveals that conditional knockout of Chd4 strengthens interactions among developmentally repressed contact domains as well as genomic loops in a manner that tightly correlates with increased accessibility, enhancer activity, and cohesin occupancy at these sites. Collectively, our findings define a role for chromatin remodeling in the control of genome architecture organization in the mammalian brain. The mechanisms underlying gene regulation and genome architecture remain poorly understood. Here, the authors investigate the role of chromatin remodelling enzyme Chd4 in granule neurons of the mouse cerebellum and find that conditional knockout of Chd4 preferentially activates enhancers and modulates genome architecture at a genome-wide level. [ABSTRACT FROM AUTHOR]

Neuronal-activity-dependent transcription couples sensory experience to adaptive responses of the brain including learning and memory. Mechanisms of activity-dependent gene expression including alterations of the epigenome have been characterized1–8. However, the fundamental question of whether sensory experience remodels chromatin architecture in the adult brain in vivo to induce neural code transformations and learning and memory remains to be addressed. Here we use in vivo calcium imaging, optogenetics and pharmacological approaches to show that granule neuron activation in the anterior dorsal cerebellar vermis has a crucial role in a delay tactile startle learning paradigm in mice. Of note, using large-scale transcriptome and chromatin profiling, we show that activation of the motor-learning-linked granule neuron circuit reorganizes neuronal chromatin including through long-distance enhancer–promoter and transcriptionally active compartment interactions to orchestrate distinct granule neuron gene expression modules. Conditional CRISPR knockout of the chromatin architecture regulator cohesin in anterior dorsal cerebellar vermis granule neurons in adult mice disrupts enhancer–promoter interactions, activity-dependent transcription and motor learning. These findings define how sensory experience patterns chromatin architecture and neural circuit coding in the brain to drive motor learning. The authors identify a role for genome architecture reorganization in anterior dorsal cerebellar vermis granule neurons in learning a conditioned startle paradigm in mice. [ABSTRACT FROM AUTHOR]

Although ubiquitin ligases have been implicated in autism, their roles and mechanisms in brain development remain incompletely understood. Here, we report that in vivo knockdown or conditional knockout of the autism-linked ubiquitin ligase RNF8 or associated ubiquitin-conjugating enzyme UBC13 in rodent cerebellar granule neurons robustly increases the number of parallel fiber presynaptic boutons and functional parallel fiber/Purkinje cell synapses. In contrast to the role of nuclear RNF8 in proliferating cells, RNF8 operates in the cytoplasm in neurons to suppress synapse differentiation in vivo. Proteomics analyses reveal that neuronal RNF8 interacts with the HECT domain protein HERC2 and scaffold protein NEURL4, and knockdown of HERC2 or NEURL4 phenocopies the inhibition of RNF8/UBC13 signaling on synapse differentiation. In behavior analyses, granule neuron-specific knockout of RNF8 or UBC13 impairs cerebellar-dependent learning. Our study defines RNF8 and UBC13 as components of a novel cytoplasmic ubiquitin-signaling network that suppresses synapse formation in the brain. [ABSTRACT FROM AUTHOR]

Perturbations of astrocytes trigger neurodegeneration in several diseases, but the glial cell-intrinsic mechanisms that induce neurodegeneration remain poorly understood. We found that a protein complex of α2-Na/K ATPase and α-adducin was enriched in astrocytes expressing mutant superoxide dismutase 1 (SOD1), which causes familial amyotrophic lateral sclerosis (ALS). Knockdown of α2-Na/K ATPase or α-adducin in mutant SOD1 astrocytes protected motor neurons from degeneration, including in mutant SOD1 mice in vivo. Heterozygous disruption of the α2-Na/K ATPase gene suppressed degeneration in vivo and increased the lifespan of mutant SOD1 mice. The pharmacological agent digoxin, which inhibits Na/K ATPase activity, protected motor neurons from mutant SOD1 astrocyte-induced degeneration. Notably, α2-Na/K ATPase and α-adducin were upregulated in spinal cord of sporadic and familial ALS patients. Collectively, our findings define chronic activation of the α2-Na/K ATPase/α-adducin complex as a critical glial cell-intrinsic mechanism of non-cell autonomous neurodegeneration, with implications for potential therapies for neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

We introduce a mass spectrometry-based method that provides residue-resolved quantitative information about protein phosphorylation. In this assay we combined our full-length expressed stable isotope-labeled protein for quantification strategy (FLEXIQuant) with a traditional kinase assay to determine the mechanisms of multikinase substrate phosphorylation such as priming-dependent kinase activities. The assay monitors the decrease in signal intensity of the substrate peptides and the concomitant increase in the (n × 80 Da)-shifted phosphorylated peptide. We analyzed the c-Jun N-terminal kinase (JNK)-dependent glycogen synthase kinase 3? (GSK3?) activity on doublecortin (DCX) revealing mechanistic details about the role of phosphorylation cross-talk in GSK3? activity and permitting an advanced model for GSK3?-mediated signaling. [ABSTRACT FROM AUTHOR]

The protein kinase calcium/calmodulin-dependent kinase II (CaMKII) predominantly consists of the ? and ? isoforms in the brain. Although CaMKII? functions have been elucidated, the isoform-specific catalytic functions of CaMKII? have remained unknown. Using knockdown analyses in primary rat neurons and in the rat cerebellar cortex in vivo, we report that CaMKII? operates at the centrosome in a CaMKII?-independent manner to drive dendrite retraction and pruning. We also find that the targeting protein PCM1 (pericentriolar material 1) localizes CaMKII? to the centrosome. Finally, we uncover the E3 ubiquitin ligase Cdc20-APC (cell division cycle 20-anaphase promoting complex) as a centrosomal substrate of CaMKII?. CaMKII? phosphorylates Cdc20 at Ser51, which induces Cdc20 dispersion from the centrosome, thereby inhibiting centrosomal Cdc20-APC activity and triggering the transition from growth to retraction of dendrites. Our findings define a new, isoform-specific function for CaMKII? that regulates ubiquitin signaling at the centrosome and thereby orchestrates dendrite patterning, with important implications for neuronal connectivity in the brain. [ABSTRACT FROM AUTHOR]

Phosphorylation and small ubiquitin-like modifier (SUMO) conjugation contribute to the spatial and temporal regulation of substrates containing phosphorylation-dependent SUMO consensus motifs (PDSMs). Myocyte-enhancement factor 2 (MEF2) is a transcription factor and PDSM substrate whose modification by SUMO drives postsynaptic dendritic differentiation. NMR analysis revealed that the human SUMO E2 interacted with model substrates for phosphorylated and nonphosphorylated MEF2 in similar extended conformations. Mutational and biochemical analysis identified a basic E2 surface that enhanced SUMO conjugation to phosphorylated PDSM substrates MEF2 and heat-shock transcription factor 1 (HSF1), but not to nonphosphorylated MEF2 or HSF1, nor the non-PDSM substrate p53. Mutant ubiquitin-conjugating enzyme UBC9 isoforms defective in promoting SUMO conjugation to phosphorylated MEF2 in vitro and in vivo also impair postsynaptic differentiation in organotypic cerebellar slices. These data support an E2-dependent mechanism that underlies phosphorylation-dependent SUMO conjugation in pathways that range from the heat-shock response to nuclear hormone signaling to brain development. [ABSTRACT FROM AUTHOR]

In the developing nervous system, Id2 (inhibitor of DNA binding 2, also known as inhibitor of differentiation 2) enhances cell proliferation, promotes tumour progression and inhibits the activity of neurogenic basic helix–loop–helix (bHLH) transcription factors. The anaphase promoting complex/cyclosome and its activator Cdh1 (APC/CCdh1) restrains axonal growth but the targets of APC/CCdh1 in neurons are unknown. Id2 and other members of the Id family are very unstable proteins that are eliminated as cells enter the quiescent state, but how they are targeted for degradation has remained elusive. Here we show that Id2 interacts with the core subunits of APC/C and Cdh1 in primary neurons. APC/CCdh1 targets Id2 for degradation through a destruction box motif (D box) that is conserved in Id1 and Id4. Depletion of Cdh1 stabilizes Id proteins in neurons, whereas Id2 D-box mutants are impaired for Cdh1 binding and remain stable in cells that exit from the cell cycle and contain active APC/CCdh1. Mutants of the Id2 D box enhance axonal growth in cerebellar granule neurons in vitro and in the context of the cerebellar cortex, and overcome the myelin inhibitory signals for growth. Conversely, activation of bHLH transcription factors induces a cluster of genes with potent axonal inhibitory functions including the gene coding for the Nogo receptor, a key transducer of myelin inhibition. Degradation of Id2 in neurons permits the accumulation of the Nogo receptor, thereby linking APC/CCdh1 activity with bHLH target genes for the inhibition of axonal growth. These findings indicate that deregulated Id activity might be useful to reprogramme quiescent neurons into the axonal growth mode. [ABSTRACT FROM AUTHOR]

Familial hemiplegic migraine is an episodic neurological disorder characterized by transient sensory and motor symptoms and signs. Mutations of the ion pump α2-Na/K ATPase cause familial hemiplegic migraine, but the mechanisms by which α2-Na/K ATPase mutations lead to the migraine phenotype remain incompletely understood. Here, we show that mice in which α2-Na/K ATPase is conditionally deleted in astrocytes display episodic paralysis. Functional neuroimaging reveals that conditional α2-Na/K ATPase knockout triggers spontaneous cortical spreading depression events that are associated with EEG low voltage activity events, which correlate with transient motor impairment in these mice. Transcriptomic and metabolomic analyses show that α2-Na/K ATPase loss alters metabolic gene expression with consequent serine and glycine elevation in the brain. A serine- and glycine-free diet rescues the transient motor impairment in conditional α2-Na/K ATPase knockout mice. Together, our findings define a metabolic mechanism regulated by astrocytic α2-Na/K ATPase that triggers episodic motor paralysis in mice. Mutations of α2-Na/K ATPase can cause familial hemiplegic migraine via unclear mechanisms. Here, the authors show that deletion of α2-Na/K ATPase in astrocytes results in gene expression and metabolic changes leading to cortical spreading depression and episodic transient motor paralysis in mice. [ABSTRACT FROM AUTHOR]

In this Letter, '≥' should be '≤' in the sentence: "Intra-chromosomal reads were further split into short-range reads (≥1 kb) and long-range reads (>1 kb)". This error has been corrected online. An amendment to this paper has been published and can be accessed via a link at the top of the paper [ABSTRACT FROM AUTHOR]