Nde1 is required for heterochromatin compaction and stability in neocortical neurons.

The NDE1 gene encodes a scaffold protein essential for brain development. Although biallelic NDE1 loss of function (LOF) causes microcephaly with profound mental retardation, NDE1 missense mutations and copy number variations are associated with multiple neuropsychiatric disorders. However, the etiology of the diverse phenotypes resulting from NDE1 aberrations remains elusive. Here we demonstrate Nde1 controls neurogenesis through facilitating H4K20 trimethylation-mediated heterochromatin compaction. This mechanism patterns diverse chromatin landscapes and stabilizes constitutive heterochromatin of neocortical neurons. We demonstrate that NDE1 can undergo dynamic liquid-liquid phase separation, partitioning to the nucleus and interacting with pericentromeric and centromeric satellite repeats. Nde1 LOF results in nuclear architecture aberrations and DNA double-strand breaks, as well as instability and derepression of pericentromeric satellite repeats in neocortical neurons. These findings uncover a pivotal role of NDE1/Nde1 in establishing and protecting neuronal heterochromatin. They suggest that heterochromatin instability predisposes a wide range of brain dysfunction.

Sensorimotor dysfunction in a mild mouse model of cortical contusion injury without significant neuronal loss is associated with increases in inflammatory proteins with innate but not adaptive immune functions.

Traumatic brain injury is a leading cause of mortality and morbidity in the United States. Acute trauma to the brain triggers chronic secondary injury mechanisms that contribute to long-term neurological impairment. We have developed a single, unilateral contusion injury model of sensorimotor dysfunction in adult mice. By targeting a topographically defined neurological circuit with a mild impact, we are able to track sustained behavioral deficits in sensorimotor function in the absence of tissue cavitation or neuronal loss in the contused cortex of these mice. Stereological histopathology and multiplex enzyme-linked immunosorbent assay proteomic screening confirm contusion resulted in chronic gliosis and the robust expression of innate immune cytokines and monocyte attractant chemokines IL-1ß, IL-5, IL-6, TNFα, CXCL1, CXCL2, CXCL10, CCL2, and CCL3 in the contused cortex. In contrast, the expression of neuroinflammatory proteins with adaptive immune functions was not significantly modulated by injury. Our data support widespread activation of innate but not adaptive immune responses, confirming an association between sensorimotor dysfunction with innate immune activation in the absence of tissue or neuronal loss in our mice.

Host sex and transplanted human induced pluripotent stem cell phenotype interact to influence sensorimotor recovery in a mouse model of cortical contusion injury.

Traumatic brain injury (TBI) is a substantial cause of disability and death worldwide. Primary head trauma triggers chronic secondary injury mechanisms in the brain that are a focus of therapeutic efforts to treat TBI. Currently, there is no successful clinical strategy to repair brain injury. Cell transplantation therapies have demonstrated promise in attenuating secondary injury mechanisms of neuronal death and dysfunction in animal models of brain injury. In this study, we used a unilateral cortical contusion injury (CCI) model of sensorimotor brain injury to examine the effects of human induced pluripotent stem cell (hiPSC) transplantation on pathology in male and female adult mice. We determined transplanted hiPSC-derived neural stem cells (NSCs) and neuroblasts but not astrocytes best tolerate the injured host environment. Surviving NSC and neuroblast cells were clustered at the site of injection within the deep layers of the cortex and underlying corpus callosum. Cell grafts extended neuritic processes that crossed the midline into the contralateral corpus callosum or continued laterally within the external capsule to enter the ipsilateral entorhinal cortex. To determine the effect of transplantation on neuropathology, we performed sensorimotor behavior testing and stereological estimation of host neurons, astrocytes, and microglia within the contused cortex. These measures did not reveal a consistent effect of transplantation on recovery post-injury. Rather the positive and negative effects of cell transplantation were dependent on the host sex, highlighting the importance of developing patient-specific approaches to treat TBI. Our study underscores the complex interactions of sex, neuroimmune responses and cell therapy in a common experimental model of TBI.

SMN-deficiency disrupts SERCA2 expression and intracellular Ca2+ signaling in cardiomyocytes from SMA mice and patient-derived iPSCs.

Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by loss of alpha motor neurons and skeletal muscle atrophy. The disease is caused by mutations of the SMN1 gene that result in reduced functional expression of survival motor neuron (SMN) protein. SMN is ubiquitously expressed, and there have been reports of cardiovascular dysfunction in the most severe SMA patients and animal models of the disease. In this study, we directly assessed the function of cardiomyocytes isolated from a severe SMA model mouse and cardiomyocytes generated from patient-derived IPSCs. Consistent with impaired cardiovascular function at the very early disease stages in mice, heart failure markers such as brain natriuretic peptide were significantly elevated. Functionally, cardiomyocyte relaxation kinetics were markedly slowed and the T50 for Ca2+ sequestration increased to 146 ± 4 ms in SMN-deficient cardiomyocytes from 126 ± 4 ms in wild type cells. Reducing SMN levels in cardiomyocytes from control patient IPSCs slowed calcium reuptake similar to SMA patent-derived cardiac cells. Importantly, restoring SMN increased calcium reuptake rate. Taken together, these results indicate that SMN deficiency impairs cardiomyocyte function at least partially through intracellular Ca2+ cycling dysregulation.

Neuroinflammatory astrocytes generated from cord blood-derived human induced pluripotent stem cells.

BACKGROUND: Astrocytes respond to central nervous system (CNS) injury and disease by transforming to a reactive astrogliosis cell state that can contribute to either CNS dysfunction or repair. Neuroinflammation is a powerful driver of a harmful A1 astrogliosis phenotype associated with in vitro neurotoxicity and histopathology in human neurodegenerative diseases. Here we report a protocol for the rapid development of a human cell culture model of neuroinflammatory astrogliosis using induced pluripotent stem cells (iPSCs). METHODS: Using RNA sequencing and in vitro cell assays, we measured transcriptional and cellular effects of chronic exposure of human iPSC-derived astrocytes to the cytokines TNFα (tumor necrosis factor alpha) or IL-1ß (interleukin-1 beta). RESULTS: We show TNFα and IL-1ß induce pro-inflammatory gene signatures but by widely different magnitudes. TNFα treatment results in 606 differential expressed genes, the suppression of glutamate-uptake, and increased phagocytic activity in astrocyte cultures. In contrast, IL-1ß effects are attenuated to 33 differential expressed genes and no significant effects on glutamate-uptake or increased phagocytic activity. CONCLUSION: Our approach demonstrates a rapid tool for modeling neuroinflammatory human astrocytic responses in nervous system trauma and disease. In particular, we reveal a model for robust TNFα-induced human astrogliosis suitable for the study of neurotoxic A1 astrocytes.

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells.

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical insult to secondary injury processes, including apoptosis and inflammation. Animal modeling has been valuable in the search to unravel injury mechanisms and evaluate potential neuroprotective therapies. This protocol describes the controlled cortical impact (CCI) model of focal, open-head TBI. Specifically, parameters for producing a mild unilateral cortical injury are described. Behavioral consequences of CCI are analyzed using the adhesive tape removal test of bilateral sensorimotor integration. Regarding experimental therapy for TBI pathology, this protocol also illustrates a process for transplanting cultured cells into the brain. Neural cell cultures derived from human induced pluripotent stem cells (hiPSCs) were chosen for their potential to show superior functional restoration in human TBI patients. Chronic survival of hiPSCs in the host mouse brain tissue is detected using a modified DAB immunohistochemical process.

Conditional deletion of Neurog1 in the cerebellum of postnatal mice delays inhibitory interneuron maturation.

The transcriptional programs that drive the generation of diverse GABAergic neuron populations from their common progenitor pools in the developing cerebellum remain unclear. Neurog1 is a pro-neural basic helix-loop-helix transcription factor expressed in GABAergic progenitor cells in the ventricular zone (VZ) of embryos and subsequently in the presumptive white matter (pWM) tracts of developing postnatal mice. Genetic inducible fate-mapping labels Purkinje cells and all inhibitory interneuron cell types of the cerebellar cortex. As conventional Neurog1Neo knockout (KO) mice are neonatal lethal, we generated Neurog1loxP mutant mice to examine the effects of conditional Neurog1 deletion on the postnatal development of the cerebellum. Targeted Neurog1 loss-of-function in the developing cerebellum does not result in significant differences in cerebellar morphology or in the number of GABAergic neurons in the cerebellar cortex of mice at postnatal day 21 (P21). To determine the effects of Neurog1 deletion on GABAergic progenitors, we quantified rates of cell proliferation and cell cycle progression or re-entry in embryonic Neurog1Neo and postnatal Neurog1loxP mutants. The data revealed no significant effect of Neurog1 loss-of-function on embryonic day 12.5 (E12.5) VZ progenitors or on P5 and P6 progenitors in the pWM at P7. However, 4-5 day pulse-labeling of P5 and P6 progenitors revealed reductions in inhibitory interneuron dispersal from the pWM to the cerebellar cortex in P10 conditional Neurog1loxP/loxP KO mice. Thus, our conditional Neurog1 KO approach reveals a requirement for Neurog1 activity in inhibitory interneuron cell dispersal from pWM tracts in the developing cerebellum of postnatal mice.

Neonatal mouse cortical but not isogenic human astrocyte feeder layers enhance the functional maturation of induced pluripotent stem cell-derived neurons in culture.

Human induced pluripotent stem (iPS) cell-derived neurons and astrocytes are attractive cellular tools for nervous system disease modeling and drug screening. Optimal utilization of these tools requires differentiation protocols that efficiently generate functional cell phenotypes in vitro. As nervous system function is dependent on networked neuronal activity involving both neuronal and astrocytic synaptic functions, we examined astrocyte effects on the functional maturation of neurons from human iPS cell-derived neural stem cells (NSCs). We first demonstrate human iPS cell-derived NSCs can be rapidly differentiated in culture to either neurons or astrocytes with characteristic cellular, molecular and physiological features. Although differentiated neurons were capable of firing multiple action potentials (APs), few cells developed spontaneous electrical activity in culture. We show spontaneous electrical activity was significantly increased by neuronal differentiation of human NSCs on feeder layers of neonatal mouse cortical astrocytes. In contrast, co-culture on feeder layers of isogenic human iPS cell-derived astrocytes had no positive effect on spontaneous neuronal activity. Spontaneous electrical activity was dependent on glutamate receptor-channel function and occurred without changes in INa , IK , Vm , and AP properties of iPS cell-derived neurons. These data demonstrate co-culture with neonatal mouse cortical astrocytes but not human isogenic iPS cell-derived astrocytes stimulates glutamatergic synaptic transmission between iPS cell-derived neurons in culture. We present RNA-sequencing data for an immature, fetal-like status of our human iPS cell-derived astrocytes as one possible explanation for their failure to enhance synaptic activity in our co-culture system.

Inhibition of the histone demethylase Kdm5b promotes neurogenesis and derepresses Reln (reelin) in neural stem cells from the adult subventricular zone of mice.

The role of epigenetic regulators in the control of adult neurogenesis is largely undefined. We show that the histone demethylase enzyme Kdm5b (Jarid1b) negatively regulates neurogenesis from adult subventricular zone (SVZ) neural stem cells (NSCs) in culture. shRNA-mediated depletion of Kdm5b in proliferating adult NSCs decreased proliferation rates and reduced neurosphere formation in culture. When transferred to differentiation culture conditions, Kdm5b-depleted adult NSCs migrated from neurospheres with increased velocity. Whole-genome expression screening revealed widespread transcriptional changes with Kdm5b depletion, notably the up-regulation of reelin (Reln), the inhibition of steroid biosynthetic pathway component genes and the activation of genes with intracellular transport functions in cultured adult NSCs. Kdm5b depletion increased extracellular reelin concentration in the culture medium and increased phosphorylation of the downstream reelin signaling target Disabled-1 (Dab1). Sequestration of extracellular reelin with CR-50 reelin-blocking antibodies suppressed the increase in migratory velocity of Kdm5b-depleted adult NSCs. Chromatin immunoprecipitation revealed that Kdm5b is present at the proximal promoter of Reln, and H3K4me3 methylation was increased at this locus with Kdm5b depletion in differentiating adult NSCs. Combined the data suggest Kdm5b negatively regulates neurogenesis and represses Reln in neural stem cells from the adult SVZ.

Neurog1 Genetic Inducible Fate Mapping (GIFM) Reveals the Existence of Complex Spatiotemporal Cyto-Architectures in the Developing Cerebellum.

Neurog1 is a pro-neural basic helix-loop-helix (bHLH) transcription factor expressed in progenitor cells located in the ventricular zone and subsequently the presumptive white matter tracts of the developing mouse cerebellum. We used genetic inducible fate mapping (GIFM) with a transgenic Neurog1-CreER allele to characterize the contributions of Neurog1 lineages to cerebellar circuit formation in mice. GIFM reveals Neurog1-expressing progenitors are fate-mapped to become Purkinje cells and all GABAergic interneuron cell types of the cerebellar cortex but not glia. The spatiotemporal sequence of GIFM is unique to each neuronal cell type. GIFM on embryonic days (E) 10.5 to E12.5 labels Purkinje cells with different medial-lateral settling patterns depending on the day of tamoxifen delivery. GIFM on E11.5 to P7 labels interneurons and the timing of tamoxifen administration correlates with the final inside-to-outside resting position of GABAergic interneurons in the cerebellar cortex. Proliferative status and long-term BrdU retention of GIFM lineages reveals Purkinje cells express Neurog1 around the time they become post-mitotic. In contrast, GIFM labels mitotic and post-mitotic interneurons. Neurog1-CreER GIFM reveals a correlation between the timing of Neurog1 expression and the spatial organization of GABAergic neurons in the cerebellar cortex with possible implications for cerebellar circuit assembly.

Cortical contusion injury disrupts olfactory bulb neurogenesis in adult mice.

BACKGROUND: Experimental brain trauma activates quiescent neural stem cells (NSCs) to increase neuronal progenitor cell proliferation in the adult rodent brain. Previous studies have shown focal brain contusion in the form of a unilateral controlled cortical impact (CCI) stimulates NSCs to bilaterally increase neurogenesis in the adult hippocampus. RESULTS: In this study we clarified the bi-lateral effects of a unilateral CCI on proliferation in the subventricular zone (SVZ) NSC niche and on neurogenesis in the olfactory bulb of adult mice. By varying the depth of impact from 1 mm to 2 mm depth, we show CCI to the left somatosensory cortex resulted in graded changes in mouse behavior and cellular pathology in the forebrain. As expected, contusion to the sensorimotor cortex resulted in motor coordination deficits in adult mice. During the first 3 days after injury, CCI increased proliferation in the impacted cortex, deeper striatum and SVZ of the forebrain ipsilateral to the CCI. In each of these regions proliferation was increased with increasing injury severity. At 30 days post-procedure, CCI resulted in a significant reduction in neurogenesis in the olfactory bulb ipsilateral to the CCI. Olfactory avoidance testing indicated disruptions in olfactory bulb neurogenesis were associated with impaired olfactory discrimination in mice post-injury. CONCLUSION: The data demonstrate a focal cortical contusion injury to the left somatosensory cortex disrupts SVZ-olfactory bulb neurogenesis and impairs olfactory discrimination and motor coordination in adult mice.

Histone deacetylase inhibitors SAHA and sodium butyrate block G1-to-S cell cycle progression in neurosphere formation by adult subventricular cells.

BACKGROUND: Histone deacetylases (HDACs) are enzymes that modulate gene expression and cellular processes by deacetylating histones and non-histone proteins. While small molecule inhibitors of HDAC activity (HDACi) are used clinically in the treatment of cancer, pre-clinical treatment models suggest they also exert neuroprotective effects and stimulate neurogenesis in neuropathological conditions. However, the direct effects of HDACi on cell cycle progression and proliferation, two properties required for continued neurogenesis, have not been fully characterized in adult neural stem cells (NSCs). In this study, we examined the effects of two broad class I and class II HDACi on adult mouse NSCs, the hydroxamate-based HDACi suberoylanilide hydroxamic acid (vorinostat, SAHA) and the short chain fatty acid HDACi sodium butyrate. RESULTS: We show that both HDACi suppress the formation of neurospheres by adult mouse NSCs grown in proliferation culture conditions in vitro. DNA synthesis is significantly inhibited in adult mouse NSCs exposed to either SAHA or sodium butyrate and inhibition is associated with an arrest in the G1 phase of the cell cycle. HDACi exposure also resulted in transcriptional changes in adult mouse NSCs. Cdk inhibitor genes p21 and p27 transcript levels are increased and associated with elevated H3K9 acetylation levels at proximal promoter regions of p21 and p27. mRNA levels for notch effector Hes genes and Spry-box stem cell transcription factors are downregulated, whereas pro-neural transcription factors Neurog1 and Neurod1 are upregulated. Lastly, we show HDAC inhibition under proliferation culture conditions leads to long-term changes in cell fate in adult mouse NSCs induced to differentiate in vitro. CONCLUSION: SAHA and sodium butyrate directly regulate cdk inhibitor transcription to control cell cycle progression in adult mouse NSCs. HDAC inhibition results in G1 arrest in adult mouse NSCs and transcriptional changes associated with activation of neuronal lineage commitment programs and a reduction of stem/progenitor state. Changes in differentiated cell state in adult mouse NSCs treated with HDACi under proliferation culture conditions suggests an intrinsic relationship between multipotency, cell cycle progression and HDAC activity in these cells.

Role of H3K4 demethylases in complex neurodevelopmental diseases.

Significant neurological disorders can result from subtle perturbations of gene regulation that are often linked to epigenetic regulation. Proteins that regulate the methylation of lysine 4 of histone H3 (H3K4) and play a central role in epigenetic regulation, and mutations in genes encoding these enzymes have been identified in both autism and Rett syndrome. The H3K4 demethylases remove methyl groups from lysine 4 leading to loss of RNA polymerase binding and transcriptional repression. When these proteins are mutated, brain development is altered. Currently, little is known regarding how these gene regulators function at the genomic level. In this article, we will discuss findings that link H3K4 demethylases to neurodevelopment and neurological disease.

Application of a translational profiling approach for the comparative analysis of CNS cell types.

Comparative analysis can provide important insights into complex biological systems. As demonstrated in the accompanying paper, translating ribosome affinity purification (TRAP) permits comprehensive studies of translated mRNAs in genetically defined cell populations after physiological perturbations. To establish the generality of this approach, we present translational profiles for 24 CNS cell populations and identify known cell-specific and enriched transcripts for each population. We report thousands of cell-specific mRNAs that were not detected in whole-tissue microarray studies and provide examples that demonstrate the benefits deriving from comparative analysis. To provide a foundation for further biological and in silico studies, we provide a resource of 16 transgenic mouse lines, their corresponding anatomic characterization, and translational profiles for cell types from a variety of central nervous system structures. This resource will enable a wide spectrum of molecular and mechanistic studies of both well-known and previously uncharacterized neural cell populations.

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.