Transcriptional maintenance of cortical somatostatin interneuron subtype identity during migration.

Although cardinal cortical interneuron identity is established upon cell-cycle exit, it remains unclear whether specific interneuron subtypes are pre-established, and if so, how their identity is maintained prior to circuit integration. We conditionally removed Sox6 (Sox6-cKO) in migrating somatostatin (Sst+) interneurons and assessed the effects on their mature identity. In adolescent mice, five of eight molecular Sst+ subtypes were nearly absent in the Sox6-cKO cortex without a reduction in cell number. Sox6-cKO cells displayed electrophysiological maturity and expressed genes enriched within the broad class of Sst+ interneurons. Furthermore, we could infer subtype identity prior to cortical integration (embryonic day 18.5), suggesting that the loss in subtype was due to disrupted subtype maintenance. Conversely, Sox6 removal at postnatal day 7 did not disrupt marker expression in the mature cortex. Therefore, Sox6 is necessary during migration for maintenance of Sst+ subtype identity, indicating that subtype maintenance requires active transcriptional programs.

Interneuron diversity in the human dorsal striatum.

Deciphering the striatal interneuron diversity is key to understanding the basal ganglia circuit and to untangle the complex neurological and psychiatric diseases affecting this brain structure. We performed snRNA-seq of postmortem human caudate nucleus and putamen samples to elucidate the diversity and abundance of interneuron populations and their transcriptional structure in the human dorsal striatum. We propose a new taxonomy of striatal interneurons with eight main classes and fourteen subclasses and provide their specific markers and some quantitative FISH validation, particularly for a novel PTHLH-expressing population. For the most abundant populations, PTHLH and TAC3, we found matching known mouse interneuron populations based on key functional genes such as ion channels and synaptic receptors. Remarkably, human TAC3 and mouse Th populations share important similarities including the expression of the neuropeptide tachykinin 3. Finally, we were able to integrate other published datasets supporting the generalizability of this new harmonized taxonomy.

Protection and Repair of the Nigrostriatal Pathway with Stem-Cell-Derived Carotid Body Glomus Cell Transplants in Chronic MPTP Parkinsonian Model.

Antiparkinsonian carotid body (CB) cell therapy has been proven to be effective in rodent and nonhuman primate models of Parkinson's disease (PD), exerting trophic protection and restoration of the dopaminergic nigrostriatal pathway. These neurotrophic actions are mediated through the release of high levels of glial-cell-line-derived neurotrophic factor (GDNF) by the CB transplant. Pilot clinical trials have also shown that CB autotransplantation can improve motor symptoms in PD patients, although its effectiveness is affected by the scarcity of the grafted tissue. Here, we analyzed the antiparkinsonian efficacy of in vitro-expanded CB dopaminergic glomus cells. Intrastriatal xenografts of rat CB neurospheres were shown to protect nigral neurons from degeneration in a chronic MPTP mouse PD model. In addition, grafts performed at the end of the neurotoxic treatment resulted in the repair of striatal dopaminergic terminals through axonal sprouting. Interestingly, both neuroprotective and reparative effects induced by in vitro-expanded CB cells were similar to those previously reported by the use of CB transplants. This action could be explained because stem-cell-derived CB neurospheres produce similar amounts of GDNF compared to native CB tissue. This study provides the first evidence that in vitro-expanded CB cells could be a clinical option for cell therapy in PD.

Postnatal Sox6 Regulates Synaptic Function of Cortical Parvalbumin-Expressing Neurons.

Cortical parvalbumin-expressing (Pvalb+) neurons provide robust inhibition to neighboring pyramidal neurons, crucial for the proper functioning of cortical networks. This class of inhibitory neurons undergoes extensive synaptic formation and maturation during the first weeks after birth and continue to dynamically maintain their synaptic output throughout adulthood. While several transcription factors, such as Nkx2-1, Lhx6, and Sox6, are known to be necessary for the differentiation of progenitors into Pvalb+ neurons, which transcriptional programs underlie the postnatal maturation and maintenance of Pvalb+ neurons' innervation and synaptic function remains largely unknown. Because Sox6 is continuously expressed in Pvalb+ neurons until adulthood, we used conditional knock-out strategies to investigate its putative role in the postnatal maturation and synaptic function of cortical Pvalb+ neurons in mice of both sexes. We found that early postnatal loss of Sox6 in Pvalb+ neurons leads to failure of synaptic bouton growth, whereas later removal in mature Pvalb+ neurons in the adult causes shrinkage of already established synaptic boutons. Paired recordings between Pvalb+ neurons and pyramidal neurons revealed reduced release probability and increased failure rate of Pvalb+ neurons' synaptic output. Furthermore, Pvalb+ neurons lacking Sox6 display reduced expression of full-length tropomyosin-receptor kinase B (TrkB), a key modulator of GABAergic transmission. Once re-expressed in neurons lacking Sox6, TrkB was sufficient to rescue the morphologic synaptic phenotype. Finally, we showed that Sox6 mRNA levels were increased by motor training. Our data thus suggest a constitutive role for Sox6 in the maintenance of synaptic output from Pvalb+ neurons into adulthood.SIGNIFICANCE STATEMENT Cortical parvalbumin-expressing (Pvalb+) inhibitory neurons provide robust inhibition to neighboring pyramidal neurons, crucial for the proper functioning of cortical networks. These inhibitory neurons undergo extensive synaptic formation and maturation during the first weeks after birth and continue to dynamically maintain their synaptic output throughout adulthood. However, it remains largely unknown which transcriptional programs underlie the postnatal maturation and maintenance of Pvalb+ neurons. Here, we show that the transcription factor Sox6 cell-autonomously regulates the synaptic maintenance and output of Pvalb+ neurons until adulthood, leaving unaffected other maturational features of this neuronal population.

Intrinsic electrophysiological properties predict variability in morphology and connectivity among striatal Parvalbumin-expressing Pthlh-cells.

Determining the cellular content of the nervous system in terms of cell types and the rules of their connectivity represents a fundamental challenge to the neurosciences. The recent advent of high-throughput techniques, such as single-cell RNA-sequencing has allowed for greater resolution in the identification of cell types and/or states. Although most of the current neuronal classification schemes comprise discrete clusters, several recent studies have suggested that, perhaps especially, within the striatum, neuronal populations exist in continua, with regards to both their molecular and electrophysiological properties. Whether these continua are stable properties, established during development, or if they reflect acute differences in activity-dependent regulation of critical genes is currently unknown. We set out to determine whether gradient-like molecular differences in the recently described Pthlh-expressing inhibitory interneuron population, which contains the Pvalb-expressing cells, correlate with differences in morphological and connectivity properties. We show that morphology and long-range inputs correlate with a spatially organized molecular and electrophysiological gradient of Pthlh-interneurons, suggesting that the processing of different types of information (by distinct anatomical striatal regions) has different computational requirements.

Probabilistic cell typing enables fine mapping of closely related cell types in situ.

Understanding the function of a tissue requires knowing the spatial organization of its constituent cell types. In the cerebral cortex, single-cell RNA sequencing (scRNA-seq) has revealed the genome-wide expression patterns that define its many, closely related neuronal types, but cannot reveal their spatial arrangement. Here we introduce probabilistic cell typing by in situ sequencing (pciSeq), an approach that leverages previous scRNA-seq classification to identify cell types using multiplexed in situ RNA detection. We applied this method by mapping the inhibitory neurons of mouse hippocampal area CA1, for which ground truth is available from extensive previous work identifying their laminar organization. Our method identified these neuronal classes in a spatial arrangement matching ground truth, and further identified multiple classes of isocortical pyramidal cell in a pattern matching their known organization. This method will allow identifying the spatial organization of closely related cell types across the brain and other tissues.

Genome-wide analysis of insomnia in 1,331,010 individuals identifies new risk loci and functional pathways.

Insomnia is the second most prevalent mental disorder, with no sufficient treatment available. Despite substantial heritability, insight into the associated genes and neurobiological pathways remains limited. Here, we use a large genetic association sample (n = 1,331,010) to detect novel loci and gain insight into the pathways, tissue and cell types involved in insomnia complaints. We identify 202 loci implicating 956 genes through positional, expression quantitative trait loci, and chromatin mapping. The meta-analysis explained 2.6% of the variance. We show gene set enrichments for the axonal part of neurons, cortical and subcortical tissues, and specific cell types, including striatal, hypothalamic, and claustrum neurons. We found considerable genetic correlations with psychiatric traits and sleep duration, and modest correlations with other sleep-related traits. Mendelian randomization identified the causal effects of insomnia on depression, diabetes, and cardiovascular disease, and the protective effects of educational attainment and intracranial volume. Our findings highlight key brain areas and cell types implicated in insomnia, and provide new treatment targets.

Biological annotation of genetic loci associated with intelligence in a meta-analysis of 87,740 individuals.

Variance in IQ is associated with a wide range of health outcomes, and 1% of the population are affected by intellectual disability. Despite a century of research, the fundamental neural underpinnings of intelligence remain unclear. We integrate results from genome-wide association studies (GWAS) of intelligence with brain tissue and single cell gene expression data to identify tissues and cell types associated with intelligence. GWAS data for IQ (N = 78,308) were meta-analyzed with a study comparing 1247 individuals with mean IQ ~170 to 8185 controls. Genes associated with intelligence implicate pyramidal neurons of the somatosensory cortex and CA1 region of the hippocampus, and midbrain embryonic GABAergic neurons. Tissue-specific analyses find the most significant enrichment for frontal cortex brain expressed genes. These results suggest specific neuronal cell types and genes may be involved in intelligence and provide new hypotheses for neuroscience experiments using model systems.

Diversity of Interneurons in the Dorsal Striatum Revealed by Single-Cell RNA Sequencing and PatchSeq.

Striatal locally projecting neurons, or interneurons, act on nearby circuits and shape functional output to the rest of the basal ganglia. We performed single-cell RNA sequencing of striatal cells enriching for interneurons. We find seven discrete interneuron types, six of which are GABAergic. In addition to providing specific markers for the populations previously described, including those expressing Sst/Npy, Th, Npy without Sst, and Chat, we identify two small populations of cells expressing Cck with or without Vip. Surprisingly, the Pvalb-expressing cells do not constitute a discrete cluster but rather are part of a larger group of cells expressing Pthlh with a spatial gradient of Pvalb expression. Using PatchSeq, we show that Pthlh cells exhibit a continuum of electrophysiological properties correlated with expression of Pvalb. Furthermore, we find significant molecular differences that correlate with differences in electrophysiological properties between Pvalb-expressing cells of the striatum and those of the cortex.

Meta-analysis of genome-wide association studies for neuroticism in 449,484 individuals identifies novel genetic loci and pathways.

Neuroticism is an important risk factor for psychiatric traits, including depression1, anxiety2,3, and schizophrenia4-6. At the time of analysis, previous genome-wide association studies7-12 (GWAS) reported 16 genomic loci associated to neuroticism10-12. Here we conducted a large GWAS meta-analysis (n = 449,484) of neuroticism and identified 136 independent genome-wide significant loci (124 new at the time of analysis), which implicate 599 genes. Functional follow-up analyses showed enrichment in several brain regions and involvement of specific cell types, including dopaminergic neuroblasts (P = 3.49 × 10-8), medium spiny neurons (P = 4.23 × 10-8), and serotonergic neurons (P = 1.37 × 10-7). Gene set analyses implicated three specific pathways: neurogenesis (P = 4.43 × 10-9), behavioral response to cocaine processes (P = 1.84 × 10-7), and axon part (P = 5.26 × 10-8). We show that neuroticism's genetic signal partly originates in two genetically distinguishable subclusters13 ('depressed affect' and 'worry'), suggesting distinct causal mechanisms for subtypes of individuals. Mendelian randomization analysis showed unidirectional and bidirectional effects between neuroticism and multiple psychiatric traits. These results enhance neurobiological understanding of neuroticism and provide specific leads for functional follow-up experiments.

Genome-wide association meta-analysis in 269,867 individuals identifies new genetic and functional links to intelligence.

Intelligence is highly heritable1 and a major determinant of human health and well-being2. Recent genome-wide meta-analyses have identified 24 genomic loci linked to variation in intelligence3-7, but much about its genetic underpinnings remains to be discovered. Here, we present a large-scale genetic association study of intelligence (n = 269,867), identifying 205 associated genomic loci (190 new) and 1,016 genes (939 new) via positional mapping, expression quantitative trait locus (eQTL) mapping, chromatin interaction mapping, and gene-based association analysis. We find enrichment of genetic effects in conserved and coding regions and associations with 146 nonsynonymous exonic variants. Associated genes are strongly expressed in the brain, specifically in striatal medium spiny neurons and hippocampal pyramidal neurons. Gene set analyses implicate pathways related to nervous system development and synaptic structure. We confirm previous strong genetic correlations with multiple health-related outcomes, and Mendelian randomization analysis results suggest protective effects of intelligence for Alzheimer's disease and ADHD and bidirectional causation with pleiotropic effects for schizophrenia. These results are a major step forward in understanding the neurobiology of cognitive function as well as genetically related neurological and psychiatric disorders.

Genetic identification of brain cell types underlying schizophrenia.

With few exceptions, the marked advances in knowledge about the genetic basis of schizophrenia have not converged on findings that can be confidently used for precise experimental modeling. By applying knowledge of the cellular taxonomy of the brain from single-cell RNA sequencing, we evaluated whether the genomic loci implicated in schizophrenia map onto specific brain cell types. We found that the common-variant genomic results consistently mapped to pyramidal cells, medium spiny neurons (MSNs) and certain interneurons, but far less consistently to embryonic, progenitor or glial cells. These enrichments were due to sets of genes that were specifically expressed in each of these cell types. We also found that many of the diverse gene sets previously associated with schizophrenia (genes involved in synaptic function, those encoding mRNAs that interact with FMRP, antipsychotic targets, etc.) generally implicated the same brain cell types. Our results suggest a parsimonious explanation: the common-variant genetic results for schizophrenia point at a limited set of neurons, and the gene sets point to the same cells. The genetic risk associated with MSNs did not overlap with that of glutamatergic pyramidal cells and interneurons, suggesting that different cell types have biologically distinct roles in schizophrenia.

Synaptic Regulator α-Synuclein in Dopaminergic Fibers Is Essentially Required for the Maintenance of Subependymal Neural Stem Cells.

Synaptic protein α-synuclein (α-SYN) modulates neurotransmission in a complex and poorly understood manner and aggregates in the cytoplasm of degenerating neurons in Parkinson's disease. Here, we report that α-SYN present in dopaminergic nigral afferents is essential for the normal cycling and maintenance of neural stem cells (NSCs) in the brain subependymal zone of adult male and female mice. We also show that premature senescence of adult NSCs into non-neurogenic astrocytes in mice lacking α-SYN resembles the effects of dopaminergic fiber degeneration resulting from chronic exposure to 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine or intranigral inoculation of aggregated toxic α-SYN. Interestingly, NSC loss in α-SYN-deficient mice can be prevented by viral delivery of human α-SYN into their sustantia nigra or by treatment with l-DOPA, suggesting that α-SYN regulates dopamine availability to NSCs. Our data indicate that α-SYN, present in dopaminergic nerve terminals supplying the subependymal zone, acts as a niche component to sustain the neurogenic potential of adult NSCs and identify α-SYN and DA as potential targets to ameliorate neurogenic defects in the aging and diseased brain.SIGNIFICANCE STATEMENT We report an essential role for the protein α-synuclein present in dopaminergic nigral afferents in the regulation of adult neural stem cell maintenance, identifying the first synaptic regulator with an implication in stem cell niche biology. Although the exact role of α-synuclein in neural transmission is not completely clear, our results indicate that it is required for stemness and the preservation of neurogenic potential in concert with dopamine.

Integrated Bayesian analysis of rare exonic variants to identify risk genes for schizophrenia and neurodevelopmental disorders.

BACKGROUND: Integrating rare variation from trio family and case-control studies has successfully implicated specific genes contributing to risk of neurodevelopmental disorders (NDDs) including autism spectrum disorders (ASD), intellectual disability (ID), developmental disorders (DDs), and epilepsy (EPI). For schizophrenia (SCZ), however, while sets of genes have been implicated through the study of rare variation, only two risk genes have been identified. METHODS: We used hierarchical Bayesian modeling of rare-variant genetic architecture to estimate mean effect sizes and risk-gene proportions, analyzing the largest available collection of whole exome sequence data for SCZ (1,077 trios, 6,699 cases, and 13,028 controls), and data for four NDDs (ASD, ID, DD, and EPI; total 10,792 trios, and 4,058 cases and controls). RESULTS: For SCZ, we estimate there are 1,551 risk genes. There are more risk genes and they have weaker effects than for NDDs. We provide power analyses to predict the number of risk-gene discoveries as more data become available. We confirm and augment prior risk gene and gene set enrichment results for SCZ and NDDs. In particular, we detected 98 new DD risk genes at FDR < 0.05. Correlations of risk-gene posterior probabilities are high across four NDDs (ρ>0.55), but low between SCZ and the NDDs (ρ<0.3). An in-depth analysis of 288 NDD genes shows there is highly significant protein-protein interaction (PPI) network connectivity, and functionally distinct PPI subnetworks based on pathway enrichment, single-cell RNA-seq cell types, and multi-region developmental brain RNA-seq. CONCLUSIONS: We have extended a pipeline used in ASD studies and applied it to infer rare genetic parameters for SCZ and four NDDs ( https://github.com/hoangtn/extTADA ). We find many new DD risk genes, supported by gene set enrichment and PPI network connectivity analyses. We find greater similarity among NDDs than between NDDs and SCZ. NDD gene subnetworks are implicated in postnatally expressed presynaptic and postsynaptic genes, and for transcriptional and post-transcriptional gene regulation in prenatal neural progenitor and stem cells.

Oligodendrocyte heterogeneity in the mouse juvenile and adult central nervous system.

Oligodendrocytes have been considered as a functionally homogeneous population in the central nervous system (CNS). We performed single-cell RNA sequencing on 5072 cells of the oligodendrocyte lineage from 10 regions of the mouse juvenile and adult CNS. Thirteen distinct populations were identified, 12 of which represent a continuum from Pdgfra(+) oligodendrocyte precursor cells (OPCs) to distinct mature oligodendrocytes. Initial stages of differentiation were similar across the juvenile CNS, whereas subsets of mature oligodendrocytes were enriched in specific regions in the adult brain. Newly formed oligodendrocytes were detected in the adult CNS and were responsive to complex motor learning. A second Pdgfra(+) population, distinct from OPCs, was found along vessels. Our study reveals the dynamics of oligodendrocyte differentiation and maturation, uncoupling them at a transcriptional level and highlighting oligodendrocyte heterogeneity in the CNS.

Chronic and progressive Parkinson's disease MPTP model in adult and aged mice.

Despite the different animal models of Parkinson's disease developed during the last years, they still present limitations modelling the slow and progressive process of neurodegeneration. Here, we undertook a histological, neurochemical and behavioural analysis of a new chronic parkinsonian mouse model generated by the subcutaneous administration of low doses of MPTP (20 mg/kg, 3 times per week) for 3 months, using both young adult and aged mice. The MPTP-induced nigrostriatal neurodegeneration was progressive and was accompanied by a decrease in striatal dopamine levels and motor impairment. We also demonstrated the characteristic neuroinflammatory changes (microglial activation and astrogliosis) associated with the neurodegenerative process. Aged animals showed both a faster time course of neurodegeneration and an altered neuroinflammatory response. The long-term systemic application of low MPTP doses did not induce any increase in mortality in either young adult or aged mice and better resembles the slow evolution of the neurodegenerative process. This treatment could be useful to model different stages of Parkinson's disease, providing a better understanding of the pathophysiology of the disease and facilitating the testing of both protective and restorative treatments. Here, we show a new chronic and progressive parkinsonian mouse model, in young and aged mice. This model produces a stable degeneration of the dopaminergic nigrostriatal pathway, continuous neuroinflammatory reaction and motor deficits. Aged animals showed a faster neurodegeneration and an altered neuroinflammatory response. This treatment could be useful to model different stages of PD and to test both protective and restorative therapeutic approaches.

BCL11B/CTIP2 is highly expressed in GABAergic interneurons of the mouse somatosensory cortex.

In the nervous system, BCL11B is crucial for the development of deep layer corticospinal projection neurons and striatal medium spiny neurons and is often used as a marker for the aforementioned cell types. However, the expression of BCL11B in subtypes of non-excitatory neurons in the primary somatosensory cortex (S1) has not been reported in the mouse. In this study we show that BCL11B is extensively expressed in S1 GABAergic interneurons, throughout the three main subgroups (somatostatin-, parvalbumin- and 5HT3a-expresssing). Almost all BCL11B positive cells in the upper S1 layers were GABAergic interneurons and surprisingly, almost 40% of the BCL11B positive neurons in layer V were GABAergic interneurons. Single cell mRNA sequencing data revealed higher Bcl11b expression in S1 interneurons compared to deep layer pyramidal neurons. The highest levels of Bcl11b expression were found within the 5HT3a population, specifically in putative neurogliaform interneuron subclasses (5HT3a-positive but not expressing vasoactive intestinal peptide). In the light of our findings we suggest caution using BCL11B as a single marker to identify neurons.

Brain structure. Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq.

The mammalian cerebral cortex supports cognitive functions such as sensorimotor integration, memory, and social behaviors. Normal brain function relies on a diverse set of differentiated cell types, including neurons, glia, and vasculature. Here, we have used large-scale single-cell RNA sequencing (RNA-seq) to classify cells in the mouse somatosensory cortex and hippocampal CA1 region. We found 47 molecularly distinct subclasses, comprising all known major cell types in the cortex. We identified numerous marker genes, which allowed alignment with known cell types, morphology, and location. We found a layer I interneuron expressing Pax6 and a distinct postmitotic oligodendrocyte subclass marked by Itpr2. Across the diversity of cortical cell types, transcription factors formed a complex, layered regulatory code, suggesting a mechanism for the maintenance of adult cell type identity.

Cellular properties and chemosensory responses of the human carotid body.

The carotid body (CB) is the major peripheral arterial chemoreceptor in mammals that mediates the acute hyperventilatory response to hypoxia. The CB grows in response to sustained hypoxia and also participates in acclimatisation to chronic hypoxaemia. Knowledge of CB physiology at the cellular level has increased considerably in recent times thanks to studies performed on lower mammals, and rodents in particular. However, the functional characteristics of human CB cells remain practically unknown. Herein, we use tissue slices or enzymatically dispersed cells to determine the characteristics of human CB cells. The adult human CB parenchyma contains clusters of chemosensitive glomus (type I) and sustentacular (type II) cells as well as nestin-positive progenitor cells. This organ also expresses high levels of the dopaminotrophic glial cell line-derived neurotrophic factor (GDNF). We found that GDNF production and the number of progenitor and glomus cells were preserved in the CBs of human subjects of advanced age. Moreover, glomus cells exhibited voltage-dependent Na(+), Ca(2+) and K(+) currents that were qualitatively similar to those reported in lower mammals. These cells responded to hypoxia with an external Ca(2+)-dependent increase of cytosolic Ca(2+) and quantal catecholamine secretion, as reported for other mammalian species. Interestingly, human glomus cells are also responsive to hypoglycaemia and together these two stimuli can potentiate each other's effects. The chemosensory responses of glomus cells are also preserved at an advanced age. These new data on the cellular and molecular physiology of the CB pave the way for future pathophysiological studies involving this organ in humans.

Neuroprotective and reparative effects of carotid body grafts in a chronic MPTP model of Parkinson's disease.

Intrastriatal transplantation of dopaminergic carotid body (CB) cells ameliorates parkinsonism in animal models and, with less efficacy, in Parkinson's disease patients. CB-based cell therapy was initially proposed because of its high dopamine content. However, later studies suggested that its beneficial effect might be due to a trophic action exerted on nigrostriatal neurons. Compatible with this concept are the high levels of neurotrophic factors encountered in CB cells. To test experimentally this idea, unilateral striatal transplants were performed with a sham graft in the contralateral striatum, as a robust internal control. Thereafter, the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6, -tetrahydropyridine was injected during 3 months. CB grafts protected from degeneration ipsilateral nigral dopaminergic neurons projecting to the transplant in a dose-dependent manner regarding size and glial cell line-derived neurotrophic factor expression. Grafts performed at different times after the onset of the neurotoxic treatment demonstrated with histological and behavioral methods protection and repair of the nigrostriatal pathway by CB transplants. This study provides a mechanistic explanation for the action of CB transplants on parkinsonian models. It should also help to improve cell therapy approaches to Parkinson's disease.