Molecular Diversity and Specializations among the Cells of the Adult Mouse Brain.

The mammalian brain is composed of diverse, specialized cell populations. To systematically ascertain and learn from these cellular specializations, we used Drop-seq to profile RNA expression in 690,000 individual cells sampled from 9 regions of the adult mouse brain. We identified 565 transcriptionally distinct groups of cells using computational approaches developed to distinguish biological from technical signals. Cross-region analysis of these 565 cell populations revealed features of brain organization, including a gene-expression module for synthesizing axonal and presynaptic components, patterns in the co-deployment of voltage-gated ion channels, functional distinctions among the cells of the vasculature and specialization of glutamatergic neurons across cortical regions. Systematic neuronal classifications for two complex basal ganglia nuclei and the striatum revealed a rare population of spiny projection neurons. This adult mouse brain cell atlas, accessible through interactive online software (DropViz), serves as a reference for development, disease, and evolution.

Complement genes contribute sex-biased vulnerability in diverse disorders.

Many common illnesses, for reasons that have not been identified, differentially affect men and women. For instance, the autoimmune diseases systemic lupus erythematosus (SLE) and Sjögren's syndrome affect nine times more women than men1, whereas schizophrenia affects men with greater frequency and severity relative to women2. All three illnesses have their strongest common genetic associations in the major histocompatibility complex (MHC) locus, an association that in SLE and Sjögren's syndrome has long been thought to arise from alleles of the human leukocyte antigen (HLA) genes at that locus3-6. Here we show that variation of the complement component 4 (C4) genes C4A and C4B, which are also at the MHC locus and have been linked to increased risk for schizophrenia7, generates 7-fold variation in risk for SLE and 16-fold variation in risk for Sjögren's syndrome among individuals with common C4 genotypes, with C4A protecting more strongly than C4B in both illnesses. The same alleles that increase risk for schizophrenia greatly reduce risk for SLE and Sjögren's syndrome. In all three illnesses, C4 alleles act more strongly in men than in women: common combinations of C4A and C4B generated 14-fold variation in risk for SLE, 31-fold variation in risk for Sjögren's syndrome, and 1.7-fold variation in schizophrenia risk among men (versus 6-fold, 15-fold and 1.26-fold variation in risk among women, respectively). At a protein level, both C4 and its effector C3 were present at higher levels in cerebrospinal fluid and plasma8,9 in men than in women among adults aged between 20 and 50 years, corresponding to the ages of differential disease vulnerability. Sex differences in complement protein levels may help to explain the more potent effects of C4 alleles in men, women's greater risk of SLE and Sjögren's syndrome and men's greater vulnerability to schizophrenia. These results implicate the complement system as a source of sexual dimorphism in vulnerability to diverse illnesses.

Schizophrenia risk from complex variation of complement component 4.

Schizophrenia is a heritable brain illness with unknown pathogenic mechanisms. Schizophrenia's strongest genetic association at a population level involves variation in the major histocompatibility complex (MHC) locus, but the genes and molecular mechanisms accounting for this have been challenging to identify. Here we show that this association arises in part from many structurally diverse alleles of the complement component 4 (C4) genes. We found that these alleles generated widely varying levels of C4A and C4B expression in the brain, with each common C4 allele associating with schizophrenia in proportion to its tendency to generate greater expression of C4A. Human C4 protein localized to neuronal synapses, dendrites, axons, and cell bodies. In mice, C4 mediated synapse elimination during postnatal development. These results implicate excessive complement activity in the development of schizophrenia and may help explain the reduced numbers of synapses in the brains of individuals with schizophrenia.

Small-molecule screen reveals pathways that regulate C4 secretion in stem cell-derived astrocytes.

In the brain, the complement system plays a crucial role in the immune response and in synaptic elimination during normal development and disease. Here, we sought to identify pathways that modulate the production of complement component 4 (C4), recently associated with an increased risk of schizophrenia. To design a disease-relevant assay, we first developed a rapid and robust 3D protocol capable of producing large numbers of astrocytes from pluripotent cells. Transcriptional profiling of these astrocytes confirmed the homogeneity of this population of dorsal fetal-like astrocytes. Using a novel ELISA-based small-molecule screen, we identified epigenetic regulators, as well as inhibitors of intracellular signaling pathways, able to modulate C4 secretion from astrocytes. We then built a connectivity map to predict and validate additional key regulatory pathways, including one involving c-Jun-kinase. This work provides a foundation for developing therapies for CNS diseases involving the complement cascade.

The microtubule-associated protein DCAMKL1 regulates osteoblast function via repression of Runx2.

Osteoblasts are responsible for the formation and mineralization of the skeleton. To identify novel regulators of osteoblast differentiation, we conducted an unbiased forward genetic screen using a lentiviral-based shRNA library. This functional genomics analysis led to the identification of the microtubule-associated protein DCAMKL1 (Doublecortin-like and CAM kinase-like 1) as a novel regulator of osteogenesis. Mice with a targeted disruption of Dcamkl1 displayed elevated bone mass secondary to increased bone formation by osteoblasts. Molecular experiments demonstrated that DCAMKL1 represses osteoblast activation by antagonizing Runx2, the master transcription factor in osteoblasts. Key elements of the cleidocranial dysplasia phenotype observed in Runx2(+/-) mice are reversed by the introduction of a Dcamkl1-null allele. Our results establish a genetic linkage between these two proteins in vivo and demonstrate that DCAMKL1 is a physiologically relevant regulator of anabolic bone formation.