Relationship between sex biases in gene expression and sex biases in autism and Alzheimer's disease.

BACKGROUND: Sex differences in the brain may play an important role in sex-differential prevalence of neuropsychiatric conditions. METHODS: In order to understand the transcriptional basis of sex differences, we analyzed multiple, large-scale, human postmortem brain RNA-Seq datasets using both within-region and pan-regional frameworks. RESULTS: We find evidence of sex-biased transcription in many autosomal genes, some of which provide evidence for pathways and cell population differences between chromosomally male and female individuals. These analyses also highlight regional differences in the extent of sex-differential gene expression. We observe an increase in specific neuronal transcripts in male brains and an increase in immune and glial function-related transcripts in female brains. Integration with single-nucleus data suggests this corresponds to sex differences in cellular states rather than cell abundance. Integration with case-control gene expression studies suggests a female molecular predisposition towards Alzheimer's disease, a female-biased disease. Autism, a male-biased diagnosis, does not exhibit a male predisposition pattern in our analysis. CONCLUSION: Overall, these analyses highlight mechanisms by which sex differences may interact with sex-biased conditions in the brain. Furthermore, we provide region-specific analyses of sex differences in brain gene expression to enable additional studies at the interface of gene expression and diagnostic differences.

We sought to understand why females have higher rates of Alzheimer's disease, and males have higher rates of autism. One idea was that the female brain at baseline may be more similar to an Alzheimer's brain, so it is easier for them to shift into that state (likewise, males may be more similar to autism). To test this, we examined gene expression differences between brains of biological males and biological females. While all people have the same ~ 25,000 genes, each gene can be on or off ('expressed') to different extents. Overall, we found that there were differences in gene expression between males and females in all brain regions tested but more differences in some brain regions than others. By looking at the role of these genes we estimate that female immune system processes might be more active in the brain. We also found female brain gene expression looked slightly more like people with Alzheimer's compared to people without Alzheimer's, which may explain why females get Alzheimer's disease more easily. However, the male brain gene expression did not look more like autism, suggesting that the reason males have higher rates of autism is complex and needs further investigation.

Relationship between sex biases in gene expression and sex biases in autism and Alzheimer's disease.

Sex differences in the brain may play an important role in sex-differential prevalence of neuropsychiatric conditions. In order to understand the transcriptional basis of sex differences, we analyzed multiple, large-scale, human postmortem brain RNA-seq datasets using both within-region and pan-regional frameworks. We find evidence of sex-biased transcription in many autosomal genes, some of which provide evidence for pathways and cell population differences between chromosomally male and female individuals. These analyses also highlight regional differences in the extent of sex-differential gene expression. We observe an increase in specific neuronal transcripts in male brains and an increase in immune and glial function-related transcripts in female brains. Integration with single-cell data suggests this corresponds to sex differences in cellular states rather than cell abundance. Integration with case-control gene expression studies suggests a female molecular predisposition towards Alzheimer's disease, a female-biased disease. Autism, a male-biased diagnosis, does not exhibit a male predisposition pattern in our analysis. Finally, we provide region specific analyses of sex differences in brain gene expression to enable additional studies at the interface of gene expression and diagnostic differences.

Extensive characterization of a Williams Syndrome murine model shows Gtf2ird1 -mediated rescue of select sensorimotor tasks, but no effect on enhanced social behavior.

Williams Syndrome is a rare neurodevelopmental disorder exhibiting cognitive and behavioral abnormalities, including increased social motivation, risk of anxiety and specific phobias along with perturbed motor function. Williams Syndrome is caused by a microdeletion of 26-28 genes on chromosome 7, including GTF2IRD1 , which encodes a transcription factor suggested to play a role in the behavioral profile of Williams Syndrome. Duplications of the full region also lead to frequent autism diagnosis, social phobias, and language delay. Thus, genes in the region appear to regulate social motivation in a dose-sensitive manner. A 'Complete Deletion' mouse, heterozygously eliminating the syntenic Williams Syndrome region, has been deeply characterized for cardiac phenotypes, but direct measures of social motivation have not been assessed. Furthermore, the role of Gtf2ird1 in these behaviors has not been addressed in a relevant genetic context. Here, we have generated a mouse overexpressing Gtf2ird1 , which can be used both to model duplication of this gene alone and to rescue Gtf2ird1 expression in the Complete Deletion mice. Using a comprehensive behavioral pipeline and direct measures of social motivation, we provide evidence that the Williams Syndrome Critical Region regulates social motivation along with motor and anxiety phenotypes, but that Gtf2ird1 complementation is not sufficient to rescue most of these traits, and duplication does not decrease social motivation. However, Gtf2ird1 complementation does rescue light-aversive behavior and performance on select sensorimotor tasks, perhaps indicating a role for this gene in sensory processing or integration.

Extensive characterization of a Williams syndrome murine model shows Gtf2ird1-mediated rescue of select sensorimotor tasks, but no effect on enhanced social behavior.

Williams syndrome is a rare neurodevelopmental disorder exhibiting cognitive and behavioral abnormalities, including increased social motivation, risk of anxiety and specific phobias along with perturbed motor function. Williams syndrome is caused by a microdeletion of 26-28 genes on chromosome 7, including GTF2IRD1, which encodes a transcription factor suggested to play a role in the behavioral profile of Williams syndrome. Duplications of the full region also lead to frequent autism diagnosis, social phobias and language delay. Thus, genes in the region appear to regulate social motivation in a dose-sensitive manner. A "complete deletion" mouse, heterozygously eliminating the syntenic Williams syndrome region, has been deeply characterized for cardiac phenotypes, but direct measures of social motivation have not been assessed. Furthermore, the role of Gtf2ird1 in these behaviors has not been addressed in a relevant genetic context. Here, we have generated a mouse overexpressing Gtf2ird1, which can be used both to model duplication of this gene alone and to rescue Gtf2ird1 expression in the complete deletion mice. Using a comprehensive behavioral pipeline and direct measures of social motivation, we provide evidence that the Williams syndrome critical region regulates social motivation along with motor and anxiety phenotypes, but that Gtf2ird1 complementation is not sufficient to rescue most of these traits, and duplication does not decrease social motivation. However, Gtf2ird1 complementation does rescue light-aversive behavior and performance on select sensorimotor tasks, perhaps indicating a role for this gene in sensory processing or integration.

Local translation in microglial processes is required for efficient phagocytosis.

Neurons, astrocytes and oligodendrocytes locally regulate protein translation within distal processes. Here, we tested whether there is regulated local translation within peripheral microglial processes (PeMPs) from mouse brain. We show that PeMPs contain ribosomes that engage in de novo protein synthesis, and these are associated with transcripts involved in pathogen defense, motility and phagocytosis. Using a live slice preparation, we further show that acute translation blockade impairs the formation of PeMP phagocytic cups, the localization of lysosomal proteins within them, and phagocytosis of apoptotic cells and pathogen-like particles. Finally, PeMPs severed from their somata exhibit and require de novo local protein synthesis to effectively surround pathogen-like particles. Collectively, these data argue for regulated local translation in PeMPs and indicate a need for new translation to support dynamic microglial functions.

Evaluation of gliovascular functions of AQP4 readthrough isoforms.

Aquaporin-4 (AQP4) is a water channel protein that links the astrocytic endfeet to the blood-brain barrier (BBB) and regulates water and potassium homeostasis in the brain, as well as the glymphatic clearance of waste products that would otherwise potentiate neurological diseases. Recently, translational readthrough was shown to generate a C-terminally extended variant of AQP4, known as AQP4x, which preferentially localizes around the BBB through interaction with the scaffolding protein α-syntrophin, and loss of AQP4x disrupts waste clearance from the brain. To investigate the function of AQP4x, we generated a novel AQP4 mouse line (AllX) to increase relative levels of the readthrough variant above the ~15% of AQP4 in the brain of wild-type (WT) mice. We validated the line and assessed characteristics that are affected by the presence of AQP4x, including AQP4 and α-syntrophin localization, integrity of the BBB, and neurovascular coupling. We compared AllXHom and AllXHet mice to WT and to previously characterized AQP4 NoXHet and NoXHom mice, which cannot produce AQP4x. An increased dose of AQP4x enhanced perivascular localization of α-syntrophin and AQP4, while total protein expression of the two was unchanged. However, at 100% readthrough, AQP4x localization and the formation of higher order complexes were disrupted. Electron microscopy showed that overall blood vessel morphology was unchanged except for an increased proportion of endothelial cells with budding vesicles in NoXHom mice, which may correspond to a leakier BBB or altered efflux that was identified in NoX mice using MRI. These data demonstrate that AQP4x plays a small but measurable role in maintaining BBB integrity as well as recruiting structural and functional support proteins to the blood vessel. This also establishes a new set of genetic tools for quantitatively modulating AQP4x levels.

Evaluation of gliovascular functions of Aqp4 readthrough isoforms.

Aquaporin-4 (AQP4) is a water channel protein that links astrocytic endfeet to the blood-brain barrier (BBB) and regulates water and potassium homeostasis in the brain, as well as the glymphatic clearance of waste products that would otherwise potentiate neurological diseases. Recently, translational readthrough was shown to generate a C-terminally extended variant of AQP4, known as AQP4x, that preferentially localizes around the BBB through interaction with the scaffolding protein α-syntrophin, and loss of AQP4x disrupts waste clearance from the brain. To investigate the function of AQP4x, we generated a novel mouse AQP4 line (AllX) to increase relative levels of the readthrough variant above the ~15% of AQP4 in the brain of wildtype (WT) mice. We validated the line and assessed characteristics that are affected by the presence of AQP4x, including AQP4 and α-syntrophin localization, integrity of the BBB, and neurovascular coupling. We compared AllXHom and AllXHet mice to wildtype, and to previously characterized AQP4 NoXHet and NoXHom mice, which cannot produce AQP4x. Increased dose of AQP4x enhanced perivascular localization of α-syntrophin and AQP4, while total protein expression of the two were unchanged. However, at 100% readthrough, AQP4x localization and formation of higher-order complexes was disrupted. Electron microscopy showed that overall blood vessel morphology was unchanged except for increased endothelial cell vesicles in NoXHom mice, which may correspond to a leakier BBB or altered efflux that was identified in NoX mice using MRI. These data demonstrate that AQP4x plays a small but measurable role in maintaining BBB integrity as well as recruiting structural and functional support proteins to the blood vessel. This also establishes a new set of genetic tools for quantitatively modulating AQP4x levels.

A Cre-dependent massively parallel reporter assay allows for cell-type specific assessment of the functional effects of non-coding elements in vivo.

The function of regulatory elements is highly dependent on the cellular context, and thus for understanding the function of elements associated with psychiatric diseases these would ideally be studied in neurons in a living brain. Massively Parallel Reporter Assays (MPRAs) are molecular genetic tools that enable functional screening of hundreds of predefined sequences in a single experiment. These assays have not yet been adapted to query specific cell types in vivo in a complex tissue like the mouse brain. Here, using a test-case 3'UTR MPRA library with genomic elements containing variants from autism patients, we developed a method to achieve reproducible measurements of element effects in vivo in a cell type-specific manner, using excitatory cortical neurons and striatal medium spiny neurons as test cases. This targeted technique should enable robust, functional annotation of genetic elements in the cellular contexts most relevant to psychiatric disease.

Activity-dependent translation dynamically alters the proteome of the perisynaptic astrocyte process.

Within eukaryotic cells, translation is regulated independent of transcription, enabling nuanced, localized, and rapid responses to stimuli. Neurons respond transcriptionally and translationally to synaptic activity. Although transcriptional responses are documented in astrocytes, here we test whether astrocytes have programmed translational responses. We show that seizure activity rapidly changes the transcripts on astrocyte ribosomes, some predicted to be downstream of BDNF signaling. In acute slices, we quantify the extent to which cues of neuronal activity activate translation in astrocytes and show that this translational response requires the presence of neurons, indicating that the response is non-cell autonomous. We also show that this induction of new translation extends into the periphery of astrocytes. Finally, synaptic proteomics show that new translation is required for changes that occur in perisynaptic astrocyte protein composition after fear conditioning. Regulation of translation in astrocytes by neuronal activity suggests an additional mechanism by which astrocytes may dynamically modulate nervous system functioning.