Local translation in neuronal processes is key to the alteration of synaptic strength necessary for long-term potentiation, learning, and memory. Here, we present evidence that regulated de novo protein synthesis occurs within distal, perisynaptic astrocyte processes. Astrocyte ribosomal proteins are found adjacent to synapses in vivo, and immunofluorescent detection of peptide elongation in acute slices demonstrates robust translation in distal processes. We have also developed a biochemical approach to define candidate transcripts that are locally translated in astrocyte processes. Computational analyses indicate that astrocyte-localized translation is both sequence-dependent and enriched for particular biological functions, such as fatty acid synthesis, and for pathways consistent with known roles for astrocyte processes, such as GABA and glutamate metabolism. These transcripts also include glial regulators of synaptic refinement, such as Sparc. Finally, the transcripts contain a disproportionate amount of a binding motif for the quaking RNA binding protein, a sequence we show can significantly regulate mRNA localization and translation in the astrocytes. Overall, our observations raise the possibility that local production of astrocyte proteins may support microscale alterations of adjacent synapses.astrocyte | local translation | synapse | TRAP | RNA-sequencing

SUMMARY Directed reprogramming of human fibroblasts into fully-differentiated neurons requires massive changes in epigenetic and transcriptional states. Induction of a chromatin environment permissive to acquiring neuronal subtype identity is therefore a major barrier to fate conversion. Here we show that the brain-enriched miRNAs miR-9/9* and miR-124 (miR-9/9*-124) trigger reconfiguration of chromatin accessibility, DNA methylation, and mRNA expression to induce a default neuronal state. MiR-9/9*-124-induced neurons (miNs) are functionally excitable and are uncommitted towards specific subtypes yet possess open chromatin at neuronal subtype-specific loci, suggesting such identity can be imparted by additional lineage-specific transcription factors. Consistently, we show ISL1 and LHX3 selectively drive conversion to a highly homogenous population of human spinal cord motor neurons. Taken together, this study shows modular synergism between miRNAs and neuronal subtype-specific transcription factors can drive lineage-specific neuronal reprogramming, thereby providing a general platform for high-efficiency generation of distinct subtypes of human neurons.

Localized translation in neurites helps regulate synaptic strength and development. Dysregulation of local translation is associated with many neurological disorders. However, due to technical limitations, study of this phenomenon has largely been limited to brain regions with laminar organization of dendrites such as the hippocampus or cerebellum. It has not been examined in the cortex, a region of importance for most neurological disorders, where dendrites of each neuronal population are densely intermingled with cell bodies of others. Therefore, we have developed a novel method, SynapTRAP, which combines synaptoneurosomal fractionation with translating ribosome affinity purification to identify ribosome-bound mRNA in processes of genetically defined cell types. We demonstrate SynapTRAP's efficacy and report local translation in the cortex of mice, where we identify a subset of mRNAs that are translated in dendrites by neuronal ribosomes. These mRNAs have disproportionately longer lengths, enrichment for FMRP binding and G-quartets, and their genes are under greater evolutionary constraint in humans. In addition, we show that alternative splicing likely regulates this phenomenon. Overall, SynapTRAP allows for rapid isolation of cell-type-specific localized translation and is applicable to classes of previously inaccessible neuronal and non-neuronal cells Instructions for making proteins are found in the genome, housed within the nucleus of each cell. These are then copied as RNA and exported to manufacture new proteins. However, in the brain, memory is thought to be encoded by strengthening individual connections (synapses) between neurons far from the nucleus. Thus, to efficiently make new proteins specifically where they are needed, neurons can transport RNAs to sites near synapses to locally produce proteins. Importantly, several mutations that cause autism disrupt this process. It has been assumed this process occurs in all brain regions, but has never been measured in the cortex. We applied a newly developed method measure to study, for the first time, local translation in cortical neurons.

21Local translation in neuronal processes is key to the alteration of synaptic strength that contributes 22

The decreasing cost of whole-genome and whole-exome sequencing has resulted in a renaissance for identifying Mendelian disease mutations, and for the first time it is possible to survey the distribution and characteristics of these mutations in large population samples. We conducted carrier screening for all autosomal-recessive (AR) mutations known to be present in members of a founder population and revealed surprisingly high carrier frequencies for many of these mutations. By utilizing the rich demographic, genetic, and phenotypic data available on these subjects and simulations in the exact pedigree that these individuals belong to, we show that the majority of mutations were most likely introduced into the population by a single founder and then drifted to the high carrier frequencies observed. We further show that although there is an increased incidence of AR diseases overall, the mean carrier burden is likely to be lower in the Hutterites than in the general population. Finally, on the basis of simulations, we predict the presence of 30 or more undiscovered recessive mutations among these subjects, and this would at least double the number of AR diseases that have been reported in this isolated population.