The Differences in Local Translatome across Distinct Neuron Types Is Mediated by Both Baseline Cellular Differences and Post-transcriptional Mechanisms.

Local translation in neurites is a phenomenon that enhances the spatial segregation of proteins and their functions away from the cell body, yet it is unclear how local translation varies across neuronal cell types. Further, it is unclear whether differences in local translation across cell types simply reflect differences in transcription or whether there is also a cell type-specific post-transcriptional regulation of the location and translation of specific mRNAs. Most of the mRNAs discovered as being locally translated have been identified from hippocampal neurons because their laminar organization facilitates neurite-specific dissection and microscopy methods. Given the diversity of neurons across the brain, studies have not yet analyzed how locally translated mRNAs differ across cell types. Here, we used the SynapTRAP method to harvest two broad cell types in the mouse forebrain: GABAergic neurons and layer 5 projection neurons. While some transcripts overlap, the majority of the local translatome is not shared across these cell types. In addition to differences driven by baseline expression levels, some transcripts also exhibit cell type-specific post-transcriptional regulation. Finally, we provide evidence that GABAergic neurons specifically localize mRNAs for peptide neurotransmitters, including somatostatin and cortistatin, suggesting localized production of these key signaling molecules in the neurites of GABAergic neurons. Overall, this work suggests that differences in local translation in neurites across neuronal cell types are poised to contribute substantially to the heterogeneity in neuronal phenotypes.

MicroRNAs Induce a Permissive Chromatin Environment that Enables Neuronal Subtype-Specific Reprogramming of Adult Human Fibroblasts.

Directed reprogramming of human fibroblasts into fully differentiated neurons requires massive changes in epigenetic and transcriptional states. Induction of a chromatin environment permissive for 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 uncommitted toward specific subtypes but possess open chromatin at neuronal subtype-specific loci, suggesting that such identity can be imparted by additional lineage-specific transcription factors. Consistently, we show that ISL1 and LHX3 selectively drive conversion to a highly homogeneous population of human spinal cord motor neurons. This study shows that modular synergism between miRNAs and neuronal subtype-specific transcription factors can drive lineage-specific neuronal reprogramming, providing a general platform for high-efficiency generation of distinct subtypes of human neurons.

Transcriptomic Analysis of Ribosome-Bound mRNA in Cortical Neurites In Vivo.

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 in vivoSIGNIFICANCE STATEMENT 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.

Astrocytes locally translate transcripts in their peripheral processes.

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.

Fmrp targets or not: long, highly brain-expressed genes tend to be implicated in autism and brain disorders.

BACKGROUND: Many studies have demonstrated a robust statistical overlap between genes whose transcripts are reported as Fragile X Mental Retardation Protein (Fmrp)-binding targets and genes implicated in various psychiatric disorders, including autism. However, it is not clear how to interpret this overlap as the Fmrp protein itself is not considered to be central to all instances of these conditions. FINDINGS: We tested whether Fmrp binding may be a proxy for some other features of these transcripts. Reviewing recent literature on the cross-linking and immunoprecipitation (CLIP)-derived targets of Fmrp in the brain, and the literature on identifying genes thought to mediate autism and other psychiatric disorders, reveals that both appear to be disproportionately made up of highly brain-expressed genes. This suggests a parsimonious explanation-that the overlap between Fmrp targets and neuropsychiatric candidate genes might be secondary to simple features such as transcript length and robust expression in the brain. Indeed, reanalyzing Fmrp high-throughput sequencing of RNAs isolated by CLIP (HITS-CLIP) data suggests that approximately 60% of CLIP tag depth can be predicted by gene expression, coding sequence length, and transcript length. Furthermore, there is a statistically significant overlap between autism candidate genes and random samples of long, highly brain-expressed genes, whether they are Fmrp targets or not. CONCLUSIONS: Comparison of known Fmrp-binding targets to candidate gene lists should be informed by both of these features.

Disclosure of genetic research results to members of a founder population.

There is currently extensive discussion and debate in the literature on how, when, and to whom genetic research results should be returned (see Genetics in Medicine, April 2012 issue). Here, we describe our experience in disclosing genetic information on Mendelian disorders discovered during the course of our research in the Hutterites. We first assessed attitudes toward the disclosure of carrier results, which revealed that many individuals wanted carrier information and that many intended to use the information in family planning. Based on this information, we developed a pilot study to test and disclose cystic fibrosis (CF) carrier status. Next, a larger scale project was developed in order to disclose genetic research results for 14 diseases to those interested in receiving the information. We developed brochures, offered a live interactive educational program, conducted a consent process, and disclosed results in letters mailed to the consented individuals. Overall, ~80% of individuals who participated in the educational program signed consent forms for the release of their results for 14 diseases. We describe our experience with returning individual genetic research results to participants in a population-based research study.

A population-based study of autosomal-recessive disease-causing mutations in a founder population.

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.

Mammary tumors induce select cognitive impairments.

Cancer, in addition to many other chronic diseases, is associated with serious and problematic behavioral symptoms, including cognitive impairments. In humans, various factors likely contribute to cancer-associated cognitive deficits including disease awareness and chemotherapy; however, the endogenous biological factors arising from tumor development may also play a causal role. In the present study, rats with mammary tumors exhibited impaired spatial reference memory on a radial arm maze and amnesia for familiar objects in an object recognition memory test. In contrast, their performance in the Morris water maze and in fear conditioning tests was comparable to that of controls. These select cognitive impairments were accompanied by elevations in hippocampal interleukin-1beta mRNA expression, but were not associated with decreases in hippocampal brain-derived neurotrophic factor gene expression. Together the results indicate that peripheral tumors alone are sufficient to induce increases in hippocampal cytokine expression and select deficits in hippocampal-dependent memory tasks.