Stress-induced translation of KCNB1 contributes to the enhanced synaptic transmission of the lateral habenula.

The lateral habenula (LHb) is a well-established brain region involved in depressive disorders. Synaptic transmission of the LHb neurons is known to be enhanced by stress exposure; however, little is known about genetic modulators within the LHb that respond to stress. Using recently developed molecular profiling methods by phosphorylated ribosome capture, we obtained transcriptome profiles of stress responsive LHb neurons during acute physical stress. Among such genes, we found that KCNB1 (Kv2.1 channel), a delayed rectifier and voltage-gated potassium channel, exhibited increased expression following acute stress exposure. To determine the roles of KCNB1 on LHb neurons during stress, we injected short hairpin RNA (shRNA) against the kcnb1 gene to block its expression prior to stress exposure. We observed that the knockdown of KCNB1 altered the basal firing pattern of LHb neurons. Although KCNB1 blockade did not rescue despair-like behaviors in acute learned helplessness (aLH) animals, we found that KCNB1 knockdown prevented the enhancement of synaptic strength in LHb neuron after stress exposure. This study suggests that KCNB1 may contribute to shape stress responses by regulating basal firing patterns and neurotransmission intensity of LHb neurons.

Translating Ribosome Affinity Purification (TRAP) of Cell Type-specific mRNA from Mouse Brain Lysates.

Mammalian tissues are highly heterogenous and complex, posing a challenge in understanding the molecular mechanisms regulating protein expression within various tissues. Recent studies have shown that translation at the level of the ribosome is highly regulated, and can vary independently of gene expression observed at a transcriptome level, as well as between cell populations, contributing to the diversity of mammalian tissues. Earlier methods that analyzed gene expression at the level of translation, such as polysomal- or ribosomal-profiling, required large amounts of starting material to isolate enough RNA for analysis by microarray or RNA-sequencing. Thus, rare or less abundant cell types within tissues were not able to be properly studied with these methods. Translating ribosome affinity purification (TRAP) utilizes the incorporation of an eGFP-affinity tag on the large ribosome subunit, driven by expression of cell-type specific Cre-lox promoters, to allow for identification and capture of transcripts from actively translating ribosomes in a cell-specific manner. As a result, TRAP offers a unique opportunity to evaluate the entire mRNA translation profile within a specific cell type, and increase our understanding regarding the cellular complexity of mammalian tissues. Graphical abstract: Schematic demonstrating TRAP protocol for identifying ribosome-bound transcripts specifically within cerebellar Purkinje cells.

Reproductive tissue-specific translatome of a rice thermo-sensitive genic male sterile line.

Translational regulation, especially tissue- or cell type-specific gene regulation, plays essential roles in plant growth and development. Thermo-sensitive genic male sterile (TGMS) lines have been widely used for hybrid breeding in rice (Oryza sativa). However, little is known about translational regulation during reproductive stage in TGMS rice. Here, we use translating ribosome affinity purification (TRAP) combined with RNA sequencing to investigate the reproductive tissue-specific translatome of TGMS rice expressing FLAG-tagged ribosomal protein L18 (RPL18) from the germline-specific promoter MEIOSIS ARRESTED AT LEPTOTENE1 (MEL1). Differentially expressed genes at the transcriptional and translational levels are enriched in pollen and anther-related formation and development processes. These contain a number of genes reported to be involved in tapetum programmed cell death (PCD) and lipid metabolism during pollen development and anther dehiscence in rice, including several encoding transcription factors and key enzymes, as well as several long non-coding RNAs (lncRNAs) that potentially affect tapetum and pollen-related genes in male sterility. This study represents the comprehensive reproductive tissue-specific characterization of the translatome in TGMS rice. These results contribute to our understanding of the molecular basis of sterility in TGMS rice and will facilitate further genetic manipulation of TGMS rice in two-line breeding systems.

Chronic copper exposure directs microglia towards degenerative expression signatures in wild-type and J20 mouse model of Alzheimer's disease.

BACKGROUND: Copper (Cu) is an essential metal mediating a variety of vital biological reactions with its redox property. Its dyshomeostasis has been associated with accelerated cognitive decline and neurodegenerative disorders, such as Alzheimer's disease (AD). However, underlying neurotoxic mechanisms elicited by dysregulated Cu remain largely elusive. We and others previously demonstrated that exposure to Cu in drinking water significantly exacerbated pathological hallmarks of AD and pro-inflammatory activation of microglia, coupled with impaired phagocytic capacity, in mouse models of AD. METHODS: In the present study, we extended our investigation to evaluate whether chronic Cu exposure to wild-type (WT) and J20 mouse model of AD perturbs homeostatic dynamics of microglia and contributes to accelerated transformation of microglia towards degenerative phenotypes that are closely associated with neurodegeneration. We further looked for evidence of alterations in the microglial morphology and spatial memory of the Cu-exposed mice to assess the extent of the Cu toxicity. RESULTS: We find that chronic Cu exposure to pre-pathological J20 mice upregulates the translation of degenerative genes and represses homeostatic genes within microglia even in the absence amyloid-beta plaques. We also observe similar expression signatures in Cu-exposed WT mice, suggesting that excess Cu exposure alone could lead to perturbed microglial homeostatic phenotypes and contribute to accelerated cognitive decline. CONCLUSION: Our findings highlight the risk of chronic Cu exposure on cognitive decline and altered microglia activation towards degenerative phenotypes. These changes may represent one of the key mechanisms linking Cu exposure or its dyshomeostasis to an increased risk for AD.

Preparing the Astrocyte Perivascular Endfeet Transcriptome to Investigate Astrocyte Molecular Regulations at the Brain-Vascular Interface.

Astrocytes send out long processes that are terminated by endfeet at the vascular surface and regulate vascular functions in particular through the expression of a specific molecular repertoire in perivascular endfeet. We recently proposed that local translation might sustain this structural and functional polarization. More specifically we showed that a subset of mRNAs is distributed in astrocyte endfeet and characterized this transcriptome. We also identified among these endfeet RNAs, the ones bound to ribosomes, the polysomal astrocyte endfeet mRNAs, which we called the endfeetome. Here, we describe experimental strategies to identify mRNAs and polysomes in astrocyte perivascular endfeet, which are based on the combination of gliovascular unit purification and astrocyte-specific translating ribosome affinity purification.