Cyclic GMP-AMP synthase (cGAS) and its downstream effector, stimulator of interferon genes (STING), form a key cytosolic DNA-sensing pathway that drives innate immune activation and proinflammatory signaling. We previously showed that cGAS is upregulated in Huntington disease (HD) cellular models, where it regulates autophagy and inflammation; however, its in vivo role remained unclear. Here, we genetically ablated cGAS in Q175DN knock-in HD mice and performed longitudinal behavioral assessments from 2 to 14 mo of age. cGAS deletion significantly improved HD-associated motor deficits, including rotarod performance and beam-walk coordination, and mitigated progressive body-weight loss. Histological analyses revealed reduced lateral ventricle enlargement and decreased striatal astrogliosis and microgliosis. While minimal effects were observed in wild-type littermates, transcriptomic profiling of HD brains lacking cGAS showed downregulation of genes involved in development and cell-cell communication, along with upregulation of genes linked to ion transport and synaptic function. Lipidomic analysis further demonstrated increased levels of immunoregulatory lipids, particularly 12-HETE and 12-HEPE, indicating a shift toward a protective lipid profile. Importantly, pharmacological inhibition of STING using H-151 improved age-dependent motor performance, reduced striatal atrophy, and attenuated glial cell activation in Q175DN mice. Collectively, these findings identify the cGAS-STING pathway as a critical driver of HD progression and support its inhibition as a promising therapeutic strategy.

Tunneling nanotubes (TNTs) are membranous structures that mediate intercellular transfer of proteins, including the pathogenic mutant Huntingtin (mHTT) protein in Huntington disease (HD). We previously identified the ras homolog enriched in the striatum (Rhes) as a key regulator of TNT formation and mHTT transmission; however, the molecular components underlying this process remained unknown. Here, using unbiased liquid chromatography-tandem mass spectrometry analysis of membrane-associated Rhes complexes, we identify Slc4a7 (solute carrier family 4 member 7), an intracellular pH sensor, as a top membrane-binding partner of Rhes. Functional studies revealed that small interfering RNA-mediated depletion or pharmacological inhibition of Slc4a7 substantially reduced Rhes-induced TNT formation and suppressed mHTT intercellular transfer. Mechanistically, Rhes directly interacts with Slc4a7 through both its amino- and carboxyl-terminal domains and modulates intracellular pH to facilitate TNT formation. This interaction does not depend on the transporter activity of Slc4a7. However, inhibition of Rhes farnesylation-a lipid modification that anchors Rhes to the membrane-disrupts its binding to Slc4a7 and abolishes TNT formation. Slc4a7 knock-out mice showed markedly reduced cell-to-cell transmission of mHTT in the striatum in vivo. Together, these findings uncover a previously unrecognized Rhes-Slc4a7 signaling axis critical for TNT-mediated mHTT transmission and highlight Slc4a7 as a potential therapeutic target to limit disease spread in HD.

Tunneling nanotubes (TNTs) are hollow tubular connections between animal cells that are membrane-enclosed and based on F-actin. They carry a range of biological cargo, including proteins involved in neurodegenerative diseases like Tau and mutant huntingtin (mHTT). Nonetheless, the molecular mechanisms underlying their formation, especially in the brain, remain ambiguous. Ras homolog abundant in the striatum (Rhes) is associated with Huntington's disease and tauopathy. We discovered that Rhes causes TNT-like protrusions and facilitates the transfer of mHTT across neuronal cells in culture and the brain. Using cell culture models, Co-immunoprecipitation assays, High resolution microscopy and mass spectrometry, we investigated the role of Rhes in TNT formation and cargo transfer. Pharmacological and knockout approaches were used to assess the impact of SLC4A7 loss on TNT formation and mHTT/Tau transfer in cellular models and the intact striatum. We discovered that Rhes causes TNT-like protrusions and facilitates the transfer of mHTT across neuronal cells in culture and the brain. The preliminary findings indicated that Rhes can transmit Tau between cultivated cells. Nonetheless, the processes remain unclear. Employing an unbiased mass spectrometry technique, we identified solute carrier family four member 7 (SLC4A7), a regulator of intracellular pH, as a novel regulator of Rhes-mediated tunneling nanotubes (TNTs). Rhes binds directly to SLC4A7 and alters intracellular pH. Pharmacological and knockout investigations indicate that the loss of SLC4A7 reduces TNTs and inhibits the dissemination of mHTT in cellular models and within the intact striatum. The preliminary findings indicate that Rhes can enhance TNT formation in microglial cultures. Rhes plays a crucial role in regulating the production of TNT and the dissemination of Tau and mHTT throughout the brain through neurons and microglia. Targeting Rhes presents a therapeutic possibility for Huntington's disease and tauopathies.

Rhes (Ras homolog enriched in the striatum), a multifunctional protein that regulates striatal functions associated with motor behaviors and neurological diseases, can shuttle from cell to cell via the formation of tunneling-like nanotubes (TNTs). However, the mechanisms by which Rhes mediates diverse functions remain unclear. Rhes is a small GTPase family member which contains a unique C-terminal Small Ubiquitin-like Modifier (SUMO) E3-like domain that promotes SUMO post-translational modification of proteins (SUMOylation) by promoting "cross-SUMOylation" of the SUMO enzyme SUMO E1 (Aos1/Uba2) and SUMO E2 ligase (Ubc-9). Nevertheless, the identity of the SUMO substrates of Rhes remains largely unknown. Here, by combining high throughput interactome and SUMO proteomics, we report that Rhes regulates the SUMOylation of nuclear proteins that are involved in the regulation of gene expression. Rhes increased the SUMOylation of histone deacetylase 1 (HDAC1) and histone 2B, while decreasing SUMOylation of heterogeneous nuclear ribonucleoprotein M (HNRNPM), protein polybromo-1 (PBRM1) and E3 SUMO-protein ligase (PIASy). We also found that Rhes itself is SUMOylated at 6 different lysine residues (K32, K110, K114, K120, K124, and K245). Furthermore, Rhes regulated the expression of genes involved in cellular morphogenesis and differentiation in the striatum, in a SUMO-dependent manner. Our findings thus provide evidence for a previously undescribed role for Rhes in regulating the SUMOylation of nuclear targets and in orchestrating striatal gene expression via SUMOylation.

ABSTRACTThe mechanistic target of rapamycin (mTOR) signaling is influenced by multiple regulatory proteins and post-translational modifications, however, underlying mechanisms remains unclear. Here, we report a novel role of small ubiquitin-like modifier (SUMO) in mTOR complex assembly and activity. By investigating the SUMOylation status of core mTOR components, we observed that the regulatory subunit, GβL, is modified by SUMO1, 2, and 3 isoforms. Using mutagenesis and mass spectrometry, we identified that GβL is SUMOylated at lysine sites K86, K215, K245, K261 and K305. We found that SUMO depletion reduces mTOR-Raptor and mTOR-Rictor complex formation and diminishes nutrient-induced mTOR signaling. Furthermore, we found that reconstitution with WT GβL but not SUMOylation defective KR mutant GβL promote mTOR signaling in GβL-depleted cells. Taken together, we report for the very first time that SUMO modifies GβL, influences the assembly of mTOR protein complexes, and regulates mTOR activity.

Huntington disease (HD) is caused by an expanded polyglutamine mutation in huntingtin (mHTT) that promotes prominent atrophy in the striatum and subsequent psychiatric, cognitive deficits, and choreiform movements. Multiple lines of evidence point to an association between HD and aberrant striatal mitochondrial functions; however, the present knowledge about whether (or how) mitochondrial mRNA translation is differentially regulated in HD remains unclear. We found that protein synthesis is diminished in HD mitochondria compared to healthy control striatal cell models. We utilized ribosome profiling (Ribo-Seq) to analyze detailed snapshots of ribosome occupancy of the mitochondrial mRNA transcripts in control and HD striatal cell models. The Ribo-Seq data revealed almost unaltered ribosome occupancy on the nuclear-encoded mitochondrial transcripts involved in oxidative phosphorylation (SDHA, Ndufv1, Timm23, Tomm5, Mrps22) in HD cells. By contrast, ribosome occupancy was dramatically increased for mitochondrially encoded oxidative phosphorylation mRNAs (mt-Nd1, mt-Nd2, mt-Nd4, mt-Nd4l, mt-Nd5, mt-Nd6, mt-Co1, mt-Cytb, and mt-ATP8). We also applied tandem mass tag-based mass spectrometry identification of mitochondrial proteins to derive correlations between ribosome occupancy and actual mature mitochondrial protein products. We found many mitochondrial transcripts with comparable or higher ribosome occupancy, but diminished mitochondrial protein products, in HD. Thus, our study provides the first evidence of a widespread dichotomous effect on ribosome occupancy and protein abundance of mitochondria-related genes in HD.

The field of neuroscience is rapidly progressing, continuously uncovering new insights and discoveries. Among the areas that have shown immense potential in research, tunneling nanotubes (TNTs) have emerged as a promising subject of study. These minute structures act as conduits for the transfer of cellular materials between cells, representing a mechanism of communication that holds great significance. In particular, the interplay facilitated by TNTs among various cell types within the brain, including neurons, astrocytes, oligodendrocytes, glial cells, and microglia, can be essential for the normal development and optimal functioning of this complex organ. The involvement of TNTs in neurodegenerative disorders, such as Alzheimer's disease, Huntington's disease, and Parkinson's disease, has attracted significant attention. These disorders are characterized by the progressive degeneration of neurons and the subsequent decline in brain function. Studies have predicted that TNTs likely play critical roles in the propagation and spread of pathological factors, contributing to the advancement of these diseases. Thus, there is a growing interest in understanding the precise functions and mechanisms of TNTs within the nervous system. This review article, based on our recent work on Rhes-mediated TNTs, aims to explore the functions of TNTs within the brain and investigate their implications for neurodegenerative diseases. Using the knowledge gained from studying TNTs could offer novel opportunities for designing targeted treatments that can stop the progression of neurodegenerative disorders.

The conserved exocyst complex regulates plasma membrane-directed vesicle fusion in eukaryotes. However, its role in stem cell proliferation has not been reported. Germline stem cell (GSC) proliferation in the nematode Caenorhabditis elegans is regulated by conserved Notch signaling. Here, we reveal that the exocyst complex regulates C. elegans GSC proliferation by modulating Notch signaling cell autonomously. Notch membrane density is asymmetrically maintained on GSCs. Knockdown of exocyst complex subunits or of the exocyst-interacting GTPases Rab5 and Rab11 leads to Notch redistribution from the GSC-niche interface to the cytoplasm, suggesting defects in plasma membrane Notch deposition. The anterior polarity (aPar) protein Par6 is required for GSC proliferation, and for maintaining niche-facing membrane levels of Notch and the exocyst complex. The exocyst complex biochemically interacts with the aPar regulator Par5 (14-3-3ζ) and Notch in C. elegans and human cells. Exocyst components are required for Notch plasma membrane localization and signaling in mammalian cells. Our study uncovers a possibly conserved requirement of the exocyst complex in regulating GSC proliferation and in maintaining optimal membrane Notch levels.

Tunneling nanotubes (TNTs) are F-actin-based, membrane-enclosed tubular connections between animal cells that transport a variety of cellular cargo. Over the last 15 years since their discovery, TNTs have come to be recognized as key players in normal cell communication and organism development, and are also exploited for the spread of various microbial pathogens and major diseases like cancer and neurodegenerative disorders. TNTs have also been proposed as modalities for disseminating therapeutic drugs between cells. Despite the rapidly expanding and wide-ranging relevance of these structures in both health and disease, there is a glaring dearth of molecular mechanistic knowledge regarding the formation and function of these important but enigmatic structures. A series of fundamental steps are essential for the formation of functional nanotubes. The spatiotemporally controlled and directed modulation of cortical actin dynamics would be required to ensure outward F-actin polymerization. Local plasma membrane deformation to impart negative curvature and membrane addition at a rate commensurate with F-actin polymerization would enable outward TNT elongation. Extrinsic tactic cues, along with cognate intrinsic signaling, would be required to guide and stabilize the elongating TNT towards its intended target, followed by membrane fusion to create a functional TNT. Selected cargoes must be transported between connected cells through the action of molecular motors, before the TNT is retracted or destroyed. This review summarizes the current understanding of the molecular mechanisms regulating these steps, also highlighting areas that deserve future attention.

Tunneling nanotubes (TNTs) mediate intercellular communication between animal cells in health and disease, but the mechanisms of their biogenesis and function are poorly understood. Here we report that the RNA-binding protein (RBP) nucleolin, which interacts with the known TNT-inducing protein MSec, is essential for TNT formation in mammalian cells. Nucleolin, through its RNA-binding domains (RBDs), binds to and maintains the cytosolic levels of 14-3-3ζ mRNA, and is, therefore, required for TNT formation. A specific region of the 3'-untranslated region (UTR) of the 14-3-3ζ mRNA is likely to be involved in its regulation by nucleolin. Functional complementation experiments suggest that nucleolin and 14-3-3ζ form a linear signaling axis that promotes the phosphorylation and inactivation of the F-actin depolymerization factor cofilin to induce TNT formation. MSec also similarly inactivates cofilin, but potentiates TNT formation independent of the nucleolin-14-3-3ζ axis, despite biochemically interacting with both proteins. We show that 14-3-3ζ and nucleolin are required for the formation of TNTs between primary mouse neurons and astrocytes and in multiple other mammalian cell types. We also report that the Caenorhabditis elegans orthologs of 14-3-3ζ and MSec regulate the size and architecture of the TNT-like cellular protrusions of the distal tip cell (DTC), the germline stem cell niche in the gonad. Our study demonstrates a novel and potentially conserved mRNA-guided mechanism of TNT formation through the maintenance of cellular 14-3-3ζ mRNA levels by the RBP nucleolin.

The molecular motor dynein is essential for mitotic spindle orientation, which defines the axis of cell division. The light intermediate chain subunits, LIC1 and LIC2, define biochemically and functionally distinct vertebrate dynein complexes, with LIC2-dynein playing a crucial role in ensuring spindle orientation. We reveal a novel, mitosis-specific interaction of LIC2-dynein with the cortical actin-bundling protein transgelin-2. Transgelin-2 is required for maintaining proper spindle length, equatorial metaphase chromosome alignment, spindle orientation and timely anaphase onset. We show that transgelin-2 stabilizes the cortical recruitment of LGN-NuMA, which together with dynein is required for spindle orientation. The opposing actions of transgelin-2 and LIC2-dynein maintain optimal cortical levels of LGN-NuMA. In addition, we show that the highly conserved serine 194 phosphorylation of LIC2 is required for proper spindle orientation, by maintaining mitotic centrosome integrity to ensure optimal astral microtubule nucleation. The work reveals two specific mechanisms through which LIC2-dynein regulates mitotic spindle orientation; namely, through a new interactor transgelin-2, which is required for engagement of LGN-NuMA with the actin cortex, and through mitotic phosphoregulation of LIC2 to control microtubule nucleation from the poles.This article has an associated First Person interview with the first author of the paper.

Tunneling nanotubes (TNTs) are membrane conduits that mediate long-distance intercellular cross-talk in several organisms and play vital roles during development, pathogenic transmission, and cancer metastasis. However, the molecular mechanisms of TNT formation and function remain poorly understood. The protein MSec (also known as TNFα-induced protein 2 (TNFAIP2) and B94) is essential for TNT formation in multiple cell types. Here, using affinity protein purification, mass spectrometric identification, and confocal immunofluorescence microscopy assays, we found that MSec interacts with the endoplasmic reticulum (ER) chaperone ERp29. siRNA-mediated ERp29 depletion in mammalian cells significantly reduces TNT formation, whereas its overexpression induces TNT formation, but in a strictly MSec-dependent manner. ERp29 stabilized MSec protein levels, but not its mRNA levels, and the chaperone activity of ERp29 was required for maintaining MSec protein stability. Subcellular ER fractionation and subsequent limited proteolytic treatment suggested that MSec is associated with the outer surface of the ER. The ERp29-MSec interaction appeared to require the presence of other bridging protein(s), perhaps triggered by post-translational modification of ERp29. Our study implicates MSec as a target of ERp29 and reveals an indispensable role for the ER in TNT formation, suggesting new modalities for regulating TNT numbers in cells and tissues.