Mutant huntingtin, which causes Huntington’s disease (HD), is ubiquitously expressed but induces preferential loss of striatal neurons by unclear mechanisms. Rab11 dysfunction mediates homeostatic disturbance of HD neurons. Here, we report that Rab11 dysfunction also underscores the striatal vulnerability in HD. We profiled the proteome of Rab11-positive endosomes of HD-vulnerable striatal cells to look for protein(s) linking Rab11 dysfunction to striatal vulnerability in HD and found XK, which triggers the selective death of striatal neurons in McLeod syndrome. XK was trafficked together with Rab11 and was diminished on the surface of immortalized HD striatal cells and striatal neurons in HD mouse brains. We found that XK participated in transporting manganese, an essential trace metal depleted in HD brains. Introducing dominantly active Rab11 into HD striatal cells improved XK dynamics and increased manganese accumulation in an XK-dependent manner. Our study suggests that impaired Rab11-based recycling of XK onto cell surfaces for importing manganese is a driver of striatal dysfunction in Huntington’s disease.
Coordinated actions of Rab and Rho are necessary for numerous essential cellular processes ranging from vesicle budding to whole cell movement. How Rab and Rho are choreographed is poorly understood. Here, we report a protein complex comprised of kalirin, a Rho guanine nucleotide exchange factor (GEF) activating Rac1, and RabGEF transport protein particle (TRAPP). Kalirin was identified in a mass spectrometry analysis of proteins precipitated by trappc4 and detected on membranous organelles containing trappc4. Acute knockdown of kalirin did not affect trappc4, but significantly reduced overall and membrane-bound levels of trappc9, which specifies TRAPP toward activating Rab11. Trappc9 deficiency led to elevated expression of kalirin in neurons. Co-localization of kalirin and Rab11 occurred at a low frequency in NRK cells under steady state and was enhanced upon expressing an inactive Rab11 mutant to prohibit the dissociation of Rab11 from the kalirin-TRAPP complex. The small RNA-mediated depletion of kalirin diminished activities in cellular membranes for activating Rab11 and resulted in a shift in size of Rab11 positive structures from small to larger ones and tubulation of recycling endosomes. Our study suggests that kalirin and TRAPP form a dual GEF complex to choreograph actions of Rab11 and Rac1 at recycling endosomes.
Huntingtin is ubiquitously expressed and enriched in the brain. Deletion of the huntingtin gene in mice is lethal during early embryonic development. The function of huntingtin is, however, not clear. Here, we report that huntingtin is important for the function of Rab11, a critical GTPase in regulating membrane traffic from recycling endosomes to the plasma membrane. In huntingtin-null embryonic stem cells, the levels of Rab11 on membranes and nucleotide exchange activity on Rab11 were significantly reduced compared with normal embryonic stem cells. In brain membranes, an antibody against huntingtin immunoprecipitated a nucleotide exchange activity on Rab11 and huntingtin was coprecipitated with Rab11 in the presence of guanosine diphosphate. These data suggest a role for huntingtin in a complex that activates Rab11.
Huntingtin is a protein of unknown function that contains a polyglutamine tract, which is expanded in patients with Huntington's disease (HD). We investigated the localization and a potential function for huntingtin in the nucleus. In human fibroblasts from normal and HD patients, huntingtin localized diffusely in the nucleus and in subnuclear compartments identified as speckles, promyelocytic leukemia protein bodies, and nucleoli. Huntingtin-positive nuclear bodies redistributed after treatment with sodium butyrate. By Western blot, purified nuclei had low levels of full-length huntingtin compared with the cytoplasm but contained high levels of N- and C-terminal huntingtin fragments, which tightly bound the nuclear matrix. Full-length huntingtin co-immunoprecipitated with the transcriptional corepressor C-terminal binding protein, and polyglutamine expansion in huntingtin reduced this interaction. Full-length wild-type and mutant huntingtin repressed transcription when targeted to DNA. Truncated N-terminal mutant huntingtin repressed transcription, whereas the corresponding wild-type fragment did not repress transcription. We speculate that wild-type huntingtin may function in the nucleus in the assembly of nuclear matrix-bound protein complexes involved with transcriptional repression and RNA processing. Proteolysis of mutant huntingtin may alter nuclear functions by disrupting protein complexes and inappropriately repressing transcription in HD.
Abstract Small interfering RNAs are a new class of drugs, exhibiting sequence-driven, potent, and sustained silencing of gene expression in vivo. We recently demonstrated that siRNA chemical architectures can be optimized to provide efficient delivery to the CNS, enabling development of CNS-targeted therapeutics. Many genetically-defined neurodegenerative disorders are dominant, favoring selective silencing of the mutant allele. In some cases, successfully targeting the mutant allele requires targeting single nucleotide polymorphism (SNP) heterozygosities. Here, we use Huntington’s disease (HD) as a model. The optimized compound exhibits selective silencing of mutant huntingtin protein in patient-derived cells and throughout the HD mouse brain, demonstrating SNP-based allele-specific RNAi silencing of gene expression in vivo in the CNS. Targeting a disease-causing allele using RNAi-based therapies could be helpful in a range of dominant CNS disorders where maintaining wild-type expression is essential.
Abstract Loss-of-function mutations of the gene encoding the trafficking protein particle complex subunit 9 (trappc9) cause intellectual disability and obesity by unknown mechanisms. Genome-wide analysis links trappc9 to non-alcoholic fatty liver disease (NAFLD). The abrogation of trappc9 in mice has been shown to alter the density of neurons containing dopamine receptor D2 (DRD2) and/or DRD1 in the striatum. Here, we report that trappc9 deficiency in mice resulted in disruption of systemic glucose homeostasis and onset of obesity and NAFLD, which were relieved upon chronic treatment combining DRD2 agonist quinpirole and DRD1 antagonist SCH23390. The homeostasis of systemic glucose in trappc9-deficient mice was restored upon administrating quinpirole alone. Transcriptomic and proteomic analyses revealed signs of impairments in neurotransmitter secretion in trappc9-deficient mice. Brain examinations showed that trappc9-deficient mice synthesized dopamine normally, but their dopamine-secreting neurons had a lower abundance of structures for releasing dopamine in the striatum. Our study suggests that trappc9 loss-of-function causes obesity and NAFLD by constraining dopamine transmission.
'%3e%3cpath fill='%23012169' d='M0 0v30h60V0z'/%3e%3cpath stroke='%23fff' stroke-width='6' d='m0 0 60 30m0-30L0 30'/%3e%3cpath stroke='%23C8102E' stroke-width='4' d='m0 0 60 30m0-30L0 30' clip-path='url(%23b)'/%3e%3cpath stroke='%23fff' stroke-width='10' d='M30 0v30M0 15h60'/%3e%3cpath stroke='%23C8102E' stroke-width='6' d='M30 0v30M0 15h60'/%3e%3c/g%3e%3c/svg%3e)