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The kynurenine pathway of tryptophan degradation is hypothesized to play an important role in Huntington disease, a neurodegenerative disorder caused by a polyglutamine expansion in the protein huntingtin. Neurotoxic metabolites of the kynurenine pathway, generated in microglia and macrophages, are present at increased levels in the brains of patients and mouse models during early stages of disease, but the mechanism by which kynurenine pathway up-regulation occurs in Huntington disease is unknown. Here we report that expression of a mutant huntingtin fragment was sufficient to induce transcription of the kynurenine pathway in yeast and that this induction was abrogated by impairing the activity of the histone deacetylase Rpd3. Moreover, numerous genetic suppressors of mutant huntingtin toxicity that are functionally unrelated converged unexpectedly on the kynurenine pathway, supporting a critical role for the kynurenine pathway in mediating mutant huntingtin toxicity in yeast. Histone deacetylase-dependent regulation of the kynurenine pathway was also observed in a mouse model of Huntington disease, in which treatment with a neuroprotective histone deacetylase inhibitor blocked activation of the kynurenine pathway in microglia expressing a mutant huntingtin fragment in vitro and in vivo. These findings suggest that a mutant huntingtin fragment can perturb transcriptional programs in microglia, and thus implicate these cells as potential modulators of neurodegeneration in Huntington disease that are worthy of further investigation.

Membrane fusion in the secretory pathway is mediated by soluble N -ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins. Different fusion steps are thought to be effected by independent sets of SNAREs, but it is unclear whether specificity is determined by an intrinsic specificity of SNARE pairing or by upstream factors. Using a newly developed microscopy-based assay, we have investigated the SNARE specificity of homotypic early endosomal fusion. We show that early endosomes contain multiple sets of SNAREs, including, in addition to the putative early endosomal SNAREs, those involved in exocytosis and in fusion of late endosomes. We demonstrate that fusion is largely mediated by a complex formed by syntaxin 13, syntaxin 6, vti1a, and VAMP4, whereas the exocytic and late endosomal SNAREs play little or no role in the reaction. In contrast, proteoliposomes reconstituted with early endosomal SNAREs promiscuously fuse with liposomes containing exocytotic or late endosomal SNAREs. We conclude that the specificity of SNARE pairing does not suffice to determine the specificity of organelle fusion.

SNARE proteins mediate membrane fusion in eukaryotic cells. They contain conserved SNARE motifs that are usually located adjacent to a C-terminal transmembrane domain. SNARE motifs spontaneously assemble into four helix bundles, with each helix belonging to a different subfamily. Liposomes containing SNAREs spontaneously fuse with each other, but it is debated how the SNAREs are distributed between the membranes. Here, we report that the SNAREs mediating homotypic fusion of early endosomes fuse liposomes in five out of seven possible combinations, in contrast to previously studied SNAREs involved in heterotypic fusion events. The crystal structure of the early endosomal SNARE complex resembles that of the neuronal and late endosomal complexes, but differs in surface side-chain interactions. We conclude that homotypic fusion reactions may proceed with multiple SNARE topologies, suggesting that the conserved SNARE structure allows for flexibility in the initial interactions needed for fusion.

Early endosomes are well-established acceptor compartments of endocytic vesicles in many cell types. Little evidence of their existence or function has been obtained in synapses, and it is generally believed that synaptic vesicles recycle without passing through an endosomal intermediate. We show here that the early endosomal SNARE proteins are enriched in synaptic vesicles. To investigate their function in the synapse, we isolated synaptic nerve terminals (synaptosomes), stimulated them in presence of different fluorescent markers to label the recycling vesicles and used these vesicles in in vitro fusion assays. The recently endocytosed vesicles underwent homotypic fusion. They also fused with endosomes from PC12 and BHK cells. The fusion process was dependent upon NSF activity. Moreover, fusion was dependent upon the early endosomal SNAREs but not upon the SNAREs involved in exocytosis. Our results thus show that at least a fraction of the vesicles endocytosed during synaptic activity are capable of fusing with early endosomes and lend support to an involvement of endosomal intermediates during recycling of synaptic vesicles.