Search

Huntington’s disease (HD) results from a CAG repeat expansion in the gene for Huntington (HTT) resulting in expansion of the polyglutamine (Q) tract in the mutant protein (mHTT). Synaptic changes are early manifestations of neuronal dysfunction in HD. However, the mechanism(s) by which mHTT impacts synapse formation and function is not well defined. Herein we explored HD pathogenesis in the BACHD and the ΔN17-BACHD mouse models of HD by examining cortical synapse formation and function in primary cultures maintained for up to 35 days (DIV35). We identified synapses by immunostaining with antibodies against pre-synaptic (Synapsin 1) and a post-synaptic (PSD95) marker. Consistent with earlier studies, cortical neurons from both WT and the HD models began to form synapses at DIV14; at this age there were no genotypic differences in synapse numbers. However, from DIV21 through DIV35 BACHD neurons showed progressively smaller numbers of synapses relative to WT neurons. Remarkably, BACHD synaptic deficits were completely rescued by treating cultures with BDNF. Building on earlier studies using reagents inspired by the chaperonin TRiC, we found that addition of the recombinant apical domain of CCT1 partially rescued synapse number. Unexpectedly, unlike BACHD cultures, synapses in ΔN17-BACHD cultures showed a progressive increase in number as compared to WT neurons, thus distinguishing synaptic changes in these HD models. Using multielectrode arrays, we discovered age-related functional deficits in BACHD cortical cultures with significant differences present by DIV28. As for synapse number, BDNF treatment prevented most synaptic deficits, including mean firing rate, spikes per burst, inter-burst interval, and synchrony. The apical domain of CCT1 showed similar, albeit less potent effects. These data are evidence that deficits in HD synapse number and function can be replicatedin vitroand that treatment with either BDNF or a TRiC-inspired reagent can prevent them. Our findings support the use of cellular models to further explicate HD pathogenesis and its treatments.

Huntington’s Disease (HD) is caused by an expanded CAG repeat in the huntingtin gene, yielding a Huntingtin protein with an expanded polyglutamine tract. Patient-derived induced pluripotent stem cells (iPSCs) can help understand disease; however, defining pathological biomarkers is challenging. Here, we used cryogenic electron tomography to visualize neurites in HD patient iPSC-derived neurons with varying CAG repeats, and primary cortical neurons from BACHD, deltaN17-BACHD, and wild-type mice. In HD models, we discovered mitochondria with enlarged granules and distorted cristae, and thin sheet aggregates in double membrane-bound organelles. We used artificial intelligence to quantify mitochondrial granules, and proteomics showed differential protein content in HD mitochondria. Knockdown of Protein Inhibitor of Activated STAT1 ameliorated aberrant phenotypes in iPSC-neurons and reduced phenotypes in BACHD neurons. We show that integrated ultrastructural and proteomic approaches may uncover early HD phenotypes to accelerate diagnostics and the development of targeted therapeutics for HD.

Recent evidence has uncovered an important role of Rab7 in regulating mitochondrial morphology and function. Missense mutation(s) of Rab7 underlies the pathogenesis of Charcot Marie Tooth 2B (CMT2B) peripheral neuropathy. Herein, we investigated how mitochondrial morphology and function were impacted by the CMT2B associated Rab7V162M mutation in fibroblasts from human CMT2B patients as well as in a knockin mouse model. In contrast to recently published results from studies of using heterologous overexpression systems, our results have demonstrated significant mitochondrial fragmentation in fibroblasts of both human CMT2B patients and CMT2B mouse embryonic fibroblasts (MEFs). Furthermore, we have shown that mitochondria were fragmented and axonal mitochondrial movement was dysregulated in primary cultured E18 dorsal root ganglion (DRG) sensory neurons, but not in E18 hippocampal and cortical primary neurons. We also show that inhibitors to either the mitochondrial fission protein Drp1 or to the nucleotide binding to Rab7 normalized the mitochondrial deficits in both MEFs and E18 cultured DRG neurons. Our study has revealed, for the first time, that expression of CMT2B Rab7 mutation at physiological level enhances Drp1 activity to promote mitochondrial fission, that may potentially underlie selective vulnerability of peripheral sensory neurons in CMT2B pathogenesis.

Brain-Derived Neurotrophic Factor (BDNF) is broadly expressed in many circuits of the central nervous system (CNS). It binds TrkB and p75 to trigger different signaling pathways, including ERK1/2 and PI3K-mTOR, to induce dendritic growth and synaptic plasticity. When binding to BDNF, TrkB and p75 are endocytosed to signaling endosomes to continue signaling inside the cell. Whether BDNF/TrkB-p75 signaling endosomes in axons are regulating long-distance signaling in cell bodies to modify neuronal morphology is unknown. Here, we studied the functional role of BDNF signaling endosomes in long-distance regulation of dendritic growth using compartmentalized cultures of rat and mouse cortical neurons derived from p75exonIII knock-out or TrkBF616A knock-in mice. By applying BDNF to distal axons, we showed the capacity of axonal BDNF to increase dendritic arborization in cell bodies. This process depended on TrkB activity, but not p75 expression. In axons, BDNF/TrkB co-localized with Rab5 endosomes and increased active Rab5. Also, dynein was required for BDNF long-distance signaling, consistent with sorting and transport of signaling endosomes. Using neurons derived from TrkBF616A knock-in mice and the 1NM-PP1 inhibitor, we were able to demonstrate that TrkB receptors activated in the axons by BDNF, were required in the neuronal cell body to increase TrkB activity and phosphorylation of CREB. Also, we were able to visualize endosomes containing activated TrkB. PI3K activity was not required in the axons for dynein dependent BDNF responses. However, dendritic arborization induced by axonal BDNF signaling required both nuclear CREB and PI3K activation in cell bodies. Consistently, axonal BDNF increased protein translation in cell bodies and CREB and PI3K and mTOR activity were required for this process. Altogether, these results show that BDNF/TrkB signaling endosomes generated in axons allows long-distance control of dendritic growth coordinating both transcription and protein translation. Our results suggest a role of BDNF-TrkB signaling endosomes wiring circuits in the CNS.