Excess Rab4 rescues synaptic and behavioral dysfunction caused by defective HTT-Rab4 axonal transport in Huntington’s disease

Abstract

Huntington's disease (HD) is characterized by protein inclusions and loss of striatal neurons which result from expanded CAG repeats in the poly-glutamine (polyQ) region of the huntingtin (HTT) gene. Both polyQ expansion and loss of HTT have been shown to cause axonal transport defects. While studies show that HTT is important for vesicular transport within axons, the cargo that HTT transports to/from synapses remain elusive. Here, we show that HTT is present with a class of Rab4-containing vesicles within axons in vivo. Reduction of HTT perturbs the bidirectional motility of Rab4, causing axonal and synaptic accumulations. In-vivo dual-color imaging reveal that HTT and Rab4 move together on a unique putative vesicle that may also contain synaptotagmin, synaptobrevin, and Rab11. The moving HTT-Rab4 vesicle uses kinesin-1 and dynein motors for its bi-directional movement within axons, as well as the accessory protein HIP1 (HTT-interacting protein 1). Pathogenic HTT disrupts the motility of HTT-Rab4 and results in larval locomotion defects, aberrant synaptic morphology, and decreased lifespan, which are rescued by excess Rab4. Consistent with these observations, Rab4 motility is perturbed in iNeurons derived from human Huntington's Disease (HD) patients, likely due to disrupted associations between the polyQ-HTT-Rab4 vesicle complex, accessory proteins, and molecular motors. Together, our observations suggest the existence of a putative moving HTT-Rab4 vesicle, and that the axonal motility of this vesicle is disrupted in HD causing synaptic and behavioral dysfunction. These data highlight Rab4 as a potential novel therapeutic target that could be explored for early intervention prior to neuronal loss and behavioral defects observed in HD.

Huntingtin and the Synapse

“…Indeed, in Drosophila , knocking out the Drosophila homolog of wtHTT was found to slow axonal trafficking of synaptotagmin-containing SVs ( Zala et al, 2013 ). More recent evidence demonstrates that wtHTT moves along the axon together with Rab4 + SVs that also contain synaptic SNARE proteins synaptotagmin and synaptobrevin ( White et al, 2020 ). Rab4 is a Rab GTPase that plays a key role synaptic homeostasis by controlling the recycling and degradation of synaptic vesicles ( Dey et al, 2017 ).…”

“…The bidirectional movement of these Rab4 + vesicles was mediated by interactions with HIP1, rather than HAP1, and the molecular motors kinesin-1 and dynein. RNAi-mediated wtHTT reduction reduced the axonal mobility of Rab4 + vesicles ( White et al, 2020 ). Together, wtHTT appears to facilitate the axonal trafficking of a variety of cargos, many of which have essential roles in presynaptic fidelity and SV maintenance.…”

Real-Time Three-Dimensional Tracking of Single Vesicles Reveals the Abnormal Motion and Vesicle Pools of Synaptic Vesicles in Neurons of Huntington’s Disease Mice

“…Recently, Rab4, which coordinates vesicle trafficking, was reported to be affected in HD (White et al, 2020). Given that the huntingtin protein can interact with many proteins, some of which may have additional binding partners (Shirasaki et al, 2012), indirect interactions with the mutant huntingtin protein may contribute-at least in part-to the observed abnormal synaptic vesicle dynamics in HD neurons.…”

Differential mitochondrial roles for α-synuclein in DRP1-dependent fission and PINK1/Parkin-mediated oxidation

“…Larvae expressing MitoTimer, Mito-roGFP2-ORP1, or Mito-roGFP2-GRX1 were dissected and imaged under physiological conditions in dissection buffer as previously described [ 71 ]. MitoTimer-568nm/MitoTimer-488nm or Mito-roGFP2-488 nm/Mito-roGFP2-405 nm were simultaneously visualized within larval segmental nerves using a NikonTE-2000E inverted fluorescence microscope with a beam splitter containing narrow single-band GFP/DsRED or YFP/GFP filters, a Cool Snap HQ cooled CCD camera, and a ProScan II high speed shutter (100 mm/s) for simultaneous imaging as previously done [ 72 , 73 ]. For each larva, four sets of movies at an imaging window frame size of 100 Microns at 150 frames were taken from the middle region of the larvae at an exposure of 500 ms.…”

“…The lysate was centrifuged at 1000 × g for 10 min at 4 °C and the resulting PNS was incubated overnight with α-syn antibody (BD Biosciences) at 4 °C. Protein A/G Magnetic Beads (Pierce) were then added, incubated at room temperature for 1 h, and eluted in low pH elution buffer (Pierce) as previously described [ 73 ]. The low pH was neutralized (Tris pH 8.8) and the concentration of the α-syn pull down was determined (Bicinchoninic acid (BCA) protein assay, Pierce).…”