UAS-Rab4-mRFP or UAS-HTT15Q-mRFP males were crossed with Appl-GAL4 or Appl-GAL4; T(2,3), CyO, TM6B, Tb1 / Pin88k virgin females. The chromosome carrying T(2:3),CyO,TM6B,Tb is referred to as B3 and carries the dominant markers, Hu, Tb and CyO. The larval Tb (tubby) marker is used to select larvae of interest. Males that were Appl-GAL4/Y;UAS-Rab4-mRFP/B3, Appl-GAL4/Y;UAS-YFP-Rab4/B3, Appl-GAL4/Y;UAS-HTT15Q-mRFP/B3 were crossed with either Drosophila htt-RNAi , Df(98E2);CG9990 , Rab4MI10530 (BDSC), klc8ex94/TM6B (Goldstein), roblk/B3 (Goldstein), UAS-HTTex1-25Q-eGFP (Perrimon), UAS-APP-YFP , UAS-Syt-GFP, UAS-nSyb-GFP/TM3, UAS-Rab11-GFP, UAS-YFP-Rab3, klp64Dk1/TM3, unc-104d11204/CyO, milt[k04704]/CyO, hip1[MI05905], nmo[P1]/TM6B, nuf[MB09772], or rip11[KG02485]/FM7c (BDSC), UAS-rip11-GFP.CT (Ready, BDSC), UAS-HTT16Q (Vitruvean, BDSC) UAS-HTT128Q (Vitruvean, BDSC), UAS-HTTex1-72Q-eGFP (Perrimon), UAS-HTTex1-103Q-eGFP (Perrimon) or UAS-HTT138Q-mRFP (Littleton) virgins. In all cases, non-tubby female 3rd instar larvae were selected. Sibling tubby larvae were evaluated as controls. Reciprocal crossings were also done to confirm observations. A comprehensive list of Drosophila strains used in this study can be found in Table S1.
In-vivo analysis of vesicle motility within whole mount larval axons
Larvae were dissected and immediately imaged under physiological conditions as previously detailed in White et al. [4, 77]. Non-tubby, female larvae were dissected and imaged under physiological conditions in dissection buffer. Motility was visualized in the red or green, channels using a Nikon TE-2000E inverted fluorescence microscope with a Cool Snap HQ cooled CCD camera (Photometrics, Tucson, AZ, USA), and a ProScan II high speed shutter (100 mm/s) (Nikon, Melville, NY, USA). From each larva, four sets of movies at an imaging window frame size of 90 μm at 150 frames were taken from the mid-region of the larva at an exposure of 500 ms using the Metamorph imaging system (Molecular Devices, Sunnyvale, CA, USA). From a total of 10 larvae a set of 40 movies were imaged for each genotype at a spatial resolution of 0.126 μm/pixel. Movies were analyzed using a MATLAB-based particle tracker program as previously detailed [25, 59, 77]. Briefly, a standard data set consisted of four movies, each lasting 75 s span a total time of 6 min recorded for 10 individual animals. A band (5 pixels in thickness) flanking the axon is extracted from each frame. Bands from all frames are pasted top-to-bottom to form a kymograph computationally. Computationally recovered vesicle trajectories were color-coded and overlaid on the kymograph; colors were selected randomly to differentiate crossing trajectories. Truncated vesicle trajectories are excluded for each movie. Full trajectories are analyzed as detailed in Reis et al. 2012  and Gunawardena et al. 2013 . Vesicle trajectories were analyzed to obtain the overall distribution of cargo populations and individual vesicle movement behaviors (velocities, pause frequencies/durations, run lengths) as previously done [25, 59, 77]. As detailed in Reis et al. 2012  and Gunawardena et al. 2013 , the anterograde (retrograde) duration-weighted segmental velocity of a cargo within its track is defined as the sum of its anterograde (retrograde) segmental velocities weighted by their durations and divided by the sum of the durations. Pause frequency details the total number of times a cargo pauses divided by its total time in movement. Pause duration evaluates the total time a cargo pauses within its directional track. Run length is a description of the total distance a cargo moves in a particular direction before pausing or changing direction.
Simultaneous dual-color in vivo imaging and co-migration analysis in whole mount larval axons
Simultaneous dual-view imaging was done as detailed previously in Banerjee et al. 2020 . Briefly, we used a NikonTE-2000E inverted fluorescence microscope with a beam splitter containing narrow single-band GFP/DsRED filters, a Cool Snap HQ cooled CCD camera, and a ProScan II high speed shutter (100 mm/s) for simultaneous imaging. Movies were taken in Dual-View mode using the split view software in Metamorph at 150 frames from the mid-region of the larvae at an exposure of 500 ms to simultaneously image RFP- and GFP- tagged vesicles. We routinely checked for bleed through by imaging each fluorophore with the individual filters, As seen in Fig. 2 not all fluorophores overlap. In some movies none of the red or green tracks overlap (Rab4-mRFP and YFP-Rab3, Rab4-mRFP and APP-YFP) indicating the specificity of our system. The Cool Snap HQ camera Dual-View mode was aligned using Metamorph software (Split-View settings) before each imaging session. Movies were split by wavelength and each kymograph for each split movie was created, merged and analyzed for co-migration. Trajectories of vesicles with co-localized tracks were identified from kymographs using Metamorph software. For each fluorescence channel, a kymograph was generated using Metamorph as previously done [25, 59, 77]. Briefly, after selecting the first channel, all frames within the time-lapse image sequence of this channel were added together to produce a summed image. To identify vesicles with co-localized signals from both channels, the kymographs were colored in red and green, respectively, and combined into a single RGB kymograph. Note that to differentiate meaningful co-localization we evaluated the co-localization of the entire trajectory of a moving particle during the entire time frame of the movie. Therefore, only particles containing the same trajectory in both red and green would show co-localization in yellow when merged and spurious co-localization observed in a one-time frame would be avoided. The total number of co-localized full trajectories for 10 kymographs across five larvae were counted for each genotype. Pearson’s correlation coefficients, percentage of co-localization between red and green trajectories, and two-color 2D intensity histograms were obtained using Coloc2 and EZ colocalization in ImageJ. Briefly, kymographs from Drosophila larval segmental nerves expressing two fluorophores were separated into red and green channels using Metamorph and analyzed using Coloc2 and EZ colocalization in ImageJ.
Immunohistochemistry in Drosophila larval axons and NMJs
Third instar Drosophila larvae were dissected and fixed in 4% paraformaldehyde. KLC (Goldstein, 1:100), DIC (Abcam 1:50), or DCSP-3 (DSHB, 1:50) antibodies were used in conjunction with secondary antibodies anti-mouse or anti-rabbit AlexaFlour®488, AlexFlour®568, or Alexaflour®647 (ThermoFisher, 1:100). Images of fixed larval segmental nerves were taken at 60X or 100x using FITC, TxRed, and Cy5 filters which were merged into a single RGB image to analyze co-localization noted as yellow or white puncta. Axonal blockages were quantified as previously done in Gunawardena et al. . For each genotype, a minimum of 5 confocal optical images across 6 larvae were imaged. Sub-pixel imaging refers to the subpixel detection accuracy of fluorescent puncta, which was previously confirmed by directly comparing Gaussian fitting of conventional microscopy data analyzed to super resolution imaging in . Szpankowski et al., 2012  showed that the same subpixel localization method implemented predicted coordinates of detected puncta accurately compared with structured illumination-based OMX (Applied Precision Instruments) super resolution analysis of the same data. For NMJ analysis, HRP-FITC or HRP-TxRED (Jackson ImmunoResearch Labs) was used (1:50). Non-tubby, female larvae were dissected, fixed, and stained with HRP. Quantification of NMJ morphology and Rab4 accumulations were performed as in Kang et al. . We examined type-1 synaptic boutons between muscles 6/7 at larval abdominal segments A4-A5 of third instar larval. Images of NMJs were collected using a Nikon Eclipse TE 2000 U microscope at 60X (Nikon, Melville, NY, USA). For each genotype, a minimum of 4 optical images across 8 larvae were imaged. Bouton number (#), bouton area (μm2), and total NMJ length (μm) were measured using NIH ImageJ software. Comprehensive list of antibodies used in this study can be found in Table S1.
Quantification of Drosophila larval motility and adult lifespan
Larval velocity and contractions were performed by visualizing third instar larval crawling patterns on 1% agarose gel, dyed blue for added contrast, that was embedded with a 0.25 cm × 0.25 cm grid. Once placed at the center of the agarose gel, a 2-min interval recording began. Each larva was recorded during three independent trials, controlling for temperature (~ 25 °C) and humidity (~ 60%). Twenty larvae were tested per genotype. Quantifications of the number of contractions and the larval velocity were measured using NIH ImageJ software. A total of 1.5 min of the 2-min recording was utilized for quantification purposes allotting the initial 15 s for larva self-adjustment after being placed in the center of the agarose gel. Lifespan analysis was performed by placing three vials of 20 female adult flies for each genotype at 25 °C, 60% humidity. Flies were counted every day for ~ 70 days to tally the number of survivors.
WT and HD iPSC cultures and neuronal differentiation
The following cell lines were obtained from the NIGMS Human Genetic Cell Repository at the Coriell Institute for Medical Research: iPCS from WT (GM23279-polyQ = 25, 26y, female) and HD (GM23225-polyQ = 72, 20y, female) patients were purchased from the Coriell Cell Repository (Camden, NJ) (Table S1). iPSCs from WT (ND38555-polyQ = 17, 48y, female) and HD (ND42222-polyQ = 109, 9y, female) patients were purchased from the NINDS Repository (Table S1). iPSCs were grown and expanded on corning matrigel (Fisher) using E8 iPSC media (Invitrogen). Pluripotency was analyzed using an antibody against OCT-3/4 (Santa Cruz, 1:200) and Hoechst was used as a nuclear staining as detailed below. After 4 passages iPSCs were differentiated into neuronal precursors (NPCs) using PSC neural induction media (Invitrogen) and published protocols (publication #MAN0008031). NPCs were identified using an antibody against Nestin (Santa Cruz, 1:200) and then differentiated into mature iNeurons using neurobasal media supplemented with 1X B27 and 2 mM glutamine (Invitrogen, ThermoFisher). Differentiated neurons, identified using antibodies against MAP2 (BD Biosciences, 1:200), βIII-tubulin (Biolegend, 1:200) and Tyrosine Hydroxylase (EMD Millipore, 1:200) exhibited an extensive neurite network after 21 days at which time they were used for electrophysiology experiments, biochemical experiments, immunofluorescence, and transfections.
Electrophysiology in iNeurons
Whole-cell patch clamp was used to record from cells with neuronal morphology. Borosilicate glass pipettes (World Precision Instruments, Inc., USA, 4–9 MΩ) were filled with (in mM): 135 k-gluconate, 7.5 KCl, 10 phosphocreatine, 10 HEPES, 2 MgATP, 0.3 Na2GTP, pH 7.3 with KOH and adjusted to 290 mOsmol using sucrose. During recording, cells were bathed in (in mM): 140 NaCl, 4 KCl, 2 CaCl2, 2 MgCl2, 10 HEPES, 10 D-glucose, 10 sucrose, pH 7.4 with NaOH. To record voltage-gated Na+/K+ channel activity, a series of depolarizing potentials from -80 mV to 80 mV were applied in 20 mV intervals for 500 msec. Series resistance, whole-cell capacitance, and pipette capacitance were compensated to minimize transient capacitive current artifacts during recording. To inhibit currents, either 100 μM tetrodotoxin (TTX) or 50 mM tetraethylammonium chloride (TEA) was added to the bath via pipette. Currents were low-pass filtered at 2 kHz (Axopatch 200B, 4-pole Bessel) and sampled at 5 kHz (Digidata 1440A). To record action potentials, cells were current-clamped to maintain a holding membrane potential of -65 mV. Cells requiring more than 100pA current injection to maintain -65 mV were discarded. A series of stepwise pulses of current were injected from 0 to + 140 pA in 10pA intervals. Membrane potentials were low-pass filtered at 10 kHz (MultiClamp 700B Commander) and sampled at 20 kHz (Digidata 1440A). All data was recorded in Clampex 10.5 and analyzed in Clampfit 10.2 (Molecular Devices). Voltage-gated Na + channel and voltage-gated K+ channel currents were quantified by peak and steady-state current values, respectively.
In vitro analysis of Rab4 motility within WT and HD iNeurons
21-day-old differentiated WT and HD iNeurons were transfected with a mammalian expression vector expressing mCherry-Rab4a-7 (Addgene#55125, Table S1) using Lipofectamine 3000 (Fisher). Two to four days post-transfection, transfected neurons expressing mCherry-Rab4 were imaged at 100x and vesicle motility was imaged, analyzed and quantified as described for in vivo analysis of vesicle motility in whole mount larval axons.
Immunohistochemistry: human iPSCs/NPCs/iNeurons
WT and HD iPSC/NPC colonies or 21-day old differentiated neurons were washed in PBS pH 7.2 and fixed in 4% paraformaldehyde. Blocking solution (1x PBST with 5% BSA) was added to cells for 60 min prior to antibody incubation. Cells were incubated in primary antibodies, OCT-4 (Santa Cruz 1:200), Nestin (Santa Cruz 1:200), MAP2 (BD Biosciences 1:200) βIII-tubulin (Biolegend 1:200), Tyrosine Hydroxylase (EMD Millipore 1:200), Rab4 (Abcam 1:200), HIP1 (Novus Biological, 1:200), DIC (Abcam, 1:200), or PolyQ (EMD Millipore, 1:200) for 16 h at 4 °C and appropriate secondary antibodies (AlexaFluor® 488 or 568, ThermoFisher) for 1 h at 25 °C. DAPI was used to stain nuclei. Cells were then imaged at 20x-40x (for iPSC and NPC) or 60x-100x (iNeurons). iNeurons were imaged on glass slide bottom dishes (In Vitro Scientific China, D29–14-1-N). As above, fixed images were taken at 100x using DAPI, FITC, TxRed, and Cy5 filters which were merged into a single RGB image to analyze co-localization noted as yellow or white puncta. Comprehensive list of reagents and antibodies used in this study can be found in Table S1.
Preparation of protein extracts from mouse brains and human iNeurons
Mouse brains (gift from K. Medler, C57BL/6) were dissected and halved. Brains were stored on dry ice and used immediately or stored at -80C for future use. Mouse brains were homogenized in homogenization buffer (10 mM HEPES, pH 7.4, 100 mM K acetate, 150 mM sucrose, 5 mM EGTA, 3 mM Mg acetate, 1 mM DTT) containing a cocktail of protease inhibitors (Roche) and 5 mM EDTA. Mouse brain extracts were then centrifuged at 1000 g for 15 min at 4 °C. The supernatant (PNS) was then used for sucrose gradient fractionation analysis as detailed below or for western blotting. WT and HD iNeurons were manually removed from 12-well plates using ice-cold homogenization buffer (as above) and blended for 30 s on ice using a motorized pestle. Neuronal extracts were then centrifuged at 1000 g for 15 min and the supernatant was analyzed using western blot analysis.
Sucrose gradient fractionations
PNS samples from mouse brain or human iNeuron extracts were further fractionated into soluble, heavy membrane (P1), and vesicle fractions (VF) by sucrose gradient ultra-centrifugations as previously done [3, 15] using lysis buffer (4 mM HEPES, 320 mM sucrose pH 7.4) containing a phosphatase and protease inhibitor cocktail (Pierce). Briefly, 300ul of PNS was combined with 300ul 62% sucrose and layered onto a sucrose gradient (35, and 8% sucrose) and centrifuged at ~ 100,000 g for 90 mins. The vesicle fraction (VF, 35/8 layer), the soluble fraction and the heavy membrane fractions were removed and used in western blot analysis. 100ul of homogenization buffer was used to dissolve the heavy membrane pellet (P1).
Isolation of Rab4 vesicles was performed as described [3, 15]. 1000μg of total protein from the mouse or human iNeuron vesicle fraction were incubated with a rabbit monoclonal antibody to Rab4 (Abcam, 1:100) for 1 h at 25 °C. Protein homogenates were then incubated with magnetic beads (Pierce Protein A/G Magnetic Beads) with rotation for 90 min and eluted with Pierce elution buffer (pH 2.8). Eluents were then analyzed by western blot analysis as detailed below. Alternatively, Co-IP was performed on protein homogenates using the Pierce™ Co-Immunoprecipitation Kit. Isolation of Rab4 vesicles using magnetic beads occurred the absence of detergents to preserve vesicular membranes. Eluents were then separated by SDS-PAGE and analyzed via western blot.
SDS-PAGE and Western blot analysis
Mouse or human iNeuron fractions in NuPage LDS sample buffer with 4 mM β-mercaptoethanol were run on 4–12% Bis-Tris gels (Invitrogen) and transferred to nitrocellulose membranes. Blots were blocked using TBST with 5% BSA for 60 mins at 25 °C and incubated with primary antibodies (SYT1 (Phosphosolutions 1:1000), Rab4 (Abcam 1:1000), KIF5C (Goldstein 1:500), DIC (Abcam 1:1000), Actin (ThermoFisher 1:1000), Tubulin (Abcam 1:2000), HTT rabbit polyclonal (Abcam 1:1000), HTT mouse monoclonal (EMD Millipore 1:1000), Hip1 (Novus Biological 1:1000), KDEL (Abcam 1:1000), Golgi (Millipore Sigma 1:1000), Cytochrome C (Santa Cruz 1:1000), HAP1 (Santa Cruz 1:1000), Rab11 (Abcam 1:1000), Rab5 (Abcam, 1:1000), or Syntaxin17 (Juhasz, 1:500) for 16 h at 4 °C. Blots were then incubated with anti-mouse or anti-rabbit secondary HRP-conjugated antibodies (ThermoFisher 1:1000) and imaged using a BioRad Chemi-doc system with Pierce ECL or diluted Femto substrate (1:5 in TBS). Images from 3 to 5 blots were quantified using ImageLab. Comprehensive list of reagents and antibodies used in this study can be found in Table S1.
The statistical analysis used for each experiment is indicated in each figure legend. First power and sample size (n) calculations were performed on minitab18 for each experimental paradigm: comparing 2 means from 2 samples, with two-tailed equality to identify the sample size that corresponds to a power of 0.9 with α = 0.01. For each experiment, a stringent significance threshold of p < 0.01 (99% confidence) was used as detailed in White et al. . Using Minitab18 calculation of sample size for a power of 0.9 and α = 0.01, the n-value for each experiment was determined based on the control data (average and standard error) in each case. For western blot quantifications, α = 0.05. The n-value refers to the number of larvae, the number of flies, the number of mouse brain lysate, or the number of induced human neurons. Individual data points for each quantification was averaged for each n and then compared.
To select the appropriate statistical test, data distributions for each transport dynamic analyzed were first checked for normality using the nortest package of R: the Lilliefors test and Anderson–Darling test as previously detailed [25, 59, 77]. Statistical significance of normal distributions was calculated by a two-sample two-tailed Student’s t-test and/or ANOVA while the non-normal segmental velocity distributions were compared using the non-parametric Wilcoxon–Mann–Whitney rank sum test in Excel and Minitab18. The global velocity averages across each larva were found to be normal distributions, as well as the global pause frequencies, pause durations and run lengths across each larva. For motility dynamic quantifications, duration-weighted segmental velocities each larva (total of 4 movies, > 500 particles) were pooled, then the average global dur-weighted segmental velocities from each larva were averaged (total 4 movies, > 500 particles) before statistical analysis as detailed in [25, 59]. Therefore, statistical analysis was performed on global velocities from each larva rather than individual particle velocities (n = 10 larvae, 4 movies per larvae). Statistical significance was determined using the two-sample two-tailed Student’s t-test.
For statistical analysis on motility dynamics from iNeurons, analysis was performed on individual particle velocities (n = 7, > 250 particles), therefore, the non-parametric Wilcoxon–Mann–Whitney rank sum test was utilized due to non-normality in the data. For immunofluorescence analysis, statistical analysis was performed in Excel and Minitab18 using the two-sample two-tailed Student’s t-test/ANOVA. Differences were considered significant at a significance level of p < 0.01, which means a 99% statistically significant correlation. Based on the power analysis, quantifications were performed across 5, 8, or 10 larvae. For western blots, quantification analysis was performed using Image Lab software. Data obtained from Image Lab was analyzed in Excel and Minitab18 using two-tailed Student’s t-test/ANOVA across three independent experiments (n = 3). Differences for western blots were considered significant at a significance level of p < 0.05. Overlaid dot plots were constructed for all figures using OriginLab / OriginPro.