Transneuronal propagation of mutant huntingtin contributes to non–cell autonomous pathology in neurons

Journal name:
Nature Neuroscience
Volume:
17,
Pages:
1064–1072
Year published:
DOI:
doi:10.1038/nn.3761
Received
Accepted
Published online

Abstract

In Huntington's disease (HD), whether transneuronal spreading of mutant huntingtin (mHTT) occurs and its contribution to non–cell autonomous damage in brain networks is largely unknown. We found mHTT spreading in three different neural network models: human neurons integrated in the neural network of organotypic brain slices of HD mouse model, an ex vivo corticostriatal slice model and the corticostriatal pathway in vivo. Transneuronal propagation of mHTT was blocked by two different botulinum neurotoxins, each known for specifically inactivating a single critical component of the synaptic vesicle fusion machinery. Moreover, healthy human neurons in HD mouse model brain slices displayed non–cell autonomous changes in morphological integrity that were more pronounced when these neurons bore mHTT aggregates. Altogether, our findings suggest that transneuronal propagation of mHTT might be an important and underestimated contributor to the pathophysiology of HD.

At a glance

Figures

  1. Maturation of hESC-derived neurons co-cultured in mouse wild-type OTBSs.
    Figure 1: Maturation of hESC-derived neurons co-cultured in mouse wild-type OTBSs.

    (a) hGFP neurons at 2, 4 and 6 weeks of co-culture in OTBSs. (b) Morphometric analysis of hGFP neurons at 2, 4 and 6 weeks of co-culture. Left, cell body diameter (*P = 0.018, t = 2.82, df = 100). Middle, number of primary (1°) and secondary (2°) neurites (unpaired Student's t test; ***P < 0.0001, t = 4, df = 100; **P = 0.0015, t = 3.27, df = 100). Right, length of the longest 1° neurite (unpaired Student's t test; ##P < 0.004, t = 2.94, df = 100; #P = 0.030, t = 2.20, df = 100; 2 and 4 weeks, n = 51 cells; 6 weeks, n = 60 cells; 12 slices from 4 mice were analyzed for each time point). Data are presented as mean ± s.e.m. (c) Expression of dcx and NeuN in hGFP neurons at 1 and 6 weeks of co-culture. (d) Quantification of c (unpaired Student's t test; dcx: ***P < 0.0001, t = 21.23, df = 105; 1 week, n = 51 cells; 6 weeks, n = 56 cells; 12 slices from 4 mice were analyzed for each time point; NeuN: ###P < 0.0001, t = 10.69, df = 146; 1 week, n = 70; 6 weeks, n = 78 cells; at least 12 slices from 4 mice were analyzed for each time point). Data are presented as mean ± s.e.m. (e) Representative current-voltage relationships recorded in a hGFP neuron co-cultured for 4 weeks. Top, recordings in current-clamp mode showing hyper- and depolarization of the membrane potential and action potential firing. Bottom, recordings showing fast inward sodium currents followed by slow outward potassium currents. This experiment was successfully repeated three times. We did not encounter any issue with repeatability. (f) Bottom left, illustration of an OTBS/hGFP neuron co-culture in which AAV-ChRII(tdimer)–infected mouse neurons are focally stimulated using blue light (blue circle) and illustrating a recording from a ChRII(tdimer)-expressing mouse neuron (representative image, top left). Right, extracellular recordings of inward currents from the mouse cell when blue light was ON (top traces), block of inward currents in presence of TTX (lower traces). This experiment was successfully repeated five times. We did not encounter any issues with repeatability. aCSF, artificial cerebrospinal fluid. (g) Bottom left, illustration as in f showing a recording from a synaptically connected hGFP neuron (representative image in top left). Right, extracellular recordings of inward currents from the mouse cell when blue light was ON (top traces), and block of inward currents in presence of CNQX and AP5 (lower traces). Quantification is shown in h. (h) Magnitude of eEPSCs (pA) recorded in three hGFP neurons synaptically connected with AAV-ChRII(tdimer)–infected mouse neurons after blue light stimulation and blocking by CNQX and AP5. Data points are presented with mean ± s.e.m. (paired Student's t test, **P < 0.001, n = 3 cells from 3 sections from 3 mice).

  2. Non-cell autonomous morphological changes in hGFP neurons cultured in R6/2 OTBSs.
    Figure 2: Non–cell autonomous morphological changes in hGFP neurons cultured in R6/2 OTBSs.

    (a) Morphometric analysis of hGFP neurons at 2, 4 and 6 weeks of co-culture in wild-type (WT) as compared with R6/2 OTBSs. Left, length of the longest 1° neurite (***P < 0.0001, t = 4.66, df = 119; wild type at 2 weeks, n = 50; 4 weeks, n = 62; 6 weeks, n = 67 cells; R6/2 at 2 weeks, n = 48; 4 weeks, n = 51; 6 weeks, n = 54 cells). Middle, numbers of 1° neurites (4 weeks (R6/2) versus 6 weeks (R6/2), **P = 0.008, t = 2.70, df = 108; 6 weeks (wild type) versus 6 weeks (R6/2), ##P = 0.0037, t = 2.97, df = 112). Right, numbers of 2° neurites (#P = 0.008, t = 2.69, df = 129; ###P < 0.0001, t = 6.13, df = 105; wild type at 2 weeks, n = 50 cells; 4 weeks, n = 62 cells; 6 weeks, n = 67 cells; R6/2 at 2 weeks, n = 48 cells; 4 weeks, n = 51 cells; 6 weeks, n = 54 cells; 12 slices from 4 mice were analyzed for each time point). (b) Colocalization of synaptophysin with hGFP neurons in R6/2 compared with wild-type OTBSs at 6 weeks of co-culture. (c) Percentage of synaptophysin-positive puncta colocalizing with hGFP neurons in wild-type and R6/2 OTBSs at 4 and 6 weeks (*P = 0.045, t = 2.01, df = 100; wild type at 4 weeks, n = 210 cells; 6 weeks, n = 200 cells; R6/2 at 4 weeks, n = 192 cells; 6 weeks, n = 151 cells; 20 slices from 5 mice were analyzed for each time point). (d) Percentage of DARPP-32+ hGFP neurons normalized to the total number of hGFP neurons in wild-type and R6/2 OTBSs at different time points (**P = 0.0063, t = 2.78, df = 108; *P = 0.034, t = 2.15, df = 110; wild type at 2 weeks, n = 51 cells; 4 weeks, n = 56 cells; 6 weeks, n = 48 cells; 8 weeks, n = 51 cells; R6/2 at 2 weeks, n = 51 cells; 4 weeks, n = 54 cells; 6 weeks, n = 50 cells; 8 weeks, n = 61 cells; at least 11 slices from 4 mice were analyzed for each time point). (e) hGFP neuron, located in the striatal region, expressing GAD65 and Tuj1. This experiment was successfully repeated four times. We did not encounter any issues with repeatability (unpaired Student's t test). Data are presented as mean ± s.e.m.

  3. Transneuronal spreading of mHTT.
    Figure 3: Transneuronal spreading of mHTT.

    (a) hGPF neurons in R6/2 OTBSs containing mHTT aggregates located either in boutons (top), the cytoplasm (middle), the nucleus (bottom, white arrow) or in close proximity to a hGFP neuron (bottom, yellow arrow). Rectangles on low-magnification images indicate areas of xyz cuts. (b) Percentages of hGFP neurons with mHTT aggregates, as normalized to the total number of hGFP neurons located in R6/2 OTBS striatum (left) and cortex (right) (striatum: 2 weeks, n = 112 cells; 3 weeks, n = 81 cells; 4 weeks, n = 125 cells; 6 weeks, n = 144 cells; 8 weeks, n = 13 cells; cortex: 2 weeks, n = 162 cells; 3 weeks, n = 137 cells; 4 weeks, n = 75 cells; 6 weeks, n = 116 cells; 8 weeks, n = 64 cells; at least 15 slices from 6 mice were analyzed for each time point). (c,d) Left, low magnification of hGFP-iPSC–derived neurons in R6/2 OTBS after 4 weeks of co-culture. Right, xyz cut of boxed area showing a mHTT aggregate located inside an hGFP-iPSC–derived neuron. Data are presented as mean ± s.e.m. This experiment was successfully repeated three times. We did not encounter any issues with repeatability.

  4. Cellular atrophy is associated with the presence of mHTT.
    Figure 4: Cellular atrophy is associated with the presence of mHTT.

    (ad) Morphometric analysis comparing hGFP-neurons cultured either in wild-type or R6/2 OTBSs, with hGFP neurons in R6/2 OTBSs that carry or lack mHTT aggregates at 4 and 6 weeks of co-culture plotted separately. We quantified cell body diameter (a; *P = 0.042, t = 2.11, df = 114; 6 weeks (wild type) versus 6 weeks (with mHTT), **P = 0.0064, t = 2.78, df = 100; 6 weeks (w/o mHTT) versus 6 weeks (with mHTT), ##P = 0.006, t = 2.81, df = 100), the number of 1° neurites (b; 4 weeks (with mHTT) versus 6 weeks (with mHTT), ***P < 0.0001, t = 4.69, df = 114; 6 weeks (wild type) versus 6 weeks (with mHTT), ***P < 0.0001, t = 4.88, df = 100; 6 weeks (w/o mHTT) versus 6 weeks (with mHTT), ***P < 0.0001, t = 4.47, df = 100), the number of 2° neurites (c; *P = 0.025, t = 1.97, df = 114; 4 weeks (with mHTT) versus 6 weeks (with mHTT), **P = 0.0045, t = 2.65, df = 114; 6 weeks (wild type) versus 6 weeks (w/o mHTT), #P = 0.0021, t = 3.16, df = 100; 6 weeks (wild type) versus 6 weeks (with mHTT), ##P = 0.0051, t = 2.86, df = 100; 6 weeks (w/o mHTT) versus 6 weeks (with mHTT), ###P = 0.01, t = 2.62, df = 100) and the length of the longest 1° neurite (d; 6 weeks (wild type) versus 6 weeks (w/o mHTT), ***P < 0.0001, t = 4.11, df = 100; 6 weeks (wild type) versus 6 weeks (with mHTT), ###P < 0.0001, t = 5.33, df = 100; at 4 weeks:wild type, n = 58 cells; R6/2 w/o mHTT, n = 54 cells; R6/2 with mHTT, n = 65 cells; at 6 weeks:wild type, n = 51 cells; R6/2 w/o mHTT, n = 51 cells; R6/2 with mHTT, n = 51 cells; at least 12 slices from 4 mice were analyzed for each time point; unpaired Student's t test). Data are presented as mean ± s.e.m.

  5. Propagation of mHTT aggregate pathology in mixed-genotype corticostriatal brain slice cultures.
    Figure 5: Propagation of mHTT aggregate pathology in mixed-genotype corticostriatal brain slice cultures.

    (a) Illustration of mixed-genotype corticostriatal brain slice cultures consisting of either wild-type cortex (WT(cx)), wild-type striatum (WT(st)), or R6/2 cortex (R6/2(cx)) and WT(st). The red pipette indicates the deep cortical layer in which glutamate was puffed and the blue pipette indicates the position in striatum from where postsynaptic responses were recorded. (b) NF staining of the corresponding mixed-genotype cultures in a. (c) Representative traces of eEPSCs recorded from striatal neurons in slice cultures from WT(cx)/WT(st) and R6/2(cx)/WT(st) as depicted in a. The pink bar reflects the 10-ms time window in which glutamate was puffed. Gray dashed lines denote basal holding currents before puffing glutamate. (d) eEPSC amplitudes (pA) recorded in WT(cx)/WT(st) and R6/2(cx)/WT(st) slices (n = 5 recordings from 4 slices from 3 mice). (e) eEPSC amplitudes recorded in R6/2(cx)/WT(st) slices before (baseline) and after bath application of TTX (1 μM) (paired Student's t test; ***P = 0.0037, t = 8.24, df = 3, n = 4 recordings from 4 slices from 3 mice). Trace on the right is an example of a single recording. (f) Presence of mHTT aggregates in R6/2(cx)/WT(st) and absence of mHTT aggregates in the corresponding WT(cx)/WT(st) mixed-genotype culture. (g) A mHTT aggregate inside a NF-positive R6/2 corticostriatal process in the striatum of a R6/2(cx)/WT(st) mixed-genotype culture. (h) A mHTT aggregate inside a DARPP-32+ MSN located in WT(st) of a mixed-genotype culture with R6/2(cx). Experiments (b,f,g,h) were successfully repeated three times. We did not encounter any issues with repeatability. Data points are presented with mean ± s.e.m.

  6. Transneuronal propagation of mHTT in vivo.
    Figure 6: Transneuronal propagation of mHTT in vivo.

    (a) Low-magnification image of a corticostriatal section showing the injection site (inj) in cortex (cx) in which two viruses (synaptophysin-GFP and Q72-Htt-Exon1) were co-delivered. The positions of high-magnification images shown in bd are indicated. (b) Expression of synaptophysin-GFP and Q72-Htt-Exon1 in cortical neurons at the injection site. (c,d) DARPP-32+ MSNs located in the striatal area innervated and marked by synaptophysin-GFP corticostriatal terminals (c). For comparison, a non-innervated striatal part is shown (d). Scale bar in d applies to image in c. (eg) xyz cuts of DARPP-32+ MSNs located in striatal areas with a rich network of synaptophysin-GFP–labeled presynaptic terminals as shown in c. Q72-Htt-Exon1 partially colocalized with (e) and was observed in close proximity to (f) synaptophysin-GFP. (f,g) Intracellular Q72-Htt-Exon1 aggregates in DARPP-32+ MSNs located in either cytoplasm (f) or nucleus (g). For better visualization of Q72-Htt-Exon1 and synaptophysin-GFP, DARPP-32 was omitted from images displayed on the right. Quantification is shown in (h). (h) Percentages of DARPP-32+ cells with mHTT aggregates, as normalized to the total number of DARPP-32+ cells located in areas with high versus low density of synaptophysin-GFP–labeled terminals (unpaired Student's t test, ***P < 0.0001, t = 14.8, df = 900; high syn-GFP, n = 575 cells from 12 sections from 3 mice; low syn-GFP, n = 327 cells from 10 sections from 3 mice). Box plots min to max show median (center line), 25–75th data percentiles (boxes) and data range (whiskers). Experiments were successfully repeated three times. We did not encounter any issues with repeatability.

  7. Transneuronal propagation of mHTT requires synaptic machinery.
    Figure 7: Transneuronal propagation of mHTT requires synaptic machinery.

    (a) High-power magnification of an hGFP neuronal process showing a mHTT aggregate located between synaptophysin and PSD-95. Values on the right indicate the z step distance for each image. (b) Three-dimensional reconstruction of images shown in a. The side view shows the localization of mHTT (red) in between PSD-95 (purple) and synaptophysin (blue). To visualize the localization of mHTT and PSD-95, an hGFP neuron (green) is shown with 40% opacity. (c) Low-magnification image showing a human neuron coexpressing GFP and RFP (arrow) and its presynaptically connected labeled neurons (red (RFP+) neurons). (d) High-magnification image of RFP+ neuron, identified as a mouse neuron by the absence of Tuj1 immunoreactivity (M). (e) Human neuron coexpressing TVA(GFP) and ΔGRabRFP(EnVA); human origin was identified by Tuj1 immunoreactivity. H indicates the position of mHTT. (f) xyz cuts show intracellular mHTT aggregate in the boxed human neuron shown in e. (g) Percentage of hGFP neurons containing mHTT aggregates in R6/2 OTBS co-cultures, either untreated (w/o) or treated with BoNT/A or BoNT/B, starting 2 weeks after initiating the co-cultures (pre-treatment, pre) (unpaired Student's t test, ***P < 0.0001, t = 4.60, df = 153; **P = 0.0013, t = 3.28, df = 158; w/o BoNT/A, n = 91 cells; with BoNT/A, n = 64 cells; w/o BoNT/B, n = 90 cells; with BoNT/B, n = 70 cells). Treatment with BoNT/A 4 weeks after starting the co-culture (post-treatment (post): w/o BoNT/A, n = 85 cells; with BoNT/A, n = 64 cells). At least 15 slices from 4 mice were analyzed for each condition. Data are presented as mean ± s.e.m. The experiment in a was successfully repeated three times. We did not encounter any issues with repeatability. The experiment in cf was successfully repeated three times. We did not encounter any issues with repeatability.

  8. Characterization of hGFP neurons
    Supplementary Fig. 1: Characterization of hGFP neurons

    (a) Schematic representation of sequential experimental steps applied to differentiate human stem cells into neuronal precursors. Nucleofection with CAG-GFP reporter plasmid occurs before starting the co-culture with mouse OTBSs. (b) (top) Schematic representation of a corticostriatal OTBS. Dotted square indicates position of low magnification image. (bottom) Image shows hGPF-neurons (green), synaptophysin (red) and vGlut1 (purple) in mouse OTBS after 4 weeks of co-culture. (c) hGFP-neurons express human antigen (HA) and pan-neuronal marker Tuj1. (d) hGFP-neurons express Tuj1 and lack expression of astroglial marker GFAP. Histogram showing quantification of Tuj1 positive or GFAP positive GFP expressing cells in wt OTBSs at 4 weeks of co-culture (n = 52 cells, from 10 slices from 3 mice). (e) At 4 weeks of co-culture hGFP-neurons formed functional connections with mouse neurons with a probability of 60% (n=5 cells, from 3 sections, from 3 mice).

  9. hGFP neurons express brain region-specific neuronal cell-type markers in 6-week-old wild-type OTBSs.
    Supplementary Fig. 2: hGFP neurons express brain region–specific neuronal cell-type markers in 6-week-old wild-type OTBSs.

    (a) hGFP-neuron located in striatum and expressing the MSN marker DARPP-32. (b) hGFP-neuron located in cortex and expressing the upper cortical layer-specific neuronal marker SATB2. (c) hGFP-neuron located in a deep cortical layer and expressing the layer 6-specific marker Tbr1. (d) hGFP-neuron located in dentate gyrus (DG) and expressing Prox1, a marker for DG granule neurons.

  10. HD-like pathology in corticostriatal R6/2 OTBSs
    Supplementary Fig. 3: HD-like pathology in corticostriatal R6/2 OTBSs

    (a) Representative images showing mHTT aggregate load in R6/2 OTBSs at 2 and 8 weeks of culture. Aggregates were detected using the mHTT-specific antibody EM48. (b) Quantification of mHTT aggregates per 0.8μm diameter surface area between weeks 2 and 8 in R6/2 OTBS striatum (left panel) and cortex (right panel). 2-8 weeks: n = 3 OTBSs. (c) Neurofilament (NF) staining in striatum (st) and cortex (cx) of wt and R6/2 OTBS at 6 weeks. (d) Semi-quantitative results showing greatly reduced neurofilament immunoreactivity in striatum but not cortex of 2, 3 and 6 weeks-old R6/2 OTBSs as compared to wt. (wt 2-6 weeks: n = 30 OTBSs; R6/2 2 weeks: n = 25 OTBSs, 3+6 weeks: n = 30 OTBSs). (e) Representative images showing co-expression of DARPP-32 and GAD65 in a hGFP-neuron located in striatum of a 6-weeks old OTBS. Data is presented as mean ± s.e.m. unpaired Student’s t-test: * p<0.05, ** p<0.01.

  11. mHTT aggregates are located inside hGFP neurons and localization gradually changes from cytoplasmatic to predominantly nuclear
    Supplementary Fig. 4: mHTT aggregates are located inside hGFP neurons and localization gradually changes from cytoplasmatic to predominantly nuclear

    (a) Low and high magnification of hGFP-neuron (green) and EM48+ mHTT aggregates before (pink) and after (red) Triton-X100 treatment. (b) Same sequence of images showing hGFP-neuron containing intracellular mHTT aggregates (red). The images below show xyz-cut illustrating intracellular location of mHTT aggregate. This experiment was successfully repeated three times. We did not encounter any issues with repeatability. (c) Representative images showing mHTT localization in hGFP-neurons after 3 and 6 weeks of co-culture in R6/2 OTBSs. (d) Bar diagram showing the percentages of hGFP-neurons in R6/2 OTBSs with cytoplasmic and nuclear mHTT aggregates at different time points. Upper graph: results for hGFP-neurons located in striatum. Lower graph: results for hGFP-neurons located in cortex. (striatum: wt 2 weeks: n = 112 cells, 3 weeks: n = 81 cells, 4 weeks: n = 125 cells, 6 weeks: n = 144 cells; 8 weeks: n = 13 cells; cortex: 2 weeks: n = 162 cells; 3 weeks: n = 137 cells, 4 weeks: n = 75 cells, 6 weeks: n = 116 cells, 8 weeks: n = 64 cells). (e) Scheme summarizing R6/2 OTBS-related non-cell autonomous morphological changes detected in hGFP-neurons with or without mHTT aggregates at 6 weeks of co-culture. hGFP-neuron in wt OTBSs (left), in R6/2 OTBS without mHTT aggregate (middle) and with mHTT aggregate (right). Morphological differences between hGFP-neurons cultured in wt versus R6/2 OTBSs, independent of the presence of mHTT aggregates, are indicated in light grey, whereas changes correlated with the presence of mHTT are shown in dark grey.

  12. Transneuronal spreading of mHTT to hGFP-iPSC-derived neurons
    Supplementary Fig. 5: Transneuronal spreading of mHTT to hGFP-iPSC–derived neurons

    (a) hGFP-iPSC-derived neuron expressing human antigen (HA). (b) hGFP-iPSC-derived neuron expressing the pan-neuronal marker Tuj1. These experiments were successfully repeated three times. We did not encounter any issues with repeatability.

  13. hGFP neurons acquire mHTT aggregates after transplantation in R6/2 mice
    Supplementary Fig. 6: hGFP neurons acquire mHTT aggregates after transplantation in R6/2 mice

    (a,b) Percentages of hGFP-neurons with mHTT aggregates as normalized to the total number of hGFP-neurons at 4 and 8 weeks post grafting (a) and their cytoplasmic versus nuclear localization (b) (4 weeks: n = 426 cells from 12 sections from 4 mice; 8 weeks: n = 329 cells from 12 sections from 3 mice).. Box plots min to max show median (center line), 25-75th data percentiles (boxes) and data range (whiskers). Unpaired Student’s t-test: * p < 0.05.

    (c) Representative images illustrate mHTT aggregates (arrow) accumulating in hGFP-neurons at 4 and 8 weeks after stereotactic injection into a R6/2 mouse brain.

  14. Propagation of HD-like pathology in mixed-genotype cultures
    Supplementary Fig. 7: Propagation of HD-like pathology in mixed-genotype cultures

    (a) Left panel: Schematic representation of different mixed-genotype cultures consisting of either wt cx and R6/2 st or R6/2 cx and R6/2 st. Middle panel: Anti-NF staining of the corresponding mixed-genotype cultures. Right panel: images illustrate the presence or absence of EM48+ mHTT aggregates in cx and st. (b) Left panel: Schematic representation of mixed-genotype cultures consisting of either wt cx and wt st, R6/2 cx and wt st, wt cx and R6/2 st. Right panels: representative images of the corresponding mixed-genotype cultures. These experiments were successfully repeated three times. We did not encounter any issues with repeatability.

  15. Colocalization of mHTT and clathrin and lack of mHTT in OTBS culture media
    Supplementary Fig. 8: Colocalization of mHTT and clathrin and lack of mHTT in OTBS culture media

    (a) Left panel: Images illustrate co-localization (arrows) of mHTT and clathrin; counterstained with DAPI. Right panel: Bar graph shows percentage of clathrin co-localizing with mHTT in hGFP-neurons normalized to total mHTT aggregates (n = 48 regions of interest from 6 OTBSs).(b) Mesoscale electrochemiluminescence detection of mHTT using 2B7 and MW1 antibodies did not reveal any trace of soluble huntingtin in the supernatant of R6/2 OTBS collected at 2, 3, 4, 5 and 6 weeks in culture (n = 3).

  16. Retrograde monosynaptic rabies viral tracing system
    Supplementary Fig. 9: Retrograde monosynaptic rabies viral tracing system

    (a) Representative images show h-neuron expressing G(V5), TVA(GFP) and ΔGRabRFP(EnVA). (b) Images of a Tuj1 positive h-neuron (arrow H) expressing TVA(GFP), which is synaptically connected with a Tuj1 negative mouse neuron (arrow M). The mouse neuron expresses RFP after retrograde transfer of ΔGRabRFP(EnVA) from the infected human neuron. These experiments were successfully repeated three times. We did not encounter any issues with repeatability.

  17. BoNT/A blocks transneuronal propagation of Alexa 594-labeled mHTTQ72
    Supplementary Fig. 10: BoNT/A blocks transneuronal propagation of Alexa 594–labeled mHTTQ72

    (a) Experimental steps performed to assess transneuronal propagation of Alexa594-labeled mHTTQ72 from mouse cells to hGFP-neurons. (b) (left) Representative image illustrating uptake of exogenously supplied Alexa594-labeled mHTTQ72 in wt OTBS. Right: wt OTBS without mHTTQ72. (c) Representative xyz-cut of mHTTQ72 aggregates located either in close proximity to (yellow arrow) or inside (white arrow) a hGFP-neuron. (d) Bar diagram shows the percentage of hGFP-neurons containing mHTTQ72 aggregates normalized to the total number of hGFP-neurons in wt OTBSs infected with mHTTQ72, treated with or without BoNT/A. (w/o: n = 141 cells, with: n = 78 cells). Data is presented as mean ± s.e.m. unpaired Student’s t-test ** p<0.01.

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Author information

  1. These authors contributed equally to this work.

    • Eline Pecho-Vrieseling &
    • Claus Rieker

Affiliations

  1. Novartis Institutes for Biomedical Research, Basel, Switzerland.

    • Eline Pecho-Vrieseling,
    • Claus Rieker,
    • Sascha Fuchs,
    • Dorothee Bleckmann,
    • Chris Goldstein,
    • Mario Bernhard,
    • Ivan Galimberti,
    • Matthias Müller,
    • Tewis Bouwmeester,
    • Herman van der Putten &
    • Francesco Paolo Di Giorgio
  2. Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

    • Maria Soledad Esposito,
    • Paolo Botta,
    • Andreas Lüthi &
    • Silvia Arber
  3. Biozentrum, University of Basel, Basel, Switzerland.

    • Maria Soledad Esposito &
    • Silvia Arber
  4. National Contest for Life (NCL) Foundation against Batten Disease Hamburg, Germany.

    • Herman van der Putten

Contributions

E.P.-V., C.R., H.v.d.P. and F.P.D. designed the study. E.P.-V. and C.R. performed the experiments with contributions from S.F., D.B., C.G. and M.M. (generation of iPSC- and hESC-derived neurons) and from I.G. and M.B. (organotypic mouse brain slice technology). M.S.E. performed in vivo stereotaxic injections of lenti-mHTT. S.A. contributed to the design of the rabies monosynaptic tracing experiments and in vivo stereotactic injection of lenti-mHTT. P.B. performed optogenetic-based experiments and recordings from mixed-culture OTBS, with input and supervision from A.L. E.P.-V., C.R., H.v.d.P. and F.P.D. wrote the manuscript with input from T.B. and S.A.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Characterization of hGFP neurons (447 KB)

    (a) Schematic representation of sequential experimental steps applied to differentiate human stem cells into neuronal precursors. Nucleofection with CAG-GFP reporter plasmid occurs before starting the co-culture with mouse OTBSs. (b) (top) Schematic representation of a corticostriatal OTBS. Dotted square indicates position of low magnification image. (bottom) Image shows hGPF-neurons (green), synaptophysin (red) and vGlut1 (purple) in mouse OTBS after 4 weeks of co-culture. (c) hGFP-neurons express human antigen (HA) and pan-neuronal marker Tuj1. (d) hGFP-neurons express Tuj1 and lack expression of astroglial marker GFAP. Histogram showing quantification of Tuj1 positive or GFAP positive GFP expressing cells in wt OTBSs at 4 weeks of co-culture (n = 52 cells, from 10 slices from 3 mice). (e) At 4 weeks of co-culture hGFP-neurons formed functional connections with mouse neurons with a probability of 60% (n=5 cells, from 3 sections, from 3 mice).

  2. Supplementary Figure 2: hGFP neurons express brain region–specific neuronal cell-type markers in 6-week-old wild-type OTBSs. (530 KB)

    (a) hGFP-neuron located in striatum and expressing the MSN marker DARPP-32. (b) hGFP-neuron located in cortex and expressing the upper cortical layer-specific neuronal marker SATB2. (c) hGFP-neuron located in a deep cortical layer and expressing the layer 6-specific marker Tbr1. (d) hGFP-neuron located in dentate gyrus (DG) and expressing Prox1, a marker for DG granule neurons.

  3. Supplementary Figure 3: HD-like pathology in corticostriatal R6/2 OTBSs (405 KB)

    (a) Representative images showing mHTT aggregate load in R6/2 OTBSs at 2 and 8 weeks of culture. Aggregates were detected using the mHTT-specific antibody EM48. (b) Quantification of mHTT aggregates per 0.8μm diameter surface area between weeks 2 and 8 in R6/2 OTBS striatum (left panel) and cortex (right panel). 2-8 weeks: n = 3 OTBSs. (c) Neurofilament (NF) staining in striatum (st) and cortex (cx) of wt and R6/2 OTBS at 6 weeks. (d) Semi-quantitative results showing greatly reduced neurofilament immunoreactivity in striatum but not cortex of 2, 3 and 6 weeks-old R6/2 OTBSs as compared to wt. (wt 2-6 weeks: n = 30 OTBSs; R6/2 2 weeks: n = 25 OTBSs, 3+6 weeks: n = 30 OTBSs). (e) Representative images showing co-expression of DARPP-32 and GAD65 in a hGFP-neuron located in striatum of a 6-weeks old OTBS. Data is presented as mean ± s.e.m. unpaired Student’s t-test: * p<0.05, ** p<0.01.

  4. Supplementary Figure 4: mHTT aggregates are located inside hGFP neurons and localization gradually changes from cytoplasmatic to predominantly nuclear (504 KB)

    (a) Low and high magnification of hGFP-neuron (green) and EM48+ mHTT aggregates before (pink) and after (red) Triton-X100 treatment. (b) Same sequence of images showing hGFP-neuron containing intracellular mHTT aggregates (red). The images below show xyz-cut illustrating intracellular location of mHTT aggregate. This experiment was successfully repeated three times. We did not encounter any issues with repeatability. (c) Representative images showing mHTT localization in hGFP-neurons after 3 and 6 weeks of co-culture in R6/2 OTBSs. (d) Bar diagram showing the percentages of hGFP-neurons in R6/2 OTBSs with cytoplasmic and nuclear mHTT aggregates at different time points. Upper graph: results for hGFP-neurons located in striatum. Lower graph: results for hGFP-neurons located in cortex. (striatum: wt 2 weeks: n = 112 cells, 3 weeks: n = 81 cells, 4 weeks: n = 125 cells, 6 weeks: n = 144 cells; 8 weeks: n = 13 cells; cortex: 2 weeks: n = 162 cells; 3 weeks: n = 137 cells, 4 weeks: n = 75 cells, 6 weeks: n = 116 cells, 8 weeks: n = 64 cells). (e) Scheme summarizing R6/2 OTBS-related non-cell autonomous morphological changes detected in hGFP-neurons with or without mHTT aggregates at 6 weeks of co-culture. hGFP-neuron in wt OTBSs (left), in R6/2 OTBS without mHTT aggregate (middle) and with mHTT aggregate (right). Morphological differences between hGFP-neurons cultured in wt versus R6/2 OTBSs, independent of the presence of mHTT aggregates, are indicated in light grey, whereas changes correlated with the presence of mHTT are shown in dark grey.

  5. Supplementary Figure 5: Transneuronal spreading of mHTT to hGFP-iPSC–derived neurons (274 KB)

    (a) hGFP-iPSC-derived neuron expressing human antigen (HA). (b) hGFP-iPSC-derived neuron expressing the pan-neuronal marker Tuj1. These experiments were successfully repeated three times. We did not encounter any issues with repeatability.

  6. Supplementary Figure 6: hGFP neurons acquire mHTT aggregates after transplantation in R6/2 mice (260 KB)

    (a,b) Percentages of hGFP-neurons with mHTT aggregates as normalized to the total number of hGFP-neurons at 4 and 8 weeks post grafting (a) and their cytoplasmic versus nuclear localization (b) (4 weeks: n = 426 cells from 12 sections from 4 mice; 8 weeks: n = 329 cells from 12 sections from 3 mice).. Box plots min to max show median (center line), 25-75th data percentiles (boxes) and data range (whiskers). Unpaired Student’s t-test: * p < 0.05.

    (c) Representative images illustrate mHTT aggregates (arrow) accumulating in hGFP-neurons at 4 and 8 weeks after stereotactic injection into a R6/2 mouse brain.

  7. Supplementary Figure 7: Propagation of HD-like pathology in mixed-genotype cultures (819 KB)

    (a) Left panel: Schematic representation of different mixed-genotype cultures consisting of either wt cx and R6/2 st or R6/2 cx and R6/2 st. Middle panel: Anti-NF staining of the corresponding mixed-genotype cultures. Right panel: images illustrate the presence or absence of EM48+ mHTT aggregates in cx and st. (b) Left panel: Schematic representation of mixed-genotype cultures consisting of either wt cx and wt st, R6/2 cx and wt st, wt cx and R6/2 st. Right panels: representative images of the corresponding mixed-genotype cultures. These experiments were successfully repeated three times. We did not encounter any issues with repeatability.

  8. Supplementary Figure 8: Colocalization of mHTT and clathrin and lack of mHTT in OTBS culture media (194 KB)

    (a) Left panel: Images illustrate co-localization (arrows) of mHTT and clathrin; counterstained with DAPI. Right panel: Bar graph shows percentage of clathrin co-localizing with mHTT in hGFP-neurons normalized to total mHTT aggregates (n = 48 regions of interest from 6 OTBSs).(b) Mesoscale electrochemiluminescence detection of mHTT using 2B7 and MW1 antibodies did not reveal any trace of soluble huntingtin in the supernatant of R6/2 OTBS collected at 2, 3, 4, 5 and 6 weeks in culture (n = 3).

  9. Supplementary Figure 9: Retrograde monosynaptic rabies viral tracing system (334 KB)

    (a) Representative images show h-neuron expressing G(V5), TVA(GFP) and ΔGRabRFP(EnVA). (b) Images of a Tuj1 positive h-neuron (arrow H) expressing TVA(GFP), which is synaptically connected with a Tuj1 negative mouse neuron (arrow M). The mouse neuron expresses RFP after retrograde transfer of ΔGRabRFP(EnVA) from the infected human neuron. These experiments were successfully repeated three times. We did not encounter any issues with repeatability.

  10. Supplementary Figure 10: BoNT/A blocks transneuronal propagation of Alexa 594–labeled mHTTQ72 (374 KB)

    (a) Experimental steps performed to assess transneuronal propagation of Alexa594-labeled mHTTQ72 from mouse cells to hGFP-neurons. (b) (left) Representative image illustrating uptake of exogenously supplied Alexa594-labeled mHTTQ72 in wt OTBS. Right: wt OTBS without mHTTQ72. (c) Representative xyz-cut of mHTTQ72 aggregates located either in close proximity to (yellow arrow) or inside (white arrow) a hGFP-neuron. (d) Bar diagram shows the percentage of hGFP-neurons containing mHTTQ72 aggregates normalized to the total number of hGFP-neurons in wt OTBSs infected with mHTTQ72, treated with or without BoNT/A. (w/o: n = 141 cells, with: n = 78 cells). Data is presented as mean ± s.e.m. unpaired Student’s t-test ** p<0.01.

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