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.
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Change history
17 July 2018
In the version of this article initially published, the catalog numbers for BoNT A and B were given in the Methods section as T0195 and T5644; the correct numbers are B8776 and B6403. The error has been corrected in the HTML and PDF versions of the article.
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Acknowledgements
We thank A. Weiss (Promidis) for providing the mHTTQ72. D. Shimshek, T. Haffner and C. Stemmelen for help with stereotactic injection, C. Schöffel-Mattes for help with electrophysiological recording, T. Doll and I. Fruh for help with cell culture, P. Caroni, F.H. Gage and K. Eggan for comments on the paper, and R. Kuhn and G. Bilbe for providing valuable scientific input and continuous support. M.S.E. was supported by a long-term fellowship of the Human Frontier Science Program, and S.A. by a European Research Council Advanced Grant and the Swiss National Science Foundation. P.B. and A.L. are supported by the Novartis Research Foundation.
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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.
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Integrated supplementary information
Supplementary Figure 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).
Supplementary Figure 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.
Supplementary Figure 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.
Supplementary Figure 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.
Supplementary Figure 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.
Supplementary Figure 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.
Supplementary Figure 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.
Supplementary Figure 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).
Supplementary Figure 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.
Supplementary Figure 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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Pecho-Vrieseling, E., Rieker, C., Fuchs, S. et al. Transneuronal propagation of mutant huntingtin contributes to non–cell autonomous pathology in neurons. Nat Neurosci 17, 1064–1072 (2014). https://doi.org/10.1038/nn.3761
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DOI: https://doi.org/10.1038/nn.3761
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