Mutant huntingtin (mHtt) may now join the ranks of pathogenic proteins that wreak havoc by spreading from one neuron to another. According to a study published July 13 in Nature Neuroscience, mHtt aggregates pass from neurons in the cortex to neurons in the striatum via synaptic vesicles. The researchers used a substantial battery of organ culture and in vivo models to document the hand-off. They reported that medium spiny neurons in the striatum withered after taking up the aggregates. The study, led by Francesco Di Giorgio at Novartis Institutes for Biomedical Research in Basel, Switzerland, opens up the possibility of targeting the spread of pathogenic Htt to slow progression of the disease.

Huntingtin Hand-Off.

After six weeks of commingling with mouse neurons expressing mutant huntingtin (mHtt), a transplanted human neuron (GFP, green) bears an mHtt aggregate (pink). [Image courtesy of Pecho-Vrieseling et al., Nature Neuroscience 2014.]

“This is an elegant series of experiments using interesting and informative models,” Lary Walker of Emory University in Atlanta wrote in an email to Alzforum. “The findings complement a growing literature demonstrating the cell-to-cell spread of pathogenic protein seeds formed by Aβ, PrP, tau, α-synuclein, SOD1, and others.” 

The movement of pathogenic proteins between cells in the brain has been reported in the context of many neurodegenerative diseases marked by the accumulation of toxic protein aggregates, including Alzheimer’s and Parkinson’s diseases and amyotrophic lateral sclerosis (see Guo and Lee, 2014). However, whether such transmission happens in Huntington’s disease (HD) had never been reported in vivo. Scientists debate exactly how the transmission would occur.

HD is an autosomal-dominant disorder caused by excessive CAG repeats in the huntingtin gene. These repeats trigger abnormal splicing of the gene and yield polyglutamine stretches in the protein. Any of the splice variants containing polyglutamine repeats ultimately wind up in aggregates—the hallmark of the disease. Huntingtin inclusions litter the brain in affected people.

Curiously, in HD medium spiny neurons in the striatum contain few Htt aggregates but take the brunt of the damage, dying off in droves (see Gutekunst et al., 1999). The relationship between aggregates and HD neuropathology is mired in controversy, and the loss of medium spiny neurons has been blamed not only on Htt inclusions within the striatum, but also on a breakdown of firing from the cortex (see Arrasate and Finkbeiner, 2012; Ehrlich 2012). Whether the cortex degrades the health of its striatal neighbor by shutting down communication or by handing off toxic mHtt proteins remained to be seen.

Co-first authors Eline Pecho-Vrieseling and Claus Rieker tested the latter. The researchers started by culturing brain slices from R6/2 mice. These express human mHtt that forms aggregates. These organotypic brain slices contained both cortex and striatum, and maintained their neural circuitry for several weeks.

To see whether aggregates could travel along functional circuits, the researchers mixed R6/2 cortex with wild-type striatum. Connections formed between the two. When the researchers stimulated cortical regions with a burst of glutamate, the striatal regions responded with excitatory postsynaptic currents, indicating that the circuits worked. After one month, human mHtt aggregates appeared in the medium spiny neurons. When the researchers used R6/2 striatum—which did not form a functional circuit with the cortex—no transfer of aggregates occurred. The smuggling of aggregates from cortex to striatum, the researchers suggested, only worked when functional circuits were in place.

The same seemed true in vivo. The researchers co-injected viruses expressing synaptophysin-GFP and a fluorescent form of mutant huntingtin into the cortex of mice. They then tracked cortical projections into the striatum as well as the location of mHtt. The researchers saw huntingtin aggregates in more than 70 percent of the MSNs in regions that made contact with cortical neurons, but not in MSNs without cortical hookups.

Did the aggregates travel from one neuron to another via synaptic vesicles? The authors found that human neurons placed into the cortical/striatal circuits in the mouse brain slices integrated into the circuits and eventually took up aggregates; therefore they were able to test if blocking synaptic vesicle release from mouse cells prevented transmission of aggregates. When they added inhibitors of SNAP-25 and VAMP2, proteins essential for vesicles’ fusion to the cell membranes, no mHtt made its way from mouse R6/2 neurons into transplanted hESC-derived human neurons. This led the authors to conclude that mutant Htt hitches a ride to neighboring neurons via synaptic vesicles.

An alternative explanation could be that by inhibiting membrane fusion and synaptic transmission, the researchers were preventing synapses from forming in the first place, according Marc Diamond of Washington University in St. Louis. “This, in turn, could prevent two neurons from forming a close association, and thus might indirectly reduce transneuronal movement of aggregates that is in fact independent of neuronal firing,” Diamond wrote to Alzforum (see full comment below).

Neil Cashman of the University of British Columbia in Vancouver noted that whether mHtt aggregates corrupt normally folded proteins after transfer remains unknown. “The authors have convincingly demonstrated synapse-mediated spread of mutant huntingtin, and have associated post-synaptic neuronal molecular and morphological abnormalities with this transmission,” Cashman wrote. “They have not demonstrated, to my reading, the key aspect of a prion mechanism—the ‘recruitment’ of wild-type huntingtin in the post-synaptic cell.”

The use of human Htt knockout cells in the co-culture experiments could be a way to address this question, Diamond added. “All in all, this is a provocative study, and it raises important questions to be tested in further work.”—Jessica Shugart

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  1. This is a very elegant study that did some nice work to show that mHtt aggregates can move between neurons in brain and organotypic slice cultures of R6/2 mice. The study finishes with the use of toxins that block vesicle fusion at the synapse to suggest that synaptic activity might be mediating transneuronal movement somehow by mHtt aggregate release at the synapse. An alternative interpretation is that these toxins, by blocking synaptic activity, are disrupting the formation of synapses. This, in turn, could prevent two neurons from forming a close association, and thus might indirectly reduce transneuronal movement of aggregates that is in fact independent of neuronal firing. Given their experimental system, it is difficult to tease apart these two problems (vesicle fusion/release of mHtt vs. synapse formation with juxtaposition of cell membranes). However, in other systems it would be possible to test directly whether firing of an existing synapse would promote transcellular movement of aggregates.

    Additionally, the authors infer that human neurons transplanted into a “toxic” environment of R6/2 brain have loss of function/anatomic structure due to transfer of mHtt proteins (Fig 1). This could be more directly tested. For example, application of antibodies against mHtt to the culture could be tested to block the induction of these abnormalities. Additionally, knockdown of wt Htt in the human neurons could be tested to determine whether a “prion-like” mechanism is required for the mHtt to have its effect in the co-culture.

    All in all, this is a provocative study, and it raises important questions to be tested in further work.

  2. This is an elegant series of experiments using interesting and informative models. The findings complement a growing literature demonstrating the cell-to-cell spread of pathogenic protein seeds formed by Aβ, PrP, tau, alpha-synuclein, SOD1 and others. Given the alacrity with which neurons take up, transport, and disseminate all sorts of cargo, including viruses and foreign proteins, it seems more and more that it would be surprising to find a neurologic proteopathy that doesn't involve these mechanisms.

References

Paper Citations

  1. . Cell-to-cell transmission of pathogenic proteins in neurodegenerative diseases. Nat Med. 2014 Feb;20(2):130-8. PubMed.
  2. . Nuclear and neuropil aggregates in Huntington's disease: relationship to neuropathology. J Neurosci. 1999 Apr 1;19(7):2522-34. PubMed.
  3. . Protein aggregates in Huntington's disease. Exp Neurol. 2012 Nov;238(1):1-11. Epub 2011 Dec 19 PubMed.
  4. . Huntington's disease and the striatal medium spiny neuron: cell-autonomous and non-cell-autonomous mechanisms of disease. Neurotherapeutics. 2012 Apr;9(2):270-84. PubMed.

Further Reading

Papers

  1. . Pathological cell-cell interactions are necessary for striatal pathogenesis in a conditional mouse model of Huntington's disease. Mol Neurodegener. 2007 Apr 30;2:8. PubMed.

Primary Papers

  1. . Transneuronal propagation of mutant huntingtin contributes to non-cell autonomous pathology in neurons. Nat Neurosci. 2014 Aug;17(8):1064-72. Epub 2014 Jul 13 PubMed.