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Something wicked this way comes: huntingtin Albert R La Spada
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© 2014 Nature America, Inc. All rights reserved.
Does cell-to-cell spreading of misfolded proteins occur in all neurodegenerative disorders? A study in this issue of Nature Neuroscience now demonstrates propagation of mutant huntingtin in brain slice cultures and in vivo, thereby extending the process of cell-to-cell propagation of misfolded proteins to Huntington’s disease. One goal of a criminal justice system is to imprison malevolent offenders so that they can no longer gain access to law-abiding citizens. An important question in the neurodegenerative disease field is whether a misfolded protein produced in one neuron can leave that cell to enter an unaffected neuron, with recent work suggesting that ‘bad actor’ misfolded proteins are not confined to the cells in which they originate, but can instead move to other cells, like unrestrained criminal agents of chaos and destruction. A study by Pecho-Vrieseling et al. in this month’s issue of Nature Neuroscience provides convincing in vitro data, as well as the first definitive in vivo evidence, for spreading of misfolded huntingtin protein between neurons1, and thus supports an emerging model of cell-to-cell propagation as an essential element of disease progression in all neurodegenerative disorders. Production of misfolded proteins is a defining feature of all neurodegenerative disorders. This paradigm-shifting realization about 20 years ago marked a turning point in our understanding of neurodegeneration, and the last two decades of research have further reinforced the view that all neurodegenerative disorders result from the accumulation of misfolded proteins that form aggregates, detectable as inclusion bodies at the light microscope level. This realization linked prion diseases, whose agents are proteins that confer their own misfolding on normal conformers through a process of templated change2, to the entire spectrum of neurodegenerative disorders, Albert R. La Spada is in the Departments of Cellular & Molecular Medicine, Neurosciences and Pediatrics, Division of Biological Sciences, Institute for Genomic Medicine, and Sanford Consortium for Regenerative Medicine, University of California, San Diego, La Jolla, California, USA, and at Rady Children’s Hospital, San Diego, California, USA. e-mail: [email protected]
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including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis and tauopathies. However, in the last decade, compelling research has also suggested that, besides accumulation of misfolded proteins, another common feature of neurodegeneration is the propensity of misfolded proteins to spread from one cell to another, and that this propagation is a key feature of disease pathogenesis (reviewed in ref. 3). Although mounting in vivo evidence for ‘prion-like’ propagation has emerged for Alzheimer’s and Parkinson’s diseases4,5, the issue of whether this propagation phenomenon might apply to Huntington’s disease and other polyglutamine repeat diseases had remained unresolved. One compelling aspect of the new study from Pecho-Vrieseling et al.1 is that they took three different, progressively more sophisticated approaches to examine cell-to-cell spreading of mutant huntingtin (mHtt) protein. The initial system was based on the introduction of human embryonic stem cells (hESCs) into organotypic brain slice cultures obtained from either wild-type mice or Huntington’s disease model R6/2 mice. In the slice culture milieu, hESCs readily differentiate into neurons and form functional connections with existing neurons, as the authors nicely demonstrate through combined optogenetic and pharmacological experimentation. Having established this powerful system, they assessed hESCderived neurons located in both the striatal and cortical regions of slice cultures and found that they developed huntingtin aggregate pathology, initially in the cytosol and subsequently in the nucleus, but only when placed in the R6/2 context. Elaboration of aggregate pathology in hESC-derived neurons was not innocuous: such neurons exhibited signs of toxicity, including reductions in neurite number and neurite length. Notably, neurite degeneration phenotypes were accentuated when hESCderived neurons were situated in R6/2 slices
containing huntingtin aggregates in comparison with R6/2 slices lacking aggregates. After completing careful studies with the hESC neuron–slice culture system, Pecho-Vrieseling et al.1 pursued a more representative brain slice system to test the hypo thesis of huntingtin propagation by dissecting out cortex or striatum from wild-type mice or R6/2 mice and then placing the cortical region of one mouse adjacent to the striatal region of a different mouse to create ‘mixed’ cortico striatal brain cultures1. Of the various possible permutations, the authors chose to carefully study R6/2 cortex and wild-type striatum versus wild-type cortex and wild-type striatum, as they could only document functional corticostriatal circuit connections when mixed corticostriatal brain slices employed wild-type striatum. Here again, mHtt aggregates materialized in wild-type striatal neurons only when placed adjacent to R6/2 cortex. Consequently, these two different brain slice culture systems revealed the capacity for mHtt to spread from one neuron to another, ‘naive’ neuron, and corroborated earlier in vitro studies in which mHtt placed in the culture medium or expressed in one cell in culture was found to gain access to normal cells not expressing mHtt6,7. However, demonstrating the capacity of mHtt to move from a diseased cell to a normal cell, even when employing sophisticated organotypic slice culture approaches, may not faithfully represent what is happening in corticostriatal circuits in vivo. Thus, Pecho-Vrieseling et al.1 ultimately performed the key experiment necessary for testing mHtt propagation by co-injecting, into the cortex of wild-type mice, a lenti virus with an expression construct carrying polyglutamine-expanded (Q72) huntingtin exon 1 and an adeno-associated virus containing a synaptophysin-GFP fusion vector (to mark transduced neurons). They then documented the presence of huntingtin
volume 17 | number 8 | august 2014 nature neuroscience
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Figure 1 Potential cellular pathways for misfolded protein propagation. (a) Adjacent spread. Neurons in functional contact with one another, as shown here, or in direct physical contact via intercellular bridges (for example, tunneling nanotubes) may be subject to cell-to-cell transmission of misfolded proteins. (b) Paracrine propagation. Neurons (or other cell types) may expel misfolded proteins in vesicles from the autophagy or endosome pathways, as shown here, or may release misfolded proteins extracellularly by exocytosis. Once proteins are released in this way, neurons in the vicinity may take up the misfolded proteins by fusion with membrane-bound structures or simply by endocytosis of diffusible extracellular protein. (c) Distant dissemination (prion-like). Certain misfolded proteins and peptides (for example, prions, amyloid-β42, -synuclein) may travel great distances, perhaps originating in non-neural peripheral tissues such as the gut, to enter the CNS by crossing the blood-brain barrier and then moving into neurons and other cells.
aggregates in the majority of striatal medium spiny neurons located in regions innervated by virally transduced cortical projection neurons that synapse with the striatal medium spiny neurons, thereby providing persuasive evidence that misfolded huntingtin conformers can propagate in the mammalian brain in a transneuronal fashion1, which is consistent with recent work demonstrating the interaction of the cortex and striatum in Huntington’s disease pathogenesis8. Although this latest advance further underscores the existence of common pathogenic processes at work in neurodegeneration, it is important to not overemphasize the commonality of the propagation phenomenon, as the underlying mechanisms driving cell-to-cell spreading still defy explanation and may vary substantially in different neurodegenerative diseases. Indeed, another interesting aspect of the work by Pecho-Vrieseling et al.1 was their attempt to define the basis for mHtt propagation in their hESC neuron–R6/2 slice culture system by treating their cultures with two different botulinum toxin strains that cleave distinct components of the synaptic vesicle docking fusion complex to prevent the release of synaptic vesicles. Either strain of botulinum
toxin markedly blunted the accumulation of huntingtin aggregates in hESC-derived neurons cultured in R6/2 slices, but each treatment was effective only if applied before the first wave of aggregation, which produced cytosolic aggregates. Botulinum toxin treatment after detection of cytosolic huntingtin aggregates had no appreciable effect on the extent of huntingtin aggregate accumulation in cocultured hESC neurons. Although the necessity of synaptic vesicle release for huntingtin protein propagation requires validation with genetic knockdown approaches, given the many possible off-target effects of botulinum toxin, and also needs to be extended to the in vivo setting, the implication of synaptic vesicle delivery as the basis for mHtt propagation raises important questions, as one can envisage at least three distinct pathways by which misfolded proteins may transit from one cell to another: adjacent spread, paracrine propagation and distant dissemination. Adjacent spread, as is reported here for huntingtin protein, is a process involving cells that are directly connected to one another physically or as part of a functional neural circuit (Fig. 1a). Thus, the transmission of misfolded protein occurs by virtue of the cells’ physical and/or
nature neuroscience volume 17 | number 8 | august 2014
functional connection (for example, tunneling nanotubes9 or synapses), as hypothesized for the Huntington’s disease corticostriatal synaptic junction in the present work. However, in a process of paracrine propagation, cell-to-cell propagation may also occur between neurons and other cells that are not functionally or physically connected, but rather simply nearby10, when a misfolded protein is released from a diseased cell (Fig. 1b). Finally, for the prion diseases, and likely also in Alzheimer’s and Parkinson’s diseases, it appears that misfolded proteins can traverse great distances, sometimes even from the gut or elsewhere in the periphery3,4,10,11, to finally end up at their ultimate neuronal destination in the CNS (Fig. 1c). This distant dissemination of misfolded proteins is a defining feature of prion disease; hence, the term prionlike should be reserved for neurodegenerative diseases that exhibit this capacity for distant dissemination. Indeed, if cell-to-cell propagation of mHtt protein in Huntington’s disease occurs only at synaptic junctions (adjacent spread), and not via other pathways, then this feature of Huntington’s disease pathogenesis and progression would have strong implications for the molecular basis of the Huntington’s disease transmission process, which may involve extracellular release and uptake, or delivery in membrane-bound structures3. As it turns out, however, analysis of fetal neural allografts in post-mortem Huntington’s disease patient brain has uncovered the presence of huntingtin aggregates in the allograft tissue, albeit only in extracellular matrix12, which suggests an extracellular release process consistent with paracrine propagation. Clearly, future work should focus on defining the underlying cellular pathways by which cell-to-cell spreading of misfolded proteins occurs in different neuro degenerative diseases. As multiple pathways for cell-to-cell spreading may be imagined, defining how misfolded protein propagation occurs in different disorders will have crucial implications for therapy development. COMPETING FINANCIAL INTERESTS The author declares no competing financial interests. 1. Pecho-Vrieseling et al. Nat. Neurosci. 17, 1064–1072 (2014). 2. Pan, K.M. et al. Proc. Natl. Acad. Sci. USA 90, 10962–10966 (1993). 3. Garden, G.A. & La Spada, A.R. Neuron 73, 886–901 (2012). 4. Eisele, Y.S. et al. Science 330, 980–982 (2010). 5. Luk, K.C. et al. Science 338, 949–953 (2012). 6. Ren, P.H. et al. Nat. Cell Biol. 11, 219–225 (2009). 7. Herrera, F. et al. PLoS Curr. 3, RRN1210 (2011). 8. Wang, N. et al. Nat. Med. 20, 536–541 (2014). 9. Gousset, K. et al. Nat. Cell Biol. 11, 328–336 (2009). 10. Steiner, J.A., Angot, E. & Brundin, P. Cell Death Differ. 18, 1425–1433 (2011). 11. Visanji, N.P., Brooks, P.L., Hazrati, L.-N. & Lang, A.E. Acta Neuropathol. Commun. 1, 2 (2013). 12. Cicchetti, F. et al. Ann. Neurol. published online, doi:10.1002/ana.24174 (6 May 2014).
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Something wicked this way comes: huntingtin Albert R La Spada
npg
© 2014 Nature America, Inc. All rights reserved.
Does cell-to-cell spreading of misfolded proteins occur in all neurodegenerative disorders? A study in this issue of Nature Neuroscience now demonstrates propagation of mutant huntingtin in brain slice cultures and in vivo, thereby extending the process of cell-to-cell propagation of misfolded proteins to Huntington’s disease. One goal of a criminal justice system is to imprison malevolent offenders so that they can no longer gain access to law-abiding citizens. An important question in the neurodegenerative disease field is whether a misfolded protein produced in one neuron can leave that cell to enter an unaffected neuron, with recent work suggesting that ‘bad actor’ misfolded proteins are not confined to the cells in which they originate, but can instead move to other cells, like unrestrained criminal agents of chaos and destruction. A study by Pecho-Vrieseling et al. in this month’s issue of Nature Neuroscience provides convincing in vitro data, as well as the first definitive in vivo evidence, for spreading of misfolded huntingtin protein between neurons1, and thus supports an emerging model of cell-to-cell propagation as an essential element of disease progression in all neurodegenerative disorders. Production of misfolded proteins is a defining feature of all neurodegenerative disorders. This paradigm-shifting realization about 20 years ago marked a turning point in our understanding of neurodegeneration, and the last two decades of research have further reinforced the view that all neurodegenerative disorders result from the accumulation of misfolded proteins that form aggregates, detectable as inclusion bodies at the light microscope level. This realization linked prion diseases, whose agents are proteins that confer their own misfolding on normal conformers through a process of templated change2, to the entire spectrum of neurodegenerative disorders, Albert R. La Spada is in the Departments of Cellular & Molecular Medicine, Neurosciences and Pediatrics, Division of Biological Sciences, Institute for Genomic Medicine, and Sanford Consortium for Regenerative Medicine, University of California, San Diego, La Jolla, California, USA, and at Rady Children’s Hospital, San Diego, California, USA. e-mail: [email protected]
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including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis and tauopathies. However, in the last decade, compelling research has also suggested that, besides accumulation of misfolded proteins, another common feature of neurodegeneration is the propensity of misfolded proteins to spread from one cell to another, and that this propagation is a key feature of disease pathogenesis (reviewed in ref. 3). Although mounting in vivo evidence for ‘prion-like’ propagation has emerged for Alzheimer’s and Parkinson’s diseases4,5, the issue of whether this propagation phenomenon might apply to Huntington’s disease and other polyglutamine repeat diseases had remained unresolved. One compelling aspect of the new study from Pecho-Vrieseling et al.1 is that they took three different, progressively more sophisticated approaches to examine cell-to-cell spreading of mutant huntingtin (mHtt) protein. The initial system was based on the introduction of human embryonic stem cells (hESCs) into organotypic brain slice cultures obtained from either wild-type mice or Huntington’s disease model R6/2 mice. In the slice culture milieu, hESCs readily differentiate into neurons and form functional connections with existing neurons, as the authors nicely demonstrate through combined optogenetic and pharmacological experimentation. Having established this powerful system, they assessed hESCderived neurons located in both the striatal and cortical regions of slice cultures and found that they developed huntingtin aggregate pathology, initially in the cytosol and subsequently in the nucleus, but only when placed in the R6/2 context. Elaboration of aggregate pathology in hESC-derived neurons was not innocuous: such neurons exhibited signs of toxicity, including reductions in neurite number and neurite length. Notably, neurite degeneration phenotypes were accentuated when hESCderived neurons were situated in R6/2 slices
containing huntingtin aggregates in comparison with R6/2 slices lacking aggregates. After completing careful studies with the hESC neuron–slice culture system, Pecho-Vrieseling et al.1 pursued a more representative brain slice system to test the hypo thesis of huntingtin propagation by dissecting out cortex or striatum from wild-type mice or R6/2 mice and then placing the cortical region of one mouse adjacent to the striatal region of a different mouse to create ‘mixed’ cortico striatal brain cultures1. Of the various possible permutations, the authors chose to carefully study R6/2 cortex and wild-type striatum versus wild-type cortex and wild-type striatum, as they could only document functional corticostriatal circuit connections when mixed corticostriatal brain slices employed wild-type striatum. Here again, mHtt aggregates materialized in wild-type striatal neurons only when placed adjacent to R6/2 cortex. Consequently, these two different brain slice culture systems revealed the capacity for mHtt to spread from one neuron to another, ‘naive’ neuron, and corroborated earlier in vitro studies in which mHtt placed in the culture medium or expressed in one cell in culture was found to gain access to normal cells not expressing mHtt6,7. However, demonstrating the capacity of mHtt to move from a diseased cell to a normal cell, even when employing sophisticated organotypic slice culture approaches, may not faithfully represent what is happening in corticostriatal circuits in vivo. Thus, Pecho-Vrieseling et al.1 ultimately performed the key experiment necessary for testing mHtt propagation by co-injecting, into the cortex of wild-type mice, a lenti virus with an expression construct carrying polyglutamine-expanded (Q72) huntingtin exon 1 and an adeno-associated virus containing a synaptophysin-GFP fusion vector (to mark transduced neurons). They then documented the presence of huntingtin
volume 17 | number 8 | august 2014 nature neuroscience
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© 2014 Nature America, Inc. All rights reserved.
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Figure 1 Potential cellular pathways for misfolded protein propagation. (a) Adjacent spread. Neurons in functional contact with one another, as shown here, or in direct physical contact via intercellular bridges (for example, tunneling nanotubes) may be subject to cell-to-cell transmission of misfolded proteins. (b) Paracrine propagation. Neurons (or other cell types) may expel misfolded proteins in vesicles from the autophagy or endosome pathways, as shown here, or may release misfolded proteins extracellularly by exocytosis. Once proteins are released in this way, neurons in the vicinity may take up the misfolded proteins by fusion with membrane-bound structures or simply by endocytosis of diffusible extracellular protein. (c) Distant dissemination (prion-like). Certain misfolded proteins and peptides (for example, prions, amyloid-β42, -synuclein) may travel great distances, perhaps originating in non-neural peripheral tissues such as the gut, to enter the CNS by crossing the blood-brain barrier and then moving into neurons and other cells.
aggregates in the majority of striatal medium spiny neurons located in regions innervated by virally transduced cortical projection neurons that synapse with the striatal medium spiny neurons, thereby providing persuasive evidence that misfolded huntingtin conformers can propagate in the mammalian brain in a transneuronal fashion1, which is consistent with recent work demonstrating the interaction of the cortex and striatum in Huntington’s disease pathogenesis8. Although this latest advance further underscores the existence of common pathogenic processes at work in neurodegeneration, it is important to not overemphasize the commonality of the propagation phenomenon, as the underlying mechanisms driving cell-to-cell spreading still defy explanation and may vary substantially in different neurodegenerative diseases. Indeed, another interesting aspect of the work by Pecho-Vrieseling et al.1 was their attempt to define the basis for mHtt propagation in their hESC neuron–R6/2 slice culture system by treating their cultures with two different botulinum toxin strains that cleave distinct components of the synaptic vesicle docking fusion complex to prevent the release of synaptic vesicles. Either strain of botulinum
toxin markedly blunted the accumulation of huntingtin aggregates in hESC-derived neurons cultured in R6/2 slices, but each treatment was effective only if applied before the first wave of aggregation, which produced cytosolic aggregates. Botulinum toxin treatment after detection of cytosolic huntingtin aggregates had no appreciable effect on the extent of huntingtin aggregate accumulation in cocultured hESC neurons. Although the necessity of synaptic vesicle release for huntingtin protein propagation requires validation with genetic knockdown approaches, given the many possible off-target effects of botulinum toxin, and also needs to be extended to the in vivo setting, the implication of synaptic vesicle delivery as the basis for mHtt propagation raises important questions, as one can envisage at least three distinct pathways by which misfolded proteins may transit from one cell to another: adjacent spread, paracrine propagation and distant dissemination. Adjacent spread, as is reported here for huntingtin protein, is a process involving cells that are directly connected to one another physically or as part of a functional neural circuit (Fig. 1a). Thus, the transmission of misfolded protein occurs by virtue of the cells’ physical and/or
nature neuroscience volume 17 | number 8 | august 2014
functional connection (for example, tunneling nanotubes9 or synapses), as hypothesized for the Huntington’s disease corticostriatal synaptic junction in the present work. However, in a process of paracrine propagation, cell-to-cell propagation may also occur between neurons and other cells that are not functionally or physically connected, but rather simply nearby10, when a misfolded protein is released from a diseased cell (Fig. 1b). Finally, for the prion diseases, and likely also in Alzheimer’s and Parkinson’s diseases, it appears that misfolded proteins can traverse great distances, sometimes even from the gut or elsewhere in the periphery3,4,10,11, to finally end up at their ultimate neuronal destination in the CNS (Fig. 1c). This distant dissemination of misfolded proteins is a defining feature of prion disease; hence, the term prionlike should be reserved for neurodegenerative diseases that exhibit this capacity for distant dissemination. Indeed, if cell-to-cell propagation of mHtt protein in Huntington’s disease occurs only at synaptic junctions (adjacent spread), and not via other pathways, then this feature of Huntington’s disease pathogenesis and progression would have strong implications for the molecular basis of the Huntington’s disease transmission process, which may involve extracellular release and uptake, or delivery in membrane-bound structures3. As it turns out, however, analysis of fetal neural allografts in post-mortem Huntington’s disease patient brain has uncovered the presence of huntingtin aggregates in the allograft tissue, albeit only in extracellular matrix12, which suggests an extracellular release process consistent with paracrine propagation. Clearly, future work should focus on defining the underlying cellular pathways by which cell-to-cell spreading of misfolded proteins occurs in different neuro degenerative diseases. As multiple pathways for cell-to-cell spreading may be imagined, defining how misfolded protein propagation occurs in different disorders will have crucial implications for therapy development. COMPETING FINANCIAL INTERESTS The author declares no competing financial interests. 1. Pecho-Vrieseling et al. Nat. Neurosci. 17, 1064–1072 (2014). 2. Pan, K.M. et al. Proc. Natl. Acad. Sci. USA 90, 10962–10966 (1993). 3. Garden, G.A. & La Spada, A.R. Neuron 73, 886–901 (2012). 4. Eisele, Y.S. et al. Science 330, 980–982 (2010). 5. Luk, K.C. et al. Science 338, 949–953 (2012). 6. Ren, P.H. et al. Nat. Cell Biol. 11, 219–225 (2009). 7. Herrera, F. et al. PLoS Curr. 3, RRN1210 (2011). 8. Wang, N. et al. Nat. Med. 20, 536–541 (2014). 9. Gousset, K. et al. Nat. Cell Biol. 11, 328–336 (2009). 10. Steiner, J.A., Angot, E. & Brundin, P. Cell Death Differ. 18, 1425–1433 (2011). 11. Visanji, N.P., Brooks, P.L., Hazrati, L.-N. & Lang, A.E. Acta Neuropathol. Commun. 1, 2 (2013). 12. Cicchetti, F. et al. Ann. Neurol. published online, doi:10.1002/ana.24174 (6 May 2014).
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