HHS Public Access Author manuscript Author Manuscript
Science. Author manuscript; available in PMC 2016 April 24. Published in final edited form as: Science. 2016 January 8; 351(6269): 125–126. doi:10.1126/science.aad9872.
Disrupted nuclear import/export in neurodegeneration Sandrine Da Cruz1 and Don W. Cleveland2,* 1Ludwig
Institute for Cancer Research, University of California at San Diego, La Jolla, CA 92093,
USA 2Department
of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, CA 92093, USA
Author Manuscript Author Manuscript
The major human neurodegenerative diseases, including Alzheimer’s (AD), amyotrophic lateral sclerosis (ALS), Parkinson’s, and Huntington’s (HD) diseases, are associated with accumulation and aggregation of misfolded proteins. In most disease examples the majority of aberrantly aggregated proteins are found in the cell cytoplasm (the site of protein synthesis), albeit in the trinucleotide repeat diseases including HD and spinocerebellar ataxia – the corresponding aggregates of proteins containing the encoded polyglutamine expansions are predominantly nuclear. The mechanisms through which misfolded or aggregated proteins generate toxicity are unsettled. Indeed, whether differences in intracellular location matters for the toxicity ultimately generated from each mutant protein has not been determined. This latter question is now taken on directly by Woerner et al. (1) on page XX, who provide a provocative demonstration that aggregate location matters, with toxicity shown to arise from cytoplasmic accumulation of a pair of artificial proteins built to mimic the properties of amyloid aggregates. In what will come as a surprise to many, forcing the same artificial aggregates to accumulate in the nucleus eliminates their toxicity.
Author Manuscript
Woerner et al (1) establish an elegant culture system where artificial β-sheet proteins, previously shown to form fibrillar amyloid aggregates (2), can be targeted to accumulate in the cytoplasm or nucleus by inclusion of a nuclear export sequence (NES) or nuclear localization sequence (NLS), respectively. With this approach, Woerner and colleagues (1) convincingly demonstrate that only the cytoplasmic aggregates, but not the nuclear counterparts, cause cell death. The authors propose that the reduced toxicity of the nuclear aggregates, despite accumulating at levels comparable to those in the cytoplasm, may be the result of the chaperone-like activity of a highly abundant nucleolar protein nucleophosmin-1 (NPM1), which is shown to interact only with the nuclear aggregates. These discoveries add to other emerging evidence that compartment-specific chaperones complexing with misfolded proteins in the cytosol (3) or nucleus (4) – may play central roles in ameliorating damage from accumulated aggregates, possibly by generating compartmentspecific conformers with different propensities for cellular toxicity. Woerner et al (1) report that nuclear aggregates have reduced solubility and weaker affinity for an amyloid-specific dye compared to their cytosolic counterparts, underscoring possible differences in aggregate
*
To whom correspondence should be addressed at: [email protected].
Da Cruz and Cleveland
Page 2
Author Manuscript
conformation between the two subcellular compartments. Whether any of the newly identified nuclear-specific chaperones (4) contribute to these possible conformational changes or whether they shield the surfaces of the nuclear amyloid-like protein aggregates making them innocuous has not been established.
Author Manuscript
So why is protein aggregation in the cytoplasm toxic? Woerner et al (1) use a proteomic approach to implicate in primary neurons a direct binding partner of the cytoplasmic β-sheet containing aggregates to be the THOC2 subunit of the THO complex involved in mRNA export. Indeed, follow up examination reveals that THOC2 is mislocalized to the cytoplasm in cells with cytoplasmic β-sheet aggregates, albeit its interaction with those is unlikely to be direct as the aggregates are quite distinct from the cytoplasmic redistributed THOC2. Components of the nuclear pore complex (NPC) and nuclear import receptors are misaccumulated in the cytoplasm, strongly implicating diminished nuclear import and export in the affected cells. Not yet determined is whether nuclear proteins or nuclear pore components, and if so which ones, are trapped by the cytoplasmic amyloid aggregates, thus preventing their proper nuclear localization and function.
Author Manuscript
Similar assays are used to show that expression of disease-linked fragments of polyglutamine-containing huntingtin or ALS-causing mutants in TDP-43 also inhibit mRNA export when expressed in cultured cells, suggesting that errors in nuclear-cytoplasmic transport may be applicable to multiple neurological conditions. That said, expression of mutant huntingtin in post-mitotic primary cortical neurons led preferentially to nuclear aggregation, which did not impair nuclear mRNA export, consistent with earlier evidence that intranuclear inclusions containing polyglutamine-containing huntingtin fragments are not toxic per se (5). Woerner et al (1) also report that there is impaired RNA export in rare neurons with aggregated huntingtin in the widely used R6/2 HD mouse model, albeit it must be admitted that not all readers will find the evidence fully persuasive. Nevertheless, consideration of Woerner et al’s findings and the consensus from analyses of human samples and most mouse models now raises the question of whether it is the much rarer cytoplasmic aggregates that are the primary contributors to toxicity in HD, rather than the more abundant intranuclear ones.
Author Manuscript
The finding that cytoplasmic aggregates diminish nuclear import and/or export adds to the growing recognition that diminished nucleocytoplasmic transport may be a common component of multiple human neurodegenerative diseases. Three independent teams (6–8) reported in 2015 that hexanucleotide expansion within the C9orf72 gene, the most frequent inherited cause of both ALS and frontal temporal dementia (FTD), disrupted import/export. How this defect arises is not firmly established, with one team identifying a direct interaction between the hexanucleotide repeat-containing RNAs and Ran-GAP, a factor required for nuclear import, and others implicating import inhibition mediated by repeat associated the non-AUG (referred to as RAN) dependent translation-produced polydipeptides encoded by expansion-containing RNAs. To this controversy, the evidence from Woerner et al (1) demonstrates that nuclear/cytoplasmic transport defects can be attributed uniquely to a proteotoxicity caused by cytoplasmic events correlated with β-sheet protein-containing aggregates. Additionally, the recent finding that RAN translation is not restricted to diseases with non-coding region repeat expansions but also occurs across
Science. Author manuscript; available in PMC 2016 April 24.
Da Cruz and Cleveland
Page 3
Author Manuscript
repeats located in an open reading frame, such as HD (9), provides a new perspective on potential mechanisms underlying toxicity in HD. A critical next step will be to determine whether RAN peptides can directly provoke nucleocytoplasmic transport defects previously reported in HD (10), and whether there is compartment-specific toxicity, as demonstrated for the β-sheet protein aggregates.
Author Manuscript
Altogether these results support the growing role of nucleocytoplasmic transport in the cellular aging process as well as in many neurological conditions including HD, AD, ALS, FTD, and Parkinson’s (10, 11), where components of the import/export machinery are mislocalized and found to interact with disease-associated mutant proteins. To the unresolved, key question of how does cytoplasmic cytoplasmic accumulation of aggregates provoke diminished nuclear import/export, it must be noted that thinking in AD (12), Huntington’s disease (HD) (5, 13) and most recently in inherited ALS (14), have reversed an initial focus of the large aggregates seen with conventional pathology. Instead, investigators have refocused onto oligomeric assemblies of the misfolded protein as most important contributors to neuronal dysfunction that leads in each case to the characteristic disease symptoms (15). Seen from this prospective, the β-sheet protein aggregates (and other aggregates in the various disorders) may actually be protective, with toxicity arising from oligomeric species that are hard to detect.
References
Author Manuscript Author Manuscript
1. Woerner AC, et al. Cytoplasmic protein aggregates interfere with nucleo-cytoplasmic transport of protein and RNA. Science. 2015 2. Olzscha H, et al. Amyloid-like aggregates sequester numerous metastable proteins with essential cellular functions. Cell. 2011; 144:67–78. [PubMed: 21215370] 3. Kaganovich D, Kopito R, Frydman J. Misfolded proteins partition between two distinct quality control compartments. Nature. 2008; 454:1088–1095. [PubMed: 18756251] 4. Miller SB, et al. Compartment-specific aggregases direct distinct nuclear and cytoplasmic aggregate deposition. The EMBO journal. 2015; 34:778–797. [PubMed: 25672362] 5. Saudou F, Finkbeiner S, Devys D, Greenberg ME. Huntingtin acts in the nucleus to induce apoptosis but death does not correlate with the formation of intranuclear inclusions. Cell. 1998; 95:55–66. [PubMed: 9778247] 6. Freibaum BD, et al. GGGGCC repeat expansion in C9orf72 compromises nucleocytoplasmic transport. Nature. 2015; 525:129–133. [PubMed: 26308899] 7. Jovicic A, et al. Modifiers of C9orf72 dipeptide repeat toxicity connect nucleocytoplasmic transport defects to FTD/ALS. Nat Neurosci. 2015; 18:1226–1229. [PubMed: 26308983] 8. Zhang K, et al. The C9orf72 repeat expansion disrupts nucleocytoplasmic transport. Nature. 2015; 525:56–61. [PubMed: 26308891] 9. Banez-Coronel M, et al. RAN Translation in Huntington Disease. Neuron. 2015; 88:667–677. [PubMed: 26590344] 10. Patel VP, Chu CT. Nuclear transport, oxidative stress, and neurodegeneration. International journal of clinical and experimental pathology. 2011; 4:215–229. [PubMed: 21487518] 11. D'Angelo MA, Raices M, Panowski SH, Hetzer MW. Age-dependent deterioration of nuclear pore complexes causes a loss of nuclear integrity in postmitotic cells. Cell. 2009; 136:284–295. [PubMed: 19167330] 12. Cohen E, Bieschke J, Perciavalle RM, Kelly JW, Dillin A. Opposing activities protect against ageonset proteotoxicity. Science. 2006; 313:1604–1610. [PubMed: 16902091]
Science. Author manuscript; available in PMC 2016 April 24.
Da Cruz and Cleveland
Page 4
Author Manuscript
13. Arrasate M, Mitra S, Schweitzer ES, Segal MR, Finkbeiner S. Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death. Nature. 2004; 431:805–810. [PubMed: 15483602] 14. Parone PA, et al. Enhancing mitochondrial calcium buffering capacity reduces aggregation of misfolded SOD1 and motor neuron cell death without extending survival in mouse models of inherited amyotrophic lateral sclerosis. J Neurosci. 2013; 33:4657–4671. [PubMed: 23486940] 15. Treusch S, Cyr DM, Lindquist S. Amyloid deposits: protection against toxic protein species? Cell Cycle. 2009; 8:1668–1674. [PubMed: 19411847]
Author Manuscript Author Manuscript Author Manuscript Science. Author manuscript; available in PMC 2016 April 24.
Da Cruz and Cleveland
Page 5
Author Manuscript Author Manuscript Author Manuscript Author Manuscript Science. Author manuscript; available in PMC 2016 April 24.
Science. Author manuscript; available in PMC 2016 April 24. Published in final edited form as: Science. 2016 January 8; 351(6269): 125–126. doi:10.1126/science.aad9872.
Disrupted nuclear import/export in neurodegeneration Sandrine Da Cruz1 and Don W. Cleveland2,* 1Ludwig
Institute for Cancer Research, University of California at San Diego, La Jolla, CA 92093,
USA 2Department
of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, CA 92093, USA
Author Manuscript Author Manuscript
The major human neurodegenerative diseases, including Alzheimer’s (AD), amyotrophic lateral sclerosis (ALS), Parkinson’s, and Huntington’s (HD) diseases, are associated with accumulation and aggregation of misfolded proteins. In most disease examples the majority of aberrantly aggregated proteins are found in the cell cytoplasm (the site of protein synthesis), albeit in the trinucleotide repeat diseases including HD and spinocerebellar ataxia – the corresponding aggregates of proteins containing the encoded polyglutamine expansions are predominantly nuclear. The mechanisms through which misfolded or aggregated proteins generate toxicity are unsettled. Indeed, whether differences in intracellular location matters for the toxicity ultimately generated from each mutant protein has not been determined. This latter question is now taken on directly by Woerner et al. (1) on page XX, who provide a provocative demonstration that aggregate location matters, with toxicity shown to arise from cytoplasmic accumulation of a pair of artificial proteins built to mimic the properties of amyloid aggregates. In what will come as a surprise to many, forcing the same artificial aggregates to accumulate in the nucleus eliminates their toxicity.
Author Manuscript
Woerner et al (1) establish an elegant culture system where artificial β-sheet proteins, previously shown to form fibrillar amyloid aggregates (2), can be targeted to accumulate in the cytoplasm or nucleus by inclusion of a nuclear export sequence (NES) or nuclear localization sequence (NLS), respectively. With this approach, Woerner and colleagues (1) convincingly demonstrate that only the cytoplasmic aggregates, but not the nuclear counterparts, cause cell death. The authors propose that the reduced toxicity of the nuclear aggregates, despite accumulating at levels comparable to those in the cytoplasm, may be the result of the chaperone-like activity of a highly abundant nucleolar protein nucleophosmin-1 (NPM1), which is shown to interact only with the nuclear aggregates. These discoveries add to other emerging evidence that compartment-specific chaperones complexing with misfolded proteins in the cytosol (3) or nucleus (4) – may play central roles in ameliorating damage from accumulated aggregates, possibly by generating compartmentspecific conformers with different propensities for cellular toxicity. Woerner et al (1) report that nuclear aggregates have reduced solubility and weaker affinity for an amyloid-specific dye compared to their cytosolic counterparts, underscoring possible differences in aggregate
*
To whom correspondence should be addressed at: [email protected].
Da Cruz and Cleveland
Page 2
Author Manuscript
conformation between the two subcellular compartments. Whether any of the newly identified nuclear-specific chaperones (4) contribute to these possible conformational changes or whether they shield the surfaces of the nuclear amyloid-like protein aggregates making them innocuous has not been established.
Author Manuscript
So why is protein aggregation in the cytoplasm toxic? Woerner et al (1) use a proteomic approach to implicate in primary neurons a direct binding partner of the cytoplasmic β-sheet containing aggregates to be the THOC2 subunit of the THO complex involved in mRNA export. Indeed, follow up examination reveals that THOC2 is mislocalized to the cytoplasm in cells with cytoplasmic β-sheet aggregates, albeit its interaction with those is unlikely to be direct as the aggregates are quite distinct from the cytoplasmic redistributed THOC2. Components of the nuclear pore complex (NPC) and nuclear import receptors are misaccumulated in the cytoplasm, strongly implicating diminished nuclear import and export in the affected cells. Not yet determined is whether nuclear proteins or nuclear pore components, and if so which ones, are trapped by the cytoplasmic amyloid aggregates, thus preventing their proper nuclear localization and function.
Author Manuscript
Similar assays are used to show that expression of disease-linked fragments of polyglutamine-containing huntingtin or ALS-causing mutants in TDP-43 also inhibit mRNA export when expressed in cultured cells, suggesting that errors in nuclear-cytoplasmic transport may be applicable to multiple neurological conditions. That said, expression of mutant huntingtin in post-mitotic primary cortical neurons led preferentially to nuclear aggregation, which did not impair nuclear mRNA export, consistent with earlier evidence that intranuclear inclusions containing polyglutamine-containing huntingtin fragments are not toxic per se (5). Woerner et al (1) also report that there is impaired RNA export in rare neurons with aggregated huntingtin in the widely used R6/2 HD mouse model, albeit it must be admitted that not all readers will find the evidence fully persuasive. Nevertheless, consideration of Woerner et al’s findings and the consensus from analyses of human samples and most mouse models now raises the question of whether it is the much rarer cytoplasmic aggregates that are the primary contributors to toxicity in HD, rather than the more abundant intranuclear ones.
Author Manuscript
The finding that cytoplasmic aggregates diminish nuclear import and/or export adds to the growing recognition that diminished nucleocytoplasmic transport may be a common component of multiple human neurodegenerative diseases. Three independent teams (6–8) reported in 2015 that hexanucleotide expansion within the C9orf72 gene, the most frequent inherited cause of both ALS and frontal temporal dementia (FTD), disrupted import/export. How this defect arises is not firmly established, with one team identifying a direct interaction between the hexanucleotide repeat-containing RNAs and Ran-GAP, a factor required for nuclear import, and others implicating import inhibition mediated by repeat associated the non-AUG (referred to as RAN) dependent translation-produced polydipeptides encoded by expansion-containing RNAs. To this controversy, the evidence from Woerner et al (1) demonstrates that nuclear/cytoplasmic transport defects can be attributed uniquely to a proteotoxicity caused by cytoplasmic events correlated with β-sheet protein-containing aggregates. Additionally, the recent finding that RAN translation is not restricted to diseases with non-coding region repeat expansions but also occurs across
Science. Author manuscript; available in PMC 2016 April 24.
Da Cruz and Cleveland
Page 3
Author Manuscript
repeats located in an open reading frame, such as HD (9), provides a new perspective on potential mechanisms underlying toxicity in HD. A critical next step will be to determine whether RAN peptides can directly provoke nucleocytoplasmic transport defects previously reported in HD (10), and whether there is compartment-specific toxicity, as demonstrated for the β-sheet protein aggregates.
Author Manuscript
Altogether these results support the growing role of nucleocytoplasmic transport in the cellular aging process as well as in many neurological conditions including HD, AD, ALS, FTD, and Parkinson’s (10, 11), where components of the import/export machinery are mislocalized and found to interact with disease-associated mutant proteins. To the unresolved, key question of how does cytoplasmic cytoplasmic accumulation of aggregates provoke diminished nuclear import/export, it must be noted that thinking in AD (12), Huntington’s disease (HD) (5, 13) and most recently in inherited ALS (14), have reversed an initial focus of the large aggregates seen with conventional pathology. Instead, investigators have refocused onto oligomeric assemblies of the misfolded protein as most important contributors to neuronal dysfunction that leads in each case to the characteristic disease symptoms (15). Seen from this prospective, the β-sheet protein aggregates (and other aggregates in the various disorders) may actually be protective, with toxicity arising from oligomeric species that are hard to detect.
References
Author Manuscript Author Manuscript
1. Woerner AC, et al. Cytoplasmic protein aggregates interfere with nucleo-cytoplasmic transport of protein and RNA. Science. 2015 2. Olzscha H, et al. Amyloid-like aggregates sequester numerous metastable proteins with essential cellular functions. Cell. 2011; 144:67–78. [PubMed: 21215370] 3. Kaganovich D, Kopito R, Frydman J. Misfolded proteins partition between two distinct quality control compartments. Nature. 2008; 454:1088–1095. [PubMed: 18756251] 4. Miller SB, et al. Compartment-specific aggregases direct distinct nuclear and cytoplasmic aggregate deposition. The EMBO journal. 2015; 34:778–797. [PubMed: 25672362] 5. Saudou F, Finkbeiner S, Devys D, Greenberg ME. Huntingtin acts in the nucleus to induce apoptosis but death does not correlate with the formation of intranuclear inclusions. Cell. 1998; 95:55–66. [PubMed: 9778247] 6. Freibaum BD, et al. GGGGCC repeat expansion in C9orf72 compromises nucleocytoplasmic transport. Nature. 2015; 525:129–133. [PubMed: 26308899] 7. Jovicic A, et al. Modifiers of C9orf72 dipeptide repeat toxicity connect nucleocytoplasmic transport defects to FTD/ALS. Nat Neurosci. 2015; 18:1226–1229. [PubMed: 26308983] 8. Zhang K, et al. The C9orf72 repeat expansion disrupts nucleocytoplasmic transport. Nature. 2015; 525:56–61. [PubMed: 26308891] 9. Banez-Coronel M, et al. RAN Translation in Huntington Disease. Neuron. 2015; 88:667–677. [PubMed: 26590344] 10. Patel VP, Chu CT. Nuclear transport, oxidative stress, and neurodegeneration. International journal of clinical and experimental pathology. 2011; 4:215–229. [PubMed: 21487518] 11. D'Angelo MA, Raices M, Panowski SH, Hetzer MW. Age-dependent deterioration of nuclear pore complexes causes a loss of nuclear integrity in postmitotic cells. Cell. 2009; 136:284–295. [PubMed: 19167330] 12. Cohen E, Bieschke J, Perciavalle RM, Kelly JW, Dillin A. Opposing activities protect against ageonset proteotoxicity. Science. 2006; 313:1604–1610. [PubMed: 16902091]
Science. Author manuscript; available in PMC 2016 April 24.
Da Cruz and Cleveland
Page 4
Author Manuscript
13. Arrasate M, Mitra S, Schweitzer ES, Segal MR, Finkbeiner S. Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death. Nature. 2004; 431:805–810. [PubMed: 15483602] 14. Parone PA, et al. Enhancing mitochondrial calcium buffering capacity reduces aggregation of misfolded SOD1 and motor neuron cell death without extending survival in mouse models of inherited amyotrophic lateral sclerosis. J Neurosci. 2013; 33:4657–4671. [PubMed: 23486940] 15. Treusch S, Cyr DM, Lindquist S. Amyloid deposits: protection against toxic protein species? Cell Cycle. 2009; 8:1668–1674. [PubMed: 19411847]
Author Manuscript Author Manuscript Author Manuscript Science. Author manuscript; available in PMC 2016 April 24.
Da Cruz and Cleveland
Page 5
Author Manuscript Author Manuscript Author Manuscript Author Manuscript Science. Author manuscript; available in PMC 2016 April 24.
