Nuclear import receptors are recruited by FG-nucleoporins to rescue hallmarks of TDP-43 proteinopathy

Background: Cytoplasmic mislocalization and aggregation of TAR DNA-binding protein-43 (TDP-43) is a hallmark of the amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) disease spectrum, causing both nuclear loss-of-function and cytoplasmic toxic gain-of-function phenotypes. While TDP-43 proteinopathy has been associated with defects in nucleocytoplasmic transport, this process is still poorly understood. Here we study the role of karyopherin-β1 (KPNB1) and other nuclear import receptors in regulating TDP-43 pathology. Methods: We used immunostaining, immunoprecipitation, biochemical and toxicity assays in cell lines, primary neuron and organotypic mouse brain slice cultures, to determine the impact of KPNB1 on the solubility, localization, and toxicity of pathological TDP-43 constructs. Postmortem patient brain and spinal cord tissue was stained to assess KPNB1 colocalization with TDP-43 inclusions. Turbidity assays were employed to study the dissolution and prevention of aggregation of recombinant TDP-43 fibrils in vitro. Fly models of TDP-43 proteinopathy were used to determine the effect of KPNB1 on their neurodegenerative phenotype in vivo. Results: We discovered that several members of the nuclear import receptor protein family can reduce the formation of pathological TDP-43 aggregates. Using KPNB1 as a model, we found that its activity depends on the prion-like C-terminal region of TDP-43, which mediates the co-aggregation with phenylalanine and glycine-rich nucleoporins (FG-Nups) such as Nup62. KPNB1 is recruited into these co-aggregates where it acts as a molecular chaperone that reverses aberrant phase transition of Nup62 and TDP-43. These findings are supported by the discovery that Nup62 and KPNB1 are also sequestered into pathological TDP-43 aggregates in ALS/FTD postmortem CNS tissue, and by the identification of the fly ortholog of KPNB1 as a strong protective modifier in Drosophila models of TDP-43 proteinopathy. Our results show that KPNB1 can rescue all hallmarks of TDP-43 pathology, by restoring its solubility and nuclear localization, and reducing neurodegeneration in cellular and animal models of ALS/FTD. Conclusion: Our findings suggest a novel NLS-independent mechanism where, analogous to its canonical role in dissolving the diffusion barrier formed by FG-Nups in the nuclear pore, KPNB1 is recruited into TDP-43/FG-Nup co-aggregates present in TDP-43 proteinopathies and therapeutically reverses their deleterious phase transition and mislocalization, mitigating neurodegeneration. Graphical Abstract: [Figure not available: see fulltext.]

Ocean Acidification-Induced Food Quality Deterioration Constrains Trophic Transfer

Our present understanding of ocean acidification (OA) impacts on marine organisms caused by rapidly rising atmospheric carbon dioxide (CO2) concentration is almost entirely limited to single species responses. OA consequences for food web interactions are, however, still unknown. Indirect OA effects can be expected for consumers by changing the nutritional quality of their prey. We used a laboratory experiment to test potential OA effects on algal fatty acid (FA) composition and resulting copepod growth. We show that elevated CO2 significantly changed the FA concentration and composition of the diatom Thalassiosira pseudonana, which constrained growth and reproduction of the copepod Acartia tonsa. A significant decline in both total FAs (28.1 to 17.4 fg cell−1) and the ratio of long-chain polyunsaturated to saturated fatty acids (PUFA:SFA) of food algae cultured under elevated (750 µatm) compared to present day (380 µatm) pCO2 was directly translated to copepods. The proportion of total essential FAs declined almost tenfold in copepods and the contribution of saturated fatty acids (SFAs) tripled at high CO2. This rapid and reversible CO2-dependent shift in FA concentration and composition caused a decrease in both copepod somatic growth and egg production from 34 to 5 eggs female−1 day−1. Because the diatom-copepod link supports some of the most productive ecosystems in the world, our study demonstrates that OA can have far-reaching consequences for ocean food webs by changing the nutritional quality of essential macromolecules in primary producers that cascade up the food web

Community interactions dampen acidification effects in a coastal plankton system

Changing seawater chemistry towards reduced pH as a result of increasing atmospheric carbon dioxide (CO2) is affecting oceanic organisms, particularly calcifying species. Responses of non-calcifying consumers are highly variable and mainly mediated through indirect ocean acidification effects induced by changing the biochemical content of their prey, as shown within single species and simple 2-trophic level systems. However, it can be expected that indirect CO2 impacts observed at the single species level are compensated at the ecosystem level by species richness and complex trophic interactions. A dampening of CO2-effects can be further expected for coastal communities adapted to strong natural fluctuations in pCO2, typical for productive coastal habitats. Here we show that a plankton community of the Kiel Fjord was tolerant to CO2 partial pressure (pCO2) levels projected for the end of this century (<1400 µatm), and only subtle differences were observed at the extremely high value of 4000 µatm. We found similar phyto- and microzooplankton biomass and copepod abundance and egg production across all CO2 treatment levels. Stoichiometric phytoplankton food quality was minimally different at the highest pCO2 treatment, but was far from being potentially limiting for copepods. These results are in contrast to studies that include only a single species, which observe strong indirect CO2 effects for herbivores and suggest limitations of biological responses at the level of organism to community. Although this coastal plankton community was highly tolerant to high fluctuations in pCO2, increase in hypoxia and CO2 uptake by the ocean can aggravate acidification and may lead to pH changes outside the range presently experienced by coastal organisms

Fatty acid composition and concentration of <i>Thalassiorira pseudonana</i> cultured at different CO₂ treatments.

<p><b>A</b>) Percentage of polyunsaturated (PUFA), monounsaturated (MUFA), and saturated (SFA) fatty acids relative to total fatty acids during the exponential growth phase cultured at low (realized value of 365 µatm <i>p</i>CO₂, n = 5) and high (realized value of 915 µatm <i>p</i>CO₂, n = 3) CO₂ treatments used as copepod food source. <b>B</b>) Change in the fatty acid composition in <i>T. pseudonana</i> after a shift from high to low <i>p</i>CO₂ conditions (n = 1 per treatment level). Time 0 are measured values before the culture media shift. Error bars indicate standard errors.</p

Regression statistics of <i>Acartia tonsa</i> egg production as a linear function of fatty acid composition.

<p>Bonferroni-corrected significance levels for multiple fatty acid comparisons were α = 0.008 (0.05/6). Significant correlations are highlighted in bold; n = 11. PUFA = polyunsaturated fatty acid; MUFA = monounsaturated fatty acid; SFA = saturated fatty acid; ARA-EPA = 20:5<i>n</i>3; DHA = docosahexaenoic acid (22:6<i>n</i>3).</p

Fatty acid composition, somatic growth and reproduction of <i>Acartia tonsa</i> across CO₂ treatment combinations.

<p><b>A</b>) Percentage of polyunsaturated (PUFA), monounsaturated (MUFA), and saturated (SFA) fatty acids relative to total fatty acids in female copepods. <b>B</b>) Principal component analysis (PCA) of fatty acid composition for the dietary algae <i>Thalassiorira pseudonana</i> and <i>A. tonsa</i> of the different treatment combinations. PCA scores 1 explained 40% of the variability (see <i>x</i>-axis of c) and was highly negatively correlated with 22:6<i>n</i>-3 (r² = 0.73), 20:4<i>n</i>-6+20:5<i>n</i>-3 (r² = 0.85), 18:3<i>n</i>-6 (r² = 0.73) and 16:1 (r² = 0.79), and positively with 22:1<i>n</i>-9 (r² = 0.25) and 18:1<i>n</i>-9<i>t</i> (r² = 0.57). PCA score 2 explained 17% of the overall variability (see <i>y</i>-axis of c) and was strongest positively correlated with 24:0 (r² = 0.84). Loadings of the PC scores are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034737#pone.0034737.s002" target="_blank">Figure S2</a>). <b>C</b>) Stage distribution of <i>A. tonsa</i> individuals at day 10. C4, C5, C6 = copepodite stage 4, 5, and adult, respectively. <b>D</b>) Egg production rate (EPR) of incubated females (n = 12 per treatment level). EPR was significantly different between treatments (F_{(3, 44)} = 18.02, <i>p</i><0.001). Different letters above bars represent significant differences from a Tukey HSD test. The bars represent the 25^th, 50^th and 75^th percentiles, whiskers stand for the 10^th and the 90^th percentiles and black points show outliers. Legend refers to treatment combinations of copepod zooplankton (Z) and phytoplankton food source (P) at low (L) and high (H) <i>p</i>CO₂.</p

Proximity proteomics of C9orf72 dipeptide repeat proteins identifies molecular chaperones as modifiers of poly-GA aggregation

The most common inherited cause of two genetically and clinico-pathologically overlapping neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), is the presence of expanded GGGGCC intronic hexanucleotide repeats in the C9orf72 gene. Aside from haploinsufficiency and toxic RNA foci, another non-exclusive disease mechanism is the non-canonical translation of the repeat RNA into five different dipeptide repeat proteins (DPRs), which form neuronal inclusions in affected patient brains. While evidence from cellular and animal models supports a toxic gain-of-function of pathologic poly-GA, poly-GR, and poly-PR aggregates in promoting deposition of TDP-43 pathology and neurodegeneration in affected brain areas, the relative contribution of DPRs to the disease process in c9FTD/ALS patients remains unclear. Here we have used the proximity-dependent biotin identification (BioID) proximity&nbsp;proteomics approach to investigate the formation and collective composition of DPR aggregates using cellular models. While interactomes of arginine rich poly-GR and poly-PR aggregates overlapped and were enriched for nucleolar and ribosomal proteins, poly-GA aggregates demonstrated a distinct association with proteasomal components, molecular chaperones (HSPA1A/HSP70, HSPA8/HSC70, VCP/p97), co-chaperones (BAG3, DNAJA1A) and other factors that regulate protein folding and degradation (SQSTM1/p62, CALR, CHIP/STUB1). Experiments in cellular models of poly-GA pathology show that molecular chaperones and co-chaperones are sequestered to the periphery of dense cytoplasmic aggregates, causing depletion from their typical cellular localization. Their involvement in the pathologic process is confirmed in autopsy brain tissue, where HSPA8, BAG3, VCP, and its adapter protein UBXN6 show a close association with poly-GA aggregates in the frontal cortex, temporal cortex, and hippocampus of c9FTLD and c9ALS cases. The association of heat shock proteins and co-chaperones with poly-GA led us to investigate their potential role in reducing its aggregation. We identified HSP40 co-chaperones of the DNAJB family as potent modifiers that increased the solubility of poly-GA, highlighting a possible novel therapeutic avenue and a central role of molecular chaperones in the pathogenesis of human C9orf72-linked diseases

Tdp-43 Pathology Disrupts Nuclear Pore Complexes And Nucleocytoplasmic Transport In Als/Ftd

The cytoplasmic mislocalization and aggregation of TAR DNA-binding protein-43 (TDP-43) is a common histopathological hallmark of the amyotrophic lateral sclerosis and frontotemporal dementia disease spectrum (ALS/FTD). However, the composition of aggregates and their contribution to the disease process remain unknown. Here we used proximity-dependent biotin identification (BioID) to interrogate the interactome of detergent-insoluble TDP-43 aggregates and found them enriched for components of the nuclear pore complex and nucleocytoplasmic transport machinery. Aggregated and disease-linked mutant TDP-43 triggered the sequestration and/or mislocalization of nucleoporins and transport factors, and interfered with nuclear protein import and RNA export in mouse primary cortical neurons, human fibroblasts and induced pluripotent stem cell-derived neurons. Nuclear pore pathology is present in brain tissue in cases of sporadic ALS and those involving genetic mutations in TARDBP and C9orf72. Our data strongly implicate TDP-43-mediated nucleocytoplasmic transport defects as a common disease mechanism in ALS/FTD

TDP-43 pathology disrupts nuclear pore complexes and nucleocytoplasmic transport in ALS/FTD

The cytoplasmic mislocalization and aggregation of TAR DNA-binding protein-43 (TDP-43) is a common histopathological hallmark of the amyotrophic lateral sclerosis and frontotemporal dementia disease spectrum (ALS/FTD). However, the composition of aggregates and their contribution to the disease process remain unknown. Here we used proximity-dependent biotin identification (BioID) to interrogate the interactome of detergent-insoluble TDP-43 aggregates and found them enriched for components of the nuclear pore complex and nucleocytoplasmic transport machinery. Aggregated and disease-linked mutant TDP-43 triggered the sequestration and/or mislocalization of nucleoporins and transport factors, and interfered with nuclear protein import and RNA export in mouse primary cortical neurons, human fibroblasts and induced pluripotent stem cell-derived neurons. Nuclear pore pathology is present in brain tissue in cases of sporadic ALS and those involving genetic mutations in TARDBP and C9orf72. Our data strongly implicate TDP-43-mediated nucleocytoplasmic transport defects as a common disease mechanism in ALS/FTD.ALS Association [17-IIP-353, 16-IIP-278]; Emory Medicine Catalyst Funding Program; Muscular Dystrophy Association [MDA348086]; NIH [K08-NS087121, P30-NS055077, AG025688, R01-NS091299, R35-NS097261, R01-NS085207, R01NS091749, R01-NS093362, R01-AG053960]; The Bluefield Project to Cure FTD; Alzheimers Drug Discovery Foundation; Alzheimers Association (ALZ); Alzheimers Research UK (ARUK); The Michael J. Fox Foundation for Parkinsons Research (MJFF); Weston Brain Institute Biomarkers Across Neurodegenerative Diseases Grant [11060]; UBRP; UA Provosts Office; ARCS Fellowship Roche Foundation Award6 month embargo; published online: 08 January 2018This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]