Cytoplasmic poly-GA aggregates impair nuclear import of TDP-43 in C9orf72 ALS/FTLD

A repeat expansion in the non-coding region of C9orf72 gene is the most common mutation causing frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Sense and antisense transcripts are translated into aggregating dipeptide repeat (DPR) proteins in all reading frames (poly-GA,-GP,-GR,-PA and -PR) through an unconventional mechanism. How these changes contribute to cytoplasmic mislocalization and aggregation of TDP-43 and thereby ultimately leading to neuron loss remains unclear. The repeat RNA itself and poly-GR/PR have been linked to impaired nucleocytoplasmic transport. Here, we show that compact cytoplasmic poly-GA aggregates impair nuclear import of a reporter containing the TDP-43 nuclear localization (NLS) signal. However, a reporter containing a non-classical PY-NLS was not affected. Moreover, poly-GA expression prevents TNF alpha induced nuclear translocation of p65 suggesting that poly-GA predominantly impairs theimportin-alpha/beta-dependent pathway. In neurons, prolonged poly-GA expression induces partial mislocalization of TDP-43 into cytoplasmic granules. Rerouting poly-GA to the nucleus prevented TDP-43 mislocalization, suggesting a cytoplasmic mechanism. In rescue experiments, expression of importin-alpha (KPNA3, KPNA4) or nucleoporins (NUP54, NUP62) restores the nuclear localization of the TDP reporter. Taken together, inhibition of nuclear import of TDP-43 by cytoplasmic poly-GA inclusions causally links the two main aggregating proteins in C9orf72 ALS/FTLD pathogenesis

Proteomics and C9orf72 neuropathology identify ribosomes as poly-GR/PR interactors driving toxicity

Frontotemporal dementia and amyotrophic lateral sclerosis patients with C9orf72 mutation show cytoplasmic poly-GR and poly-PR aggregates. Short poly-(Gly-Arg) and poly-(Pro-Arg) (poly-GR/PR) repeats localizing to the nucleolus are toxic in various model systems, but no interactors have been validated in patients. Here, the neuronal interactomes of cytoplasmic GFP-(GR)(149) and nucleolar (PR)(175)-GFP revealed overlapping RNA-binding proteins, including components of stress granules, nucleoli, and ribosomes. Overexpressing the poly-GR/PR interactors STAU1/2 and YBX1 caused cytoplasmic aggregation of poly-GR/PR in large stress granule-like structures, whereas NPM1 recruited poly-GR into the nucleolus. Poly-PR expression reduced ribosome levels and translation consistent with reduction of synaptic proteins detected by proteomics. Surprisingly, truncated GFP-(GR)(53), but not GFP-(GR)(149), localized to the nucleolus and reduced ribosome levels and translation similar to poly-PR, suggesting that impaired ribosome biogenesis may be driving the acute toxicity observed in vitro. In patients, only ribosomes and STAU2 co-aggregated with poly-GR/PR. Partial sequestration of ribosomesmay chronically impair protein synthesis even in the absence of nucleolar localization and contribute to pathogenesis

Active poly-GA vaccination prevents microglia activation and motor deficits in a C9orf72 mouse model

The C9orf72 repeat expansion is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia (FTD). Non-canonical translation of the expanded repeat results in abundant poly-GA inclusion pathology throughout the CNS. (GA)(149)-CFP expression in mice triggers motor deficits and neuroinflammation. Since poly-GA is transmitted between cells, we investigated the therapeutic potential of anti-GA antibodies by vaccinating (GA)(149)-CFP mice. To overcome poor immunogenicity, we compared the antibody response of multivalent ovalbumin-(GA)(10) conjugates and pre-aggregated carrier-free (GA)(15). Only ovalbumin-(GA)(10) immunization induced a strong anti-GA response. The resulting antisera detected poly-GA aggregates in cell culture and patient tissue. Ovalbumin-(GA)(10) immunization largely rescued the motor function in (GA)(149)-CFP transgenic mice and reduced poly-GA inclusions. Transcriptome analysis showed less neuroinflammation in ovalbumin-(GA)(10)-immunized poly-GA mice, which was corroborated by semiquantitative and morphological analysis of microglia/macrophages. Moreover, cytoplasmic TDP-43 mislocalization and levels of the neurofilament light chain in the CSF were reduced, suggesting neuroaxonal damage is reduced. Our data suggest that immunotherapy may be a viable primary prevention strategy for ALS/FTD in C9orf72 mutation carriers

Annelid symbiont assemblage and European stone crayfish − a deterministic relationship with implications for conservation management

The protected European stone crayfish, Austropotamobius torrentium, is a host to epibionts inhabiting its exoskeleton. Despite evidence of the close association of these epibionts to the crayfish and the beneficial services provided for the crayfish, the main factors influencing their occurrence, distribution and abundance are still poorly understood. In order to investigate the stone crayfish epibiont community, the ecological requirements of individual species, and aspects of the host-epibiont relationship we collected data at several crayfish populations in Austria, Switzerland and Germany. At each location, we recorded the epibiont position on the crayfish host along with a number of biotic and abiotic parameters. Apart from recording the Branchiobdella species B. parasita, B. hexadonta, B. pentadonta and B. balcanica, we detected for the first time the polychaete Hystricosoma chappuisi in the European Alps. Species-specific biogeographical and habitat factors such as river catchment borders, crayfish length, water temperature and nutrients concentration were identified to be important for the occurrence and abundance of epibionts. Branchiobdella species were strongly linked to certain host body areas, providing evidence for the existence of different functional traits within the annelid assemblage. With this study we demonstrate that the crayfish–epibionts relationship includes structural and functional complexities that can be important for defining management units in future conservation policies. Due to benefits for the mutualistic relationship, restocking and reintroduction actions are likely to be more successful, if ecological requirements of both the host and the epibiont species are considered

Maternal and fetal genetic effects on birth weight and their relevance to cardio-metabolic risk factors.

Birth weight variation is influenced by fetal and maternal genetic and non-genetic factors, and has been reproducibly associated with future cardio-metabolic health outcomes. In expanded genome-wide association analyses of own birth weight (n = 321,223) and offspring birth weight (n = 230,069 mothers), we identified 190 independent association signals (129 of which are novel). We used structural equation modeling to decompose the contributions of direct fetal and indirect maternal genetic effects, then applied Mendelian randomization to illuminate causal pathways. For example, both indirect maternal and direct fetal genetic effects drive the observational relationship between lower birth weight and higher later blood pressure: maternal blood pressure-raising alleles reduce offspring birth weight, but only direct fetal effects of these alleles, once inherited, increase later offspring blood pressure. Using maternal birth weight-lowering genotypes to proxy for an adverse intrauterine environment provided no evidence that it causally raises offspring blood pressure, indicating that the inverse birth weight-blood pressure association is attributable to genetic effects, and not to intrauterine programming.The Fenland Study is funded by the Medical Research Council (MC_U106179471) and Wellcome Trust

Cell-to-cell transmission of C9orf72 poly-(Gly-Ala) triggers key features of ALS/FTD.

The C9orf72 repeat expansion causes amyotrophic lateral sclerosis and frontotemporal dementia, but the poor correlation between C9orf72-specific pathology and TDP-43 pathology linked to neurodegeneration hinders targeted therapeutic development. Here, we addressed the role of the aggregating dipeptide repeat proteins resulting from unconventional translation of the repeat in all reading frames. Poly-GA promoted cytoplasmic mislocalization and aggregation of TDP-43 non-cell-autonomously, and anti-GA antibodies ameliorated TDP-43 mislocalization in both donor and receiver cells. Cell-to-cell transmission of poly-GA inhibited proteasome function in neighboring cells. Importantly, proteasome inhibition led to the accumulation of TDP-43 ubiquitinated within the nuclear localization signal (NLS) at lysine 95. Mutagenesis of this ubiquitination site completely blocked poly-GA-dependent mislocalization of TDP-43. Boosting proteasome function with rolipram reduced both poly-GA and TDP-43 aggregation. Our data from cell lines, primary neurons, transgenic mice, and patient tissue suggest that poly-GA promotes TDP-43 aggregation by inhibiting the proteasome cell-autonomously and non-cell-autonomously, which can be prevented by inhibiting poly-GA transmission with antibodies or boosting proteasome activity with rolipram

Congenic expression of poly-GA but not poly-PR in mice triggers selective neuron loss and interferon responses found in C9orf72 ALS

Expansion of a (G(4)C(2))(n)repeat inC9orf72causes amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but the link of the five repeat-encoded dipeptide repeat (DPR) proteins to neuroinflammation, TDP-43 pathology, and neurodegeneration is unclear. Poly-PR is most toxic in vitro, but poly-GA is far more abundant in patients. To directly compare these in vivo, we created congenic poly-GA and poly-PR mice. 40% of poly-PR mice were affected with ataxia and seizures, requiring euthanasia by 6 weeks of age. The remaining poly-PR mice were asymptomatic at 14 months of age, likely due to an 80% reduction of the transgene mRNA in this subgroup. In contrast, all poly-GA mice showed selective neuron loss, inflammation, as well as muscle denervation and wasting requiring euthanasia before 7 weeks of age. In-depth analysis of peripheral organs and blood samples suggests that peripheral organ failure does not drive these phenotypes. Although transgene mRNA levels were similar between poly-GA and affected poly-PR mice, poly-GA aggregated far more abundantly than poly-PR in the CNS and was also found in skeletal muscle. In addition, TDP-43 and other disease-linked RNA-binding proteins co-aggregated in rare nuclear inclusions in the hippocampus and frontal cortex only in poly-GA mice. Transcriptome analysis revealed activation of an interferon-responsive pro-inflammatory microglial signature in end-stage poly-GA but not poly-PR mice. This signature was also found in all ALS patients and enriched inC9orf72cases. In summary, our rigorous comparison of poly-GA and poly-PR toxicity in vivo indicates that poly-GA, but not poly-PR at the same mRNA expression level, promotes interferon responses inC9orf72disease and contributes to TDP-43 abnormalities and neuron loss selectively in disease-relevant regions