PARylation regulates stress granule dynamics, phase separation, and neurotoxicity of disease-related RNA-binding proteins.

Mutations in RNA-binding proteins (RBPs) localized in ribonucleoprotein (RNP) granules, such as hnRNP A1 and TDP-43, promote aberrant protein aggregation, which is a pathological hallmark of various neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Protein posttranslational modifications (PTMs) are known to regulate RNP granules. In this study, we investigate the function of poly(ADP-ribosyl)ation (PARylation), an important PTM involved in DNA damage repair and cell death, in RNP granule-related neurodegeneration. We reveal that PARylation levels are a major regulator of the assembly-disassembly dynamics of RNP granules containing disease-related RBPs, hnRNP A1 and TDP-43. We find that hnRNP A1 can both be PARylated and bind to PARylated proteins or poly(ADP-ribose) (PAR). We further uncover that PARylation of hnRNP A1 at K298 controls its nucleocytoplasmic transport, whereas PAR-binding via the PAR-binding motif (PBM) of hnRNP A1 regulates its association with stress granules. Moreover, we reveal that PAR not only dramatically enhances the liquid-liquid phase separation of hnRNP A1, but also promotes the co-phase separation of hnRNP A1 and TDP-43 in vitro and their interaction in vivo. Finally, both genetic and pharmacological inhibition of PARP mitigates hnRNP A1- and TDP-43-mediated neurotoxicity in cell and Drosophila models of ALS. Together, our findings suggest a novel and crucial role for PARylation in regulating the dynamics of RNP granules, and that dysregulation in PARylation and PAR levels may contribute to ALS disease pathogenesis by promoting protein aggregation.

Distinct multilevel misregulations of Parkin and PINK1 revealed in cell and animal models of TDP-43 proteinopathy.

Parkin and PINK1 play an important role in mitochondrial quality control, whose malfunction may also be involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). Excessive TDP-43 accumulation is a pathological hallmark of ALS and is associated with Parkin protein reduction in spinal cord neurons from sporadic ALS patients. In this study, we reveal that Parkin and PINK1 are differentially misregulated in TDP-43 proteinopathy at RNA and protein levels. Using knock-in flies, mouse primary neurons, and TDP-43Q331K transgenic mice, we further unveil that TDP-43 downregulates Parkin mRNA, which involves an unidentified, intron-independent mechanism and requires the RNA-binding and the protein-protein interaction functions of TDP-43. Unlike Parkin, TDP-43 does not regulate PINK1 at an RNA level. Instead, excess of TDP-43 causes cytosolic accumulation of cleaved PINK1 due to impaired proteasomal activity, leading to compromised mitochondrial functions. Consistent with the alterations at the molecular and cellular levels, we show that transgenic upregulation of Parkin but downregulation of PINK1 suppresses TDP-43-induced degenerative phenotypes in a Drosophila model of ALS. Together, these findings highlight the challenge associated with the heterogeneity and complexity of ALS pathogenesis, while pointing to Parkin-PINK1 as a common pathway that may be differentially misregulated in TDP-43 proteinopathy.