Dawson, Valina L.

Arginine-rich dipeptide repeat proteins (R-DPRs), abnormal translational products of a GGGGCC hexanucleotide repeat expansion in C9ORF72 , play a critical role in C9ORF72 -related amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), the most common genetic form of the disorders (c9ALS/FTD). R-DPRs form liquid condensates in vitro, induce stress granule formation in cultured cells, aggregate, and sometimes coaggregate with TDP-43 in postmortem tissue from patients with c9ALS/FTD. However, how these processes are regulated is unclear. Here, we show that loss of poly(ADP-ribose) (PAR) suppresses neurodegeneration in c9ALS/FTD fly models and neurons differentiated from patient-derived induced pluripotent stem cells. Mechanistically, PAR induces R-DPR condensation and promotes R-DPR–induced stress granule formation and TDP-43 aggregation. Moreover, PAR associates with insoluble R-DPR and TDP-43 in postmortem tissue from patients. These findings identified PAR as a promoter of R-DPR toxicity and thus a potential target for treating c9ALS/FTD.

Abstract Background: Parkinson’s disease (PD) is the second most prevalent neurodegenerative disorder. Biomarkers that can help monitor the progression of PD or response to disease-modifying agents will be invaluable in making appropriate therapeutic decisions. Further, biomarkers that could be used to distinguish PD from other related disorders with PD-like symptoms will be useful for accurate diagnosis and treatment. C-Abl tyrosine kinase is activated in PD resulting in increased phosphorylation of the tyrosine residue at position 39 (Y39) of a-synuclein (a-syn) (pY39 a-syn), which contributes to the death of dopaminergic neurons. Because pY39 a-syn may be pathogenic, monitoring pY39 a-syn could allow us to diagnose presymptomatic PD and help monitor disease progression and response to treatment. We sought to investigate if increased phosphorylation of pY39 a-syn can be detected in the cerebrospinal fluid (CSF) of PD patients by targeted mass spectrometry. Methods: Here, we report a two-step enrichment method in which phosphotyrosine peptides were first enriched with an anti-phosphotyrosine antibody followed by a second round of enrichment by titanium dioxide (TiO2) beads to detect EGVLpYVGSK sequence derived from tyrosine 39 region of α- and β-synuclein (aβ-syn). Accurate quantification was achieved by adding a synthetic heavy version of pY39 aβ-syn peptide before enzymatic digestion. Results: Using the developed enrichment methods and optimized parallel reaction monitoring (PRM) assays, we detected pY39 aβ-syn peptide in human CSF and demonstrated that the ratio of pY39 aβ-syn to Y39 aβ-syn was significantly increased in the CSF of patients with PD.Conclusions: We anticipate that this optimized two-step enrichment-based PRM detection method will help monitor c-Abl activation in PD patients and can also be used to quantify other phosphotyrosine peptides of low abundance in biological samples.

Abstract Background: Parkinson’s disease (PD) is the second most prevalent neurodegenerative disorder. Biomarkers that can help monitor the progression of PD or response to disease-modifying agents will be invaluable in making appropriate therapeutic decisions. Further, biomarkers that could be used to distinguish PD from other related disorders with PD-like symptoms will be useful for accurate diagnosis and treatment. C-Abl tyrosine kinase is activated in PD resulting in increased phosphorylation of the tyrosine residue at position 39 of a-synuclein (a-syn) (pY39 a-syn), which contributes to the death of dopaminergic neurons. Because pY39a-syn may be pathogenic, monitoring pY39 a-syn could allow us to diagnose presymptomatic PD and help monitor disease progression and response to treatment. We sought to investigate if increased phosphorylation of pY39 a-syn can be detected in the CSF of PD patients by targeted mass spectrometry. Methods: Here, we report a two-step enrichment method in which phosphotyrosine peptides including pY39 a-syn containing peptide were first enriched with an anti-phosphotyrosine antibody followed by a second round of enrichment by titanium oxide (TiO 2 ) beads. Accurate quantification was achieved by the addition of a synthetic heavy version of pY39 a-syn peptide added before enzymatic digestion. Results: Using the developed enrichment methods and optimized parallel reaction monitoring assays, we detected pY39 a-syn peptide in human CSF and demonstrate that the ratio of pY39 a-syn to total a-syn was significantly increased in the CSF of patients with PD. Conclusions: We anticipate that this optimized two-step enrichment-based PRM detection method will help monitor c-Abl activation in PD patients and can also be used to quantify other phosphotyrosine peptides of low abundance in biological samples.

Abstract Background: Parkinson’s disease (PD) is the second most prevalent neurodegenerative disorder. Biomarkers that can help monitor the progression of PD or response to disease-modifying agents will be invaluable in making appropriate therapeutic decisions. Further, biomarkers that could be used to distinguish PD from other related disorders with PD-like symptoms will be useful for accurate diagnosis and treatment. C-Abl tyrosine kinase is activated in PD resulting in increased phosphorylation of the tyrosine residue at position 39 of a-synuclein (a-syn) (pY39 a-syn), which contributes to the death of dopaminergic neurons. Because pY39a-syn may be pathogenic, monitoring pY39 a-syn could allow us to diagnose presymptomatic PD and help monitor disease progression and response to treatment. We sought to investigate if increased phosphorylation of pY39 a-syn can be detected in the CSF of PD patients by targeted mass spectrometry. Methods: Here, we report a two-step enrichment method in which phosphotyrosine peptides including pY39 a-syn containing peptide were first enriched with an anti-phosphotyrosine antibody followed by a second round of enrichment by titanium oxide (TiO2) beads. Accurate quantification was achieved by the addition of a synthetic heavy version of pY39 a-syn peptide added before enzymatic digestion. Results: Using the developed enrichment methods and optimized parallel reaction monitoring assays, we detected pY39 a-syn peptide in human CSF and demonstrate that the ratio of pY39 a-syn to total a-syn was significantly increased in the CSF of patients with PD. Conclusions: We anticipate that this optimized two-step enrichment-based PRM detection method will help monitor c-Abl activation in PD patients and can also be used to quantify other phosphotyrosine peptides of low abundance in biological samples.

Neural cells derived from induced pluripotent stem cells from patients with genetic forms of Parkinson’s disease provide insights into disease pathogenesis.

Mutation in leucine-rich repeat kinase-2 ( LRRK2 ) is the most common cause of late-onset Parkinson's disease (PD). Although most cases of PD are sporadic, some are inherited, including those caused by LRRK2 mutations. Because these mutations may be associated with a toxic gain of function, controlling the expression of LRRK2 may decrease its cytotoxicity. Here we show that the carboxyl terminus of HSP70-interacting protein (CHIP) binds, ubiquitinates, and promotes the ubiquitin proteasomal degradation of LRRK2. Overexpression of CHIP protects against and knockdown of CHIP exacerbates toxicity mediated by mutant LRRK2. Moreover, HSP90 forms a complex with LRRK2, and inhibition of HSP90 chaperone activity by 17AAG leads to proteasomal degradation of LRRK2, resulting in increased cell viability. Thus, increasing CHIP E3 ligase activity and blocking HSP90 chaperone activity can prevent the deleterious effects of LRRK2. These findings point to potential treatment options for LRRK2-associated PD.