HSP90 Inhibitor, NVP-AUY922, Improves Myelination in Vitro and Supports the Maintenance of Myelinated Axons in Neuropathic Mice.

Hereditary demyelinating neuropathies linked to peripheral myelin protein 22 (PMP22) involve the disruption of normal protein trafficking and are therefore relevant targets for chaperone therapy. Using a small molecule HSP90 inhibitor, EC137, in cell culture models, we previously validated the chaperone pathway as a viable target for therapy development. Here, we tested five commercially available inhibitors of HSP90 and identified BIIB021 and AUY922 to support Schwann cell viability and enhance chaperone expression. AUY922 showed higher efficacy, compared to BIIB021, in enhancing myelin synthesis in dorsal root ganglion explant cultures from neuropathic mice. For in vivo testing, we randomly assigned 2-3 month old C22 and 6 week old Trembler J (TrJ) mice to receive two weekly injections of either vehicle or AUY922 (2 mg/kg). By the intraperitoneal (i.p.) route, the drug was well-tolerated by all mice over the 5 month long study, without influence on body weight or general grooming behavior. AUY922 improved the maintenance of myelinated nerves of both neuropathic models and attenuated the decline in rotarod performance and peak muscle force production in C22 mice. These studies highlight the significance of proteostasis in neuromuscular function and further validate the HSP90 pathway as a therapeutic target for hereditary neuropathies

Evaluation of Current Methods to Detect Cellular Leucine-Rich Repeat Kinase 2 (LRRK2) Kinase Activity.

Background: Coding variation in the Leucine rich repeat kinase 2 gene linked to Parkinson’s disease (PD) promotes enhanced activity of the encoded LRRK2 kinase, particularly with respect to autophosphorylation at S1292 and/or phosphorylation of the heterologous substrate RAB10. Objective: To determine the inter-laboratory reliability of measurements of cellular LRRK2 kinase activity in the context of wildtype or mutant LRRK2 expression using published protocols. Methods: Benchmark western blot assessments of phospho-LRRK2 and phospho-RAB10 were performed in parallel with in situ immunological approaches in HEK293T, mouse embryonic fibroblasts, and lymphoblastoid cell lines. Rat brain tissue, with or without adenovirus-mediated LRRK2 expression, and human brain tissues from subjects with or without PD, were also evaluated for LRRK2 kinase activity markers. Results: Western blots were able to detect extracted LRRK2 activity in cells and tissue with pS1292-LRRK2 or pT73-RAB10 antibodies. However, while LRRK2 kinase signal could be detected at the cellular level with over-expressed mutant LRRK2 in cell lines, we were unable to demonstrate specific detection of endogenous cellular LRRK2 activity in cell culture models or tissues that we evaluated. Conclusion: Further development of reliable methods that can be deployed in multiple laboratories to measure endogenous LRRK2 activities are likely required, especially at cellular resolution.This research was supported in part by the Intramural Research Program of the NIH, National Institute on Aging and by NIH NINDS grants R01 NS064934 and P50NS108675 (to A.B.W.), the Alexander and Eva Nemeth Foundation and MJFF grant (17358) (to R.J.N. and T.J.M.), MJFF grants (12753 and 18287) (to D.J.M.), MJFF grant (10255.02) (to S.H. and M.C.C.H.) and Intramural Funds from Rutgers University (to S.H.).Peer reviewe

Evaluation of Current Methods to Detect Cellular Leucine-Rich Repeat Kinase 2 (LRRK2) Kinase Activity

Background: Coding variation in the Leucine rich repeat kinase 2 gene linked to Parkinson’s disease (PD) promotes enhanced activity of the encoded LRRK2 kinase, particularly with respect to autophosphorylation at S1292 and/or phosphorylation of the heterologous substrate RAB10. Objective: To determine the inter-laboratory reliability of measurements of cellular LRRK2 kinase activity in the context of wildtype or mutant LRRK2 expression using published protocols. Methods: Benchmark western blot assessments of phospho-LRRK2 and phospho-RAB10 were performed in parallel with in situ immunological approaches in HEK293T, mouse embryonic fibroblasts, and lymphoblastoid cell lines. Rat brain tissue, with or without adenovirus-mediated LRRK2 expression, and human brain tissues from subjects with or without PD, were also evaluated for LRRK2 kinase activity markers. Results: Western blots were able to detect extracted LRRK2 activity in cells and tissue with pS1292-LRRK2 or pT73-RAB10 antibodies. However, while LRRK2 kinase signal could be detected at the cellular level with over-expressed mutant LRRK2 in cell lines, we were unable to demonstrate specific detection of endogenous cellular LRRK2 activity in cell culture models or tissues that we evaluated. Conclusion: Further development of reliable methods that can be deployed in multiple laboratories to measure endogenous LRRK2 activities are likely required, especially at cellular resolution

Genetic and Environmental Factors Influence the Pleomorphy of LRRK2 Parkinsonism

Missense mutations in the LRRK2 gene were first identified as a pathogenic cause of Parkinson’s disease (PD) in 2004. Soon thereafter, a founder mutation in LRRK2, p.G2019S (rs34637584), was described, and it is now estimated that there are approximately 100,000 people worldwide carrying this risk variant. While the clinical presentation of LRRK2 parkinsonism has been largely indistinguishable from sporadic PD, disease penetrance and age at onset can be quite variable. In addition, its neuropathological features span a wide range from nigrostriatal loss with Lewy body pathology, lack thereof, or atypical neuropathology, including a large proportion of cases with concomitant Alzheimer’s pathology, hailing LRRK2 parkinsonism as the “Rosetta stone” of parkinsonian disorders, which provides clues to an understanding of the different neuropathological trajectories. These differences may result from interactions between the LRRK2 mutant protein and other proteins or environmental factors that modify LRRK2 function and, thereby, influence pathobiology. This review explores how potential genetic and biochemical modifiers of LRRK2 function may contribute to the onset and clinical presentation of LRRK2 parkinsonism. We review which genetic modifiers of LRRK2 influence clinical symptoms, age at onset, and penetrance, what LRRK2 mutations are associated with pleomorphic LRRK2 neuropathology, and which environmental modifiers can augment LRRK2 mutant pathophysiology. Understanding how LRRK2 function is influenced and modulated by other interactors and environmental factors—either increasing toxicity or providing resilience—will inform targeted therapeutic development in the years to come. This will allow the development of disease-modifying therapies for PD- and LRRK2-related neurodegeneration

Inducible HSP70 Is Critical in Preventing the Aggregation and Enhancing the Processing of PMP22

Chaperones, also called heat shock proteins (HSPs), transiently interact with proteins to aid their folding, trafficking, and degradation, thereby directly influencing the transport of newly synthesized molecules. Induction of chaperones provides a potential therapeutic approach for protein misfolding disorders, such as peripheral myelin protein 22 (PMP22)-associated peripheral neuropathies. Cytosolic aggregates of PMP22, linked with a demyelinating Schwann cell phenotype, result in suppression of proteasome activity and activation of proteostatic mechanisms, including the heat shock pathway. Although the beneficial effects of chaperones in preventing the aggregation and improving the trafficking of PMP22 have been repeatedly observed, the requirement for HSP70 in events remains elusive. In this study, we show that activation of the chaperone pathway in fibroblasts from PMP22 duplication-associated Charcot–Marie–Tooth disease type 1A patient with an FDA-approved small molecule increases HSP70 expression and attenuates proteasome dysfunction. Using cells from an HSP70.1/3 −/− (inducible HSP70) mouse model, we demonstrate that under proteotoxic stress, this chaperone is critical in preventing the aggregation of PMP22, and this effect is aided by macroautophagy. When examined at steady-state, HSP70 appears to play a minor role in the trafficking of wild-type-PMP22, while it is crucial for preventing the buildup of the aggregation-prone Trembler-J-PMP22. HSP70 aids the processing of Trembler-J-PMP22 through the Golgi and its delivery to lysosomes via Rab7-positive vesicles. Together, these results demonstrate a key role for inducible HSP70 in aiding the processing and hindering the accumulation of misfolded PMP22, which in turn alleviates proteotoxicity within the cells

Inducible HSP70 Is Critical in Preventing the Aggregation and Enhancing the Processing of PMP22

Chaperones, also called heat shock proteins (HSPs), transiently interact with proteins to aid their folding, trafficking, and degradation, thereby directly influencing the transport of newly synthesized molecules. Induction of chaperones provides a potential therapeutic approach for protein misfolding disorders, such as peripheral myelin protein 22 (PMP22)-associated peripheral neuropathies. Cytosolic aggregates of PMP22, linked with a demyelinating Schwann cell phenotype, result in suppression of proteasome activity and activation of proteostatic mechanisms, including the heat shock pathway. Although the beneficial effects of chaperones in preventing the aggregation and improving the trafficking of PMP22 have been repeatedly observed, the requirement for HSP70 in events remains elusive. In this study, we show that activation of the chaperone pathway in fibroblasts from PMP22 duplication-associated Charcot–Marie–Tooth disease type 1A patient with an FDA-approved small molecule increases HSP70 expression and attenuates proteasome dysfunction. Using cells from an HSP70.1/3 −/− (inducible HSP70) mouse model, we demonstrate that under proteotoxic stress, this chaperone is critical in preventing the aggregation of PMP22, and this effect is aided by macroautophagy. When examined at steady-state, HSP70 appears to play a minor role in the trafficking of wild-type-PMP22, while it is crucial for preventing the buildup of the aggregation-prone Trembler-J-PMP22. HSP70 aids the processing of Trembler-J-PMP22 through the Golgi and its delivery to lysosomes via Rab7-positive vesicles. Together, these results demonstrate a key role for inducible HSP70 in aiding the processing and hindering the accumulation of misfolded PMP22, which in turn alleviates proteotoxicity within the cells