Comprehensive characterization and optimization of anti-LRRK2 (leucine-rich repeat kinase 2) monoclonal antibodies.

Missense mutations in LRRK2 (leucine-rich repeat kinase 2) are a major cause of PD (Parkinson's disease). Several antibodies against LRRK2 have been developed, but results using these polyclonal antibodies have varied widely leading to conflicting conclusions. To address this challenge, the Michael J. Fox Foundation for Parkinson's Research generated a number of monoclonal antibodies targeting epitopes across the LRRK2 protein. In the present paper, we report optimized protocols and results for ten monoclonal antibodies for immunoblotting, immunohistochemistry, immunoprecipitation and kinase activity assays, in rat, mouse and human brain tissue. Several efficacious antibodies were identified, but results demonstrate that the mouse monoclonal N241A/34 is suitable for most applications, with the best overall rabbit monoclonal antibody being c41-2. These antibodies produced a dominant band of the expected size via immunoblotting and a lack of labelling in tissue derived from LRRK2-knockout animals under optimized conditions. A significant proportion of LRRK2 protein localizes to insoluble fractions and no evidence of truncated LRRK2 protein was detected in any fraction from rodent or human tissues. An assay was developed for the robust detection of LRRK2 kinase activity directly from frozen mouse and human brain tissue, but precipitous declines in activity were observed that corresponded to increasing post-mortem intervals and processing times. Finally, we demonstrate the highest levels of brain-localized LRRK2 in the striatum, but note differential expression patterns between rat and mouse in both striatum and cortex. Anti-LRRK2 monoclonal antibodies that are unlimited in availability together with the proposed standardized protocols should aid in the definition of LRRK2 function in both health and disease.

Effects of gene replacement therapy with resamirigene bilparvovec (AT132) on skeletal muscle pathology in X-linked myotubular myopathy: results from a substudy of the ASPIRO open-label clinical trial.

BACKGROUND: X-linked myotubular myopathy (XLMTM) is a rare, life-threatening congenital muscle disease caused by mutations in the MTM1 gene that result in profound muscle weakness, significant respiratory insufficiency, and high infant mortality. There is no approved disease-modifying therapy for XLMTM. Resamirigene bilparvovec (AT132; rAAV8-Des-hMTM1) is an investigational adeno-associated virus (AAV8)-mediated gene replacement therapy designed to deliver MTM1 to skeletal muscle cells and achieve long-term correction of XLMTM-related muscle pathology. The clinical trial ASPIRO (NCT03199469) investigating resamirigene bilparvovec in XLMTM is currently paused while the risk:benefit balance associated with this gene therapy is further investigated. METHODS: Muscle biopsies were taken before treatment and 24 and 48 weeks after treatment from ten boys with XLMTM in a clinical trial of resamirigene bilparvovec (ASPIRO; NCT03199469). Comprehensive histopathological analysis was performed. FINDINGS: Baseline biopsies uniformly showed findings characteristic of XLMTM, including small myofibres, increased internal or central nucleation, and central aggregates of organelles. Biopsies taken at 24 weeks post-treatment showed marked improvement of organelle localisation, without apparent increases in myofibre size in most participants. Biopsies taken at 48 weeks, however, did show statistically significant increases in myofibre size in all nine biopsies evaluated at this timepoint. Histopathological endpoints that did not demonstrate statistically significant changes with treatment included the degree of internal/central nucleation, numbers of triad structures, fibre type distributions, and numbers of satellite cells. Limited (predominantly mild) treatment-associated inflammatory changes were seen in biopsy specimens from five participants. INTERPRETATION: Muscle biopsies from individuals with XLMTM treated with resamirigene bilparvovec display statistically significant improvement in organelle localisation and myofibre size during a period of substantial improvements in muscle strength and respiratory function. This study identifies valuable histological endpoints for tracking treatment-related gains with resamirigene bilparvovec, as well as endpoints that did not show strong correlation with clinical improvement in this human study. FUNDING: Astellas Gene Therapies (formerly Audentes Therapeutics, Inc.).

Intrahepatic Cholestasis Is a Clinically Significant Feature Associated with Natural History of X-Linked Myotubular Myopathy (XLMTM): A Case Series and Biopsy Report.

X-linked myotubular myopathy (XLMTM) is a rare, life-threatening congenital myopathy characterized by profound skeletal muscle weakness, respiratory distress, and motor dysfunction. However, pathology is not limited to muscle and can be associated with life-threatening hepatic peliosis. Hepatobiliary disease has been reported in up to 17% of XLMTM patients but has not been extensively characterized. We report on five XLMTM patients who experienced intrahepatic cholestasis in their disease natural history, illustrating the need to further investigate these manifestations. These patients shared presentations that included pruritus, hypertransaminemia, and hyperbilirubinemia with normal gamma-glutamyl transferase, following infection or vaccination. Three patients who had genetic testing showed no evidence of genetic mutations associated with familial cholestasis. In one patient, progression to cirrhotic, decompensated liver disease occurred. Further investigations into the molecular pathomechanism underpinning these clinical observations in XLMTM patients will be important for informing patient care.

INCEPTUS Natural History, Run-in Study for Gene Replacement Clinical Trial in X-Linked Myotubular Myopathy.

BACKGROUND: X-linked myotubular myopathy (XLMTM) is a life-threatening congenital myopathy that, in most cases, is characterized by profound muscle weakness, respiratory failure, need for mechanical ventilation and gastrostomy feeding, and early death. OBJECTIVE: We aimed to characterize the neuromuscular, respiratory, and extramuscular burden of XLMTM in a prospective, longitudinal study. METHODS: Thirty-four participants < 4 years old with XLMTM and receiving ventilator support enrolled in INCEPTUS, a prospective, multicenter, non-interventional study. Disease-related adverse events, respiratory and motor function, feeding, secretions, and quality of life were assessed. RESULTS: During median (range) follow-up of 13.0 (0.5, 32.9) months, there were 3 deaths (aspiration pneumonia; cardiopulmonary failure; hepatic hemorrhage with peliosis) and 61 serious disease-related events in 20 (59%) participants, mostly respiratory (52 events, 18 participants). Most participants (80%) required permanent invasive ventilation (>16 hours/day); 20% required non-invasive support (6-16 hours/day). Median age at tracheostomy was 3.5 months (95% CI: 2.5, 9.0). Thirty-three participants (97%) required gastrostomy. Thirty-one (91%) participants had histories of hepatic disease and/or prospectively experienced related adverse events or laboratory or imaging abnormalities. CHOP INTEND scores ranged from 19-52 (mean: 35.1). Seven participants (21%) could sit unsupported for≥30 seconds (one later lost this ability); none could pull to stand or walk with or without support. These parameters remained static over time across the INCEPTUS cohort. CONCLUSIONS: INCEPTUS confirmed high medical impact, static respiratory, motor and feeding difficulties, and early death in boys with XLMTM. Hepatobiliary disease was identified as an under-recognized comorbidity. There are currently no approved disease-modifying treatments.

Flotillin-mediated endocytic events dictate cell type-specific responses to semaphorin 3A.

Cortical efferents growing in the same environment diverge early in development. The expression of particular transcription factors dictates the trajectories taken, presumably by regulating responsiveness to guidance cues via cellular mechanisms that are not yet known. Here, we show that cortical neurons that are dissociated and grown in culture maintain their cell type-specific identities defined by the expression of transcription factors. Using this model system, we sought to identify and characterize mechanisms that are recruited to produce cell type-specific responses to Semaphorin 3A (Sema3A), a guidance cue that would be presented similarly to cortical axons in vivo. Axons from presumptive corticofugal neurons lacking the transcription factor Satb2 and expressing Ctip2 or Tbr1 respond far more robustly to Sema3A than those from presumptive callosal neurons expressing Satb2. Both populations of axons express similar levels of Sema3A receptors (neuropilin-1, cell adhesion molecule L1, and plexinA4), but significantly, axons from neurons lacking Satb2 internalize more Sema3A, and they do so via a raft-mediated endocytic pathway. We used an in silico approach to identify the endocytosis effector flotillin-1 as a Sema3A signaling candidate. We tested the contributions of flotillin-1 to Sema3A endocytosis and signaling, and show that raft-mediated Sema3A endocytosis is defined by and depends on the recruitment of flotillin-1, which mediates LIM domain kinase activation and regulates axon responsiveness to Sema3A in presumptive corticofugal axons.

Short- and long-term effects of LRRK2 on axon and dendrite growth.

Mutations in leucine-rich repeat kinase 2 (LRRK2) underlie an autosomal-dominant form of Parkinson's disease (PD) that is clinically indistinguishable from idiopathic PD. The function of LRRK2 is not well understood, but it has become widely accepted that LRRK2 levels or its kinase activity, which is increased by the most commonly observed mutation (G2019S), regulate neurite growth. However, growth has not been measured; it is not known whether mean differences in length correspond to altered rates of growth or retraction, whether axons or dendrites are impacted differentially or whether effects observed are transient or sustained. To address these questions, we compared several developmental milestones in neurons cultured from mice expressing bacterial artificial chromosome transgenes encoding mouse wildtype-LRRK2 or mutant LRRK2-G2019S, Lrrk2 knockout mice and non-transgenic mice. Over the course of three weeks of development on laminin, the data show a sustained, negative effect of LRRK2-G2019S on dendritic growth and arborization, but counter to expectation, dendrites from Lrrk2 knockout mice do not elaborate more rapidly. In contrast, young neurons cultured on a slower growth substrate, poly-L-lysine, show significantly reduced axonal and dendritic motility in Lrrk2 transgenic neurons and significantly increased motility in Lrrk2 knockout neurons with no significant changes in length. Our findings support that LRRK2 can regulate patterns of axonal and dendritic growth, but they also show that effects vary depending on growth substrate and stage of development. Such predictable changes in motility can be exploited in LRRK2 bioassays and guide exploration of LRRK2 function in vivo.