Functional Analyses of Two Novel LRRK2 Pathogenic Variants in Familial Parkinson's Disease.

BACKGROUND: Pathogenic variants in the LRRK2 gene are a common monogenic cause of Parkinson's disease. However, only seven variants have been confirmed to be pathogenic. OBJECTIVES: We identified two novel LRRK2 variants (H230R and A1440P) and performed functional testing. METHODS: We transiently expressed wild-type, the two new variants, or two known pathogenic mutants (G2019S and R1441G) in HEK-293 T cells, with or without LRRK2 kinase inhibitor treatment. We characterized the phosphorylation and kinase activity of the mutants by western blotting. Thermal shift assays were performed to determine the folding and stability of the LRRK2 proteins. RESULTS: The two variants were found in two large families and segregate with the disease. They display altered LRRK2 phosphorylation and kinase activity. CONCLUSIONS: We identified two novel LRRK2 variants which segregate with the disease. The results of functional testing lead us to propose these two variants as novel causative mutations for familial Parkinson's disease. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

A Phosphosite Mutant Approach on LRRK2 Links Phosphorylation and Dephosphorylation to Protective and Deleterious Markers, Respectively.

The Leucine Rich Repeat Kinase 2 (LRRK2) gene is a major genetic determinant of Parkinson's disease (PD), encoding a homonymous multi-domain protein with two catalytic activities, GTPase and Kinase, involved in intracellular signaling and trafficking. LRRK2 is phosphorylated at multiple sites, including a cluster of autophosphorylation sites in the GTPase domain and a cluster of heterologous phosphorylation sites at residues 860 to 976. Phosphorylation at these latter sites is found to be modified in brains of PD patients, as well as for some disease mutant forms of LRRK2. The main aim of this study is to investigate the functional consequences of LRRK2 phosphorylation or dephosphorylation at LRRK2's heterologous phosphorylation sites. To this end, we generated LRRK2 phosphorylation site mutants and studied how these affected LRRK2 catalytic activity, neurite outgrowth and lysosomal physiology in cellular models. We show that phosphorylation of RAB8a and RAB10 substrates are reduced with phosphomimicking forms of LRRK2, while RAB29 induced activation of LRRK2 kinase activity is enhanced for phosphodead forms of LRRK2. Considering the hypothesis that PD pathology is associated to increased LRRK2 kinase activity, our results suggest that for its heterologous phosphorylation sites LRRK2 phosphorylation correlates to healthy phenotypes and LRRK2 dephosphorylation correlates to phenotypes associated to the PD pathological processes.

A ß-Secretase Modulator Decreases Tau Pathology and Preserves Short-Term Memory in a Mouse Model of Neurofibrillary Degeneration.

Identifying which among several in cellulo pharmacological activities is necessary for the proper in vivo activity is essential for further drug development against Alzheimer's disease pathophysiological processes. An in-depth structure-activity relationship-based study has been carried out, and two molecules, named MAGS02-14 and PEL24-199, that share a ß-secretase modulatory effect associated or not to a lysosomotropic activity in cellulo have been identified. In terms of chemical formulas, MAGS02-14 and PEL24-199 only differ from each other by a single nitrogen atom. The study aimed to elucidate the in vivo pharmacological effects of lysosomotropic and/or the ß-secretase modulatory activity in a tau pathology mouse model. To address this question, the THY-Tau22 transgenic model of tauopathy was treated with both compounds for 6 weeks in a curative paradigm. Short-term memory, tau burden, and inflammatory processes were analyzed using orthogonal methods, and PEL24-199, but not MAGS02-14, was shown to restore the short-term memory and reduce the neurofibrillary degenerating process. These effects were associated with a reduced phosphorylation of tau, an increased phosphatase expression, and decreased astrogliosis. Our results, therefore, suggest that the lysosomotropic activity may be nonessential for the effect on tau pathology.

Mind the Gap: LRRK2 Phenotypes in the Clinic vs. in Patient Cells.

Mutations in the Parkinson's disease (PD) protein Leucine Rich Repeat Kinase 2 (LRRK2) have been under study for more than 15 years and our understanding of the cellular phenotypes for the pathogenic mutant forms of LRRK2 has significantly advanced. In parallel to research on LRRK2 mutations in experimental systems, clinical characterization of patients carrying LRRK2 mutations has advanced, as has the analysis of cells that are derived from these patients, including fibroblasts, blood-derived cells, or cells rendered pluripotent. Under the hypothesis that patient clinical phenotypes are a consequence of a cascade of underlying molecular mechanisms gone astray, we currently have a unique opportunity to compare findings from patients and patient-derived cells to ask the question of whether the clinical phenotype of LRRK2 Parkinson's disease and cellular phenotypes of LRRK2 patient-derived cells may be mutually informative. In this review, we aim to summarize the available information on phenotypes of LRRK2 mutations in the clinic, in patient-derived cells, and in experimental models in order to better understand the relationship between the three at the molecular and cellular levels and identify trends and gaps in correlating the data.