Kinase inhibition of G2019S-LRRK2 restores autolysosome formation and function to reduce endogenous alpha-synuclein intracellular inclusions

Abstract

words)The Parkinson´s disease (PD)-associated kinase Leucine-Rich Repeat Kinase 2 (LRRK2) is a potent modulator of autophagy and impacts on lysosome biology and function, but unclarity exists on the precise mechanics of its role and the direction of this modulation. LRRK2 is also involved in the degradation of pathological alpha-synuclein, with pathogenic mutations precipitating neuropathology in cellular and animal models of PD, and most LRRK2 familial cases manifesting with Lewy neuropathology. Defects in autophagic processing and lysosomal degradation of alpha-synuclein have been postulated to underlie its accumulation and onset of neuropathology. Thus, it is critical to reconcile these independent pieces of information to obtain a comprehensive knowledge on LRRK2-associated pathology that could also be generalized to the idiopathic disease. Here, we report a focused investigation on the role of PD-causing G2019S-LRRK2 in the autophagy-lysosome pathway in a recombinant cell line model. Initially, we evaluated the effect of LRRK2 expression on autophagy-related transcriptome. Then, we found that G2019S-LRRK2 leads to accumulation of autophagosomes with no net effect on autophagy induction. This is linked to abnormalities in lysosome morphology and proteolytic activity that are associated with a decrease in the successful formation of autolysosomes. Despite some of these features being shared by WT-LRRK2, alpha-synuclein intracellular inclusions are specifically found in G2019S-LRRK2 cells. Pharmacological kinase inhibition is capable of rescuing defects in the autophagy-lysosome pathway and reducing the number of inclusions. Notably, this effect is prevented by upstream blockade of autophagosome-lysosome fusion events, highlighting this step of the process as critical for alpha-synuclein clearance. Mutations in the Lrrk2 gene cause late-onset, familial Parkinson´s disease (PD) with variable penetrance that is clinically indistinguishable from idiopathic PD (iPD) but with pleomorphic pathology 1 . However, the G2019S mutation is singularly the most common genetic cause of PD worldwide, with an incidence up to 40% in specific populations 2,3 , and mostly presents with alpha-synuclein (aSyn) Lewy neuropathology at autopsy 4 . The gene codes for Leucine-Rich Repeat Kinase 2 (LRRK2), a large multidomain protein with two distinct enzymatic domains (GTPase and kinase) in close vicinity to each other 5 . The PDlinked mutations reside in this enzymatic core with G2019S located in the kinase domain and reported to increase kinase activity 6 . The cellular roles impacted by LRRK2 are varied, with stronger consensus on synaptic transmission 7 , vesicle trafficking 8 and autophagy 9 . Interestingly, these pathways might converge in neuronal biology and function 10 . Several independent investigations demonstrated that LRRK2 acts at different stages of the autophagy-lysosome pathway, with some conflicting results on the direction of this modulation 9 . Indications include a kinase-dependent role of LRRK2 in the modulation of...

“LRRK2: Autophagy and Lysosomal Activity”

“…However, the results presented by Henderson et al (2019) and Volpicelli-Daley et al (2016) taken together could suggest that it is mutated LRRK2 that increases the progression of pathological α-synuclein inclusions by increasing a pool of αsynuclein that is more susceptible to forming inclusions. These observations along with studies demonstrating α-synuclein is processed by lysosomes in cell models overexpressing LRRK2 (Hu et al, 2018;Obergasteiger et al, 2019), underline the prospects of targetting lysosomal function as novel drugdeveloping avenues worth pursuing for LRRK2-related PD.…”

“…Given the LRRK2-G2019S induced inhibitory effect on Cathepsins B and L was not dependent on kinase activity (Hu et al, 2018), it would be interesting to verify this finding in other LRRK2 mutations, in addition to confirming it in neuronal cell models. A transcriptomic analysis of autophagy related genes in SH-SY5Y cells overexpressing LRRK2-WT or LRRK2-G2019S also found altered mRNA levels of CTSB, which encodes for Cathepsin B (Obergasteiger et al, 2019). Overexpression of LRRK2-G2019S in cultured neurons and DAn in the rat SNpc exhibited an accumulation of α-synuclein inclusions after exposure to sonicated α-synuclein fibrils, when compared to nTG and to LRRK2-WT.…”

“…Proteolytic activity of lysosomes assessed with DQ-Red-BSA assay demonstrated LRRK2-G2019S induced a defect in lysosomal activity, culminating in an accumulation of endogenous α-synuclein inclusions. Defects in autophagosome-lysosomal fusion, proteolytic impairment and α-synuclein accumulation phenotypes were rescued after treatment with the PF-475 LRRK2 kinase inhibitor, highlighting the importance of LRRK2 in autophagy-mediated α-synuclein degradation (Obergasteiger et al, 2019).…”

“…Caution should be taken given that autophagy has been reported to be upregulated in H4 neuroglioma cancer cells to overcome stress induced apoptosis (Zhou et al, 2016), unlike in PD neuronal models where autophagy is deficient. On the other hand, a study by Obergasteiger et al (2019) using SH-SY5Y cells overexpressing LRRK2-WT or LRRK2-G2019S shows that LRRK2-G2019S neurons present with increased LC3-II protein levels and LC3B puncta, indicating an autophagosome accumulation. Measurement of autolysosome production using a double-tagged GFP-LC3-mCherry construct in this model showed autolysosomes were reduced in LRRK2-G2019S when compared to LRRK2-WT, suggesting a defective autophagosomelysosome fusion (Figure 1).…”

“…• Deficient autophagosome-lysosome fusion (Saez-Atienzar et al, 2014;di Domenico et al, 2019;Obergasteiger et al, 2019) • Deficient autophagosome-lysosome fusion Lysosomal function…”