Parkinson´s disease is a devastating neurodegenerative disorder. Whilst the majority of Parkinson´s disease cases are sporadic, around five to ten percent of cases are inherited, and causative mutations have been identified in a variety of genes. Autosomal-dominant point mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of familial late-onset Parkinson´s disease.  Additionally, common protein-coding and non-protein-coding variants at the LRRK2 locus comprise one of the major genetic susceptibility factors for sporadic Parkinson´s disease, suggesting that LRRK2 is key to the pathogenesis of the entire disease spectrum. All currently described pathogenic LRRK2 mutations have been shown to increase the LRRK2 kinase activity, suggesting that targeting this activity may comprise a possible disease-modifying strategy. Recent studies have also identified a subset of Rab GTPases as substrates for the LRRK2 kinase activity, and these substrates implicate LRRK2 in various intracellular membrane trafficking steps.

The Hilfiker laboratory aims to elucidate the mechanism(s) by which disease-associated LRRK2 mutations and variants induce molecular and cellular alterations which lead to eventual neurodegeneration in inherited and sporadic forms of Parkinson´s disease. The lab uses a multidisciplinary and translational approach employing cell biological, molecular and biochemical techniques in various model systems including human cell lines, primary cell cultures from transgenic rodent models, various human-derived peripheral cells as well as human postmortem brain tissue. Our goal is to understand the molecular pathogenesis of Parkinson´s disease, so as to aid in the development of novel therapies to treat or prevent this devastating disorder.

Our current work is centered around three missions:

1. Understand how pathogenic LRRK2 affects endolysosomal trafficking events relevant for Parkinson´s disease

Proper endolysosomal/autophagic trafficking is crucial towards maintaining cellular homeostasis, and alterations in this process are associated with the appearance of intracellular protein aggregates including alpha-synuclein, a pathological hallmark of Parkinson´s disease. We found that pathogenic LRRK2 causes deficits in autophagic/endolysosomal trafficking in a manner dependent on altered endolysosomal calcium levels and a decrease in the activity of Rab7, a small GTPase crucial for membrane trafficking steps to and from late endosomes/lysosomes. In addition, we recently discovered that knockdown of one of the Rab LRRK2 kinase substrates leads to the same deficits in endolysosomal trafficking as observed with pathogenic LRRK2, consistent with the idea that the LRRK2-mediated phosphorylation of this Rab protein causes its inactivation. We are working towards understanding the contribution of other LRRK2 kinase substrates on endolysosomal dyshomeostasis, and the possible beneficial effects of LRRK2 kinase inhibitors as well as other potential modulators on reversing this cellular phenotype.

Figure 1: Pathogenic LRRK2 causes the mistrafficking of receptors into endocytic recycling compartments.

2. Understand how pathogenic LRRK2 causes centrosomal alterations relevant for Parkinson´s disease

LRRK2 phosphorylation of Rab8a and Rab10 has been recently described to block primary cilia formation in cultured cells as well as in certain regions of the brain, which may impair an important neuroprotective pathway for the neurons which undergo cellular demise in Parkinson´s disease. We recently described that the phosphorylated versions of Rab8a and Rab10 accumulate around the mother centriole, and such accumulation causes both deficits in ciliogenesis as well as deficits in centrosome cohesion in dividing cells. We are trying to understand the mechanism by which such aberrant phospho-Rab protein accumulation causes the observed centrosomal and ciliogenesis alterations, as well as the respective contribution of other LRRK2 kinase substrates to these phenotypes, with the hope that such mechanistic insight will lead to the identification of additional proteins/pathways whose modulation may be beneficial in the context of at least LRRK2-related Parkinson´s disease.

Figure 2: Deficit in centrosome cohesion and ciliogenesis in primary astrocytes from G2019S LRRK2 knockin mice as compared to controls.

Figure 3: Schematics of mechanism by which pathogenic LRRK2 may cause deficits in centrosome cohesion and ciliogenesis.

3. Establish sensitive cellular assay(s) for LRRK2 kinase activity endpoint detection and/or for patient stratification purposes

Our work aims to establish cellular detection methods for altered LRRK2 activity using various peripheral human-derived cells from healthy control, sporadic and LRRK2-Parkinson´s disease patients. We have recently described centrosomal alterations in immortalized peripheral cells not only from all mutant LRRK2 patients analyzed, but also from a subset of sporadic Parkinson´s disease patients, suggesting that this readout may aid in the stratification of sporadic Parkinson´s disease patients who may benefit from LRRK2 kinase inhibitor treatments. We are expanding upon those studies to develop highly sensitive assays which may allow for the detection of altered LRRK2 kinase activity and serve to stratify patients with neurodegenerative disorders who may benefit from a LRRK2-directed treatment approach.

Figure 4: Hyvolution confocal image of duplicated centrosomes in a lymphoblastoid cell from a mutant LRRK2 Parkinson´s disease patient, stained for two distinct centrosomal markers (red and green).