LRRK-2 gene in Parkinson's Disease research paper
I would like to share a literature review on a protein involved in Parkinson’s Disease that I wrote while working as a directed study student in Cathy Rankin’s lab at the UBC Center for Brain Health.
Abstract
This review summarizes the current understanding of leucine-rich repeat kinase domain (LRRK2)’s role in Parkinson’s Disease (PD) progression regarding its biochemical activity, molecular interactions, and alterations in mutant phenotypes. Evidence from many types of animal models and cell lines is used to build general conclusions and speculations on the cellular pathways LRRK2 and its different mutations are involved in, and how these depict LRRK2’s role in PD. Emphasis on recent findings in c. elegans will be useful in determining future research avenues to help find viable cures. The occurring themes of vesicle trafficking, cytoskeletal dynamics, protein translation and autophagy disruption and their key pathogenic protein interactions and pathways are discussed. Contributions of alpha-synuclein to LRRK-2 PD symptoms are intriguing and deserve further investigation.
Keywords: Parkinson’s Disease, LRRK2, vesicle trafficking, autophagy, cytoskeletal dynamics, protein translation, kinase, genetic risk
The role of LRRK-2 in Parkinson’s Disease (PD)
Parkinson’s Disease (PD) remains the second most common neurodegenerative disease. Given that age is the greatest risk factor for developing the disease (Collier et al., 2011), and that Canada has an aging population, Parkinson’s disease prevalence is expected to double over 25 years to reach 8.7-9.3 million by 2030 (Dorsey, 2007). PD is characterized clinically by resting tremor, rigidity and hypokinesia, and presents pathologically with loss of dopaminergic neurons in the substantia nigra, and usually lewy body formation containing aggregated alpha-synuclein and or tau proteins. There are currently no therapies that can slow or prevent the disease. While 90% of cases are sporadic, genetic factors have been found to play a role in 5-10% of cases. There are three familial genes known to be associated with PD: alpha-synuclein (SNCA), leucine-rich repeat kinase-2 (LRRK2), and vacuolar protein sorting-associated protein 35 (VPS35) (Zimprich et al., 2004)(Rideout, 2017). It is interesting that SNCA PD is fully penetrant, while LRRK2 mutations have reduced and age-dependent penetrance (Healy et al., 2008; Clark et al., 2006; Goldwurm et al., 2007; Ozelius et al., 2006; Ruiz-Martinez et al., 2010). Perhaps not surprisingly, the disease pathology most commonly found in LRRK2 carriers is lewy-bodies, composed of alpha-synuclein aggregates (Hardy, 2009). However, not all animal models and humans with classic PD symptoms present with lewy body pathology, making LRRK2 a promising candidate in the search for the root cause of most forms of PD. Mutations in the LRRK2 (Park8) gene are the most common cause of the genetic form of the disease and drive pathogenesis in both familial and non-familial PD (Kumari and Tan, 2009). Thus, this review focuses mainly on LRRK2 and how it may be involved with other genetic risk factors in driving PD pathogenesis. Both familial and non-familial forms of LRRK-2 PD have been found to have an almost indistinguishable clinicopathology regarding incomplete penetrance, age of onset, the likely (although not required) presence of Lewy bodies (LBs), and motor and nonmotor symptoms (Healy et al., 2008; Kasten et al., 2010; Silveira-Moriyama et al. 2008; Haugarvoll et al., 2008). Current findings suggest that LRRK-2 plays an important role in the dysregulation of protein translation, vesicle trafficking, neurite outgrowth, autophagy, and cytoskeletal dynamics.
LRRK2 Structure and Function
LRRK2 is a large, multi-domain protein composed of 2527 amino acids (289 kDa). It contains a kinase domain sequence, a Ras of complex catalytic protein domain (ROC) and the regulatory C-terminal of ROC (COR) domain that are predicted to bind and hydrolyze GTP similarly to the ROCO protein family (Gotthardt et al., 2008). These three domains are considered the catalytic core of LRRK2. Additionally, LRRK2 has Ankyrin repeats, Leucine-rich repeats (LRR) and a WD40 domain that is important for protein folding, and thus kinase activity, and predominantly serves as binding sites for protein-protein interactions and structural scaffolds for different signaling processes. Thus, unsurprisingly, the entire protein, including the C-terminal domain is required to produce acute toxic effects in neuronal culture (Jorgensen et al., 2009). This implies that only a functional LRRK2 protein will induce toxicity and that the C-terminal domain plays a key role in maintaining its structure. The normal function of LRRK2 is unknown, however is implicated in vesicle trafficking, protein synthesis, cytoskeletal dynamics, and immune function.
LRRK2 Mutations
LRRK2 mutations are predominantly found in the kinase (G2019S, I2020T) and the ROC-COR GTP-ase domains (R1441C/G/H, Y1699C), implying that these enzymatic activities are crucial for pathogenesis (Rudenko and Cookson, 2014). There are seven missense mutations known to be truly pathogenic: G2019S, R1441C/G/H, I2020T, Y1699C, and G2385R (Aasly et al., 2010). Defining the functional characteristics of theses mutants is ongoing; current findings are summarized in Table 1. The seven pathogenic mutations are shown in relation to the entire LRRK2 protein in Figure 1. The G2019S kinase domain mutation is the most common LRRK2 mutation leading to PD, and activates the kinase two- to threefold compared to wild-type (WT) (West et al., 2005; Khan, 2005; Jaleel et al., 2007; Healy et al., 2008). Increased LRRK2 kinase activity has been shown to retard neuritic outgrowth and extension in several primary neuronal cultures (Smith et al., 2006; Macleod et al., 2006; Smith et al., 2005; Greggio et al., Dachsel et al., 2010) Additionally, many in vivo invertebrate models have shown similar evidence, building support for the toxic effect of increased LRRK2 kinase activity (Imai et al., 2008; Yao et al., 2010; Liu et al. 2008). Transgenic G2019S LRRK2 mice display progressive degeneration of substantia nigra pars compacta dopaminergic neurons and motor dysfunction, as well as disrupted vesicle trafficking, suggesting that this mutation may be functionally relevant to the disease (Chen et al., 2009)(Pan et al., 2017).
LRKK2 mutations associated with sporadic Parkinson’s Disease
The R1441 residue is the second most common spot of pathogenic LRRK2 mutations with three substitutions: R1441C, R1441G, and R1441H (Healy, 2008). While the location of these mutations is in the ROC-COR GTPase domain, they have generally been observed to increase kinase activity, although results vary. This is likely due to intramolecular regulation between the GTPase and kinase domains. The R1441H mutation exhibits slowed GTP hydrolysis and increased affinity for GTP (Saha et al., 2014). Y1699C is also in the GTPase domain and exhibits similar kinase activity levels as other 1441 mutants. I2020T has been associated with both increased and decreased kinase activity which may depend on the substrate used in the assay (Ray S et al., 2014). All of these mutations have been shown to have enhanced vulnerability to mitochondrial dysfunction, inhibition of autophagy, and neurodegeneration, but their biochemical functional effects are not well characterized (Rideout, 2017). G2385R has recently been discovered to be a partial loss of function mutant that regulates tethering of synaptic vesicles (Carion et al., 2017). It binds many proteins with its WD40 C-terminal domain involved in synaptic vesicle exocytosis in pre-synaptic membranes. It is genetically linked to PD in Asian and Korean families and while it is associated with similar clinical symptoms to idiopathic PD, it significantly lowers the expected age of onset (Tan et al., 2009). This finding complicates the requirements for LRRK2 toxicity, and does not align with the G2019S toxic gain of function theory. Perhaps, a more intricate balance of LRRK2 activity is important for cell survival, as increased or decreased presence of the protein seems to influence toxicity. This is very perplexing and deserves further study.
Effects of over-expression and knock-out of LRRK2
Over-expression of WT, mutants and knock-out models of LRRK2 have also been used to gain insight into its function. WT LRRK2 seems to have a toxicity protective effect; LRRK2 was found to have a protective effect on dopamine (DA) neurons in C. elegans from the toxicity of 6-hydroxydopamine and human α-synuclein (Yuaun et al., 2011). However, when LRRK2 WT is overexpressed, cellular dysfunctions such as fragmentation of the Golgi complex and dopaminergic degeneration occur (Lin et al., 2009; Xiong et al., 2012). Additionally, G2019S-LRRK2 overexpression results in mitochondrial uncoupling (Papkovskaia et al., 2012 ). The observations that over-expression of LRRK2, but not knock-out, leads to increased dopaminergic neurodegeneration (Xiong et al., 2012) suggest that PD-causing mutations of LRRK2 are primarily toxic gain of function mutations. Thus, the majority of findings support the idea that higher levels of the protein are more toxic, likely due to its kinase activity.