Enzymatic activity of LRRK2

LRRK2 harbors both a GTPase and kinase domain, an extremely rare arrangement found only in LRRK1 and possibly the DAPK1 protein in mammals. A solid precedent for
GTPase activity dependent modification of protein kinase activity suggests that the enzymatic domains encoded inLRRK2 may function as a self-regulatory apparatus. The most common LRRK2 mutations localize to the kinase domain (amino acid G2019) and the GTPase domain (amino acid R1441), implicating enzymatic output as critical to PD. Beyond a domain-prediction analysis that helps define conserved features and potentially critical residues, in silico approaches do not further predict LRRK2 function in cells. The LRRK2 kinase domain possesses highest sequence homology
to the mixed-lineage kinase (MLK) subfamily of MAPKKK proteins but differs in the critical amino acids that define MLK proteins, whereas the GTPase domain displays
distinct architecture reminiscent of Rab-like GTPases. As one of the largest protein kinases in the mammalian kinome, LRRK2 will necessarily resist characterization by the usual gauntlet of biochemical assays that have historically well served the characterization of other protein kinases. Nevertheless, tremendous advances in a short time have outlined a surprising consensus story for the impact of PD causing
mutations on protein function. Before a functional description of the LRRK2 protein, a functional description of LRRK1 suggested that intrinsic kinase activity was stimulated on GTPase activation. This phenomenon holds true for LRRK2 as well, in which application of GTP or GTPgS spurs modest increases in kinase output. However, a clear relation between kinase activity and GTPase activity is defined through the us of specific mutations that ablate either GTPase activity, kinase activity, or both activities. Mutations in the conserved ATP binding pocket site, in the consensus residues of the activation loop, or mutation within the proton-acceptor site have no apparent effect on GTP binding. In contrast, mutations in the
GTPase domain thoroughly ablate kinase activity. Thus, LRRK2 functions as a signal-transduction pathway encoded into a single protein. The activity of LRRK2 is
perhaps an ancient design, because the orthologue GbpC in slime mold operates in a similar manner, although GbpC also encodes GEF domains upstream of GTPase activity.
Through functional descriptions of GbpC, LRRK1, and LRRK2 protein, the encoded domains function in a signal F2c cascade ultimately to regulate kinase output.In an initial functional description of the LRRK2 protein, the most common PD-associated mutations, G2019S and R1441C, enhanced kinase activity but failed to alter other
basic biochemical properties, such as protein localization and turnover. PD-causing mutations in the GTPase domain likewise enhance the proportion of GTP-bound protein in pull-down experiments, whereas mutations in the kinase domain have no effect on the proportion of GTP-bound protein. Additional studies demonstrate that PD-causing
mutations in the GTPase domain do not result in a higher affinity for GTP; rather, GTP hydrolysis is disrupted because of PD-associated mutations, thereby prolonging GTP-bound states and an activated GTPase domain. Missense mutations in the LRRK2 gene that segregate with disease in families and are therefore likely pathogenic variants all result in enhanced kinase activity in vitro, although not all
laboratories demonstrate a significant difference between kinase activity associated with wild-type protein and that containing PD-causing mutations. Differences in assay
protocols and the lack of a relevant substrate and robust kinase-dependent phenotype in cells combine to prevent the assumption that kinase output is the true and only possible functional link between LRRK2 protein and PD. However, the available data, when taken as a whole, suggest that kinase activity represents the final output of activity responsible for pathogenesis.

LRRk2 expression and localization

LRRK2 mRNA displays near ubiquitous localization throughout the mouse and human brain, with particular concentration within neurons of the cortex, striatum, and
hippocampus. Protein distributes in a likewise manner, detectable in the vulnerable neurons of the substantia nigra pars compacta. LRRK2 displays particularly high expression in the kidney and appears to increase in expression on organogenesis and cell maturation. LRRK2 adopts a punctate intracellular cytosolic localization
that associates with various membranous structures including vesicles, mitochondria, Golgi, and the ER. Biochemical fractionation suggests that LRRK2 resides on the
cytoplasmic side of membrane-containing organelles without evidence of nuclear or mitochondrial internalization. Similar to a-syn, clear orthologues to LRRK2 have been
identified in all described mammalian genomes, but in invertebrates, orthologous genes may show closer homology to mammalian LRRK1 than to the LRRK2 gene. Close sequence homology and overlapping expression profiles between the LRRK1 and LRRK2 genes suggests redundancy in function. LRRK2-deficient mice are viable with no dramatic abnormalities, although detailed reports have not been published. Loss of the LRRK orthologues in Drosophila seems to produce a strain-dependent phenotype, and additional studies will help resolve the controversy. Loss of
LRRK orthologues in nematodes produces defects in vesicle sorting, whereas loss in slime mold produces defects in chemotaxis.

LRRK2 structure and function

As opposed to a-syn, LRRK2 had not been cloned or thoroughly annotated when missense mutations were identified in PD cases. Bioinformatic prediction strongly suggested that LRRK2 encodes a GTPase-like domain together with a kinase domain, thereby placing LRRK2 within the fraction of the proteome considered modifiable with small molecules or intervention therapies. The potential for LRRK2 as a therapeutic target depends not only on activities of the protein associated with PD, but on normal function and the relation between health and disease. For example, generalized inhibition of LRRK2 activity may not be compatible with normal cellular function, or inhibition of one particular aspect of LRRK2 activity may not influence another activity that underlies
disease mechanisms. Deep insight into LRRK2-related biology will provide the background necessary for rationally designed therapeutic approaches.

Autosomal dominant genes in PD

LRRK2.
A genetic locus initially described as associated with late-onset PD in a large Japanese kindred demonstrated association with late-onset autosomal-dominant PD in several additional families, and missense mutations in the LRRK2 gene were identified. Sequence analysis of the encoded enzymatic domains revealed an alteration within the activation loop of the kinase domain responsible for a
significant percentage of PD disease in many case populations studied. In general, common genetic variation in LRRK2 does not appear to contribute heavily to PD susceptibility, with the exception of some Asian populations, but in some populations, the missense mutations themselves account for more than one third of PD cases. Mutations that clearly segregate with disease in well described
families and are overrepresented in PD cases versus age-matched controls represent a fraction of described LRRK2 variants, and the difficulty in distinguishing benign versus pathogenic variants prevents resolution of the true frequency
of LRRK2 mutations in PD.

a-Syn. The first genetic cause for PD was described in a large Italian family that inherited early-onset PD in an autosomal dominant fashion. Subsequently, missense mutations in the a-syn gene were also identified in Greek, German, and
Spanish families, with missense mutations localized to the N-terminal half of the protein. As opposed to missense mutations in LRRK2, pathogenic missense mutations
in the a-syn gene seem confined to only a handful of PD cases worldwide. Again in contrast with LRRK2, genetic variation in the a-syn promoter and other regions of the gene appears to modify susceptibility to PD. The identification of genomic multiplications that include a-syn and are causative for PD solidifies the importance of a-syn dosage in PD. The main strength that suggests a critical involvement for a-syn in PD does not necessarily lie with human genetic studies; rather, a-syn represents the major protein component of the pathologic structures that define PD-associated lesions in affected regions of the brain.

Autosomal-recessive parkinsonism

Families that inherit PD in a manner compatible with autosomal-recessive disease have long been described, with disease usually manifesting earlier than that in the majority of PD cases. Mutations in the parkin gene were described in
Japanese juvenile-onset cases of disease, and genetic variation in parkin clearly associates with early-onset PD in multiple ethnicities. Apart from the large deletions and rearrangements that inactivate expression, pathogenic point
mutations impart deleterious function compatible with a general loss-of-function disease mechanism. Likewise, loss-of-function mutations that include nonsense
mutations, genomic rearrangements, and missense mutations AU1c have also been described in the PINK1 (PTEN-induced kinase-1) and DJ-1 genes in early-onset cases. In general, overexpression of these proteins usually imparts cytoprotective
properties to cells from a variety of insults. Because most effective drug therapies for a variety of human illnesses involve the ablation or reduction of activity of associated targets, dealing with loss-of-function mechanisms usually imparts additional technical demands on therapeutic strategies. Although the autosomal-recessive genes encode clearly relevant proteins in PD, additional work that characterizes underlying cellular pathways in relevant disease models seems necessary before parkin, PINK1, and DJ-1 can be fully realized as potential therapeutic targets.

Genetic Susceptibilities in Parkinson’s Disease

The current understanding of PD includes a long initial
phase, often dubbed ‘‘preclinical,’’ in which non–movement disorder
symptoms are common but heterogeneous in presentation. Early twin studies suggest that genetic susceptibilities might not play a large role in PD etiology. More
recent studies show that, in some populations, a single point
mutation in the LRRK2 gene causes more than one third of PD
cases, with the ancient G2019S mutation reported at a higher
frequency in patients without knowledge of PD in their family
than in patients aware of a family history of disease, in some
populations. Genetic studies have therefore illuminated
specific causes of PD that promise to aid in the delineation
of pathogenesis and the development of therapeutics.

3 prominent questions on LRRK2

A recent review article by Cookson et al have pointed out 3 prominent questions about LRRK2. These are some burning issues and answers to these questions would lay the fundamental foundation stone in understanding the LRRK2 biology at the molecular level in a better way. It will also help in designing therapeutics for LRRK2, which itself has been a good kinase target.
The 3 questions that the authors fundamentally wish to address are:

1. What is the normal function of LRRK2.

2. How mutations impact LRRk2 function.

3. Possible mechanisms of LRRK2 mediated neurotoxicity.