The pathogenetic mechanisms leading to typical PD remain unknown. Prevailing concepts have favored either an environmental factor or genetic contributors as the primary event in its aetiology, but few models have explored the impact of both in in vivo models (Aleyasin et al. 2010
). The discovery that late-onset PD could be caused by the inheritance of a mutation in the LRRK2
gene leading to familial as well as sporadic forms has provided us with an opportunity to explore the pathophysiological events underlying this complex disease. However, as summarized in the introduction, more questions have arisen than been answered surrounding the function(s) of LRRK2
(reviewed in: Cookson 2010
); most poignantly, to date no model has adequately explained how a gene, which is expressed at exceedingly low levels in brain nuclei that are severely affected by the process that leads to PD (eg, S. nigra
), can promote a neurological disorder when mutated.
Here, we provided experimental evidence to support the following two conclusions: one, Lrrk2 is a relatively abundant protein in mammalian leukocytes of circulating blood and organs of the immune system; and two, lrrk2
expression is up-regulated in cultured macrophages following their recognition of microbial structures. We arrived at these two conclusions based on several, complementary approaches; our findings are in agreement with the recently published, elegant study by Gardet et al. These authors demonstrated the involvement of LRRK2
in the IFN-γ response after leukocyte invasion and pointed out how it possibly relates to CD pathogenesis (Gardet et al. 2010
). In their study, LRRK2
was up-regulated in intestinal tissues of CD patients with expression detected in macrophages, B-cells and CD103+ dendritic cells of inflamed intestines. Gardet et al further showed that exogenous expression of Lrrk2 promoted transcriptional activation of the NF-κB pathway, and that its down-regulation facilitated enhanced bacterial survival in a S. typhimurium
cell infection assay (Gardet et al. 2010
). However, we believe our data further expand on these findings by adding three additional observations regarding Lrrk2 biology: one, we demonstrated –to our knowledge for the first time- that the expression of R1441C
mutant lrrk2 in a primary, non-neural cell model (ie, BMDM) reveals an autophagy defect; two, we showed that the up-regulation of lrrk2
also occurs in response to viral transduction of a host cell by lentiviral particles (and likely, in B-lymphocytes transformed by EBV); and three, when screening for a lrrk2
genotype-dependent effect on IL-6 and KC cytokine release, we found that these two signaling molecules are not regulated by leukocytic lrrk2.
Below, we mention five specific limitations of this study followed by a discussion of five reasons why we consider our Lrrk2 findings relevant to the pathogenesis of PD. One, although we consistently demonstrated the expression of LRRK2
in CD19+ B-cells, CD4+ T-cells (in contrast to Gardet et al. 2010
; Makaewa et al. 2010), CD8+ T-cells, CD14+ monocytes, macrophages and dendritic cells, we obtained highly variable -and thus, inconclusive- results regarding granulocytes, which were: mostly negative in staining of human blood smears; positive in cellular isolates from murine spleen and mesenteric lymph nodes analyzed by FACS; and negative in genotyped spleen sections examined by IHC. These differences may be due to low LRRK2
expression rates in healthy donors, species differences in accessible epitopes of granulocytic Lrrk2, or subtle changes in the binding strength of the antibodies used in our different protocols. In future work, we will revisit the degree of LRRK2
expression in granulocytes focusing on mRNA-based techniques (eg, rt-qPCR).
Two, in our IHC work of brain tissue, where we used a protocol that includes formalin-fixed, paraffin-embedded material that included two antigen retrieval steps, we have not yet identified staining of microglia (or of any other glia) with our panel of anti-Lrrk2 antibodies, in contrast to a report by Miklossy et al. 2006
. As before, differences in antibody specificity and antigen accessibility could account for this mismatch in results. To convincingly answer the question of expression of lrrk2
in resting or activated microglia, which are derived from the monocytic lineage, and to exclude any false positive signals due to antibody cross-reactivity, future studies will employ the induction of microglial activation in WT lrrk2 versus lrrk2-deficient mice using established inflammatory paradigms (Mount et al. 2007
Three, although we recorded a mean, ≥31% reduction in basal LC3-II (but not LC3-I) levels in BMDM from R1441C
lrrk2-expressing mice when compared with age-matched WT littermates processed at the same time, more autophagy markers need to be examined in primary cells (and tissue) using a larger series of animals and comparing unstimulated vs stimulated conditions than we did here (mice, n=6). If our data were to be confirmed, ie, reduced autophagy rates can be seen in R1441C
lrrk2-expressing BMDM and tissue homogenates as concluded from our LC3-II results, then the effect of this point mutation could be similar to the recently observed dysregulation of autophagy described by Tong et al in lrrk2-deficient mice (Tong et al. 2010
). Such a reduction in autophagic efficiency due to the loss of dimeric WT Lrrk2 activity is principally consistent with the results of Gardet et al., who recorded increased bacterial survival rates in macrophages after lrrk2
knock-down (Gardet et al. 2010
). Effective phagocytosis following pathogen opsonization requires intact autophagy. In parallel, future studies need to systematically screen additional cytokines (and signaling pathways) beyond those carried out so far to better define the full complement of cellular changes downstream of lrrk2
gene expression in activated immune cells.
Four, in our work we have not yet defined the post-translational nature of the HMW lrrk2 band(s) seen with multiple specific antibodies in PBMC and, even more strikingly, in BMDM. We performed our SDS/PAGE experiments under reducing and denaturing conditions, thereby precluding dimer (or higher order multimer) stability. We predict that the relative abundance of Lrrk2 protein in the monocyte/macrophage lineage will significantly aid its biochemical dissection in the future, including the delineation of post-translational modifications, of its still elusive binding partners, and of authentic substrates for its kinase activity (Cookson 2010
Fifth, one could argue that the precise single nucleotide polymorphisms at or near the LRRK2
locus, which were associated with CD and leprosy risk in genome-wide association studies, have not yet been independently validated, thereby theoretically minimizing the general applicability of our cellular studies. Intriguingly, most recently LRRK2/MUC19
was identified as one of seven CD loci (out of 30 examined) that was positively associated with risk modulation for yet another human condition, ie, ankylosing spondylitis, which often occurs together with CD (Danoy et al. 2010
). Importantly, the dysregulation of monocytes, macrophages, B-cells and T-cells plays a pivotal role in the pathogenesis of CD, ankylosing spondylitis and leprosy (Schurr and Gros 2009
Importantly, five arguments can be made in favor of studying LRRK2
biology in leukocytes as a suitable cell model for PD and to functionally compare it with other CD- and leprosy-associated genes in suitable paradigms. One, in vivo studies and post-mortem analyses of PD subjects have generated robust evidence from multiple centers in favor of ongoing inflammation (Hirsch and Hunot 2009
; Simon-Sanchez 2009
; Reale et al. 2009
). Furthermore, the infiltration by CD4+ and CD8+ lymphocytes and the activation of microglia in PD brain have been well documented (Brochard et al. 2009
). In addition, the analysis of peripheral cytokine concentrations in PD subjects has revealed abnormal levels for MCP-1 (monocyte chemoattractant protein-1), MIP-1 (macrophage inflammatory protein-1), IFN-γ, IL-8, IL-1β, IL-6, and TNF-α (Reale et al. 2009
; Chen et al. 2008
) among others, thereby justifying the systematic exploration as to the possible reason(s) for their dysregulation in immune cells.
Two, the mammalian immune system undergoes significant changes during the process of ageing including for example in the rate of lrrk2
expression (Maekawa et al. 2010
) and by inference, in WT lrrk2 activity, which could further influence the age-of-onset, the expressivity of the PD phenotype, or its progression rate.
Three, the two central themes in the aetiology of typical, late-onset PD, which Jellinger recently referred to as a “progressive multisystem (multiorgan) disorder” (Jellinger 2011
), have not yet been successfully integrated. However, its environmental and genetic contributors are not mutually exclusive, but rather represent the foundation of many human disorders (Klein and Schlossmacher 2007
; Klein et al. 2011
). The immune system governs the host’s susceptibility to pathogen invasion (eg, viruses, bacteria, fungi etc.), controls its tolerance of possible colonization, and determines the response mounted by the host to eliminate any threat. Nucleotide exchange domain- and LRR domain-carrying proteins (“NLR”) encoded by the genome play an evolutionarily conserved role in immune surveillance from plants to mammals (reviewed in: Takken and Tameling 2009
; Taxman et al. 2010
). Together with TLR proteins, NLR molecules are functionally understood as pattern recognition receptors. NLR proteins oversee inflammasome assembly and control the pathogen-induced regulation of cytokine release (Lamkanfi and Dixit 2010
). Based on the functional domain similarities between Nod2 and Lrrk2, we seek to further test the hypothesis of a NLR-type function for Lrrk2.
Fourth, if established successfully, such a LRRK2
-dependent regulation of immune cell function during the interaction between host and environment would be expected to significantly vary based on geography. This scenario –together with other factors- could help explain several known, previously difficult to explain aspects of LRRK2
-linked PD, eg, variable rates of penetrance in carriers of the same mutation in different regions of the world, differences in the phenolconversion rate among members of the same family, variable rates of disease progression, and possibly, even the pleomorphic pathology (Zimprich et al, 2004
) that could have been triggered by distinct agents in the environment.
Lastly, and most compellingly, a primary role for Lrrk2 at the interface between host and environment, which we propose here, could provide a new platform to revisit Braak’s hypothesis as it relates to the pathogenesis of typical PD. Braak et al postulated that an elusive environmental pathogen (microbial or otherwise) is essential for pathology to ensue (Braak et al. 2003). At stage 0, these authors described the earliest signs of synucleinopathy in the submucosal plexus of the gut and in the olfactory bulb, two structures in close proximity to the environment. The convergence of several independent pieces of evidence from the fields of CD pathogenesis, leprosy genetics and LRRK2 research in leukocyte function promises to bring together five previously irreconcilable elements in the aetiology of PD: one, the importance of the environment; two, the evolution of its neuropathology; three, the significance of genetic susceptibility; four, the role of the immune system; and five, the progress of ageing in a long lived host.