Analysis of macroautophagy related proteins in G2019S LRRK2 Parkinson's disease brains with Lewy body pathology.

LRRK2, the gene encoding the multidomain kinase Leucine-Rich Repeat Kinase 2 (LRRK2), has been linked to familial and sporadic forms of Parkinson's disease (PD), as well as cancer, leprosy and Crohn's disease, establishing it as a target for discovery therapeutics. LRRK2 has been associated with a range of cellular processes, however its physiological and pathological functions remain unclear. The most prevalent LRRK2 mutations in PD have been shown to affect macroautophagy in various cellular models while a role in autophagy signalling has been recapitulated in vivo. Dysregulation of autophagy has been implicated in PD pathology, and this raises the possibility that differential autophagic activity is relevant to disease progression in PD patients carrying LRRK2 mutations. To examine the relevance of LRRK2 to the regulation of macroautophagy in a disease setting we examined the levels of autophagic markers in the basal ganglia of G2019S LRRK2 PD post-mortem tissue, in comparison to pathology-matched idiopathic PD (iPD), using immunoblotting (IB). Significantly lower levels of p62 and LAMP1 were observed in G2019S LRRK2 PD compared to iPD cases. Similarly, an increase in ULK1 was observed in iPD but was not reflected in G2019S LRRK2 PD cases. Furthermore, examination of p62 by immunohistochemistry (IH) recapitulated a distinct signature for G2019S PD. IH of LAMP1, LC3 and ULK1 broadly correlated with the IB results. Our data from a small but pathologically well-characterized cases highlights a divergence of G2019S PD carriers in terms of autophagic response in alpha-synuclein pathology affected brain regions compared to iPD.

Endoplasmic Reticulum Stress Markers and Their Possible Implications in Leprosy's Pathogenesis.

Mycobacterium leprae causes leprosy, a dermatoneurological disease which affects the skin and peripheral nerves. One of several cellular structures affected during M. leprae infection is the endoplasmic reticulum (ER). Infection by microorganisms can result in ER stress and lead to the accumulation of unfolded or poorly folded proteins. To restore homeostasis in the cell, the cell induces a series of signaling cascades known as the unfolded protein response called UPR (unfolded protein response). The present work is aimed at investigating the in situ expression of these markers in cutaneous lesions of clinical forms of leprosy and establish possible correlation expression patterns and types of lesion. A total of 43 samples from leprosy patients were analyzed by immunohistochemistry with monoclonal antibodies against GRP78/BiP, PERK, IRE1α, and ATF6. A statistically significant difference between the indeterminate, tuberculoid, and lepromatous clinical forms was detected, with high expression of GRP78/BiP, PERK, IRE1α, and ATF6 in tuberculoid forms (TT) when compared to lepromatous leprosy (LL) and indeterminate (I) leprosy. These results represent the first evidence of ER stress in samples of skin lesions from leprosy patients. We believe that they will provide better understanding of the complex pathogenesis of the disease and facilitate further characterization of the cascade of molecular events elicited during infection.

LRRK2 and RAB7L1 coordinately regulate axonal morphology and lysosome integrity in diverse cellular contexts.

Leucine-rich repeat kinase 2 (LRRK2) has been linked to several clinical disorders including Parkinson's disease (PD), Crohn's disease, and leprosy. Furthermore in rodents, LRRK2 deficiency or inhibition leads to lysosomal pathology in kidney and lung. Here we provide evidence that LRRK2 functions together with a second PD-associated gene, RAB7L1, within an evolutionarily conserved genetic module in diverse cellular contexts. In C. elegans neurons, orthologues of LRRK2 and RAB7L1 act coordinately in an ordered genetic pathway to regulate axonal elongation. Further genetic studies implicated the AP-3 complex, which is a known regulator of axonal morphology as well as of intracellular protein trafficking to the lysosome compartment, as a physiological downstream effector of LRRK2 and RAB7L1. Additional cell-based studies implicated LRRK2 in the AP-3 complex-related intracellular trafficking of lysosomal membrane proteins. In mice, deficiency of either RAB7L1 or LRRK2 leads to prominent age-associated lysosomal defects in kidney proximal tubule cells, in the absence of frank CNS pathology. We hypothesize that defects in this evolutionarily conserved genetic pathway underlie the diverse pathologies associated with LRRK2 in humans and in animal models.

Leucine-rich repeat kinase 2 deficiency is protective in rhabdomyolysis-induced kidney injury.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common known genetic cause of Parkinson's disease, and LRRK2 is also linked to Crohn's and Hansen's disease. LRRK2 is expressed in many organs in mammals but is particularly abundant in the kidney. We find that LRRK2 protein is predominantly localized to collecting duct cells in the rat kidney, with much lower expression in other kidney cells. While genetic knockout (KO) of LRRK2 expression is well-tolerated in mice and rats, a unique age-dependent pathology develops in the kidney. The cortex and medulla of LRRK2 KO rat kidneys become darkly pigmented in early adulthood, yet aged animals display no overt signs of kidney failure. Accompanying the dark pigment we find substantial macrophage infiltration in LRRK2 KO kidneys, suggesting the presence of chronic inflammation that may predispose to kidney disease. Unexpectedly, the dark kidneys of the LRRK2 KO rats are highly resistant to rhabdomyolysis-induced acute kidney injury compared with wild-type rats. Biochemical profiling of the LRRK2 KO kidneys using immunohistochemistry, proteomic and lipidomic analyses show a massive accumulation of hemoglobin and lipofuscin in renal tubules that account for the pigmentation. The proximal tubules demonstrate a corresponding up-regulation of the cytoprotective protein heme oxygenase-1 (HO-1) which is capable of mitigating acute kidney injury. The unusual kidney pathology of LRRK2 KO rats highlights several novel physiological roles for LRRK2 and provides indirect evidence for HO-1 expression as a protective mechanism in acute kidney injury in LRRK2 deficiency.

Differential role of HAMP-like linkers in regulating the functionality of the group III histidine kinase DhNik1p.

Nik1 orthologs are sensor kinases that function upstream of the high osmolarity glycerol/p38 MAPK pathway in fungi. They contain a poly-HAMP module at their N terminus, which plays a pivotal role in osmosensing as well as fungal death upon exposure to fludioxonil. DhNik1p is a typical member of this class that contains five HAMP domains and four HAMP-like linkers. We investigated the contribution of each of the HAMP-like linker regions to the functionality of DhNik1p and found that the HAMP4b linker was essential as its deletion resulted in the complete loss of activity. Replacement of this linker with flexible peptide sequences did not restore DhNik1p activity. Thus, the HAMP-like sequence and possibly structural features of this linker region are indispensable for the kinase activity of DhNik1p. To gain insight into the global shape of the poly-HAMP module in DhNik1p (HAMP1­5), multi-angle laser light and small angle x-ray scattering studies were carried out. Those data demonstrate that the maltose-binding protein-tagged HAMP1­5 protein exist as a dimer in solution with an elongated shape of maximum linear dimension ∼365 Å. Placement of a sequence similarity based model of the HAMP1­5 protein inside experimental data-based models showed how two chains of HAMP1­5 are entwined on each other and the overall structure retained a periodicity. Normal mode analysis of the structural model is consistent with the H4b linker being a key to native-like collective motion in the protein. Overall, our shape-function studies reveal how different elements in the HAMP1­5 structure mediate its function.

Deciphering the function of leucine-rich repeat kinase 2 and targeting its dysfunction in disease.

LRRK2 (leucine-rich repeat kinase 2) is a gene of unknown function that has been linked to a number a human diseases, including PD (Parkinson's disease), IBD (inflammatory bowel disease), leprosy and cancer. The papers from the LRRK2: Function and Dysfunction meeting in this issue of Biochemical Society Transactions explore our growing knowledge of LRRK2's normal function, the role that it plays in disease and emerging strategies to exploit LRRK2 as a therapeutic target.

LRRK2 and autophagy: a common pathway for disease.

LRRK2 (leucine-rich repeat kinase 2) is an enzyme implicated in human disease, containing kinase and GTPase functions within the same multidomain open reading frame. Dominant mutations in the LRRK2 gene are the most common cause of familial PD (Parkinson's disease). Additionally, in genome-wide association studies, the LRRK2 locus has been linked to risk of PD, Crohn's disease and leprosy, and LRRK2 has also been linked with cancer. Despite its association with human disease, very little is known about its pathophysiology. Recent reports suggest a functional association between LRRK2 and autophagy. Implications of this set of data for our understanding of LRRK2's role in physiology and disease are discussed in the present paper.

Number and brightness analysis of LRRK2 oligomerization in live cells.

Leucine-rich repeat kinase 2 (LRRK2) is a large multidomain protein that contains enzymatically functional GTPase and kinase domains. Several noncoding LRRK2 gene polymorphisms have been associated with susceptibility to Parkinson's disease (PD), Crohn's disease, and leprosy. Many LRRK2 coding polymorphisms have been associated with or causally linked to PD. The G2019S point mutation within the LRRK2 kinase domain is the most common cause of familial PD. The G2019S mutation appears to alter LRRK2 kinase activity. Some but not all studies have reported that LRRK2 kinase activity is dependent upon LRRK2 dimerization and membrane localization. It is important to define the oligomeric state(s) of LRRK2 in living cells, which to date have only been characterized in vitro. Here we use confocal and total internal reflection microscopy coupled with number and brightness analysis to study the oligomeric states of LRRK2 within the cytosol and on the plasma membrane of live CHO-K1 cells. Our results show, for the first time to our knowledge, that LRRK2 is predominantly monomeric throughout the cytosol of living cells, but attains predominately higher oligomeric states in the plasma membrane.

Parkinson's disease and immune system: is the culprit LRRKing in the periphery?

Leucine-rich repeat kinase 2 (LRRK2) is a large multidomain kinase/GTPase that has been recently linked to three pathological conditions: Parkinson's disease; Crohn's disease; and leprosy. Although LRRK2 physiological function is poorly understood, a potential role in inflammatory response is suggested by its high expression in immune cells and tissues, its up-regulation by interferon γ, and its function as negative regulator of the immune response transcription factor NFAT1. In this review we discuss the most recent findings regarding how LRRK2 could be a player in the inflammatory response and we propose a scenario where the detrimental effects mediated by Parkinson's disease LRRK2 mutations may initiate in the periphery and extend to the central nervous system as a consequence of increased levels of pro-inflammatory factors permeable to the blood brain barrier.

LRRK2 and human disease: a complicated question or a question of complexes?

Leucine-rich repeat kinase 2 (LRRK2) is linked to various diseases, including Parkinson's disease, cancer, and leprosy. Data from LRRK2 knockout mice has highlighted a possible role for LRRK2 in regulating signaling pathways that are linked to the pathogenesis of Crohn's disease. Here, we examine how LRRK2's role as a signaling hub in the cell could lead to diverse pathologies.

Parkinson's disease-linked LRRK2 is expressed in circulating and tissue immune cells and upregulated following recognition of microbial structures.

Sequence variants at or near the leucine-rich repeat kinase 2 (LRRK2) locus have been associated with susceptibility to three human conditions: Parkinson's disease (PD), Crohn's disease and leprosy. As all three disorders represent complex diseases with evidence of inflammation, we hypothesized a role for LRRK2 in immune cell functions. Here, we report that full-length Lrrk2 is a relatively common constituent of human peripheral blood mononuclear cells (PBMC) including affinity isolated, CD14(+) monocytes, CD19(+) B cells, and CD4(+) as well as CD8(+) T cells. Up to 26% of PBMC from healthy donors and up to 43% of CD14(+) monocytes were stained by anti-Lrrk2 antibodies using cell sorting. PBMC lysates contained full-length (>260 kDa) and higher molecular weight Lrrk2 species. The expression of LRRK2 in circulating leukocytes was confirmed by microscopy of human blood smears and in sections from normal midbrain and distal ileum. Lrrk2 reactivity was also detected in mesenteric lymph nodes and spleen (including in dendritic cells), but was absent in splenic mononuclear cells from lrrk2-null mice, as expected. In cultured bone marrow-derived macrophages from mice we made three observations: (i) a predominance of higher molecular weight lrrk2; (ii) the reduction of autophagy marker LC3-II in (R1441C)lrrk2-mutant cells (<31%); and (iii) a significant up-regulation of lrrk2 mRNA (>fourfold) and protein after exposure to several microbial structures including bacterial lipopolysaccharide and lentiviral particles. We conclude that Lrrk2 is a constituent of many cell types in the immune system. Following the recognition of microbial structures, stimulated macrophages respond with altered lrrk2 gene expression. In the same cells, lrrk2 appears to co-regulate autophagy. A pattern recognition receptor-type function for LRRK2 could explain its locus' association with Crohn's disease and leprosy risk. We speculate that the role of Lrrk2 in immune cells may also be relevant to the susceptibility of developing PD or its progression.

C-type lectin DC-SIGN modulates Toll-like receptor signaling via Raf-1 kinase-dependent acetylation of transcription factor NF-kappaB.

Adaptive immune responses by dendritic cells (DCs) are critically controlled by Toll-like receptor (TLR) function. Little is known about modulation of TLR-specific signaling by other pathogen receptors. Here, we have identified a molecular signaling pathway induced by the C-type lectin DC-SIGN that modulates TLR signaling at the level of the transcription factor NF-kappaB. We demonstrated that pathogens trigger DC-SIGN on human DCs to activate the serine and threonine kinase Raf-1, which subsequently leads to acetylation of the NF-kappaB subunit p65, but only after TLR-induced activation of NF-kappaB. Acetylation of p65 both prolonged and increased IL10 transcription to enhance anti-inflammatory cytokine responses. We demonstrated that different pathogens such as Mycobacterium tuberculosis, M. leprae, Candida albicans, measles virus, and human immunodeficiency virus-1 interacted with DC-SIGN to activate the Raf-1-acetylation-dependent signaling pathway to modulate signaling by different TLRs. Thus, this pathway is involved in regulation of adaptive immunity by DCs to bacterial, fungal, and viral pathogens.

Tissue-specific down-regulation of RIPK 2 in Mycobacterium leprae-infected nu/nu mice.

RIPK 2 is adapter molecule in the signal pathway involved in Toll-like receptors. However, there has been no reported association between receptor-interacting serine/threonine kinase 2 (RIPK 2) expression and the infectious diseases involving mycobacterial infection. This study found that its expression was down-regulated in the footpads and skin but was up-regulated in the liver of Mycobacterium leprae-infected nu/nu mice compared with those of the M. leprae non-infected nu/nu mice. It was observed that the interlukin-12p40 and interferon-gamma genes involved in the susceptibility of M. leprae were down-regulated in the skin but were up-regulated in the liver. Overall, this suggests that regulation of RIPK 2 expression is tissue-specifically associated with M. leprae infection.