LRRK2 kinase in Parkinson's disease Alessi, Dario R; Sammler, Esther
Science (American Association for the Advancement of Science),
04/2018, Letnik:
360, Številka:
6384
Journal Article
Recenzirano
Odprti dostop
Defects in vesicular trafficking and immune responses are found in Parkinson's disease
Despite intensive research, attempts to pause or even just slow the progression of Parkinson's disease (PD) have ...thus far failed. Although most cases of PD are idiopathic and with largely unknown aetiology, mutations in ∼20 genes, including
LRRK2
(leucine-rich repeat kinase 2), cause rare genetic Parkinsonism. All pathogenic mutations in
LRRK2
result in hyperactivation of the LRRK2 kinase, offering the prospect of elaborating disease-modifying treatments. Indeed, LRRK2 inhibitors have entered phase 1 clinical trials. Data are also emerging for LRRK2 involvement in idiopathic PD, suggesting that inhibitors may benefit patients beyond those carrying
LRRK2
mutations. Recent advances point toward a role for LRRK2 in regulating autophagy, an intracellular process that delivers cytoplasmic constituents to the lysosome for degradation and recycling. LRRK2 phosphorylates a subgroup of RAB proteins and regulates their ability to bind cognate effector proteins. Additionally, LRRK2 is highly expressed in immune cells. Intriguing research indicates that, in early life, increased LRRK2 activity may protect against opportunistic pathogenic infection but then later increases the risk of developing PD, a concept called antagonistic pleiotropy.
There is compelling evidence for the role of the leucine-rich repeat kinase 2 (LRRK2) and in particular its kinase function in Parkinson's disease. Orally bioavailable, brain penetrant and potent ...LRRK2 kinase inhibitors are in the later stages of clinical development. Here, we describe a facile and robust assay to quantify LRRK2 kinase pathway activity by measuring LRRK2-mediated phosphorylation of Rab10 in human peripheral blood neutrophils. We use the selective MJFF-pRab10 monoclonal antibody recognising the Rab10 Thr73 phospho-epitope that is phosphorylated by LRRK2. We highlight the feasibility and practicability of using our assay in the clinical setting by studying a few patients with G2019S LRRK2 associated and sporadic Parkinson's as well as healthy controls. We suggest that peripheral blood neutrophils are a valuable resource for LRRK2 research and should be considered for inclusion in Parkinson's bio-repository collections as they are abundant, homogenous and express relatively high levels of LRRK2 as well as Rab10. In contrast, the widely used peripheral blood mononuclear cells are heterogeneous and only a minority of cells (monocytes and contaminating neutrophils) express LRRK2. While our LRRK2 kinase pathway assay could assist in patient stratification based on LRRK2 kinase activity, we envision that it may find greater utility in pharmacodynamic and target engagement studies in future LRRK2 inhibitor trials.
Pathogenic mutations in the Leucine-rich repeat kinase 2 (LRRK2) increase its activity leading to increased phosphorylation of Rab proteins. Here we introduce a sensitive and accurate assay to ...measure increased phospho Rab levels using synthetic stable isotope-labeled analogues for both phosphorylated and non-phosphorylated tryptic peptides surrounding Rab10-Thr73. Compared to healthy controls, carriers of mutated LRRK2 had 1.9-fold and those with VPS35 mutation 3.7-fold increased pRab10 levels in their neutrophils. Our generic MS-based assay helps to stratify PD patient and determine LRRK2 inhibitor efficiency in clinical trials.
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Highlights
•MS-based clinical assay that accurately determines phospho Rab10 occupancy.•Stable isotope labeled phosphopeptide injected as a standard with endogenous tryptic phospho Rab peptide for accurate ratio determination.•Determination of pRab levels in neutrophils of Parkinson disease patients.•Relevance of pRab levels as marker of PD.
Pathogenic mutations in the Leucine-rich repeat kinase 2 (LRRK2) are the predominant genetic cause of Parkinson's disease (PD). They increase its activity, resulting in augmented Rab10-Thr73 phosphorylation and conversely, LRRK2 inhibition decreases pRab10 levels. Currently, there is no assay to quantify pRab10 levels for drug target engagement or patient stratification. To meet this challenge, we developed an high accuracy and sensitivity targeted mass spectrometry (MS)-based assay for determining Rab10-Thr73 phosphorylation stoichiometry in human samples. It uses synthetic stable isotope-labeled (SIL) analogues for both phosphorylated and nonphosphorylated tryptic peptides surrounding Rab10-Thr73 to directly derive the percentage of Rab10 phosphorylation from attomole amounts of the endogenous phosphopeptide. The SIL and the endogenous phosphopeptides are separately admitted into an Orbitrap analyzer with the appropriate injection times. We test the reproducibility of our assay by determining Rab10-Thr73 phosphorylation stoichiometry in neutrophils of LRRK2 mutation carriers before and after LRRK2 inhibition. Compared with healthy controls, the PD predisposing mutation carriers LRRK2 G2019S and VPS35 D620N display 1.9-fold and 3.7-fold increased pRab10 levels, respectively. Our generic MS-based assay further establishes the relevance of pRab10 as a prognostic PD marker and is a powerful tool for determining LRRK2 inhibitor efficacy and for stratifying PD patients for LRRK2 inhibitor treatment.
Mutations that increase the protein kinase activity of LRRK2 are one of the most common causes of familial Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases within their Switch-II ...motif, impacting interaction with effectors. We describe and validate a new, multiplexed targeted mass spectrometry assay to quantify endogenous levels of LRRK2-phosphorylated Rab substrates (Rab1, Rab3, Rab8, Rab10, Rab35 and Rab43) as well as total levels of Rabs, LRRK2 and LRRK2-phosphorylated at the Ser910 and Ser935 biomarker sites. Exploiting this assay, we quantify for the first time the relative levels of each of the pRab proteins in different cells (mouse embryonic fibroblasts, human neutrophils) and mouse tissues (brain, kidney, lung and spleen). We define how these components are impacted by Parkinson's pathogenic mutations (LRRK2R1441C and VPS35D620N) and LRRK2 inhibitors. We find that the VPS35D620N, but not LRRK2R1441C mutation, enhances Rab1 phosphorylation in a manner blocked by administration of an LRRK2 inhibitor, providing the first evidence that endogenous Rab1 is a physiological substrate for LRRK2. We exploit this assay to demonstrate that in Parkinson's patients with VPS35D620N mutations, phosphorylation of multiple Rab proteins (Rab1, Rab3, Rab8, Rab10 and Rab43) is elevated. We highlight the benefits of this assay over immunoblotting approaches currently deployed to assess LRRK2 Rab signalling pathway.
Missense mutations in the LRRK2 (Leucine-rich repeat protein kinase-2) and VPS35 genes result in autosomal dominant Parkinson's disease. The VPS35 gene encodes for the cargo-binding component of the ...retromer complex, while LRRK2 modulates vesicular trafficking by phosphorylating a subgroup of Rab proteins. Pathogenic mutations in LRRK2 increase its kinase activity. It is not known how the only thus far described pathogenic VPS35 mutation, p.D620N exerts its effects. We reveal that the VPS35D620N knock-in mutation strikingly elevates LRRK2-mediated phosphorylation of Rab8A, Rab10, and Rab12 in mouse embryonic fibroblasts. The VPS35D620N mutation also increases Rab10 phosphorylation in mouse tissues (the lung, kidney, spleen, and brain). Furthermore, LRRK2-mediated Rab10 phosphorylation is increased in neutrophils as well as monocytes isolated from three Parkinson's patients with a heterozygous VPS35D620N mutation compared with healthy donors and idiopathic Parkinson's patients. LRRK2-mediated Rab10 phosphorylation is significantly suppressed by knock-out or knock-down of VPS35 in wild-type, LRRK2R1441C, or VPS35D620N cells. Finally, VPS35D620N mutation promotes Rab10 phosphorylation more potently than LRRK2 pathogenic mutations. Available data suggest that Parkinson's patients with VPS35D620N develop the disease at a younger age than those with LRRK2 mutations. Our observations indicate that VPS35 controls LRRK2 activity and that the VPS35D620N mutation results in a gain of function, potentially causing PD through hyperactivation of the LRRK2 kinase. Our findings suggest that it may be possible to elaborate compounds that target the retromer complex to suppress LRRK2 activity. Moreover, patients with VPS35D620N associated Parkinson's might benefit from LRRK2 inhibitor treatment that have entered clinical trials in humans.
Hyperpolarizing synaptic inhibition through GABAA and glycine receptors depends on the presence of the neuronal cation-chloride-cotransporter protein, KCC2. Several transcriptional and ...post-transcriptional mechanisms have been shown to regulate KCC2 and thereby influence the polarity and efficacy of inhibitory synaptic transmission. It is unclear however whether regulation of KCC2 enables the transporter to attain different levels of activity thus allowing a neuron to modulate the strength of inhibitory synaptic transmission to its changing requirements. We therefore investigated whether phosphorylation can allow KCC2 to achieve distinct levels of Cl−i in neurons. We generated a variety of KCC2 alanine dephosphorylation mimics and used NH4+-induced pHi shifts in cultured hippocampal neurons to quantify the rate of KCC2 transport activity exhibited by these mutants. To explore the relationship between KCC2 transport and GABAA receptor-mediated current amplitudes we performed gramicidine perforated-patch recordings. The correlation between EGABA and NH4+-induced pHi shifts enabled an estimate of the range of chloride extrusion possible by kinase/phosphatase regulation of KCC2.
Our results demonstrate that KCC2 transport can vary considerably in magnitude depending on the combination of alanine mutations present on the protein. Transport can be enhanced to sufficiently high levels that hyperpolarizing GABAA responses may be obtained even in neurons with an extremely negative resting membrane potential and at high extracellular K+ concentrations. Our findings highlight the significant potential for regulating the inhibitory tone by KCC2-mediated chloride extrusion and suggest that cellular signaling pathways may act combinatorially to alter KCC2 phosphorylation/dephosphorylation and thereby tune the strength of synaptic inhibition.
•Dephosphorylation mimicking mutations of KCC2 were introduced in cultured neurons.•Distinct levels of transport activity depended upon mutation site and combination.•Vmax of transport activity correlated linearly with EGABA.•Regulation of KCC2 activity may effect minute changes in Cli.•This may allow to tune GABAergic inhibitory strength to meet varying neural demands.
Neuroacanthocytosis (NA) refers to a group of heterogenous, rare genetic disorders, namely chorea acanthocytosis (ChAc), McLeod syndrome (MLS), Huntington's disease-like 2 (HDL2) and pantothenate ...kinase associated neurodegeneration (PKAN), that mainly affect the basal ganglia and are associated with similar neurological symptoms. PKAN is also assigned to a group of rare neurodegenerative diseases, known as NBIA (neurodegeneration with brain iron accumulation), associated with iron accumulation in the basal ganglia and progressive movement disorder. Acanthocytosis, the occurrence of misshaped erythrocytes with thorny protrusions, is frequently observed in ChAc and MLS patients but less prevalent in PKAN (about 10%) and HDL2 patients. The pathological factors that lead to the formation of the acanthocytic red blood cell shape are currently unknown. The aim of this study was to determine whether NA/NBIA acanthocytes differ in their functionality from normal erythrocytes. Several flow-cytometry-based assays were applied to test the physiological responses of the plasma membrane, namely drug-induced endocytosis, phosphatidylserine exposure and calcium uptake upon treatment with lysophosphatidic acid. ChAc red cell samples clearly showed a reduced response in drug-induced endovesiculation, lysophosphatidic acid-induced phosphatidylserine exposure, and calcium uptake. Impaired responses were also observed in acanthocyte-positive NBIA (PKAN) red cells but not in patient cells without shape abnormalities. These data suggest an "acanthocytic state" of the red cell where alterations in functional and interdependent membrane properties arise together with an acanthocytic cell shape. Further elucidation of the aberrant molecular mechanisms that cause this acanthocytic state may possibly help to evaluate the pathological pathways leading to neurodegeneration.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Electrical synapses (gap junctions) rapidly transmit signals between neurons and are composed of connexins. In neurons, connexin36 (Cx36) is the most abundant isoform; however, the mechanisms ...underlying formation of Cx36-containing electrical synapses are unknown. We focus on homocellular and heterocellular gap junctions formed by an AII amacrine cell, a key interneuron found in all mammalian retinas. In mice lacking native Cx36 but expressing a variant tagged with enhanced green fluorescent protein at the C-terminus (KO-Cx36-EGFP), heterocellular gap junctions formed between AII cells and ON cone bipolar cells are fully functional, whereas homocellular gap junctions between two AII cells are not formed. A tracer injected into an AII amacrine cell spreads into ON cone bipolar cells but is excluded from other AII cells. Reconstruction of Cx36-EGFP clusters on an AII cell in the KO-Cx36-EGFP genotype confirmed that the number, but not average size, of the clusters is reduced - as expected for AII cells lacking a subset of electrical synapses. Our studies indicate that some neurons exhibit at least two discriminatory mechanisms for assembling Cx36. We suggest that employing different gap-junction-forming mechanisms could provide the means for a cell to regulate its gap junctions in a target-cell-specific manner, even if these junctions contain the same connexin.
Loss-of-function variants in the low-density lipoprotein receptor-related protein 10 (
LRP10
) gene have been associated with autosomal-dominant Parkinson’s disease (PD), PD dementia, and dementia ...with Lewy bodies (DLB). Moreover,
LRP10
variants have been found in individuals diagnosed with progressive supranuclear palsy and amyotrophic lateral sclerosis. Despite this genetic evidence, little is known about the expression and function of LRP10 protein in the human brain under physiological or pathological conditions. To better understand how
LRP10
variants lead to neurodegeneration, we first performed an in-depth characterisation of LRP10 expression in post-mortem brains and human-induced pluripotent stem cell (iPSC)-derived astrocytes and neurons from control subjects. In adult human brain, LRP10 is mainly expressed in astrocytes and neurovasculature but undetectable in neurons. Similarly, LRP10 is highly expressed in iPSC-derived astrocytes but cannot be observed in iPSC-derived neurons. In astrocytes, LRP10 is present at trans-Golgi network, plasma membrane, retromer, and early endosomes. Interestingly, LRP10 also partially co-localises and interacts with sortilin-related receptor 1 (SORL1). Furthermore, although LRP10 expression and localisation in the substantia nigra of most idiopathic PD and DLB patients and
LRP10
variant carriers diagnosed with PD or DLB appeared unchanged compared to control subjects, significantly enlarged LRP10-positive vesicles were detected in a patient carrying the LRP10 p.Arg235Cys variant. Last, LRP10 was detected in Lewy bodies (LB) at late maturation stages in brains from idiopathic PD and DLB patients and in
LRP10
variant carriers. In conclusion, high LRP10 expression in non-neuronal cells and undetectable levels in neurons of control subjects indicate that LRP10-mediated pathogenicity is initiated via cell non-autonomous mechanisms, potentially involving the interaction of LRP10 with SORL1 in vesicle trafficking pathways. Together with the specific pattern of LRP10 incorporation into mature LBs, these data support an important mechanistic role for disturbed vesicle trafficking and loss of LRP10 function in neurodegenerative diseases.
A harmless high? Sammler, Esther M, MD; Foley, Peter L, MRCP; Lauder, Gavin D, MSc ...
The Lancet (British edition),
08/2010, Letnik:
376, Številka:
9742
Journal Article
Recenzirano
Brain CT scan was unremarkable; however, the cerebrospinal fluid (CSF) opening pressure during lumbar puncture in the lateral decubitus position was raised at 350 mm of water. ... mephedrone has ...recently been implicated in teenage morbidity and mortality, resulting in a heated media and public debate which has expedited a change in legislation.2,3 As of April 16, 2010, possessing or supplying mephedrone is illegal in the UK.3 Little is known about mephedrone's mechanism of action, spectrum of clinical signs and symptoms, and in particular, potential neurotoxic effects.2 Given its chemical structure, mephedrone is thought to stimulate release and inhibit reuptake of monoamine neurotransmitters.2 There is ample evidence that other amphetamines such as ecstasy are associated with clinically significant hyponatraemia.4,5 Excessive sweating with electrolyte loss and increased fluid intake may be confounding factors, but there is also evidence from human and animal studies of ecstasyinduced secretion of antidiuretic hormone (ADH) mediated via serotonin.4,5 We think that mephedrone, like ecstasy, promotes serotonin-mediated ADH release, and that a mephedrone-induced syndrome of inappropriate ADH secretion resulted in our patient's hyponatraemia and altered mental status.
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