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INTRODUCTION: The cytoprotective PTEN-induced kinase 1 (PINK1)-parkin RBR E3 ubiquitin protein ligase (PRKN) pathway selectively labels damaged mitochondria with phosphorylated ubiquitin (pS65-Ub) for their autophagic removal (mitophagy). Because dysfunctions of mitochondria and degradation pathways are early features of Alzheimer's disease (AD), mitophagy impairments may contribute to the pathogenesis. METHODS: Morphology, levels, and distribution of the mitophagy tag pS65-Ub were evaluated by biochemical analyses combined with tissue and single cell imaging in AD autopsy brain and in transgenic mouse models. RESULTS: Analyses revealed significant increases of pS65-Ub levels in AD brain, which strongly correlated with granulovacuolar degeneration (GVD) and early phospho-tau deposits, but were independent of amyloid beta pathology. Single cell analyses revealed predominant co-localization of pS65-Ub with mitochondria, GVD bodies, and/or lysosomes depending on the brain region analyzed. DISCUSSION: Our study highlights mitophagy alterations in AD that are associated with early tau pathology, and suggests that distinct mitochondrial, autophagic, and/or lysosomal failure may contribute to the selective vulnerability in disease.
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Parkinson's disease (PD) is caused by neurodegeneration of nigrostriatal neurons, resulting in dopamine (DA) stimulated motor deficits. Like brain derived neurotrophic factor (BDNF), 7,8-dihydroxyflavone (DHF) is an agonist of the tropomyosin receptor kinase-B (TrkB) and stimulates the same secondary cascades that promote neuronal growth, survival and differentiation. We used our progressive mouse model of PD by administering increasing doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) over 4â¯weeks (5â¯days/week), and then treated mice with DHF for 4â¯weeks after the cessation of the toxin injections (i.e., restoration). Mice treated with DHF recovered motorically, even after MPTP administration. Despite a 75% loss of tyrosine hydroxylase (TH) expression in the dorsolateral (DL) striatum in the MPTP group, mice treated with DHF had a recovery comparable to that found in the respective control. There was no recovery of DA tissue levels within the DL striatum. In both the DL striatum and substantia nigra (SN)/midbrain, phosphorylated TrkB and secondary messengers were significantly increased following DHF compared to the MPTP only group. Expression of the sprouting biomarker, superior cervical ganglion 10 (SCG10), was increased â¼20% in the DL striatum and 66% in the SN/midbrain in mice treated with DHF compared to the MPTP only group. We report that after 4â¯weeks of progressive MPTP administration, DHF can restore motor deficits and TH within the DL striatum in a TrkB-dependent manner. Our data suggests that DHF may help alleviate motor symptoms of PD and restore the loss of DA terminals within the striatum.
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Doença de Parkinson , Transtornos Parkinsonianos , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina , Animais , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Flavonas , Marcha , Camundongos , Camundongos Endogâmicos C57BL , Transtornos Parkinsonianos/tratamento farmacológico , Substância Negra/metabolismo , Tirosina , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Mutations in the LRRK2 kinase are the most common cause of familial Parkinson's disease, and variants increase risk for the sporadic form of the disease. LRRK2 phosphorylates multiple RAB GTPases including RAB8A and RAB10. Phosphorylated RAB10 is recruited to centrosome-localized RILPL1, which may interfere with ciliogenesis in a disease-relevant context. Our previous studies indicate that the centrosomal accumulation of phosphorylated RAB8A causes centrosomal cohesion deficits in dividing cells, including in peripheral patient-derived cells. Here, we show that both RAB8 and RAB10 contribute to the centrosomal cohesion deficits. Pathogenic LRRK2 causes the centrosomal accumulation not only of phosho-RAB8 but also of phospho-RAB10, and the effects on centrosomal cohesion are dependent on RAB8, RAB10 and RILPL1. Conversely, the pathogenic LRRK2-mediated ciliogenesis defects correlate with the centrosomal accumulation of both phospho-RAB8 and phospho-RAB10. LRRK2-mediated centrosomal cohesion and ciliogenesis alterations are observed in patient-derived peripheral cells, as well as in primary astrocytes from mutant LRRK2 mice, and are reverted upon LRRK2 kinase inhibition. These data suggest that the LRRK2-mediated centrosomal cohesion and ciliogenesis defects are distinct cellular readouts of the same underlying phospho-RAB8/RAB10/RILPL1 nexus and highlight the possibility that either centrosomal cohesion and/or ciliogenesis alterations may serve as cellular biomarkers for LRRK2-related PD.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Centrossomo/metabolismo , Ciliopatias/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Ciliopatias/enzimologia , Ciliopatias/genética , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Fosforilação , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Mutations in leucine-rich repeat kinase 2 (LRRK2) segregate with familial Parkinson's disease (PD) and genetic variation around LRRK2 contributes to risk of sporadic disease. Although knockout (KO) of Lrrk2 or knock-in of pathogenic mutations into the mouse germline does not result in a PD phenotype, several defects have been reported in the kidneys of Lrrk2 KO mice. To understand LRRK2 function in vivo, we used an unbiased approach to determine which protein pathways are affected in LRRK2 KO kidneys. We nominated changes in cytoskeletal-associated proteins, lysosomal proteases, proteins involved in vesicular trafficking and in control of protein translation. Changes were not seen in mice expressing the pathogenic G2019S LRRK2 mutation. Using cultured epithelial kidney cells, we replicated the accumulation of lysosomal proteases and demonstrated changes in subcellular distribution of the cation-independent mannose-6-phosphate receptor. These results show that loss of LRRK2 leads to co-ordinated responses in protein translation and trafficking and argue against a dominant negative role for the G2019S mutation.
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Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Doença de Parkinson/genética , Biossíntese de Proteínas/genética , Proteômica , Animais , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Regulação da Expressão Gênica , Humanos , Rim/metabolismo , Rim/patologia , Camundongos , Camundongos Knockout , Mutação , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Fenótipo , Proteólise , Receptor IGF Tipo 2/genética , Transdução de SinaisRESUMO
Parkinson's disease-linked mutations in LRRK2 enhance the kinase activity of the protein, therefore targeting LRRK2 kinase activity is a promising therapeutic approach. Phosphorylation at S935 of LRRK2 and of its Rab GTPase substrates have proven very useful biomarkers to monitor its kinase activity. Complementary to these approaches autophosphorylation of LRRK2 can be used as a direct kinase activity readout but to date detection of autophosphorylation at endogenous levels in vivo has been limited. We developed a fractionation-based enrichment method to successfully detect endogenous S1292 LRRK2 autophosphorylation in mouse tissues and highlight S1292 as a physiological readout candidate for LRRK2 kinase activity in vivo.
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Missense mutations in the multi-domain kinase LRRK2 cause late onset familial Parkinson's disease. They most commonly with classic proteinopathy in the form of Lewy bodies and Lewy neurites comprised of insoluble α-synuclein, but in rare cases can also manifest tauopathy. The normal function of LRRK2 has remained elusive, as have the cellular consequences of its mutation. Data from LRRK2 null model organisms and LRRK2-inhibitor treated animals support a physiological role for LRRK2 in regulating lysosome function. Since idiopathic and LRRK2-linked PD are associated with the intraneuronal accumulation of protein aggregates, a series of critical questions emerge. First, how do pathogenic mutations that increase LRRK2 kinase activity affect lysosome biology in neurons? Second, are mutation-induced changes in lysosome function sufficient to alter the metabolism of α-synuclein? Lastly, are changes caused by pathogenic mutation sensitive to reversal with LRRK2 kinase inhibitors? Here, we report that mutation of LRRK2 induces modest but significant changes in lysosomal morphology and acidification, and decreased basal autophagic flux when compared to WT neurons. These changes were associated with an accumulation of detergent-insoluble α-synuclein and increased neuronal release of α-synuclein and were reversed by pharmacologic inhibition of LRRK2 kinase activity. These data demonstrate a critical and disease-relevant influence of native neuronal LRRK2 kinase activity on lysosome function and α-synuclein homeostasis. Furthermore, they also suggest that lysosome dysfunction, altered neuronal α-synuclein metabolism, and the insidious accumulation of aggregated protein over decades may contribute to pathogenesis in this late-onset form of familial PD.
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Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Lisossomos/metabolismo , Mutação , Neurônios/metabolismo , alfa-Sinucleína/metabolismo , Animais , Autofagia , Células Cultivadas , Humanos , Concentração de Íons de Hidrogênio , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/antagonistas & inibidores , Lisossomos/patologia , Camundongos Transgênicos , Neurônios/patologia , Doença de Parkinson/genética , Doença de Parkinson/metabolismoRESUMO
BACKGROUND: LRRK2 G2019S mutation is associated with increased kinase activity and is the most common mutation associated with late-onset PD. However, the transgenic mouse model has not recapitulated cardinal PD-related motor phenotypes. Non-motor symptoms of PD including cognitive impairments are very common and may appear earlier than the motor symptoms. The objective of this study was to determine whether human LRRK2 with G2019S mutation causes hippocampus-dependent cognitive deficits in mice. RESULTS: Male (LRRK2-G2019S) LRRK2-Tg mice showed impairments in the early portion of the Two-day radial arm water maze acquisition trial as well as in the reversal learning on the third day. However, their performance was similar to Non-Tg controls in the probe trial. LRRK2-Tg mice also displayed impairments in the novel arm discrimination test but not in the spontaneous alternation test in Y-maze. Interestingly, there was no statistically significant locomotor impairment during any of these cognitive test, nor in the locomotor tests including open field, accelerating rotarod and pole tests. Expression of the postsynaptic protein PSD-95 but not the presynaptic protein synaptophysin was lower in hippocampal homogenates of LRRK2-Tg mice. CONCLUSION: Consistent with previous reports in human LRRK2 G2019S carriers, the current data suggests that cognitive dysfunctions are present in LRRK2-Tg mice even in the absence of locomotor impairment. LRRK2 G2019S mutation represses the postsynaptic protein PSD-95 but not the presynaptic protein synaptophysin. This study also suggests that mild cognitive impairment may appear earlier than motor dysfunctions in LRRK2-G2019S mutation carriers.
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Disfunção Cognitiva/genética , Proteína 4 Homóloga a Disks-Large/metabolismo , Hipocampo/metabolismo , Aprendizagem/fisiologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Atividade Motora/fisiologia , Animais , Modelos Animais de Doenças , Humanos , Masculino , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Transgênicos , Mutação , Reversão de Aprendizagem/fisiologiaRESUMO
BACKGROUND: α-Synuclein (αSYN) has been genetically implicated in familial and sporadic Parkinson's disease (PD), and is associated with disease susceptibility, progression and pathology. Excess amounts of αSYN are toxic to neurons. In the brain, microglial αSYN clearance is closely related to neuronal survival. Leucine-rich repeat kinase 2 (LRRK2) is the one of the other genes implicated in familial and sporadic PD. While LRRK2 is known to be expressed in microglia, its true function remains to be elucidated. In this study, we investigated αSYN clearance by microglia isolated from LRRK2-knockout (KO) mice. RESULTS: In LRRK2-KO microglia, αSYN was taken up in larger amounts and cleared from the supernatant more effectively than for microglia isolated from wild-type (WT) mice. This higher clearance ability of LRRK2-KO microglia was thought to be due to an increase of Rab5-positive endosomes, but not Rab7- or Rab11-positive endosomes. Increased engagement between Rab5 and dynamin 1 was also observed in LRRK2-KO microglia. CONCLUSION: LRRK2 negatively regulates the clearance of αSYN accompanied by down-regulation of the endocytosis pathway. Our findings reveal a new functional role of LRRK2 in microglia and offer a new insight into the mechanism of PD pathogenesis.
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Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Microglia/metabolismo , alfa-Sinucleína/metabolismo , Animais , Western Blotting , Encéfalo/metabolismo , Encéfalo/patologia , Células Cultivadas , Dinamina I/metabolismo , Endocitose/fisiologia , Endossomos/metabolismo , Imunofluorescência , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/patologia , Proteínas Recombinantes/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismoAssuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Monócitos/metabolismo , Transtornos Parkinsonianos/metabolismo , Idoso , Animais , Linfócitos B/metabolismo , Proteínas Ligadas por GPI/metabolismo , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/deficiência , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Receptores de Lipopolissacarídeos/metabolismo , Camundongos Transgênicos , Receptores de IgG/metabolismo , Baço/metabolismoRESUMO
LRRK2 is the causal molecule of autosomal dominant familial Parkinson's disease. B2 cells express a much higher LRRK2 mRNA level than B1 cells. To reveal the function of LRRK2 in B cells, we analyzed B cell functions in LRRK2-knockout (LRRK2(-/-)) mice. LRRK2(-/-) mice had significantly higher counts of peritoneal B1 cells than wild-type mice. After BCR stimulation, phosphor-Erk1/2 of splenic B2 cells was enhanced to a higher degree in LRRK2(-/-) mice. LRRK2(-/-) mice had a significantly higher serum IgA level, and TNP-Ficoll immunization increased the titer of serum anti-TNP IgM antibody. LRRK2 may play important roles in B cells.
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Linfócitos B/metabolismo , Homeostase/genética , Imunoglobulina A/sangue , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Fatores Etários , Animais , Antígenos CD/metabolismo , Linfócitos B/classificação , Ensaio de Imunoadsorção Enzimática , Ficoll/análogos & derivados , Ficoll/imunologia , Citometria de Fluxo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Sistema de Sinalização das MAP Quinases/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Cavidade Peritoneal/citologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais/genética , Baço/citologia , Fator de Crescimento Transformador beta1/sangue , Trinitrobenzenos/imunologiaRESUMO
Pathogenic mutations and risk variants in LRRK2 (leucine-rich repeat kinase 2) represent the most common genetic cause of familial and sporadic PD (Parkinson's disease). LRRK2 protein is widely expressed throughout the brain and the periphery. Structurally, LRRK2 contains several functional domains, including a dual enzymatic core consisting of a kinase and GTPase domain. Disease-linked variants are found in both these enzymatic domains as well as in the COR [C-terminal of ROC (Ras of complex proteins)] and WD40 protein-protein binding domain. The kinase domain is widely believed to be linked to toxicity, and thus the thrust of pharmaceutical effort has focused on developing LRRK2 kinase inhibitors. However, recent data have suggested that inhibition of LRRK2 activity results in reduced LRRK2 levels and peripheral side effects, which are similar to those observed in homozygous LRRK2-knockout and LRRK2 kinase-dead rodent models. In a recent issue of the Biochemical Journal, a study led by Nichols reveals that dephosphorylation of LRRK2 cellular phosphorylation sites (Ser(910)/Ser(935)/Ser(955)/Ser(973)) triggers its ubiquitination and subsequent degradation and thus may account for the loss of function phenotypes observed in peripheral tissues in LRRK2-knockout/kinase-dead or inhibitor-treated rodents and primates. Albeit negative from a kinase inhibitor standpoint, the data open new avenues for LRRK2 biology and therapeutic approaches to counteract LRRK2 toxicity.
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Proteínas 14-3-3/metabolismo , Citoplasma/metabolismo , Mutação , Proteínas Serina-Treonina Quinases/metabolismo , Animais , HumanosRESUMO
Alzheimer's disease (AD) is a neurological disorder characterized by profound memory loss and progressive dementia. Accumulating evidence suggests that Type 2 diabetes mellitus, a metabolic disorder characterized by insulin resistance and glucose intolerance, significantly increases the risk for developing AD. Whereas amyloid-ß (Aß) deposition and neurofibrillary tangles are major histological hallmarks of AD, impairment of cerebral glucose metabolism precedes these pathological changes during the early stage of AD and likely triggers or exacerbates AD pathology. However, the mechanisms linking disturbed insulin signaling/glucose metabolism and AD pathogenesis remain unclear. The low-density lipoprotein receptor-related protein 1 (LRP1), a major apolipoprotein E receptor, plays critical roles in lipoprotein metabolism, synaptic maintenance, and clearance of Aß in the brain. Here, we demonstrate that LRP1 interacts with the insulin receptor ß in the brain and regulates insulin signaling and glucose uptake. LRP1 deficiency in neurons leads to impaired insulin signaling as well as reduced levels of glucose transporters GLUT3 and GLUT4. Consequently, glucose uptake is reduced. By using an in vivo microdialysis technique sampling brain glucose concentration in freely moving mice, we further show that LRP1 deficiency in conditional knock-out mice resulted in glucose intolerance in the brain. We also found that hyperglycemia suppresses LRP1 expression, which further exacerbates insulin resistance, glucose intolerance, and AD pathology. As loss of LRP1 expression is seen in AD brains, our study provides novel insights into insulin resistance in AD. Our work also establishes new targets that can be explored for AD prevention or therapy.
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Encéfalo , Diabetes Mellitus Experimental/patologia , Glucose/metabolismo , Insulina/metabolismo , Neurônios/metabolismo , Receptores de LDL/deficiência , Proteínas Supressoras de Tumor/deficiência , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/fisiopatologia , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/fisiopatologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Linhagem Celular Transformada , Diabetes Mellitus Experimental/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Intolerância à Glucose/genética , Transportador de Glucose Tipo 3/genética , Transportador de Glucose Tipo 3/metabolismo , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Insulina/farmacologia , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Camundongos , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Transporte Proteico/genética , Interferência de RNA/fisiologia , Receptores de LDL/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Mutations in leucine-rich repeat kinase 2 (Lrrk2) are the most common genetic cause of Parkinson's disease (PD), a neurodegenerative disorder affecting 1-2% of those >65 years old. The neurophysiology of LRRK2 remains largely elusive, although protein loss suggests a role in glutamatergic synapse transmission and overexpression studies show altered dopamine release in aged mice. We show that glutamate transmission is unaltered onto striatal projection neurons (SPNs) of adult LRRK2 knockout mice and that adult animals exhibit no detectable cognitive or motor deficits. Basal synaptic transmission is also unaltered in SPNs of LRRK2 overexpressing mice, but they do exhibit clear alterations to D2-receptor-mediated short-term synaptic plasticity, behavioral hypoactivity and impaired recognition memory. These phenomena are associated with decreased striatal dopamine tone and abnormal dopamine- and cAMP-regulated phosphoprotein 32 kDa signal integration. The data suggest that LRRK2 acts at the nexus of dopamine and glutamate signaling in the adult striatum, where it regulates dopamine levels, presynaptic glutamate release via D2-dependent synaptic plasticity and dopamine-receptor signal transduction.
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Dopamina/metabolismo , Memória , Atividade Motora , Neostriado/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Animais , Glutamatos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Masculino , Camundongos , Camundongos Transgênicos , Plasticidade Neuronal , Neurônios/metabolismo , Doença de Parkinson/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Transmissão SinápticaRESUMO
Mutations in leucine-rich repeat kinase 2 (LRRK2) are found in a significant proportion of late-onset Parkinson's disease (PD) patients. Elucidating the neuroanatomical localization of LRRK2 will further define LRRK2 function and the molecular basis of PD. Here, we utilize recently characterized monoclonal antibodies to evaluate LRRK2 expression in rodent brain regions relevant to PD. In both mice and rats, LRRK2 is highly expressed in the cortex and striatum, particularly in pyramidal neurons of layer V and in medium spiny neurons within striosomes. Overall, rats have a more restricted distribution of LRRK2 compared with mice. Mice, but not rats, show high levels of LRRK2 expression in the substantia nigra pars compacta. Expression of the pathogenic LRRK2-G2019S protein from mouse bacterial artificial chromosome (BAC) constructs closely mimics endogenous LRRK2 distribution in the mouse brain. However, LRRK2-G2019S expression derived from human BAC constructs causes LRRK2 to be expressed in additional neuron subtypes in the rat such as striatal cholinergic interneurons and the substantia nigra pars compacta. The distribution of LRRK2 from human BAC constructs more closely resembles descriptions of LRRK2 in humans and nonhuman primates. Computational analyses of DNA regulatory elements in LRRK2 show a primate-specific promoter sequence that does not exist in lower mammalian species. These noncoding regions may be involved in directing neuronal expression patterns. Together, these studies will aid in understanding the normal function of LRRK2 in the brain and will assist in model selection for future studies.
Assuntos
Córtex Cerebral/fisiologia , Corpo Estriado/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Substância Negra/fisiologia , Animais , Cromossomos Artificiais Bacterianos/genética , Técnicas de Inativação de Genes , Interneurônios/fisiologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/genética , Células Piramidais/fisiologia , Ratos Long-Evans , Ratos Sprague-Dawley , Ratos Transgênicos , Alinhamento de Sequência , Especificidade da EspécieRESUMO
Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of familial Parkinson's disease (PD). The neuropathology of LRRK2-related PD is heterogeneous and can include aberrant tau phosphorylation or neurofibrillary tau pathology. Recently, LRRK2 has been shown to phosphorylate tau in vitro; however, the major epitopes phosphorylated by LRRK2 and the physiological or pathogenic consequences of these modifications in vivo are unknown. Using mass spectrometry, we identified multiple sites on recombinant tau that are phosphorylated by LRRK2 in vitro, including pT149 and pT153, which are phospho-epitopes that to date have been largely unexplored. Importantly, we demonstrate that expression of transgenic LRRK2 in a mouse model of tauopathy increased the aggregation of insoluble tau and its phosphorylation at T149, T153, T205, and S199/S202/T205 epitopes. These findings indicate that tau can be a LRRK2 substrate and that this interaction can enhance salient features of human disease.
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Proteínas Serina-Treonina Quinases/metabolismo , Tauopatias/metabolismo , Proteínas tau/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Epitopos/genética , Epitopos/metabolismo , Feminino , Células HEK293 , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Tauopatias/genética , Tauopatias/patologiaRESUMO
Gaucher disease is an autosomal recessively inherited storage disorder caused by deficiency of the lysosomal hydrolase, acid ß-glucosidase. The disease manifestations seen in Gaucher patients are highly heterogeneous as is the responsiveness to therapy. The elucidation of the precise factors responsible for this heterogeneity has been challenging as the development of clinically relevant animal models of Gaucher disease has been problematic. Although numerous murine models for Gaucher disease have been described each has limitations in their specific utility. We describe here, transgenic murine models of Gaucher disease that will be particularly useful for the study of pharmacological chaperones. We have produced stable transgenic mouse strains that individually express wild type, N370S and L444P containing human acid ß-glucosidase and show that each of these transgenic lines rescues the lethal phenotype characteristic of acid ß-glucosidase null mice. Both the N370S and L444P transgenic models show early and progressive elevations of tissue sphingolipids with L444P mice developing progressive splenic Gaucher cell infiltration. We demonstrate the potential utility of these new transgenic models for the study of Gaucher disease pathogenesis. In addition, since these mice produce only human enzyme, they are particularly relevant for the study of pharmacological chaperones that are specifically targeted to human acid ß-glucosidase and the common mutations underlying Gaucher disease.
Assuntos
Doença de Gaucher/enzimologia , Doença de Gaucher/genética , Expressão Gênica , Variação Genética , Glucosilceramidase/genética , Glucosilceramidase/metabolismo , Ambroxol/farmacologia , Animais , Cerebelo/metabolismo , Córtex Cerebral/metabolismo , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Feminino , Genótipo , Humanos , Imino Piranoses/farmacologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Mutação , Fenótipo , Baço/metabolismo , Baço/patologiaRESUMO
Missense mutations in LRRK2 (leucine-rich repeat kinase 2) are a major cause of PD (Parkinson's disease). Several antibodies against LRRK2 have been developed, but results using these polyclonal antibodies have varied widely leading to conflicting conclusions. To address this challenge, the Michael J. Fox Foundation for Parkinson's Research generated a number of monoclonal antibodies targeting epitopes across the LRRK2 protein. In the present paper, we report optimized protocols and results for ten monoclonal antibodies for immunoblotting, immunohistochemistry, immunoprecipitation and kinase activity assays, in rat, mouse and human brain tissue. Several efficacious antibodies were identified, but results demonstrate that the mouse monoclonal N241A/34 is suitable for most applications, with the best overall rabbit monoclonal antibody being c41-2. These antibodies produced a dominant band of the expected size via immunoblotting and a lack of labelling in tissue derived from LRRK2-knockout animals under optimized conditions. A significant proportion of LRRK2 protein localizes to insoluble fractions and no evidence of truncated LRRK2 protein was detected in any fraction from rodent or human tissues. An assay was developed for the robust detection of LRRK2 kinase activity directly from frozen mouse and human brain tissue, but precipitous declines in activity were observed that corresponded to increasing post-mortem intervals and processing times. Finally, we demonstrate the highest levels of brain-localized LRRK2 in the striatum, but note differential expression patterns between rat and mouse in both striatum and cortex. Anti-LRRK2 monoclonal antibodies that are unlimited in availability together with the proposed standardized protocols should aid in the definition of LRRK2 function in both health and disease.
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Proteínas Serina-Treonina Quinases/imunologia , Animais , Encéfalo/enzimologia , Linhagem Celular , Mapeamento de Epitopos , Humanos , Immunoblotting , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Masculino , Camundongos , Camundongos Knockout , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Coelhos , RatosRESUMO
Mutations in the LRRK2 gene are the most common cause of genetic Parkinson's disease. Although the mechanisms behind the pathogenic effects of LRRK2 mutations are still not clear, data emerging from in vitro and in vivo models suggests roles in regulating neuronal polarity, neurotransmission, membrane and cytoskeletal dynamics and protein degradation.We created mice lacking exon 41 that encodes the activation hinge of the kinase domain of LRRK2. We have performed a comprehensive analysis of these mice up to 20 months of age, including evaluation of dopamine storage, release, uptake and synthesis, behavioral testing, dendritic spine and proliferation/neurogenesis analysis.Our results show that the dopaminergic system was not functionally comprised in LRRK2 knockout mice. However, LRRK2 knockout mice displayed abnormal exploratory activity in the open-field test. Moreover, LRRK2 knockout mice stayed longer than their wild type littermates on the accelerated rod during rotarod testing. Finally, we confirm that loss of LRRK2 caused degeneration in the kidney, accompanied by a progressive enhancement of autophagic activity and accumulation of autofluorescent material, but without evidence of biphasic changes.
Assuntos
Dopamina/metabolismo , Mutação/genética , Doença de Parkinson/genética , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Autofagia/genética , Comportamento Animal , Rim/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Proteínas Serina-Treonina Quinases/deficiênciaRESUMO
Genome-wide analysis of a multi-incident family with autosomal-dominant parkinsonism has implicated a locus on chromosomal region 3q26-q28. Linkage and disease segregation is explained by a missense mutation c.3614G>A (p.Arg1205His) in eukaryotic translation initiation factor 4-gamma (EIF4G1). Subsequent sequence and genotype analysis identified EIF4G1 c.1505C>T (p.Ala502Val), c.2056G>T (p.Gly686Cys), c.3490A>C (p.Ser1164Arg), c.3589C>T (p.Arg1197Trp) and c.3614G>A (p.Arg1205His) substitutions in affected subjects with familial parkinsonism and idiopathic Lewy body disease but not in control subjects. Despite different countries of origin, persons with EIF4G1 c.1505C>T (p.Ala502Val) or c.3614G>A (p.Arg1205His) mutations appear to share haplotypes consistent with ancestral founders. eIF4G1 p.Ala502Val and p.Arg1205His disrupt eIF4E or eIF3e binding, although the wild-type protein does not, and render mutant cells more vulnerable to reactive oxidative species. EIF4G1 mutations implicate mRNA translation initiation in familial parkinsonism and highlight a convergent pathway for monogenic, toxin and perhaps virally-induced Parkinson disease.
Assuntos
Cromossomos Humanos Par 3/genética , Fator de Iniciação Eucariótico 4G/genética , Doença de Parkinson/genética , Biossíntese de Proteínas/genética , Sequência de Bases , Clonagem Molecular , Variações do Número de Cópias de DNA , Análise Mutacional de DNA , Citometria de Fluxo , Ligação Genética , Genótipo , Humanos , Imunoprecipitação , Mitocôndrias/fisiologia , Dados de Sequência Molecular , Mutação de Sentido Incorreto/genética , LinhagemRESUMO
The identification of genetic causes for Mendelian disorders has been based on the collection of multi-incident families, linkage analysis, and sequencing of genes in candidate intervals. This study describes the application of next-generation sequencing technologies to a Swiss kindred presenting with autosomal-dominant, late-onset Parkinson disease (PD). The family has tremor-predominant dopa-responsive parkinsonism with a mean onset of 50.6 ± 7.3 years. Exome analysis suggests that an aspartic-acid-to-asparagine mutation within vacuolar protein sorting 35 (VPS35 c.1858G>A; p.Asp620Asn) is the genetic determinant of disease. VPS35 is a central component of the retromer cargo-recognition complex, is critical for endosome-trans-golgi trafficking and membrane-protein recycling, and is evolutionarily highly conserved. VPS35 c.1858G>A was found in all affected members of the Swiss kindred and in three more families and one patient with sporadic PD, but it was not observed in 3,309 controls. Further sequencing of familial affected probands revealed only one other missense variant, VPS35 c.946C>T; (p.Pro316Ser), in a pedigree with one unaffected and two affected carriers, and thus the pathogenicity of this mutation remains uncertain. Retromer-mediated sorting and transport is best characterized for acid hydrolase receptors. However, the complex has many types of cargo and is involved in a diverse array of biologic pathways from developmental Wnt signaling to lysosome biogenesis. Our study implicates disruption of VPS35 and retromer-mediated trans-membrane protein sorting, rescue, and recycling in the neurodegenerative process leading to PD.