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Development of a physiologically relevant and easily scalable LUHMES cell-based model of G2019S LRRK2-driven Parkinson's disease.

ABSTRACT: Parkinson's disease (PD) is a fatal neurodegenerative disorder that is primarily caused by the degeneration and loss of dopaminergic neurons of the substantia nigra in the ventral midbrain. Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of late-onset PD identified to date, with G2019S being the most frequent LRRK2 mutation, which is responsible for up to 1-2% of sporadic PD and up to 6% of familial PD cases. As no treatment is available for this devastating disease, developing new therapeutic strategies is of foremost importance. Cellular models are commonly used for testing novel potential neuroprotective compounds. However, current cellular PD models either lack physiological relevance to dopaminergic neurons or are too complex and costly for scaling up the production process and for screening purposes. In order to combine biological relevance and throughput, we have developed a PD model in Lund human mesencephalic (LUHMES) cell-derived dopaminergic neurons by overexpressing wild-type (WT) and G2019S LRRK2 proteins. We show that these cells can differentiate into dopaminergic-like neurons and that expression of mutant LRRK2 causes a range of different phenotypes, including reduced nuclear eccentricity, altered mitochondrial and lysosomal morphologies, and increased dopaminergic cell death. This model could be used to elucidate G2019S LRRK2-mediated dopaminergic neural dysfunction and to identify novel molecular targets for disease intervention. In addition, our model could be applied to high-throughput and phenotypic screenings for the identification of novel PD therapeutics.

SUBMITTER: Calamini B

PROVIDER: S-EPMC8214734 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:Mutations in leucine rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's disease (PD). Mitochondrial and autophagic dysfunction has been described as etiologic factors in different experimental models of PD. We aimed to study the role of mitochondria and autophagy in LRRK2 G2019S -mutation, and its relationship with the presence of PD-symptoms. METHODS:Fibroblasts from six non-manifesting LRRK2 G2019S -carriers (NM-LRRK2 G2019S ) and seven patients with LRRK2 G2019S -associated PD (PD-LRRK2 G2019S ) were compared to eight healthy controls (C). An exhaustive assessment of mitochondrial performance and autophagy was performed after 24-h exposure to standard (glucose) or mitochondrial-challenging environment (galactose), where mitochondrial and autophagy impairment may be heightened. RESULTS:A similar mitochondrial phenotype of NM-LRRK2 G2019S and controls, except for an early mitochondrial depolarization (54.14% increased, p?=?0.04), was shown in glucose. In response to galactose, mitochondrial dynamics of NM-LRRK2 G2019S improved (-?17.54% circularity, p?=?0.002 and +?42.53% form factor, p?=?0.051), probably to maintain ATP levels over controls. A compromised bioenergetic function was suggested in PD-LRRK2 G2019S when compared to controls in glucose media. An inefficient response to galactose and worsened mitochondrial dynamics (-?37.7% mitochondrial elongation, p?=?0.053) was shown, leading to increased oxidative stress. Autophagy initiation (SQTSM/P62) was upregulated in NM-LRRK2 G2019S when compared to controls (glucose +?118.4%, p?=?0.014; galactose +?114.44%, p?=?0.009,) and autophagosome formation increased in glucose media. Despite of elevated SQSTM1/P62 levels of PD-NM G2019S when compared to controls (glucose +?226.14%, p?=?0.04; galactose +?78.5%, p?=?0.02), autophagosome formation was deficient in PD-LRRK2 G2019S when compared to NM-LRRK2 G2019S (-?71.26%, p?=?0.022). CONCLUSIONS:Enhanced mitochondrial performance of NM-LRRK2 G2019S in mitochondrial-challenging conditions and upregulation of autophagy suggests that an exhaustion of mitochondrial bioenergetic and autophagic reserve, may contribute to the development of PD in LRRK2 G2019S mutation carriers.

Project description:UnlabelledMutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) can cause Parkinson's disease (PD), and the most common disease-associated mutation, G2019S, increases kinase activity. Because LRRK2 expression levels rise during synaptogenesis and are highest in dorsal striatal spiny projection neurons (SPNs), we tested the hypothesis that the LRRK2-G2019S mutation would alter development of excitatory synaptic networks in dorsal striatum. To circumvent experimental confounds associated with LRRK2 overexpression, we used mice expressing LRRK2-G2019S or D2017A (kinase-dead) knockin mutations. In whole-cell recordings, G2019S SPNs exhibited a fourfold increase in sEPSC frequency compared with wild-type SPNs in postnatal day 21 mice. Such heightened neural activity was increased similarly in direct- and indirect-pathway SPNs, and action potential-dependent activity was particularly elevated. Excitatory synaptic activity in D2017A SPNs was similar to wild type, indicating a selective effect of G2019S. Acute exposure to LRRK2 kinase inhibitors normalized activity, supporting that excessive neural activity in G2019S SPNs is mediated directly and is kinase dependent. Although dendritic arborization and densities of excitatory presynaptic terminals and postsynaptic dendritic spines in G2019S SPNs were similar to wild type, G2019S SPNs displayed larger spines that were matched functionally by a shift toward larger postsynaptic response amplitudes. Acutely isolating striatum from overlying neocortex normalized sEPSC frequency in G2019S mutants, supporting that abnormal corticostriatal activity is involved. These findings indicate that the G2019S mutation imparts a gain-of-abnormal function to SPN activity and morphology during a stage of development when activity can permanently modify circuit structure and function.Significance statementMutations in the kinase domain of leucine-rich repeat kinase 2 (LRRK2) follow Parkinson's disease (PD) heritability. How such mutations affect brain function is poorly understood. LRRK2 expression levels rise after birth at a time when synapses are forming and are highest in dorsal striatum, suggesting that LRRK2 regulates development of striatal circuits. During a period of postnatal development when activity plays a large role in permanently shaping neural circuits, our data show how the most common PD-causing LRRK2 mutation dramatically alters excitatory synaptic activity and the shape of postsynaptic structures in striatum. These findings provide new insight into early functional and structural aberrations in striatal connectivity that may predispose striatal circuitry to both motor and nonmotor dysfunction later in life.

Project description:BackgroundThe clinicopathological phenotype of G2019S LRRK2-associated Parkinson's disease (L2PD) is similar to idiopathic Parkinson's disease (iPD), and G2019S LRRK2 nonmanifesting carriers (L2NMCs) are at increased risk for development of PD. With various therapeutic strategies in the clinical and preclinical pipeline, there is an urgent need to identify biomarkers that can aid early diagnosis and patient enrichment for ongoing and future LRRK2-targeted trials.ObjectiveThe objective of this work was to investigate differential protein and phospho-protein changes related to G2019S mutant LRRK2 in peripheral blood mononuclear cells from G2019S L2PD patients and G2019S L2NMCs, identify specific phospho-protein changes associated with the G2019S mutation and with disease status, and compare findings with patients with iPD.MethodsWe performed an unbiased phospho-proteomic study by isobaric label-based mass spectrometry using peripheral blood mononuclear cell group pools from a LRRK2 cohort from Spain encompassing patients with G2019S L2PD (n = 20), G2019S L2NMCs (n = 20), healthy control subjects (n = 30), patients with iPD (n = 15), patients with R1441G L2PD (n = 5), and R1441G L2NMCs (n = 3) (total N = 93).ResultsComparing G2019S carriers with healthy controls, we identified phospho-protein changes associated with the G2019S mutation. Moreover, we uncovered a specific G2019S phospho-signature that changes with disease status and can discriminate patients with G2019S L2PD, G2019S L2NMCs, and healthy controls. Although patients with iPD showed a differential phospho-proteomic profile, biological enrichment analyses revealed similar changes in deregulated pathways across the three groups.ConclusionsWe found a differential phospho-signature associated with LRRK2 G2019S for which, consistent with disease status, the phospho-profile from PD at-risk G2019S L2NMCs was more similar to healthy controls than patients with G2019S L2PD with the manifested disease. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Project description:Late-onset Parkinson's disease (PD) is dominated clinically and experimentally by a focus on dopamine neuron degeneration and ensuing motor system abnormalities. There are, additionally, a number of non-motor symptoms - including cognitive and psychiatric - that can appear much earlier in the course of the disease and also significantly impair quality of life. The neurobiology of such cognitive and psychiatric non-motor symptoms is poorly understood. The recognition of genetic forms of late-onset PD, which are clinically similar to idiopathic forms in both motor and non-motor symptoms, raises the perspective that brain cells and circuits - and the behaviors they support - differ in significant ways from normal by virtue of the fact that these mutations are carried throughout life, including especially early developmental critical periods where circuit structure and function is particularly susceptible to the influence of experience-dependent activity. In this focused review, we support this central thesis by highlighting studies of LRRK2-G2019S mouse models. We describe work that shows that in G2019S mutants, corticostriatal activity and plasticity are abnormal by P21, the end of a period of excitatory synaptogenesis in striatum. Moreover, by young adulthood, impaired striatal synaptic and non-synaptic forms of plasticity likely underlie altered and variable performance by mutant mice in validated tasks that test for depression-like and anhedonia-like behaviors. Mechanistically, deficits in cellular, synaptic and behavioral plasticity may be unified by mutation-linked defects in trafficking of AMPAR subunits and other membrane channels, which in turn may reflect impairment in the function of the Rab family of GTPases, a major target of LRRK2 phosphorylation. These findings underscore the need to better understand how PD-related mutant proteins influence brain structure and function during an extended period of brain development, and offer new clues for future therapeutic strategies to target non-motor cognitive or psychiatric symptoms of PD.

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Development of a physiologically relevant and easily scalable LUHMES cell-based model of G2019S LRRK2-driven Parkinson's disease.

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