Missense mutations in leucine-rich repeat kinase 2 (LRRK2) cause familial Parkinson's disease (PD). However, a potential role of wild-type LRRK2 in idiopathic PD (iPD) remains unclear. Here, we ...developed proximity ligation assays to assess Ser1292 phosphorylation of LRRK2 and, separately, the dissociation of 14-3-3 proteins from LRRK2. Using these proximity ligation assays, we show that wild-type LRRK2 kinase activity was selectively enhanced in substantia nigra dopamine neurons in postmortem brain tissue from patients with iPD and in two different rat models of the disease. We show that this occurred through an oxidative mechanism, resulting in phosphorylation of the LRRK2 substrate Rab10 and other downstream consequences including abnormalities in mitochondrial protein import and lysosomal function. Our study suggests that, independent of mutations, wild-type LRRK2 plays a role in iPD. LRRK2 kinase inhibitors may therefore be useful for treating patients with iPD who do not carry LRRK2 mutations.
Abstract Mutations in leucine-rich repeated kinase 2 (LRRK2) cause autosomal dominant late-onset Parkinson's disease (PD), and the G2019S mutation in the kinase domain of LRRK2 is the most common ...genetic cause of familial PD. Enhanced kinase activity of G2019S LRRK2 is a suspected mechanism for carriers to develop PD but pathophysiological function of G2019S LRRK2 is not clear. The objective of the present study was to characterize a bacterial artificial chromosome rat expressing human G2019S LRRK2. Immunoblotting analysis showed that G2019S LRRK2 expression was approximately 5–8 times higher than wild-type rat LRRK2. At ages of 4, 8, and 12 months, our characterization showed that expression of G2019S LRRK2 induced oxidative stress in striatum and substantia nigra, increased inducible nitric oxide synthase expression in nigral dopamine neurons, and abnormal morphology of nigral dopaminergic neurons in transgenic rats compared with wild-type, without inducing overt neurodegeneration in nigrostriatal dopaminergic neurons. Thus, we conclude that although this model does not reproduce the key features of end-stage PD, important preclinical features of the disease are evident, which may be useful in studying the earliest stages of PD and for gene-environment interaction studies.
Endocannabinoids (eCBs) and serotonin (5-HT) play a neuromodulatory role in the central nervous system. Both eCBs and 5-HT regulate neuronal excitability and their pharmacological potentiation has ...been shown to control seizures in pre-clinical and human studies. Compelling evidence indicates that eCB and 5-HT systems interact to modulate several physiological and pathological brain functions, such as food intake, pain, drug addiction, depression, and anxiety. Nevertheless, there is no evidence of an eCB/5-HT interaction in experimental and human epilepsies, including status epilepticus (SE). Here, we performed video-EEG recording in behaving rats treated with the pro-convulsant agent pilocarpine (PILO), in order to study the effect of the activation of CB1/5-HT2 receptors and their interaction on SE. Synthetic cannabinoid agonist WIN55,212–2 (WIN) decreased behavioral seizure severity of PILO-induced SE at 2 mg/kg (but not at 1 and 5 mg/kg, i.p.), while 5-HT2B/2C receptor agonist RO60–0175 (RO; 1, 3, 10 mg/kg, i.p.) was devoid of any effect. RO 3 mg/kg was instead capable of potentiating the effect of WIN 2 mg/kg on the Racine scale score. Surprisingly, neither WIN 2 mg/kg nor RO 3 mg/kg had any effect on the incidence and the intensity of EEG seizures when administered alone. However, WIN+RO co-administration reduced the incidence and the severity of EEG SE and increased the latency to SE onset after PILO injection. WIN+RO effects were blocked by the selective CB1R antagonist AM251 and the 5-HT2BR antagonist RS127445, but not by the 5-HT2CR antagonist SB242084 or the 5-HT2AR antagonist MDL11,939.
These data revealed a synergistic interaction between CB1R/5-HT2BR in the expression of PILO-induced SE.
•CB1R agonist WIN55,212-2 reduces the behavioral SE but has no effect on the incidence and severity of the electrographic SE.•5-HT2B/2CR agonist RO60-0175 has no effect on either behavioral SE or electrographic SE.•WIN55,212-2+RO60-0175 administration reduced both behavioral and electrographic SE incidence and severity.•WIN55,212-2+RO60-0175 antiseizure effects are mediated by synergistic activation of CB1R and 5-HT2BR.
Alpha-synuclein (αS) is a conformationally plastic protein that reversibly binds to cellular membranes. It aggregates and is genetically linked to Parkinson’s disease (PD). Here, we show that αS ...directly modulates processing bodies (P-bodies), membraneless organelles that function in mRNA turnover and storage. The N terminus of αS, but not other synucleins, dictates mutually exclusive binding either to cellular membranes or to P-bodies in the cytosol. αS associates with multiple decapping proteins in close proximity on the Edc4 scaffold. As αS pathologically accumulates, aberrant interaction with Edc4 occurs at the expense of physiologic decapping-module interactions. mRNA decay kinetics within PD-relevant pathways are correspondingly disrupted in PD patient neurons and brain. Genetic modulation of P-body components alters αS toxicity, and human genetic analysis lends support to the disease-relevance of these interactions. Beyond revealing an unexpected aspect of αS function and pathology, our data highlight the versatility of conformationally plastic proteins with high intrinsic disorder.
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•αS toxicity linked to P-bodies through yeast, fly, and human genetics•Physiologic binding of αS N terminus to either membranes or P-body decapping module•Pathologic αSyn accumulation disrupts the decapping module in PD neurons and brain•Pathologic αSyn disrupts mRNA stability in PD iPSC neurons
Alpha-synuclein modulates mRNA stability to regulate gene expression with implications for understanding both normal cellular physiology and vulnerability to Parkinson’s disease and related disorders.
LRRK2 has been implicated in endolysosomal function and likely plays a central role in idiopathic Parkinson's disease (iPD). In iPD, dopaminergic neurons within the substantia nigra are characterized ...by increased LRRK2 kinase activity, endolysosomal deficits, and accumulation of autophagic vesicles with incompletely degraded substrates, including α-synuclein. Although LRRK2 has been implicated in endolysosomal and autophagic function, it remains unclear whether inhibition of LRRK2 kinase activity can prevent endolysosomal deficits or reduce dopaminergic neurodegeneration. In this study, we characterized the endolysosomal and autophagic defects in surviving dopaminergic neurons of iPD patient brain tissue. We next showed that these defects could be reproduced reliably in vivo using the rotenone model of iPD. Results suggested that there was impaired endosomal maturation, resulting in lysosomal dysfunction and deficits in protein degradation. A highly selective, brain-penetrant LRRK2 kinase inhibitor not only improved apparent endosomal maturation and lysosomal function, but also prevented rotenone-induced neurodegeneration in vivo. The fact that a LRRK2 kinase inhibitor was capable of preventing the neuropathological and endolysosomal abnormalities observed in human iPD suggests that LRRK2 inhibitors may have broad therapeutic utility in iPD, not only in those who carry a LRRK2 mutation.
•Endolysosomal dysfunction occurs in dopamine neurons of PD patients.•Rotenone recapitulates neuropathologies observed in PD patients.•Endolysosomal deficits occur prior to autophagy deficits.•LRRK2 kinase inhibitor prevents endolysosomal deficits.•LRRK2 kinase inhibitor is neuroprotective in vivo.
Glucocorticoids (GCs) are used to treat pregnant women at risk for preterm delivery; however, prenatal exposure to GCs may trigger adverse neurological side effects due to reduced neural progenitor ...cell (NPC) proliferation. Whereas many established cell-cycle regulators impact NPC proliferation, other signaling molecules, such as the gap junction protein connexin-43 (Cx43), also influence proliferation. Gap junction intercellular communication (GJIC) is influenced by GCs in some cells, but such hormone effects have not been examined in coupled stem cells. We found that both continuous and transient exposure of embryonic day 14.5 mouse neurosphere cultures to dexamethasone (DEX) limits proliferation of coupled NPCs, which is manifested by both a reduction in S-phase progression and enhanced cell-cycle exit. A short (i.e., 1-h) DEX treatment also reduced GJIC as measured by live-cell fluorescence recovery after photobleaching, and altered the synchrony of spontaneous calcium transients in coupled NPCs. GC effects on GJIC in NPCs are transcription-independent and mediated through plasma membrane glucocorticoid receptors (GRs). This nongenomic pathway operates through lipid raft-associated GRs via a site-specific, MAPK-dependent phosphorylation of Cx43, which is linked to GR via caveolin-1 (Cav-1) and c-src. Cav-1 is essential for this nongenomic action of GR, as DEX effects on GJIC, Cx43 phosphorylation, and MAPK activation are not observed in Cav-1 knockout NPCs. As transient pharmacologic inhibition of GJIC triggers reduced S-phase progression but not enhanced cell-cycle exit, the nongenomic GR signaling pathway may operate via distinct downstream effectors to alter the proliferative capacity of NPCs.
Mitochondrial dysfunction and oxidative stress are strongly implicated in Parkinson's disease (PD) pathogenesis and there is evidence that mitochondrially-generated superoxide can activate NADPH ...oxidase 2 (NOX2). Although NOX2 has been examined in the context of PD, most attention has focused on glial NOX2, and the role of neuronal NOX2 in PD remains to be defined. Additionally, pharmacological NOX2 inhibitors have typically lacked specificity. Here we devised and validated a proximity ligation assay for NOX2 activity and demonstrated that in human PD and two animal models thereof, both neuronal and microglial NOX2 are highly active in substantia nigra under chronic conditions. However, in acute and sub-acute PD models, we observed neuronal, but not microglial NOX2 activation, suggesting that neuronal NOX2 may play a primary role in the early stages of the disease. Aberrant NOX2 activity is responsible for the formation of oxidative stress-related post-translational modifications of α-synuclein, and impaired mitochondrial protein import in vitro in primary ventral midbrain neuronal cultures and in vivo in nigrostriatal neurons in rats. In a rat model, administration of a brain-penetrant, highly specific NOX2 inhibitor prevented NOX2 activation in nigrostriatal neurons and its downstream effects in vivo, such as activation of leucine-rich repeat kinase 2 (LRRK2). We conclude that NOX2 is an important enzyme that contributes to progressive oxidative damage which in turn can lead to α-synuclein accumulation, mitochondrial protein import impairment, and LRRK2 activation. In this context, NOX2 inhibitors hold potential as a disease-modifying therapy in PD.
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•We developed a novel assay to detect NADPH oxidase 2 activity with cellular resolution.•Neuronal and microglial NADPH oxidase 2 are active in PD patients.•Neuronal NADPH oxidase 2 activation occurs before microglial NADPH oxidase 2.•PD-related redox unbalance relies on a mitochondrial/ NADPH oxidase 2 interplay.•Targeting NADPH oxidase 2 may represent a promising therapeutic approach in PD.
...in most cases, these studies ultimately evaluated the downstream clinical manifestations, failing to monitor early, specific molecular epileptogenic events. ...elucidation of the underlying ...mechanisms of epileptogenesis, are essential. Consistently with these findings obtained in genetic animal models of epilepsy, studies conducted in animal models of acquired epilepsy addressed the critical role of vesicular neurotransmitters transporters (VNTs) (Van Liefferinge et al., 2013) and non-neuronal potassium channel (Kir4.1) (Nagao et al., 2013) expression during epileptogenesis. The findings reported in this topic support the idea that repeated seizures mediate neuronal necrosis and apoptosis prevalently associated to the activation of certain distinct anti/pro-apoptotic Bcl-2 family factors. ...epileptogenesis elicits apoptotic events by means of a specific pattern of Bcl-2 family proteins, which might represent a possible target of intervention to protect against the epileptic damage (Henshall and Engel, 2013). Recent findings provide novel insights in the implication of circadian rhythm in modulating transcription factors governing clock genes expression, and the mTOR signaling pathway, one of the most relevant signaling pathway in epilepsy (Cho, 2012).
Several types of brain injuries are causes of acquired temporal lobe epilepsy (TLE). The seizure-free "latent period" that often follows the brain injury is of unknown mechanistic significance but is ...commonly considered as the "epileptogenic" period characterized by gradual pathogenic processes leading to the onset of clinically detectable epilepsy. Acute convulsive status epilepticus (SE) is often associated with an adverse developmental outcome characterized by learning disabilities related to the cumulative effects of seizures and development of TLE. The symptomatic manifestations of TLE appear only after a widespread irreversible damage of entorhinal cortex, and hippocampus, the brain area most affected by this disease. These pathological features of TLE reduce the possibility of successful therapeutic approaches, often rendering the disease refractory. The difficult clinical management of chronic TLE and the limited success rate of surgical approaches, increase the incapacitating nature of this specific epileptic disorder. Prevention of TLE with an appropriate intervention after a known inciting event (in the case of acquired epilepsy) might represent the most ambitious goal in the clinical treatment of this epileptic disorder, but has been largely unsuccessful to this point. Clinical trials aimed at prevention of chronic epilepsy have often produced negative, disappointing results. However, in most cases, these studies ultimately evaluated the downstream clinical manifestations, failing to monitor early, specific molecular epileptogenic events. Therefore, elucidation of the underlying mechanisms of epileptogenesis, and their time course(s) are essential. The primary purpose of this topic is to collect scientific contributions providing novel insights in the cellular and molecular mechanisms of epileptogenesis as potential targets for innovative therapeutic approaches aimed at preventing the chronic epileptic disorder.
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