Unravelling the signaling cascade mediating microglial pro-inflammatory responses in Parkinsonian Syndrome

In vitro cell culture, ex vivo brain slice culture, and in vivo model systems of Parkinson’s disease (PD), as well as postmortem brains from PD patients, have implicated microglial hyperactivation, mediated by inflammatory ligands, in the loss of dopaminergic neurons in the substantia nigra region of the brain. The signaling pathways leading to this chronic, sustained microglial activation in response to environmental stressors or disease-associated molecular patterns (DAMPs) are not clearly understood. We show herein the role of mitochondrial dysfunction in mediating pro-inflammatory signaling cascades in microglial cells in response to environmental factors such as pesticides and metals. Exposure to the pesticides rotenone and tebufenpyrad as well as the neurotoxic divalent metal manganese (Mn) leads to the activation of the NLRP3 pro-inflammatory signaling cascade in microglial cells. Mitochondrial superoxide generation plays a key role in activation of the NLRP3 inflammasome. Furthermore, activation of NLRP3 produces neurotoxic factors, like IL-1β and IL-18, that lead to neurodegeneration. The novel mitochondria-targeted antioxidant mito-apocynin was able to reduce mitochondrial superoxide generation and NLRP3 inflammasome activation. We also demonstrated that Mn leads to mitochondrial dysfunction by downregulation of mitochondrial fusion protein (Mfn2). Lastly, we show that Mn exposure leads to the propagation of the NLRP3 inflammasome through the mechanism of exosomal release of ASC, an inflammasome component. This finding has high translational relevance since exosomes isolated from welder cohorts known to have been exposed to Mn fumes carry a higher exosomal load of ASC. Collectively, we demonstrated the gene-environment crosstalk which modulates neuroinflammation and neurodegeneration. Alpha-synuclein aggregates are a major component of Lewy bodies and neurites, which are pathological hallmarks of PD. Classical activation of microglial cells has been shown to be mediated by pre-formed fibrillar α-synuclein (αSynAgg), but the signaling mechanism is not well understood. We sought to identify the role of microglial potassium channels in regulating αSynAgg-induced neuroinflammation. We show conclusively, in both cell culture and animal models of PD, that αSynAgg-induced neuroinflammation is associated with upregulation of the voltage-gated potassium channel Kv1.3. Remarkably, Kv1.3 was also highly induced in post-mortem PD patient tissues, as well as in peripheral blood mononuclear cells (PBMC) isolated from PD patients. We show that Fyn kinase, a src family kinase, regulates Kv1.3 transcriptionally through the p38 MAPK and NFκB pathways. Fyn was also observed to directly bind to Kv1.3, phosphorylating tyrosine 139 to modulate the channel’s activity. Lastly, we demonstrate that Kv1.3 inhibition reduces neuroinflammation and neurodegeneration in vitro and in preclinical setups. Overall, we identify key mechanistic pathways in response to both environmental stressors and DAMPs which strongly contribute to the chronic microglial pro-inflammatory responses that characterize PD-associated neuroinflammation

Fyn kinase regulates microglial neuroinflammatory responses in cell culture and animal models of parkinson’s disease

Sustained neuroinflammation mediated by resident microglia is recognized as a key pathophysiological contributor to many neurodegenerative diseases, including Parkinson’s disease (PD), but the key molecular signaling events regulating persistent microglial activation have yet to be clearly defined. In the present study, we examined the role of Fyn, a non-receptor tyrosine kinase, in microglial activation and neuroinflammatory mechanisms in cell culture and animal models of PD. The well-characterized inflammogens LPS and TNFɑ rapidly activated Fyn kinase in microglia. Immunocytochemical studies revealed that activated Fyn preferentially localized to the microglial plasma membrane periphery and the nucleus. Furthermore, activated Fyn phosphorylated PKCδ at tyrosine residue 311, contributing to an inflammogen-induced increase in its kinase activity. Notably, the Fyn-PKCδ signaling axis further activated the LPSand TNFɑ-induced MAP kinase phosphorylation and activation of the NFB pathway, implying that Fyn is a major upstream regulator of proinflammatory signaling. Functional studies in microglia isolated from wild-type (Fyn) and Fyn knock-out (Fyn) mice revealed that Fyn is required for proinflammatory responses, including cytokine release as well as iNOS activation. Interestingly, a prolonged inflammatory insult induced Fyn transcript and protein expression, indicating that Fyn is upregulated during chronic inflammatory conditions. Importantly, in vivo studies using MPTP, LPS, or 6-OHDA models revealed a greater attenuation of neuroinflammatory responses in Fyn and PKCδ mice compared with wild-type mice. Collectively, our data demonstrate that Fyn is a major upstream signaling mediator of microglial neuroinflammatory processes in PD

Mito-Apocynin Prevents Mitochondrial Dysfunction, Microglial Activation, Oxidative Damage, and Progressive Neurodegeneration in MitoPark Transgenic Mice

Aims: Parkinson\u27s disease (PD) is a neurodegenerative disorder characterized by progressive motor deficits and degeneration of dopaminergic neurons. Caused by a number of genetic and environmental factors, mitochondrial dysfunction and oxidative stress play a role in neurodegeneration in PD. By selectively knocking out mitochondrial transcription factor A (TFAM) in dopaminergic neurons, the transgenic MitoPark mice recapitulate many signature features of the disease, including progressive motor deficits, neuronal loss, and protein inclusions. In the present study, we evaluated the neuroprotective efficacy of a novel mitochondrially targeted antioxidant, Mito-apocynin, in MitoPark mice and cell culture models of neuroinflammation and mitochondrial dysfunction. Results: Oral administration of Mito-apocynin (10 mg/kg, thrice a week) showed excellent central nervous system bioavailability and significantly improved locomotor activity and coordination in MitoPark mice. Importantly, Mito-apocynin also partially attenuated severe nigrostriatal degeneration in MitoPark mice. Mechanistic studies revealed that Mito-apo improves mitochondrial function and inhibits NOX2 activation, oxidative damage, and neuroinflammation. Innovation: The properties of Mito-apocynin identified in the MitoPark transgenic mouse model strongly support potential clinical applications for Mito-apocynin as a viable neuroprotective and anti-neuroinflammatory drug for treating PD when compared to conventional therapeutic approaches. Conclusion: Collectively, our data demonstrate, for the first time, that a novel orally active apocynin derivative improves behavioral, inflammatory, and neurodegenerative processes in a severe progressive dopaminergic neurodegenerative model of PD. Antioxid. Redox Signal. 27, 1048–1066

Unravelling the signaling cascade mediating microglial pro-inflammatory responses in Parkinsonian Syndrome

In vitro cell culture, ex vivo brain slice culture, and in vivo model systems of Parkinson’s disease (PD), as well as postmortem brains from PD patients, have implicated microglial hyperactivation, mediated by inflammatory ligands, in the loss of dopaminergic neurons in the substantia nigra region of the brain. The signaling pathways leading to this chronic, sustained microglial activation in response to environmental stressors or disease-associated molecular patterns (DAMPs) are not clearly understood. We show herein the role of mitochondrial dysfunction in mediating pro-inflammatory signaling cascades in microglial cells in response to environmental factors such as pesticides and metals. Exposure to the pesticides rotenone and tebufenpyrad as well as the neurotoxic divalent metal manganese (Mn) leads to the activation of the NLRP3 pro-inflammatory signaling cascade in microglial cells. Mitochondrial superoxide generation plays a key role in activation of the NLRP3 inflammasome. Furthermore, activation of NLRP3 produces neurotoxic factors, like IL-1β and IL-18, that lead to neurodegeneration. The novel mitochondria-targeted antioxidant mito-apocynin was able to reduce mitochondrial superoxide generation and NLRP3 inflammasome activation. We also demonstrated that Mn leads to mitochondrial dysfunction by downregulation of mitochondrial fusion protein (Mfn2). Lastly, we show that Mn exposure leads to the propagation of the NLRP3 inflammasome through the mechanism of exosomal release of ASC, an inflammasome component. This finding has high translational relevance since exosomes isolated from welder cohorts known to have been exposed to Mn fumes carry a higher exosomal load of ASC. Collectively, we demonstrated the gene-environment crosstalk which modulates neuroinflammation and neurodegeneration. Alpha-synuclein aggregates are a major component of Lewy bodies and neurites, which are pathological hallmarks of PD. Classical activation of microglial cells has been shown to be mediated by pre-formed fibrillar α-synuclein (αSynAgg), but the signaling mechanism is not well understood. We sought to identify the role of microglial potassium channels in regulating αSynAgg-induced neuroinflammation. We show conclusively, in both cell culture and animal models of PD, that αSynAgg-induced neuroinflammation is associated with upregulation of the voltage-gated potassium channel Kv1.3. Remarkably, Kv1.3 was also highly induced in post-mortem PD patient tissues, as well as in peripheral blood mononuclear cells (PBMC) isolated from PD patients. We show that Fyn kinase, a src family kinase, regulates Kv1.3 transcriptionally through the p38 MAPK and NFκB pathways. Fyn was also observed to directly bind to Kv1.3, phosphorylating tyrosine 139 to modulate the channel’s activity. Lastly, we demonstrate that Kv1.3 inhibition reduces neuroinflammation and neurodegeneration in vitro and in preclinical setups. Overall, we identify key mechanistic pathways in response to both environmental stressors and DAMPs which strongly contribute to the chronic microglial pro-inflammatory responses that characterize PD-associated neuroinflammation.</p

α-synuclein impairs autophagosome maturation through abnormal actin stabilization.

Vesicular trafficking defects, particularly those in the autophagolysosomal system, have been strongly implicated in the pathogenesis of Parkinson's disease and related α-synucleinopathies. However, mechanisms mediating dysfunction of membrane trafficking remain incompletely understood. Using a Drosophila model of α-synuclein neurotoxicity with widespread and robust pathology, we find that human α-synuclein expression impairs autophagic flux in aging adult neurons. Genetic destabilization of the actin cytoskeleton rescues F-actin accumulation, promotes autophagosome clearance, normalizes the autophagolysosomal system, and rescues neurotoxicity in α-synuclein transgenic animals through an Arp2/3 dependent mechanism. Similarly, mitophagosomes accumulate in human α-synuclein-expressing neurons, and reversal of excessive actin stabilization promotes both clearance of these abnormal mitochondria-containing organelles and rescue of mitochondrial dysfunction. These results suggest that Arp2/3 dependent actin cytoskeleton stabilization mediates autophagic and mitophagic dysfunction and implicate failure of autophagosome maturation as a pathological mechanism in Parkinson's disease and related α-synucleinopathies

Comparative proteomic analysis highlights metabolic dysfunction in α-synucleinopathy

© 2020, The Author(s). The synaptic protein α-synuclein is linked through genetics and neuropathology to the pathogenesis of Parkinson’s disease and related disorders. However, the mechanisms by which α-synuclein influences disease onset and progression are incompletely understood. To identify pathogenic pathways and therapeutic targets we performed proteomic analysis in a highly penetrant new Drosophila model of α-synucleinopathy. We identified 476 significantly upregulated and 563 significantly downregulated proteins in heads from α-synucleinopathy model flies compared to controls. We then used multiple complementary analyses to identify and prioritize genes and pathways within the large set of differentially expressed proteins for functional studies. We performed Gene Ontology enrichment analysis, integrated our proteomic changes with human Parkinson’s disease genetic studies, and compared the α-synucleinopathy proteome with that of tauopathy model flies, which are relevant to Alzheimer’s disease and related disorders. These approaches identified GTP cyclohydrolase (GCH1) and folate metabolism as candidate mediators of α-synuclein neurotoxicity. In functional validation studies, we found that the knockdown of Drosophila Gch1 enhanced locomotor deficits in α-synuclein transgenic flies, while folate supplementation protected from α-synuclein toxicity. Our integrative analysis suggested that mitochondrial dysfunction was a common downstream mediator of neurodegeneration. Accordingly, Gch1 knockdown enhanced metabolic dysfunction in α-synuclein transgenic fly brains while folate supplementation partially normalized brain bioenergetics. Here we outline and implement an integrative approach to identify and validate potential therapeutic pathways using comparative proteomics and genetics and capitalizing on the facile genetic and pharmacological tools available in Drosophila

Prokineticin-2 promotes chemotaxis and alternative A2 reactivity of astrocytes

Astrocyte reactivity is disease- and stimulus-dependent, adopting either a proinflammatory A1 phenotype or a protective, anti-inflammatory A2 phenotype. Recently, we demonstrated, using cell culture, animal models and human brain samples, that dopaminergic neurons produce and secrete higher levels of the chemokine-like signaling protein Prokineticin-2 (PK2) as a compensatory protective response against neurotoxic stress. As astrocytes express a high level of PK2 receptors, herein, we systematically characterize the role of PK2 in astrocyte structural and functional properties. PK2 treatment greatly induced astrocyte migration, which was accompanied by a shift in mitochondrial energy metabolism, a reduction in proinflammatory factors, and an increase in the antioxidant genes Arginase-1 and Nrf2. Overexpression of PK2 in primary astrocytes or in the in vivo mouse brain induced the A2 astrocytic phenotype with upregulation of key protective genes and A2 reactivity markers including Arginase-1 and Nrf2, PTX3, SPHK1, and TM4SF1. A small-molecule PK2 agonist, IS20, not only mimicked the protective effect of PK2 in primary cultures, but also increased glutamate uptake by upregulating GLAST. Notably, IS20 blocked not only MPTP-induced reductions in the A2 phenotypic markers SPHK1 and SCL10a6 but also elevation of the of A1 marker GBP2. Collectively, our results reveal that PK2 regulates a novel neuron-astrocyte signaling mechanism by promoting an alternative A2 protective phenotype in astrocytes, which could be exploited for development of novel therapeutic strategies for PD and other related chronic neurodegenerative diseases. PK2 signals through its receptors on astrocytes and promotes directed chemotaxis. PK2-induced astrocyte reactivity leads to an increase in antioxidant and anti-inflammatory proteins while increasing glutamate uptake, along with decreased inflammatory factors

MitoPark transgenic mouse model recapitulates the gastrointestinal dysfunction and gut-microbiome changes of Parkinson’s disease

Gastrointestinal (GI) disturbances are one of the earliest symptoms affecting most patients with Parkinson’s disease (PD). In many cases, these symptoms are observed years before motor impairments become apparent. Hence, the molecular and cellular underpinnings that contribute to this early GI dysfunction in PD have actively been explored using a relevant animal model. The MitoPark model is a chronic, progressive mouse model recapitulating several key pathophysiological aspects of PD. However, GI dysfunction and gut microbiome changes have not been categorized in this model. Herein, we show that decreased GI motility was one of the first non-motor symptoms to develop, evident as early as 8 weeks with significantly different transit times from 12 weeks onwards. These symptoms were observed well before motor symptoms developed, thereby paralleling PD progression in humans. At age 24 weeks, we observed increased colon transit time and reduced fecal water content, indicative of constipation. Intestinal inflammation was evidenced with increased expression of iNOS and TNFα in the small and large intestine. Specifically, iNOS was observed mainly in the enteric plexi, indicating enteric glial cell activation. A pronounced loss of tyrosine hydroxylase-positive neurons occurred at 24 weeks both in the mid-brain region as well as the gut, leading to a corresponding decrease in dopamine (DA) production. We also observed decreased DARPP-32 expression in the colon, validating the loss of DAergic neurons in the gut. However, the total number of enteric neurons did not significantly differ between the two groups. Metabolomic gas chromatography-mass spectrometry analysis of fecal samples showed increased sterol, glycerol, and tocopherol production in MitoPark mice compared to age-matched littermate controls at 20 weeks of age while 16 s microbiome sequencing showed a transient temporal increase in the genus Prevotella. Altogether, the data shed more light on the role of the gut dopaminergic system in maintaining intestinal health. Importantly, this model recapitulates the chronology and development of GI dysfunction along with other non-motor symptoms and can become an attractive translational animal model for pre-clinical assessment of the efficacy of new anti-Parkinsonian drugs that can alleviate GI dysfunction in PD.This is a manuscript of an article published as Ghaisas, Shivani, Monica R. Langley, Bharathi N. Palanisamy, Somak Dutta, Kirthi Narayanaswamy, Paul J. Plummer, Souvarish Sarkar et al. "MitoPark transgenic mouse model recapitulates the gastrointestinal dysfunction and gut-microbiome changes of Parkinson’s disease." Neurotoxicology 75 (2019): 186-199. DOI: 10.1016/j.neuro.2019.09.004. Copyright 2019 Elsevier B.V. Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0). Posted with permission