Arthi Kanthasamy, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): Stroke is the third leading cause of morbidity and mortality in the US, yet effective therapeutic interventions for the treatment of ischemia-induced brain damage are not currently available. Numerous studies in experimental models of ischemia have implicated oxidative stress and apoptosis as key mediators of ischemia-induced cell death. The majority of these studies have focused on either cortical or hippocampal regions; however, the cellular mechanisms underlying ischemic damage to the striatum, a dopamine enriched brain region that is highly susceptible to oxidative damage, is unclear. Although dopamine is considered to be a key mediator of oxidative stress-induced neuronal injury to the striatum, its exact role in ischemia-induced cell death signaling pathways in the striatum remain to be clarified. Likewise, ROS generation, mitochondrial dysfunction and caspase-3 activation have been implicated in ischemia- induced brain damage; however, the downstream cellular events that lead to DNA fragmentation subsequent to caspase-3 activation are currently not well established. As outlined in the preliminary data, we have identified that protein kinase C-delta, a member of the novel PKC isoform family and an oxidative stress kinase is highly expressed in the striatal neurons and serves as a key substrate for caspase-3. Furthermore, the proteolytic activation of PKC-delta by caspase-3 results in the permanent dissociation of regulatory and catalytic subunits of the kinase, thereby resulting in persistently increased kinase activity, and potentiation of oxidative stress-induced apoptotic damage. Therefore, in the present proposal we will extend our preliminary findings, by investigating the following specific aims: 1) To systematically characterize the caspase-3 dependent proteolytic activation of PKC-delta cell death pathways in primary striatal neuronal cultures following oxygen-glucose deprivation (OGD). 2) To investigate whether dopamine-induced oxidative insult is a potential trigger for PKC-delta mediated cell death pathways in striatal neurons subjected to OGD. Pharmacological, cellular and molecular approaches will be utlilized to delineate these specific aims. The proposed studies will not only provide insights into the mechanism of PKC-delta mediated cell death and the influence of DA in modulating ischemia-induced striatal cellular collapse but may also lead to the development of novel thereapeutic interventions for the treatment of cerebral ischemia. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Neuroinflammation is emerging as a central pathophysiological mediator in the neurodegenerative processes of many chronic neurodegenerative disorders including amyotrophic lateral sclerosis, Alzheimer's disease, multiple sclerosis, and Parkinson's disease. In particular, neuroinflammatory mechanisms pertaining to the progression of Parkinson's disease are current focal points of investigation since mitochondrial dysfunction and oxidative stress can not completely explain the chronic degenerative progression in the nigrostriatal dopaminergic system that ultimately results in irreversible debilitating motor deficits. Interestingly, postmortem analysis of the Parkinson's disease brain reveals signs of inflammatory processes including microglial activation and induction of proinflammatory factors such as TNFa and NF-kB along with loss of dopaminergic neurons in the nigra. Similarly, recent evidence from experimental models of Parkinson's disease clearly demonstrates microglial activation and cytokine release in the nigrostriatal region during exposure to neurotoxic insults. However, the cellular events which regulate the neuroinflammatory cascade in microglia during neurotoxic insults and the role of microglia mediated inflammation in the progression of nigral degenerative processes are not completely understood. As depicted in the preliminary data, we have identified a member of the novel PKC isoform family, protein kinase C-delta (PKCd), as a critical regulator of release of a key proinflammatory cytokine TNFa from microglial cells. We also demonstrated that the kinase also serves as a proapoptotic kinase in dopaminergic neurons during inflammatory insults. In this proposal, we will extend our preliminary findings by pursuing the following specific aims: (i) To determine the differential mode of activation of PKCd in neuronal and microglial cells using cell culture models of Parkinson's disease and to examine the role of PKCd in regulating the production of proinflammatory cytokines (e.g., TNFa) in microglial cells;ii) To examine the role of PKCd in cytokine production and microglial activation in the nigrostriatal dopaminergic system using animal models of Parkinson's disease;iii) To determine whether neuroinflammatory cytokines upregulate the expression of PKCd in microglia to sustain and amplify the inflammatory cascade in a NF-kB dependent manner;and iv) To determine whether neuroinflammatory response from microglia can induce degeneration of nigral dopaminergic neurons through proteolytic activation of PKCd in animal models of Parkinson's disease. These specific objectives will be explored using various cellular and molecular biological approaches in neuroinflammatory models of Parkinson's disease. This systematic approach will help to unravel the key molecular mechanisms that regulate the neuroinflammatory cascade in microglia during the progressive degeneration of nigral dopaminergic neurons in Parkinson's disease. Ultimately, these mechanistic studies will translate into innovative therapeutic interventions for this progressively debilitating neurodegenerative disorder. PUBLIC HEALTH RELEVANCE: Parkinson's disease is a chronic and progressive neurodegenerative disorder affecting millions worldwide. The cellular mechanisms underlying the course of progression of nigral dopaminergic neurons is not yet understood. This proposal focuses on a protein kinase isoform specific cell signaling pathway that might play a role in amplification of the neuroinflammatory cascade in microglial cells in the nigrostriatal system. Understanding the cellular mechanisms of proinflammatory events responsible for progression of nigral degeneration will ultimately result in development of therapeutic strategies that will delay or halt the course of Parkinson's disease.

DESCRIPTION (provided by applicant): Neuroinflammation is now recognized as a key degenerative mechanism in several neurodegenerative diseases including Parkinson's disease (PD). The severity of neuronal damage caused by neuroinflammatory stress is dependent on the degree of dysregulation of inflammatory and anti-inflammatory pathways in brain. Most studies to date are focused on identifying major pro-inflammatory pathways that are activated during neuroinflammatory insult. Understanding the anti-inflammatory mechanisms associated with various stages of brain inflammation will provide new insights into disease processes associated with neurodegenerative diseases. In this proposal, we aim to delineate a novel anti-inflammatory protective response in the nigrostriatal dopaminergic neurons mediated by the recently discovered mammalian protein homolog of mamba snake venom: Prokineticin-2 (PK2). Unexpectedly, we observed a dramatic up-regulation of PK2 protein and its release from dopaminergic neuronal cells during inflammatory TNF¿ insult. Further observation of increased PK2 expression in nigral dopaminergic neurons in an animal model of PD as well as in the nigral samples from postmortem PD patients provides credence for the clinical significance of our findings. Interestingly, recombinant PK2 significantly protected against apoptotic neuronal cell death and TH positive dopaminergic neuronal loss induced by neuroinflammatory insults. Surprisingly, PK2 treatment also promoted migration of both astrocytes and microglial cells. Therefore, in this proposal, we intend to expand our preliminary observations by pursuing the following specific aims: (i) To characterize the PK2 induction, release and function in dopaminergic neurons following inflammatory insults in primary cell culture and animal models, (ii) To determine the effect of PK2 induction on astroglial and microglial migration and function following inflammatory stimuli, (iii) To investigate the molecular mechanisms of PK2 up-regulation in neuronal cells, and (iv) To demonstrate the neuroprotective effect of PK2 in primary cell culture and animal models of PD. Cellular, molecular and neurochemical approaches will be used to delineate these specific aims. Together, understanding the role of PK2 up-regulation and release during inflammatory stress in dopaminergic neurons will not only provide new insights about neurodegenerative mechanisms underlying nigral dopaminergic degeneration but may also yield novel therapeutic strategies for treatment of Parkinson's disease.

DESCRIPTION (provided by applicant): Neuroinflammation has been implicated as a major pathophysiological process of Parkinson's disease (PD) in recent years. Among various neuroinflammatory triggers, protein aggregates have been shown to be a predominant pathological trigger for microglial activation and subsequent proinflammatory cytokine and chemokine production in the brain, which in turn con tributes to the accelerated progression of neurodegenerative processes. Also, emerging evidence indicates that aggregated pathogenic proteins, including ?-synuclein (?Syn), are packaged into exosomes, which propagate protein aggregates from affected neurons to other brain cells, including microglial cells, through a non-cellular autonomous process, leading to a heightened neuroinflammatory response. Despite these advances, the cellular mechanisms underlying microglia-mediated neuroinflammatory events following stimulation with ?Syn aggregates and ?Syn-containing exosomes are yet to be defined. While studying kinase signaling in PD models, we unexpectedly discovered that the major non-receptor tyrosine kinase Fyn is rapidly activated in primary microglia within a few minutes of stimulation with the known inflammogen LPS. Interestingly, Fyn activation triggers proinflammatory responses, including cytokine/chemokine release from microglia. In addition, our preliminary findings revealed that aggregated ?Syn also induced a rapid activation of Fyn kinase and the NLRP3 inflammasome. To further expand our novel preliminary results, we will systematically pursue the following specific aims: (i) to characterize the mechanism of Fyn kinase activation and its role in the regulation of NLRRP2/3 inflammasomes in microglia and astrocytes during inflammatory stress induced by ?Syn aggregates and exosomes containing ?Syn aggregates and to determine the proinflammatory role of Fyn in dopaminergic neuronal cell death, (ii) to define the molecular mechanisms underlying Fyn upregulation in microglia and astroglia during sustained inflammatory responses induced by ?Syn aggregates and ?Syn exosomes in animal models of PD, and (iii) to determine the role of Fyn in mediating the proinflammatory response in the nigrostriatal dopaminergic system during ?Syn protein aggregation in animal models of PD as well as in postmortem PD brain tissues. Biochemical, cellular and neurochemical approaches will be used to achieve these specific aims. Taken together, delineating the role of Fyn kinase in ?Syn protein aggregation-induced microglial activation will not only provide novel mechanistic insights into the progression of neurodegenerative processes in PD, but may also be useful for translating mechanistic outcomes into effective therapies for PD.

Abstract Recent studies have begun to uncover the central role of microglia-mediated neuroinflammation in Parkinson?s disease (PD) pathogenesis. Increasing evidence suggests that microglia-driven innate immunity could further potentiate deleterious ?-synuclein (?Syn) aggregation and progressive neurodegeneration. However, we lack an in-depth understanding of the cellular mechanisms regulating ?Syn-induced innate immunity. Therefore, identifying signaling mechanisms that regulate microglial function in response to Parkinsonian pathology may lead to the development of novel immunomodulatory therapies for PD. We recently discovered that the transcript and protein expression levels of the calcium-activated potassium channel KCa3.1, best known for its role in immune cell calcium signaling, are elevated in activated microglia in both postmortem PD brains and in preclinical models of PD. We further identified that disruption of either FYN or STAT1 dampens reactive microglia activation responses via modulation of inflammatory mediators in aggregated ?Syn (?Synagg)-stimulated primary microglia. Importantly, the highly selective and orally active KCa3.1 inhibitor Senicapoc reduced neuroinflammation and nigral dopamin(DA)ergic neurotoxicity in a preclinical mouse model of PD, suggesting that KCa3.1 plays a multifaceted role by governing disease pathology. Despite these encouraging findings, the exact cellular mechanisms by which KCa3.1 regulates microglial function in the context of synucleinopathy remain poorly characterized. Herein, we propose three integrated aims to test the central hypothesis that KCa3.1 promotes ?Synagg-mediated progressive nigral DAergic neurodegenerative processes via activation of the microglial Fyn- STAT1 signaling axis and that the in vivo inhibition of KCa3.1 restores microglial homeostasis and affords DAergic neuroprotection in the context of synucleinopathy. In Aim-1, we will test the hypothesis that upregulation of KCa3.1 induces the proinflammatory microglial activation phenotype and nigral DAergic neuronal loss in the context of synucleinopathy. In Aim-2, we will test the hypothesis that the Fyn-STAT1 signaling axis drives microglial responses to PD-like pathology in a KCa3.1-dependent manner. In Aim-3, we will test the hypothesis that inhibiting KCa3.1 activation is efficacious in reducing reactive microglial activation and progressive PD-like disease pathology. The proposed studies are innovative, utilizing a combination of transcriptomic profiling, RNA in situ hybridization (ISH), imaging analysis, the RT QuIC assay for ?Synagg seeding, CRISPR/Cas9 KCNN4 knockout (KO) mice, transgenic conditional KO mouse models, and electrophysiological recordings to test how microglial KCa3.1 influences progressive neurodegenerative processes in PD. These studies address key mechanistic aspects regarding the functional roles of KCa3.1 in PD pathogenesis and may aid in the identification of new molecular determinants that can be targeted for slowing or halting PD progression and/or repurposing Senicapoc for PD therapy. 1