Interplay between mitochondrial dysfunction and epigenetic alterations in environmental linked Parkinson\u27s Disease

Parkinson\u27s disease (PD) is a chronic age progressive neurodegenerative disease that affects about 1% of people over the age of 60. The hallmark pathological features of PD is characterized by the loss of 60-70% dopaminergic neurons, formation of Lewy bodies and aggregation of α-synuclein in the substantia nigra of the midbrain region. There are multiple factors that contribute to the origin of the disease and also these factors further trigger an avalanche of pathways, thus prominently increasing the risk of incurring PD. Although the etiopathology of PD is not very well understood, growing evidences on mitochondrial dysfunction and oxidative stress suggests to serve as one of the critical cause of PD. Many years of research has shown that incessant chronic exposure to several classes of pesticides lead to mitochondria dysfunction and degeneration of dopamingeric neuronal cells leading to PD. The primary target and goal of my Ph.D thesis was to particularly understand the specific role of mitochondrial complex I class of pesticides in causing apoptotic death of dopaminergic neurons. Recently we have shown the green house pesticides, tebufenpyrad and pyridaben mediate noteworthy effect on causing mitochondrial toxicity in the N27 dopaminergic neuronal cells. These pesticides are classified as mitochondrial complex I inhibitors and are functionally analogous to the Parkinsonian toxicant rotenone, in terms of target and mode of action. Herein, we demonstrate that acute exposure of tebufenpyrad or pyridaben to the N27 neuronal cells, induced significant mitochondrial structural and functional dysfunction by significant impairment of mitochondrial respiration and ATP production. Suggesting that the pesticides toxicity is associated with oxidative damage. Now on a novel direction of trying to understand the dopamingeric neuronal cell death post complex I inhibitor exposure, we looked into PKCδ a pro-apoptotic signaling kinase and its downstream target of phosphorylation. In this study, we show that tebufenpyrad exposure to the dopaminergic neuronal cells, lead to the activation of PKCδ in a caspase-3 dependent manner, which was marked by the cleavage of PKCδ and phosphorylation of PKCδ at T505. Importantly, we identified that activated PKCδ potentiated the phosphorylation of laminB1 at T575, leading to damage of the nuclear membrane integrity. Moreover the activation of PKCδ, phosphorylation of laminB1 at T575 and laminB1 loss was also observed in the PD substantia nigra brain tissues, but not in the control brains. Thereby noticeably extrapolating translational evidence and importance of our findings. Next we had ventured onto a complete novel pathway that involves the interplay between mitochondrial dysfunction and epigenetic modification such as hyperacetylation of histones H3 and H4 post mitochondrial complex 1 targeting pesticides in the dopamingeric neuronal cells. Herein, post exposure of N27 cells to pyridaben we demonstrate a dose and time-dependent increase in acetylation of histones H3 and H4. Also, further analysis performed using immuno-fluorescence staining and confocal microscopy, confirmed the site specific AcH3(K23) and AcH4(K5) up-regulation prior to rotenone or pyridaben exposure in the N27 neuronal cells. Interestingly, pyridaben or rotenone treated nuclei also demonstrate a nuclear structural defect in the form of topological alteration or chromatin declustering. Adding on, this suspected nuclear morphological change was marked by a time dependent loss of HP-1α, a very well studied marker of heterochromatin, suggesting the involvement of chromatin remodeling like process post the mitochondrial inhibiting pesticides exposure in the N27 cells. Transgenic mitochondrial defective in vitro model of TFAM CRISPR/Cas9 knockdown N27 cells also show hyperacetylation of histones H3 and H4 compared to the control CRISPR/Cas9. Furthermore, the Mitopark transgenic animal model of PD in addition demonstrated an age progressive acetylation of histones H3 and H4, and also AcH3(K23) and AcH4(K5) up-regulation and nuclear declustering morphology. Finally, our data was conclusively confirmed by the analysis of post-mortem human substantia nigra tissues, which demonstrated hyperacetylation of H3 and H4 and formation of vivid nuclear declustered structures in the PD brains. The amalgamative interpretation of these results suggests that histone hyperacetylation is a key epigenetic mechanism in the nigral dopaminergic neuronal cells following mitochondrial dysfunction and neurotoxicity in the neuronal models of PD. Overall, our results suggests that exposure of dopaminergic neuronal cells to mitochondrial complex I inhibiting pesticides leads to accentuated PKCδ kinase activity, nuclear membrane damage and histone hyperacetylation, resulting in apoptotic dopaminergic neuronal cell death. Thus intervening such mechanisms, merits the development of strategies for diagnosis and drug based treatments for PD

Mito-metformin protects against mitochondrial dysfunction and dopaminergic neuronal degeneration by activating upstream PKD1 signaling in cell culture and MitoPark animal models of Parkinson’s disease

Impaired mitochondrial function and biogenesis have strongly been implicated in the pathogenesis of Parkinson’s disease (PD). Thus, identifying the key signaling mechanisms regulating mitochondrial biogenesis is crucial to developing new treatment strategies for PD. We previously reported that protein kinase D1 (PKD1) activation protects against neuronal cell death in PD models by regulating mitochondrial biogenesis. To further harness the translational drug discovery potential of targeting PKD1-mediated neuroprotective signaling, we synthesized mito-metformin (Mito-Met), a mitochondria-targeted analog derived from conjugating the anti-diabetic drug metformin with a triphenylphosphonium functional group, and then evaluated the preclinical efficacy of Mito-Met in cell culture and MitoPark animal models of PD. Mito-Met (100–300 nM) significantly activated PKD1 phosphorylation, as well as downstream Akt and AMPKα phosphorylation, more potently than metformin, in N27 dopaminergic neuronal cells. Furthermore, treatment with Mito-Met upregulated the mRNA and protein expression of mitochondrial transcription factor A (TFAM) implying that Mito-Met can promote mitochondrial biogenesis. Interestingly, Mito-Met significantly increased mitochondrial bioenergetics capacity in N27 dopaminergic cells. Mito-Met also reduced mitochondrial fragmentation induced by the Parkinsonian neurotoxicant MPP+ in N27 cells and protected against MPP+-induced TH-positive neurite loss in primary neurons. More importantly, Mito-Met treatment (10 mg/kg, oral gavage for 8 week) significantly improved motor deficits and reduced striatal dopamine depletion in MitoPark mice. Taken together, our results demonstrate that Mito-Met possesses profound neuroprotective effects in both in vitro and in vivo models of PD, suggesting that pharmacological activation of PKD1 signaling could be a novel neuroprotective translational strategy in PD and other related neurocognitive diseases

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

Interplay between mitochondrial dysfunction and epigenetic alterations in environmental linked Parkinson's Disease

Parkinson's disease (PD) is a chronic age progressive neurodegenerative disease that affects about 1% of people over the age of 60. The hallmark pathological features of PD is characterized by the loss of 60-70% dopaminergic neurons, formation of Lewy bodies and aggregation of α-synuclein in the substantia nigra of the midbrain region. There are multiple factors that contribute to the origin of the disease and also these factors further trigger an avalanche of pathways, thus prominently increasing the risk of incurring PD. Although the etiopathology of PD is not very well understood, growing evidences on mitochondrial dysfunction and oxidative stress suggests to serve as one of the critical cause of PD. Many years of research has shown that incessant chronic exposure to several classes of pesticides lead to mitochondria dysfunction and degeneration of dopamingeric neuronal cells leading to PD. The primary target and goal of my Ph.D thesis was to particularly understand the specific role of mitochondrial complex I class of pesticides in causing apoptotic death of dopaminergic neurons. Recently we have shown the green house pesticides, tebufenpyrad and pyridaben mediate noteworthy effect on causing mitochondrial toxicity in the N27 dopaminergic neuronal cells. These pesticides are classified as mitochondrial complex I inhibitors and are functionally analogous to the Parkinsonian toxicant rotenone, in terms of target and mode of action. Herein, we demonstrate that acute exposure of tebufenpyrad or pyridaben to the N27 neuronal cells, induced significant mitochondrial structural and functional dysfunction by significant impairment of mitochondrial respiration and ATP production. Suggesting that the pesticides toxicity is associated with oxidative damage. Now on a novel direction of trying to understand the dopamingeric neuronal cell death post complex I inhibitor exposure, we looked into PKCδ a pro-apoptotic signaling kinase and its downstream target of phosphorylation. In this study, we show that tebufenpyrad exposure to the dopaminergic neuronal cells, lead to the activation of PKCδ in a caspase-3 dependent manner, which was marked by the cleavage of PKCδ and phosphorylation of PKCδ at T505. Importantly, we identified that activated PKCδ potentiated the phosphorylation of laminB1 at T575, leading to damage of the nuclear membrane integrity. Moreover the activation of PKCδ, phosphorylation of laminB1 at T575 and laminB1 loss was also observed in the PD substantia nigra brain tissues, but not in the control brains. Thereby noticeably extrapolating translational evidence and importance of our findings. Next we had ventured onto a complete novel pathway that involves the interplay between mitochondrial dysfunction and epigenetic modification such as hyperacetylation of histones H3 and H4 post mitochondrial complex 1 targeting pesticides in the dopamingeric neuronal cells. Herein, post exposure of N27 cells to pyridaben we demonstrate a dose and time-dependent increase in acetylation of histones H3 and H4. Also, further analysis performed using immuno-fluorescence staining and confocal microscopy, confirmed the site specific AcH3(K23) and AcH4(K5) up-regulation prior to rotenone or pyridaben exposure in the N27 neuronal cells. Interestingly, pyridaben or rotenone treated nuclei also demonstrate a nuclear structural defect in the form of topological alteration or chromatin declustering. Adding on, this suspected nuclear morphological change was marked by a time dependent loss of HP-1α, a very well studied marker of heterochromatin, suggesting the involvement of chromatin remodeling like process post the mitochondrial inhibiting pesticides exposure in the N27 cells. Transgenic mitochondrial defective in vitro model of TFAM CRISPR/Cas9 knockdown N27 cells also show hyperacetylation of histones H3 and H4 compared to the control CRISPR/Cas9. Furthermore, the Mitopark transgenic animal model of PD in addition demonstrated an age progressive acetylation of histones H3 and H4, and also AcH3(K23) and AcH4(K5) up-regulation and nuclear declustering morphology. Finally, our data was conclusively confirmed by the analysis of post-mortem human substantia nigra tissues, which demonstrated hyperacetylation of H3 and H4 and formation of vivid nuclear declustered structures in the PD brains. The amalgamative interpretation of these results suggests that histone hyperacetylation is a key epigenetic mechanism in the nigral dopaminergic neuronal cells following mitochondrial dysfunction and neurotoxicity in the neuronal models of PD. Overall, our results suggests that exposure of dopaminergic neuronal cells to mitochondrial complex I inhibiting pesticides leads to accentuated PKCδ kinase activity, nuclear membrane damage and histone hyperacetylation, resulting in apoptotic dopaminergic neuronal cell death. Thus intervening such mechanisms, merits the development of strategies for diagnosis and drug based treatments for PD.</p

Interleukin-6 and lactate dehydrogenase expression in a novel ex vivo rocking model of equine corneal epithelial wound healing

Purpose To establish a physiologically relevant ex vivo model of equine corneal epithelial wound healing. Methods Fourteen equine corneas were randomly assigned to one of two groups: wounded (n = 8) or unwounded (n = 6) controls. In the wounded group, the axial corneal epithelium was removed by applying a 6 mm filter paper disk soaked in 1N-NaOH for 60 s. Corneas were subsequently cultured using an air-liquid interface model. Evaluation of corneal healing was performed daily, and culture medium was collected. Corneas were randomly assigned to undergo processing via histopathology and RNAscope in situ hybridization for interleukin-6 (IL-6) and alpha-smooth muscle actin (αSMA) expression at T24, T48, and T72 h after wounding. Media of the cultured corneas were evaluated for the presence of lactate dehydrogenase (LDH) by a colorimetric assay. Results The ulcerated area of the wounded corneas decreased over time and all corneas healed within 72 h. Histologically, normal corneal architecture was observed including healthy epithelium (in areas other than the ulcerated ones), minimal stromal edema, intact endothelium, and Descemet's membrane. IL-6 expression was increased in wounded corneas compared with unwounded controls. LDH expression was elevated for both wounded and unwounded corneas at T24 but decreased substantially and was not detected at T48 in media from wounded and unwounded corneas, respectively. No αSMA expression was detected from either wounded or unwounded corneas. Conclusions The equine air-liquid interface, ex vivo, corneal epithelial wound healing model is effective and physiologically relevant. This model can be used in future studies evaluating various corneal therapies.This is the published version of the following article: Wehrman, Rita Fay, Ulrike Genschel, Adhithiya Charli, Anumantha G. Kanthasamy, Rachel Anne Allbaugh, and Gil Ben‐Shlomo. "Interleukin‐6 and lactate dehydrogenase expression in a novel ex vivo rocking model of equine corneal epithelial wound healing." Veterinary Ophthalmology 24, no. 5 (2021): 509-519. DOI: 10.1111/vop.12935. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivsLicense, which permits use and distribution in anymedium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.© 2021 The Authors. Posted with permission

Organic dust-induced mitochondrial dysfunction could be targeted via cGAS-STING or cytoplasmic NOX-2 inhibition using microglial cells and brain slice culture models

Organic dust (OD) exposure in animal production industries poses serious respiratory and other health risks. OD consisting of microbial products and particulate matter and OD exposure–induced respiratory inflammation are under investigation. However, the effect of OD exposure on brain remains elusive. We show that OD exposure of microglial cells induces an inflammatory phenotype with the release of mitochondrial DNA (mt-DNA). Therefore, we tested a hypothesis that OD exposure–induced secreted mt-DNA signaling drives the inflammation. A mouse microglial cell line was treated with medium or organic dust extract (ODE, 1% v/v) along with either phosphate-buffered saline (PBS) or mitoapocynin (MA, 10 µmol). Microglia treated with control or anti-STING siRNA were exposed to medium or ODE. Mouse organotypic brain slice cultures (BSCs) were exposed to medium or ODE with or without MA. Various samples were processed to quantify mitochondrial reactive oxygen species (mt-ROS), mt-DNA, cytochrome c, TFAM, mitochondrial stress markers and mt-DNA-induced signaling via cGAS-STING and TLR9. Data were analyzed and a p value of ≤ 0.05 was considered significant. MA treatment decreased the ODE-induced mt-DNA release into the cytosol. ODE increased MFN1/2 and PINK1 but not DRP1 and MA treatment decreased the MFN2 expression. MA treatment decreased the ODE exposure–induced mt-DNA signaling via cGAS-STING and TLR9. Anti-STING siRNA decreased the ODE-induced increase in IRF3, IFN-β and IBA-1 expression. In BSCs, MA treatment decreased the ODE-induced TNF-α, IL-6 and MFN1. Therefore, OD exposure–induced mt-DNA signaling was curtailed through cytoplasmic NOX-2 inhibition or STING suppression to reduce brain microglial inflammatory response.This pre-print of the article does not reflect post-acceptance improvements or any corrections. The Version of Record is available online at DOI: 10.1007/s00441-021-03422-x. Copyright 2021 The Author(s). Posted with permission

Fyn kinase mediates pro-inflammatory response in a mouse model of endotoxemia: Relevance to translational research

Systemic inflammation resulting from the release of pro-inflammatory cytokines and the chronic activation of the innate immune system remains a major cause of morbidity and mortality in the United States. After having demonstrated that Fyn, a Src family kinase, regulates microglial neuroinflammatory responses in cell culture and animal models of Parkinson's disease, we investigate here its role in modulating systemic inflammation using an endotoxic mouse model. Fyn knockout (KO) and their wild-type (WT) littermate mice were injected once intraperitoneally with either saline or 5 mg/kg lipopolysaccharide (LPS) and were killed 48 h later. LPS-induced mortality, endotoxic symptoms and hypothermia were significantly attenuated in Fyn KO, but not WT, mice. LPS reduced survival in Fyn WT mice to 49% compared to 84% in Fyn KO mice. Fyn KO mice were also protected from LPS-induced deficits in horizontal and vertical locomotor activities, total distance traveled and stereotypic movements. Surface body temperatures recorded at 24 h and 48 h post-LPS dropped significantly in Fyn WT, but not in KO, mice. Importantly, endotoxemia-associated changes to levels of the serum pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), splenocyte apoptosis and inducible nitric oxide synthase (iNOS) production in hepatocytes were also significantly attenuated in Fyn KO mice. Likewise, pharmacologically inhibiting Fyn with 10 mg/kg dasatinib (oral) significantly attenuated LPS-induced increases in plasma TNF-α and IL-6 protein levels and hepatic pro-IL-1β messenger ribonucleic acids (mRNAs). Collectively, these results indicate that genetic knockdown or pharmacological inhibition of Fyn dampens systemic inflammation, demonstrating for the first time that Fyn kinase plays a critical role in mediating the endotoxic inflammatory response

Prokineticin-2 upregulation during neuronal injury mediates a compensatory protective response against dopaminergic neuronal degeneration.

Prokineticin-2 (PK2), a recently discovered secreted protein, regulates important physiological functions including olfactory biogenesis and circadian rhythms in the CNS. Interestingly, although PK2 expression is low in the nigral system, its receptors are constitutively expressed on nigrostriatal neurons. Herein, we demonstrate that PK2 expression is highly induced in nigral dopaminergic neurons during early stages of degeneration in multiple models of Parkinson's disease (PD), including PK2 reporter mice and MitoPark mice. Functional studies demonstrate that PK2 promotes mitochondrial biogenesis and activates ERK and Akt survival signalling pathways, thereby driving neuroprotection. Importantly, PK2 overexpression is protective whereas PK2 receptor antagonism exacerbates dopaminergic degeneration in experimental PD. Furthermore, PK2 expression increased in surviving nigral dopaminergic neurons from PD brains, indicating that PK2 upregulation is clinically relevant to human PD. Collectively, our results identify a paradigm for compensatory neuroprotective PK2 signalling in nigral dopaminergic neurons that could have important therapeutic implications for PD

Prokineticin-2 upregulation during neuronal injury mediates a compensatory protective response against dopaminergic neuronal degeneration.

Prokineticin-2 (PK2), a recently discovered secreted protein, regulates important physiological functions including olfactory biogenesis and circadian rhythms in the CNS. Interestingly, although PK2 expression is low in the nigral system, its receptors are constitutively expressed on nigrostriatal neurons. Herein, we demonstrate that PK2 expression is highly induced in nigral dopaminergic neurons during early stages of degeneration in multiple models of Parkinson's disease (PD), including PK2 reporter mice and MitoPark mice. Functional studies demonstrate that PK2 promotes mitochondrial biogenesis and activates ERK and Akt survival signalling pathways, thereby driving neuroprotection. Importantly, PK2 overexpression is protective whereas PK2 receptor antagonism exacerbates dopaminergic degeneration in experimental PD. Furthermore, PK2 expression increased in surviving nigral dopaminergic neurons from PD brains, indicating that PK2 upregulation is clinically relevant to human PD. Collectively, our results identify a paradigm for compensatory neuroprotective PK2 signalling in nigral dopaminergic neurons that could have important therapeutic implications for PD