Vanillic acid induces mitochondrial biogenesis in SH-SY5Y cells.

BACKGROUND: Mitochondrial dysfunction has been recognized as an important mechanism of neurodegeneration. Accumulating evidence now suggests that defects in mitochondrial biogenesis can cause mitochondrial dysfunction in neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Therefore, identifying small molecules that can stimulate mitochondrial biogenesis may represent a therapeutic strategy for neuroprotection. The aim of this study was to investigate the effects of a natural compound vanillic acid (VA) on mitochondrial biogenesis and the expression of PD-related genes in SH-SY5Y cells. METHODS AND RESULTS: After determining the IC50 and non-toxic concentrations of VA, SH-SY5Y cells were exposed to a non-toxic dose of VA (300 µM) for 18 h. VA treatment resulted in significant increases in the mRNA expressions of mitochondrial biogenesis markers, PGC-1α and TFAM. Moreover, treatment of SH-SY5Y cells with 300 µM VA for 24 h significantly elevated the mitochondrial DNA (mtDNA) copy number and mitochondrial mass. Furthermore, the effects of VA on the expression of PD-related genes were analyzed using a real-time PCR array. The PCR array analysis revealed that VA can induce the expression of some genes involved in neuronal differentiation and also affect the expression of two PARK genes, PARK2 and LRRK2, whose mutations cause familial PD. CONCLUSIONS: Together, these findings indicate that VA could serve as a potential neuroprotective agent by virtue of its ability to stimulate mitochondrial biogenesis in neuronal cells and to alter the expression of some genes related to the pathogenesis of PD and neuronal differentiation.

Molecular mechanisms underlying protective effects of quercetin against mitochondrial dysfunction and progressive dopaminergic neurodegeneration in cell culture and MitoPark transgenic mouse models of Parkinson's Disease.

Quercetin, one of the major flavonoids in plants, has been recently reported to have neuroprotective effects against neurodegenerative processes. However, since the molecular signaling mechanisms governing these effects are not well clarified, we evaluated quercetin's effect on the neuroprotective signaling events in dopaminergic neuronal models and further tested its efficacy in the MitoPark transgenic mouse model of Parkinson's disease (PD). Western blot analysis revealed that quercetin significantly induced the activation of two major cell survival kinases, protein kinase D1 (PKD1) and Akt in MN9D dopaminergic neuronal cells. Furthermore, pharmacological inhibition or siRNA knockdown of PKD1 blocked the activation of Akt, suggesting that PKD1 acts as an upstream regulator of Akt in quercetin-mediated neuroprotective signaling. Quercetin also enhanced cAMP response-element binding protein phosphorylation and expression of the cAMP response-element binding protein target gene brain-derived neurotrophic factor. Results from qRT-PCR, Western blot analysis, mtDNA content analysis, and MitoTracker assay experiments revealed that quercetin augmented mitochondrial biogenesis. Quercetin also increased mitochondrial bioenergetics capacity and protected MN9D cells against 6-hydroxydopamine-induced neurotoxicity. To further evaluate the neuroprotective efficacy of quercetin against the mitochondrial dysfunction underlying PD, we used the progressive dopaminergic neurodegenerative MitoPark transgenic mouse model of PD. Oral administration of quercetin significantly reversed behavioral deficits, striatal dopamine depletion, and TH neuronal cell loss in MitoPark mice. Together, our findings demonstrate that quercetin activates the PKD1-Akt cell survival signaling axis and suggest that further exploration of quercetin as a promising neuroprotective agent for treating PD may offer clinical benefits.

Molecular cloning, epigenetic regulation, and functional characterization of Prkd1 gene promoter in dopaminergic cell culture models of Parkinson's disease.

We recently identified a compensatory survival role for protein kinase D1 (PKD1) in protecting dopaminergic neurons from oxidative insult. To investigate the molecular mechanism of Prkd1 gene expression, we cloned the 5'-flanking region (1620-bp) of the mouse Prkd1 gene. Deletion analyses revealed that the -250/+113 promoter region contains full promoter activity in MN9D dopaminergic neuronal cells. In silico analysis of the Prkd1 promoter uncovered binding sites for key redox transcription factors including Sp1 and NF-κB. Over-expression of Sp1, Sp3, and NF-κB-p65 proteins stimulated Prkd1 promoter activity. Binding of Sp3 and NF-κB-p65 to the Prkd1 promoter was confirmed using chromatin immunoprecipitation. Treatment with the Sp inhibitor mithramycin A significantly attenuated Prkd1 promoter activity and PKD1 mRNA and protein expression. Further mechanistic studies revealed that inhibition of histone deacetylation and DNA methylation up-regulated PKD1 mRNA expression. Importantly, negative modulation of PKD1 signaling by pharmacological inhibition or shRNA knockdown increased dopaminergic neuronal sensitivity to oxidative damage in a human mesencephalic neuronal cell model. Collectively, our findings demonstrate that Sp1, Sp3, and NF-κB-p65 can transactivate the mouse Prkd1 promoter and that epigenetic mechanisms, such as DNA methylation and histone modification, are key regulatory events controlling the expression of pro-survival kinase PKD1 in dopaminergic neuronal cells. Previously, we demonstrated that protein kinase D1 (PKD1) plays a survival role during the early stage of oxidative stress in dopaminergic neuronal cells.

Seminar: Functional Exposomics and Mechanisms of Toxicity-Insights from Model Systems and NAMs.

BACKGROUND: Significant progress has been made over the past decade in measuring the chemical components of the exposome, providing transformative population-scale frameworks in probing the etiologic link between environmental factors and disease phenotypes. While the analytical technologies continue to evolve with reams of data being generated, there is an opportunity to complement exposome-wide association studies (ExWAS) with functional analyses to advance etiologic search at organismal, cellular, and molecular levels. OBJECTIVES: Exposomics is a transdisciplinary field aimed at enabling discovery-based analysis of the nongenetic factors that contribute to disease, including numerous environmental chemical stressors. While advances in exposure assessment are enhancing population-based discovery of exposome-wide effects and chemical exposure agents, functional screening and elucidation of biological effects of exposures represent the next logical step toward precision environmental health and medicine. In this work, we focus on the use, strategies, and prospects of alternative approaches and model systems to enhance the current human exposomics framework in biomarker search and causal understanding, spanning from bench-based nonmammalian organisms and cell culture to computational new approach methods (NAMs). DISCUSSION: We visit the definition of the functional exposome and exposomics and discuss a need to leverage alternative models as opposed to mammalian animals for delineating exposome-wide health effects. Under the "three Rs" principle of reduction, replacement, and refinement, model systems such as roundworms, fruit flies, zebrafish, and induced pluripotent stem cells (iPSCs) are advantageous over mammals (e.g., rodents or higher vertebrates). These models are cost-effective, and cell-specific genetic manipulations in these models are easier and faster, compared to mammalian models. Meanwhile, in silico NAMs enhance hazard identification and risk assessment in humans by bridging the translational gaps between toxicology data and etiologic inference, as represented by in vitro to in vivo extrapolation (IVIVE) and integrated approaches to testing and assessment (IATA) under the adverse outcome pathway (AOP) framework. Together, these alternatives offer a strong toolbox to support functional exposomics to study toxicity and causal mediators underpinning exposure-disease links. https://doi.org/10.1289/EHP13120.

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.

Evaluation of fisetin as a potential inducer of mitochondrial biogenesis in SH-SY5Y neuronal cells.

Objectives: Increasing evidence implicates impaired mitochondrial biogenesis as an important contributor to mitochondrial dysfunction, which plays a central role in the pathogenesis of neurodegenerative diseases including Parkinson's disease (PD). For this reason, targeting mitochondrial biogenesis may present a promising therapeutic strategy for PD. The present study attempted to investigate the effects of fisetin, a dietary flavonoid, on mitochondrial biogenesis and the expression of PD-associated genes in neuronal cells. Materials and Methods: The effects of fisetin on mitochondrial biogenesis were evaluated by three different approaches; PGC-1α and TFAM mRNA expressions by RT-qPCR, mitochondrial DNA (mtDNA) content by quantitative PCR and mitochondrial mass by MitoTracker staining. Next, a PCR array was performed to evaluate the effects of fisetin on the expression profile of PD-associated genes. Finally, the common targets of fisetin and PD were analyzed by in silico analyses. Results: The results demonstrated that fisetin treatment can increase PGC-1α and TFAM mRNA levels, mtDNA copy number, and mitochondrial mass in neuronal cells. Fisetin also altered the expressions of some PD-related genes involved in neuroprotection and neuronal differentiation. Moreover, the bioinformatics analyses suggested that the AKT1-GSK3B signaling might be responsible for the potential neuroprotective effects of fisetin. Conclusion: Collectively, these results imply that fisetin could modulate some neuroprotective mechanisms including mitochondrial biogenesis, and may serve as a potential drug candidate for PD.

Characterization of nonmotor behavioral impairments and their neurochemical mechanisms in the MitoPark mouse model of progressive neurodegeneration in Parkinson's disease.

Mitochondrial dysfunction has been implicated as a key player in the pathogenesis of Parkinson's disease (PD). The MitoPark mouse, a transgenic mitochondrial impairment model developed by specific inactivation of TFAM in dopaminergic neurons, spontaneously exhibits progressive motor deficits and neurodegeneration, recapitulating several features of PD. Since nonmotor symptoms are now recognized as important features of the prodromal stage of PD, we comprehensively assessed the clinically relevant motor and nonmotor deficiencies from ages 8-24 wk in both male and female MitoPark mice and their littermate controls. As expected, motor deficits in MitoPark mice began around 12-14 wk and became severe by 16-24 wk. Interestingly, MitoPark mice exhibited olfactory deficits in the novel and social scent tests as early as 10-12 wk as compared to age-matched littermate controls. Additionally, male MitoPark mice showed spatial memory deficits before female mice, beginning at 8 wk and becoming most severe at 16 wk, as determined by the Morris water maze. MitoPark mice between 16 and 24 wk spent more time immobile in forced swim and tail suspension tests, and made fewer entries into open arms of the elevated plus maze, indicating a depressive and anxiety-like phenotype, respectively. Importantly, depressive behavior as determined by immobility in forced swim test was reversible by antidepressant treatment with desipramine. Neurochemical and mechanistic studies revealed significant changes in CREB phosphorylation, BDNF, and catecholamine levels as well as neurogenesis in key brain regions. Collectively, our results indicate that MitoPark mice progressively exhibit deficits in olfactory discrimination, cognitive learning and memory, and anxiety- and depression-like behaviors as well as key neurochemical signaling associated with nonmotor deficits in PD. Thus, MitoPark mice can serve as an invaluable model for studying nonmotor deficits in addition to studying the motor deficits related to pathology in PD.

Kv1.3 modulates neuroinflammation and neurodegeneration in Parkinson's disease.

Characterization of the key cellular targets contributing to sustained microglial activation in neurodegenerative diseases, including Parkinson's disease (PD), and optimal modulation of these targets can provide potential treatments to halt disease progression. Here, we demonstrated that microglial Kv1.3, a voltage-gated potassium channel, was transcriptionally upregulated in response to aggregated α-synuclein (αSynAgg) stimulation in primary microglial cultures and animal models of PD, as well as in postmortem human PD brains. Patch-clamp electrophysiological studies confirmed that the observed Kv1.3 upregulation translated to increased Kv1.3 channel activity. The kinase Fyn, a risk factor for PD, modulated transcriptional upregulation and posttranslational modification of microglial Kv1.3. Multiple state-of-the-art analyses, including Duolink proximity ligation assay imaging, revealed that Fyn directly bound to Kv1.3 and posttranslationally modified its channel activity. Furthermore, we demonstrated the functional relevance of Kv1.3 in augmenting the neuroinflammatory response by using Kv1.3-KO primary microglia and the Kv1.3-specific small-molecule inhibitor PAP-1, thus highlighting the importance of Kv1.3 in neuroinflammation. Administration of PAP-1 significantly inhibited neurodegeneration and neuroinflammation in multiple animal models of PD. Collectively, our results imply that Fyn-dependent regulation of Kv1.3 channels plays an obligatory role in accentuating the neuroinflammatory response in PD and identify Kv1.3 as a potential therapeutic target for PD.

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.

Manganese exposure exacerbates progressive motor deficits and neurodegeneration in the MitoPark mouse model of Parkinson's disease: Relevance to gene and environment interactions in metal neurotoxicity.

Parkinson's disease (PD) is now recognized as a neurodegenerative condition caused by a complex interplay of genetic and environmental influences. Chronic manganese (Mn) exposure has been implicated in the development of PD. Since mitochondrial dysfunction is associated with PD pathology as well as Mn neurotoxicity, we investigated whether Mn exposure augments mitochondrial dysfunction and neurodegeneration in the nigrostriatal dopaminergic system using a newly available mitochondrially defective transgenic mouse model of PD, the MitoPark mouse. This unique PD model recapitulates key features of the disease including progressive neurobehavioral changes and neuronal degeneration. We exposed MitoPark mice to a low dose of Mn (10mg/kg, p.o.) daily for 4 weeks starting at age 8 wks and then determined the behavioral, neurochemical and histological changes. Mn exposure accelerated the rate of progression of motor deficits in MitoPark mice when compared to the untreated MitoPark group. Mn also worsened olfactory function in this model. Most importantly, Mn exposure intensified the depletion of striatal dopamine and nigral TH neuronal loss in MitoPark mice. The neurodegenerative changes were accompanied by enhanced oxidative damage in the striatum and substantia nigra (SN) of MitoPark mice treated with Mn. Furthermore, Mn-treated MitoPark mice had significantly more oligomeric protein and IBA-1-immunoreactive microglia cells, suggesting Mn augments neuroinflammatory processes in the nigrostriatal pathway. To further confirm the direct effect of Mn on impaired mitochondrial function, we also generated a mitochondrially defective dopaminergic cell model by knocking out the TFAM transcription factor by using a CRISPR-Cas9 gene-editing method. Seahorse mitochondrial bioenergetic analysis revealed that Mn decreases mitochondrial basal and ATP-linked respiration in the TFAM KO cells. Collectively, our results reveal that Mn can augment mitochondrial dysfunction to exacerbate nigrostriatal neurodegeneration and PD-related behavioral symptoms. Our study also demonstrates that the MitoPark mouse is an excellent model to study the gene-environment interactions associated with mitochondrial defects in the nigral dopaminergic system as well as to evaluate the contribution of potential environmental toxicant interactions in a slowly progressive model of Parkinsonism.

Neurotoxicity of Vanadium.

Vanadium (V) is a transition metal that presents in multiple oxidation states and numerous inorganic compounds and is also an ultra-trace element considered to be essential for most living organisms. Despite being one of the lightest metals, V offers high structural strength and good corrosion resistance and thus has been widely adopted for high-strength steel manufacturing. High doses of V exposure are toxic, and inhalation exposure to V adversely affects the respiratory system. The neurotoxicological properties of V are just beginning to be identified. Recent studies by our group and others demonstrate the neurotoxic potential of this metal in the nigrostriatal system and other parts of the central nervous system (CNS). The neurotoxic effects of V have been mainly attributed to its ability to induce the generation of reactive oxygen species (ROS). It is noteworthy that the neurotoxicity induced by occupational V exposure commonly occurs with co-exposure to other metals, especially manganese (Mn). This review focuses on the chemistry, pharmacology, toxicology, and neurotoxicity of V.

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

AIMS: Parkinson's 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.

Protein kinase D1 (PKD1) phosphorylation promotes dopaminergic neuronal survival during 6-OHDA-induced oxidative stress.

Oxidative stress is a major pathophysiological mediator of degenerative processes in many neurodegenerative diseases including Parkinson's disease (PD). Aberrant cell signaling governed by protein phosphorylation has been linked to oxidative damage of dopaminergic neurons in PD. Although several studies have associated activation of certain protein kinases with apoptotic cell death in PD, very little is known about protein kinase regulation of cell survival and protection against oxidative damage and degeneration in dopaminergic neurons. Here, we characterized the PKD1-mediated protective pathway against oxidative damage in cell culture models of PD. Dopaminergic neurotoxicant 6-hydroxy dopamine (6-OHDA) was used to induce oxidative stress in the N27 dopaminergic cell model and in primary mesencephalic neurons. Our results indicated that 6-OHDA induced the PKD1 activation loop (PKD1S744/S748) phosphorylation during early stages of oxidative stress and that PKD1 activation preceded cell death. We also found that 6-OHDA rapidly increased phosphorylation of the C-terminal S916 in PKD1, which is required for PKD1 activation loop (PKD1S744/748) phosphorylation. Interestingly, negative modulation of PKD1 activation by RNAi knockdown or by the pharmacological inhibition of PKD1 by kbNB-14270 augmented 6-OHDA-induced apoptosis, while positive modulation of PKD1 by the overexpression of full length PKD1 (PKD1WT) or constitutively active PKD1 (PKD1S744E/S748E) attenuated 6-OHDA-induced apoptosis, suggesting an anti-apoptotic role for PKD1 during oxidative neuronal injury. Collectively, our results demonstrate that PKD1 signaling plays a cell survival role during early stages of oxidative stress in dopaminergic neurons and therefore, positive modulation of the PKD1-mediated signal transduction pathway can provide a novel neuroprotective strategy against PD.