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Background: An unmet clinical need in Parkinson's disease (PD) is to identify biomarkers for diagnosis, preferably in peripherally accessible tissues such as skin. Immunohistochemical studies have detected pathological α‐synuclein (αSyn) in skin biopsies from PD patients albeit sensitivity needs to be improved. Objective: Our study provides the ultrasensitive detection of pathological αSyn present in the skin of PD patients, and thus, pathological αSyn in skin could be a potential biomarker for PD. Methods: The real‐time quaking‐induced conversion assay was used to detect pathological αSyn present in human skin tissues. Further, we optimized this ultra‐sensitive and specific assay for both frozen and formalin‐fixed paraffin‐embedded sections of skin tissues. We determined the seeding kinetics of the αSyn present in the skin from autopsied subjects consisting of frozen skin tissues from 25 PD and 25 controls and formalin‐fixed paraffin‐embedded skin sections from 12 PD and 12 controls. Results: In a blinded study of skin tissues from autopsied subjects, we correctly identified 24/25 PD and 24/25 controls using frozen skin tissues (96% sensitivity and 96% specificity) compared to 9/12 PD and 10/12 controls using formalin‐fixed paraffin‐embedded skin sections (75% sensitivity and 83% specificity). Conclusions: Our blinded study results clearly demonstrate the feasibility of using skin tissues for clinical diagnosis of PD by detecting pathological αSyn. Moreover, this peripheral biomarker discovery study may have broader translational value in detecting misfolded proteins in skin samples as a longitudinal progression marker. © 2020 International Parkinson and Movement Disorder Society [ABSTRACT FROM AUTHOR]

Background: Identification of a peripheral biomarker is a major roadblock in the diagnosis of PD. Immunohistological identification of p-serine 129 α-synuclein in the submandibular gland tissues of PD patients has been recently reported.Objective: We report on a proof-of-principle study for using an ultra-sensitive and specific, real-time quaking-induced conversion assay to detect pathological α-synuclein in the submandibular gland tissues of PD patients.Methods: The α-synuclein real-time quaking-induced conversion assay was used to detect and quantify pathological α-synuclein levels in PD, incidental Lewy body disease, and control submandibular gland tissues as well as in formalin-fixed paraffin-embedded sections.Results: We determined the quantitative seeding kinetics of pathological α-synuclein present in submandibular gland tissues from autopsied subjects using the α-synuclein real-time quaking-induced conversion assay. A total of 32 cases comprising 13 PD, 3 incidental Lewy body disease, and 16 controls showed 100% sensitivity and 94% specificity. Interestingly, both PD and incidental Lewy body disease tissues showed 100% concordance for elevated levels of pathological α-synuclein seeding activity compared to control tissues. End-point dilution kinetic analyses revealed that the submandibular gland had a wide dynamic range of pathological α-synuclein seeding activity.Conclusions: Our results are the first to demonstrate the utility of using the real-time quaking-induced conversion assay on peripherally accessible submandibular gland tissues and formalin-fixed paraffin-embedded tissue sections to detect PD-related pathological changes with high sensitivity and specificity. Additionally, the detection of seeding activity from incidental Lewy body disease cases containing immunohistochemically undetected pathological α-synuclein demonstrates the α-synuclein real-time quaking-induced conversion assay's potential utility for identifying prodromal PD in submandibular gland tissues. © 2019 International Parkinson and Movement Disorder Society. [ABSTRACT FROM AUTHOR]

Hydrogen sulfide (H2S) is a highly neurotoxic gas. Acute exposure can lead to neurological sequelae among survivors. A drug for treating neurological sequelae in survivors of acute H2S intoxication is needed. Using a novel mouse model we evaluated the efficacy of cobinamide (Cob) for increasing survival of, and reducing neurological sequalae in, mice exposed to sublethal doses of H2S. There were two objectives: (1) to determine the dose-response efficacy of Cob and (2) to determine the effective therapeutic time window of Cob. To explore objective 1, mice were injected intramuscularly with Cob at 0, 50, or 100 mg/kg at 2 min after H2S exposure. For objective 2, mice were injected intramuscularly with 100 mg/kg Cob at 2, 15, and 30 min after H2S exposure. For both objectives, mice were exposed to 765 ppm of H2S gas. Cob significantly reduced H2S-induced lethality in a dose-dependent manner ( P < 0.05). Cob-treated mice exhibited significantly fewer seizures and knockdowns compared with the H2S-exposed group. Cob also reversed H2S-induced weight loss, behavioral deficits, neurochemical changes, cytochrome c oxidase enzyme inhibition, and neurodegeneration in a dose- and time-dependent manner ( P < 0.01). Overall, these findings show that Cob increases survival and is neuroprotective in a mouse model of H2S-induced neurological sequelae. [ABSTRACT FROM AUTHOR]

Hydrogen sulfide (H2S) is a highly neurotoxic gas. It is the second most common cause of gas-induced deaths. Beyond mortality, surviving victims of acute exposure may suffer long-term neurological sequelae. There is a need to develop countermeasures against H2S poisoning. However, no translational animal model of H2S-induced neurological sequelae exists. Here, we describe a novel mouse model of H2S-induced neurotoxicity for translational research. In paradigm I, C57/BL6 mice were exposed to 765 ppm H2S for 40 min on day 1, followed by 15-min daily exposures for periods ranging from 1 to 6 days. In paradigm II, mice were exposed once to 1000 ppm H2S for 60 minutes. Mice were assessed for behavioral, neurochemical, biochemical, and histopathological changes. H2S intoxication caused seizures, dyspnea, respiratory depression, knockdowns, and death. H2S-exposed mice showed significant impairment in locomotor and coordinated motor movement activity compared with controls. Histopathology revealed neurodegenerative lesions in the collicular, thalamic, and cortical brain regions. H2S significantly increased dopamine and serotonin concentration in several brain regions and caused time-dependent decreases in GABA and glutamate concentrations. Furthermore, H2S significantly suppressed cytochrome c oxidase activity and caused significant loss in body weight. Overall, male mice were more sensitive than females. This novel translational mouse model of H2S-induced neurotoxicity is reliable, reproducible, and recapitulates acute H2S poisoning in humans. [ABSTRACT FROM AUTHOR]

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 si RNA 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, mt DNA 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. [ABSTRACT FROM AUTHOR]

Hydrogen sulfide (H2S), the gas with the odor of rotten eggs, was formally discovered in 1777, over 239 years ago. For many years, it was considered an environmental pollutant and a health concern only in occupational settings. Recently, however, it was discovered that H2S is produced endogenously and plays critical physiological roles as a gasotransmitter. Although at low physiological concentrations it is physiologically beneficial, exposure to high concentrations of H2S is known to cause brain damage, leading to neurodegeneration and long-term neurological sequelae or death. Neurological sequelae include motor, behavioral, and cognitive deficits, which are incapacitating. Currently, there are concerns about accidental or malicious acute mass civilian exposure to H2S. There is a major unmet need for an ideal neuroprotective treatment, for use in the field, in the event of mass civilian exposure to high H2S concentrations. This review focuses on the neuropathology of high acute H2S exposure, knowledge gaps, and the challenges associated with development of effective neuroprotective therapy to counteract H2S-induced neurodegeneration. [ABSTRACT FROM AUTHOR]

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 m RNA and protein expression. Further mechanistic studies revealed that inhibition of histone deacetylation and DNA methylation up-regulated PKD1 m RNA expression. Importantly, negative modulation of PKD1 signaling by pharmacological inhibition or sh RNA 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. [ABSTRACT FROM AUTHOR]

Proteins tagged with lysine (Lys, K) 48 polyubiquitins chains are destined for degradation by the 26S proteasomal system. Impairment of the ubiquitin proteasome system (UPS) function culminates in the accumulation of polyubiquitinated proteins in many neurodegenerative conditions including Parkinson’s disease (PD). Nevertheless, the cellular mechanisms underlying cell death induced by an impaired UPS are still not clear. Intriguingly, recent studies indicate that several proteins associated with familial PD are capable of promoting the assembly of Lys-63 polyubiquitin chains. Therefore, the objective of this study was to examine the role of K48 and K63 ubiquitination in mitochondria-mediated apoptosis in in vitro models of dopaminergic degeneration. Exposure of the widely used proteasome inhibitor MG-132 to dopaminergic neuronal cell line (N27) induced a rapid accumulation of polyubiquitinated proteins in the mitochondria. This appears to result in the preferential association of ubiquitin conjugates in the outer membrane and polyubiquitination of outer membrane proteins. Interestingly, the ubiquitinK48R mutant effectively rescued cells from MG-132-induced mitochondrial apoptosis without altering the antioxidant status of cells; whereas the ubiqutinK63R mutant augmented the proapoptotic effect of MG-132. Herein, we report a novel conclusion that polyubiquitinated proteins, otherwise subjected to proteasomal degradation, preferentially accumulate in the mitochondria during proteolytic stress; and that polyubiquitination of Lys-48 and Lys-63 are key determinants of mitochondria-mediated cell death during proteasomal dysfunction. Together, these findings yield novel insights into a crosstalk between the UPS and mitochondria in dopaminergic neuronal cells. [ABSTRACT FROM AUTHOR]

Emerging evidence implicates impaired protein degradation by the ubiquitin proteasome system (UPS) in Parkinson's disease; however, cellular mechanisms underlying dopaminergic degeneration during proteasomal dysfunction are yet to be characterized. In the present study, we identified that the novel PKC isoform PKCδ plays a central role in mediating apoptotic cell death following UPS dysfunction in dopaminergic neuronal cells. Inhibition of proteasome function by MG-132 in dopaminergic neuronal cell model (N27 cells) rapidly depolarized mitochondria independent of ROS generation to activate the apoptotic cascade involving cytochrome c release, and caspase-9 and caspase-3 activation. PKCδ was a key downstream effector of caspase-3 because the kinase was proteolytically cleaved by caspase-3 following exposure to proteasome inhibitors MG-132 or lactacystin, resulting in a persistent increase in the kinase activity. Notably, MG-132 treatment resulted in translocation of proteolytically cleaved PKCδ fragments to mitochondria in a time-dependent fashion, and the PKCδ inhibition effectively blocked the activation of caspase-9 and caspase-3, indicating that the accumulation of the PKCδ catalytic fragment in the mitochondrial fraction possibly amplifies mitochondria-mediated apoptosis. Overexpression of the kinase active catalytic fragment of PKCδ (PKCδ-CF) but not the regulatory fragment (RF), or mitochondria-targeted expression of PKCδ-CF triggers caspase-3 activation and apoptosis. Furthermore, inhibition of PKCδ proteolytic cleavage by a caspase-3 cleavage-resistant mutant (PKCδ-CRM) or suppression of PKCδ expression by siRNA significantly attenuated MG-132-induced caspase-9 and -3 activation and DNA fragmentation. Collectively, these results demonstrate that proteolytically activated PKCδ has a significant feedback regulatory role in amplification of the mitochondria-mediated apoptotic cascade during proteasome dysfunction in dopaminergic neuronal cells. [ABSTRACT FROM AUTHOR]

Oxidative stress has been implicated as a key event in the degenerative process of dopaminergic neurons; however, the cellular mechanisms underlying chronic oxidative stress–induced neurodegeneration remain to be established. In this study, N27 cells, a dopaminergic neuronal cell line derived from rat mesencephalon, exposed to low doses of H2O2 (0–30 μM for 12–24 hr) exhibited dose- and time-dependent increases in cytotoxicity and ROS generation. In addition, the H2O2-induced neurotoxicity was accompanied by increased caspase-3 activity and PKCδ cleavage. Notably, treatment with antioxidants Trolox and MnTBAP or PKCδ cleavage inhibitor z-DIPD-fmk significantly protected against oxidative stress–induced apoptotic cell death. These results demonstrate that the N27 cell line is a useful model for the study of the chronic low-dose oxidative stress–induced apoptotic cell death cascade and that caspase-3-dependent PKCδ proteolytic activation may be important in the apoptotic process in dopaminergic neurons undergoing chronic oxidative insult. [ABSTRACT FROM AUTHOR]

Autophagy is a phylogenetically conserved process that plays a critical role in the degradation of oxidatively damaged proteins and organelle turnover. The role of oxidative stress and apoptosis in methamphetamine (METH)-induced neurotoxicity is well known; however, the potential contribution of autophagy to METH-induced oxidative damage in dopaminergic neuronal systems remains unclear. The goals of the present article were twofold: ( a) to develop an in vitro dopaminergic cell culture model to study cellular and molecular mechanisms underlying METH-ind-uced autophagy and apoptosis, and ( b) to determine whether lysosomal protease cathepsin-D activation, resulting from the loss of lysosomal membrane integrity, contributes to METH-induced apoptosis. To accomplish these goals, we characterized morphological and biochemical changes in an immortalized mesencephalic dopaminergic neuronal cell line (N27 cells) following treatment with METH. Exposure of METH (2 mM) to N27 cells resulted in the appearance of cytoplasmic vacuolar structures reminiscent of autophagic vacuoles within 3 h. In order to ascertain the identity of the vacuolar structures that are formed following METH exposure, immunohistochemical staining for markers of autophagy were performed. LAMP 2, a classical marker of autophagolysosomes, revealed an extensive punctuate pattern of distribution on the vacuolar membrane surface, with exclusive localization in the cytoplasm. Additionally, using DNA fragmentation analysis we showed a dose-dependent increase in fragmented DNA in METH treated N27 cells. Since METH-induced autophagy preceded DNA fragmentation, we tested whether dysfunction of the autophagolysosomal system contributes to nuclear damage. Immunofluorescence studies with cathepsin-d demonstrated a granular pattern of staining in untreated cells, whereas an increased cathepsin- D immunoreactivity with a globular pattern of staining was observed in METH-treated cells. Nevertheless, blockade of cathepsin-D activation by pepstatin-A, cathepsin-D inhibitor, failed to alter METH-induced DNA fragmentation. Collectively, these results demonstrate that N27 dopaminergic neuronal cell model may serve as an excellent in vitro model to study the mechanisms of METH-induced autophagy and apoptosis. Furthermore, it is less likely that cathepsin-D may serve as a trigger for the induction of apoptosis subsequent to exposure of N27 dopaminergic neuronal cells to METH. [ABSTRACT FROM AUTHOR]

Abstract 1-Methyl-4-phenylpyridinium (MPP[sup +] ), the neurotoxic metabolite of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), induces apoptosis in dopaminergic neurons; however, the cellular mechanisms underlying the degenerative process are not well understood. In the present study, we demonstrate that caspase-3 mediated proteolytic activation of protein kinase Cδ (PKCδ) is critical in MPP[sup +] -induced oxidative stress and apoptosis. MPP[sup +] exposure in rat dopaminergic neuronal cells resulted in time-dependent increases in reactive oxygen species generation, cytochrome c release, and caspase-9 and caspase-3 activation. Interestingly, MPP[sup +] induced proteolytic cleavage of PKCδ (72–74 kDa) into a 41-kDa catalytic and a 38-kDa regulatory subunit, resulting in persistently increased kinase activity. The caspase-3 inhibitor Z-DEVD-fmk effectively blocked MPP[sup +] -induced PKCδ cleavage and kinase activity, suggesting that the proteolytic activation is caspase-3 mediated. Similar results were seen in MPP[sup +] -treated rat midbrain slices. Z-DEVD-fmk and the PKCδ specific inhibitor rottlerin almost completely blocked MPP[sup +] -induced DNA fragmentation. The superoxide dismutase mimetic, MnTBAP also effectively attenuated MPP[sup +] -induced caspase-3 activation, PKCδ cleavage, and DNA fragmentation. Furthermore, rottlerin attenuated MPP[sup +] -induced caspase-3 activity without affecting basal activity, suggesting positive feedback activation of caspase-3 by PKCδ. Intracellular delivery of catalytically active recombinant PKCδ significantly increased caspase-3 activity, further indicating that PKCδ regulates caspase-3 activity. Finally, over-expression of a kinase inactive PKCδ[sup K376R] mutant prevented MPP[sup +] -induced caspase activation and DNA fragmentation, confirming the pro-apoptotic function of PKCδ in dopaminergic cell death. Together, we demonstrate for the first time that MPP[sup +] -induced oxidative stress proteolytically activates PKCδ in a caspase-3-dependent manner to induce apoptosis and up-regulate the caspase cascade in dopaminergic neuronal cells. [ABSTRACT FROM AUTHOR]

Proteins tagged with lysine (Lys, K) 48 polyubiquitins chains are destined for degradation by the 26S proteasomal system. Impairment of the ubiquitin proteasome system (UPS) function culminates in the accumulation of polyubiquitinated proteins in many neurodegenerative conditions including Parkinson's disease (PD). Nevertheless, the cellular mechanisms underlying cell death induced by an impaired UPS are still not clear. Intriguingly, recent studies indicate that several proteins associated with familial PD are capable of promoting the assembly of Lys-63 polyubiquitin chains. Therefore, the objective of this study was to examine the role of K48 and K63 ubiquitination in mitochondria-mediated apoptosis in in vitro models of dopaminergic degeneration. Exposure of the widely used proteasome inhibitor MG-132 to dopaminergic neuronal cell line (N27) induced a rapid accumulation of polyubiquitinated proteins in the mitochondria. This appears to result in the preferential association of ubiquitin conjugates in the outer membrane and polyubiquitination of outer membrane proteins. Interestingly, the ubiquitin(K48R) mutant effectively rescued cells from MG-132-induced mitochondrial apoptosis without altering the antioxidant status of cells; whereas the ubiquitin(K63R) mutant augmented the proapoptotic effect of MG-132. Herein, we report a novel conclusion that polyubiquitinated proteins, otherwise subjected to proteasomal degradation, preferentially accumulate in the mitochondria during proteolytic stress; and that polyubiquitination of Lys-48 and Lys-63 are key determinants of mitochondria-mediated cell death during proteasomal dysfunction. Together, these findings yield novel insights into a crosstalk between the UPS and mitochondria in dopaminergic neuronal cells.

Emerging evidence implicates impaired protein degradation by the ubiquitin proteasome system (UPS) in Parkinson's disease; however cellular mechanisms underlying dopaminergic degeneration during proteasomal dysfunction are yet to be characterized. In the present study, we identified that the novel PKC isoform PKCdelta plays a central role in mediating apoptotic cell death following UPS dysfunction in dopaminergic neuronal cells. Inhibition of proteasome function by MG-132 in dopaminergic neuronal cell model (N27 cells) rapidly depolarized mitochondria independent of ROS generation to activate the apoptotic cascade involving cytochrome c release, and caspase-9 and caspase-3 activation. PKCdelta was a key downstream effector of caspase-3 because the kinase was proteolytically cleaved by caspase-3 following exposure to proteasome inhibitors MG-132 or lactacystin, resulting in a persistent increase in the kinase activity. Notably MG-132 treatment resulted in translocation of proteolytically cleaved PKCdelta fragments to mitochondria in a time-dependent fashion, and the PKCdelta inhibition effectively blocked the activation of caspase-9 and caspase-3, indicating that the accumulation of the PKCdelta catalytic fragment in the mitochondrial fraction possibly amplifies mitochondria-mediated apoptosis. Overexpression of the kinase active catalytic fragment of PKCdelta (PKCdelta-CF) but not the regulatory fragment (RF), or mitochondria-targeted expression of PKCdelta-CF triggers caspase-3 activation and apoptosis. Furthermore, inhibition of PKCdelta proteolytic cleavage by a caspase-3 cleavage-resistant mutant (PKCdelta-CRM) or suppression of PKCdelta expression by siRNA significantly attenuated MG-132-induced caspase-9 and -3 activation and DNA fragmentation. Collectively, these results demonstrate that proteolytically activated PKCdelta has a significant feedback regulatory role in amplification of the mitochondria-mediated apoptotic cascade during proteasome dysfunction in dopaminergic neuronal cells.