Pirh2 modulates the mitochondrial function and cytochrome c-mediated neuronal death during Alzheimer's disease.

Pirh2 is an E3 ubiquitin ligase known to regulate the DNA damage responses through ubiquitylation of various participating signaling factors. DNA damage is a key pathological contributor to Alzheimer's disease (AD), therefore, the role of Pirh2 was investigated in streptozotocin and oligomer Aß1-42 induced rodent experimental model of AD. Pirh2 protein abundance increased during AD conditions, and transient silencing of Pirh2 inhibited the disease-specific pathological markers like level of p-Tau, ßamyloid, acetylcholinesterase activity, and neuronal death. Biochemically, Pirh2 silencing significantly attenuated the oxidative stress, depleted mitochondrial membrane potential, cytochrome c translocation from mitochondria to cytosol, and depleted mitochondrial complex-I activity, and ATP level. Pirh2 silencing also inhibited the altered level of VDAC1, hsp75, hexokinase1, t-Bid, caspase-9, and altered level of apoptotic proteins (Bcl-2, Bax). MALDI-TOF/TOF, co-immunoprecipitation, and UbcH13-linked ubiquitylation assay confirmed the interaction of Pirh2 with cytochrome c and the role of Pirh2 in ubiquitylation of cytochrome c, along with Pirh2-dependent altered proteasome activity. Additionally, Pirh2 silencing further inhibited the translocation of mitochondrion-specific endonuclease G and apoptosis-inducing factors to the nucleus and DNA damage. In conclusion, findings suggested the significant implication of Pirh2 in disease pathogenesis, particularly through impaired mitochondrial function, including biochemical alterations, translocation of cytochrome c, endonuclease G and apoptosis-inducing factor, DNA damage, and neuronal apoptosis.

Pirh2 modulates amyloid-ß aggregation through the regulation of glucose-regulated protein 78 and chaperone-mediated signaling.

Amyloid-ß (Aß) protein aggregation in the brain is a pathological hallmark of Alzheimer's disease (AD) however, the underlying molecular mechanisms regulating amyloid aggregation are not well understood. Here, we studied the propitious role of E3 ubiquitin ligase Pirh2 in Aß protein aggregation in view of its regulatory ligase activity in the ubiquitin-proteasome system employing both cellular and sporadic rodent models of AD. Pirh2 protein abundance was significantly increased during Streptozotocin (STZ) induced AD conditions, and transient silencing of Pirh2 significantly inhibited the Aß aggregation and modified the dendrite morphology along with the substantial decrease in choline level in the differentiated neurons. MALDI-TOF/TOF, coimmunoprecipitation, and UbcH7-linked in vitro ubiquitylation analysis confirmed the high interaction of Pirh2 with chaperone GRP78. Furthermore, Pirh2 silencing inhibits the STZ induced altered level of endoplasmic reticulum stress and intracellular Ca2+ levels in neuronal N2a cells. Pirh2 silencing also inhibited the AD conditions related to the altered protein abundance of HSP90 and its co-chaperones which may collectively involve in the reduced burden of amyloid aggregates in neuronal cells. Pirh2 silencing further stabilized the nuclear translocation of phospho-Nrf2 and inhibited the altered level of autophagy factors. Taken together, our data indicated that Pirh2 is critically involved in STZ induced AD pathogenesis through its interaction with ER-chaperone GRP78, improves the neuronal connectivity, affects the altered level of chaperones, co-chaperones, & autophagic markers, and collectively inhibits the Aß aggregation.

UBA52 Attunes VDAC1-Mediated Mitochondrial Dysfunction and Dopaminergic Neuronal Death.

Mitochondrial homeostasis regulates energy metabolism, calcium buffering, cell function, and apoptosis. The present study has been conducted to investigate the implications of the ubiquitin-encoding gene UBA52 in mitochondrial physiology. Transient expression of Myc-UBA52 in neurons significantly inhibited the rotenone-induced increase in reactive oxygen species generation, nitrite level, and depleted glutathione level. Mass spectrometric and coimmunoprecipitation data suggested the profound interaction of UBA52 with mitochondrial outer membrane channel protein, VDAC1 in both the wild-type and Myc-α-synuclein overexpressed neuronal cells and in the Parkinson's disease (PD)-specific substantia nigra and striatal region of the rat brain. In vitro ubiquitylation assay revealed that UBA52 participates in the ubiquitylation of VDAC1 through E3 ligase CHIP. Myc-UBA52 overexpression in neurons further improved the mitochondrial functionality and cell viability by preventing the alteration in mitochondrial membrane potential, mitochondrial complex I activity, and translocation of cytochrome c and p-Nrf2 along with the effect on intracellular calcium uptake, thus collectively inhibiting the opening of mitochondrial permeability transition pore. Additionally, Myc-UBA52 expression in neuronal cells offered protection against apoptotic and autophagic cell death. Altogether, our findings delineate a functional association between UBA52 and mitochondrial homeostasis, providing new insights into the deterrence of dopaminergic cell death during acute PD pathogenesis.

UBA52 Is Crucial in HSP90 Ubiquitylation and Neurodegenerative Signaling during Early Phase of Parkinson's Disease.

Protein aggregation is one of the major pathological events in age-related Parkinson's disease (PD) pathology, predominantly regulated by the ubiquitin-proteasome system (UPS). UPS essentially requires core component ubiquitin; however, its role in PD pathology is obscure. This study aimed to investigate the role of ubiquitin-encoding genes in sporadic PD pathology. Both cellular and rat models of PD as well as SNCA C57BL/6J-Tg (Th-SNCA*A30P*A53T)39 Eric/J transgenic mice showed a decreased abundance of UBA52 in conjunction with significant downregulation of tyrosine hydroxylase (TH) and neuronal death. In silico predictions, mass spectrometric analysis, and co-immunoprecipitation findings suggested the protein-protein interaction of UBA52 with α-synuclein, HSP90 and E3-ubiquitin ligase CHIP, and its co-localization with α-synuclein in the mitochondrion. Next, in vitro ubiquitylation assay indicated an imperative requirement of the lysine-63 residue of UBA52 in CHIP-mediated HSP90 ubiquitylation. Myc-UBA52 expressed neurons inhibited alteration in PD-specific markers such as α-synuclein and TH protein along with increased proteasome activity in diseased conditions. Furthermore, Myc-UBA52 expression inhibited the altered protein abundance of HSP90 and its various client proteins, HSP75 (homolog of HSP90 in mitochondrion) and ER stress-related markers during early PD. Taken together, the data highlights the critical role of UBA52 in HSP90 ubiquitylation in parallel to its potential contribution to the modulation of various disease-related neurodegenerative signaling targets during the early phase of PD pathology.

4-Phenylbutyrate Mitigates the Motor Impairment and Dopaminergic Neuronal Death During Parkinson's Disease Pathology via Targeting VDAC1 Mediated Mitochondrial Function and Astrocytes Activation.

Parkinson's disease (PD) is a progressive motor neurodegenerative disorder significantly associated with protein aggregation related neurodegenerative mechanisms. In view of no disease modifying drugs, the present study was targeted to investigate the therapeutic effects of pharmacological agent 4-phenylbutyric acid (4PBA) in PD pathology. 4PBA is an FDA approved monocarboxylic acid with inhibitory activity towards histone deacetylase and clinically treats urea cycle disorder. First, we observed the significant protective effects of 4PBA on PD specific neuromuscular coordination, level of tyrosine hydroxylase, α-synuclein level and neurotransmitter dopamine in both substantia nigra and striatal regions of the experimental rat model of PD. Further results revealed that treatment with 4PBA drug exhibited significant protection against disease related oxidative stress and augmented nitrite levels. The disease pathology-related depletion in mitochondrial membrane potential and augmented level of calcium as well as mitochondrion membrane located VDAC1 protein level and cytochrome-c translocation were also significantly attenuated with 4PBA administration. Inhibited neuronal apoptosis and restored neuronal morphology were also observed with 4PBA treatment as measured by level of pro-apoptotic proteins t-Bid, Bax and cleaved caspase-3 along with cresyl violet staining in both substantia nigra and striatal regions. Lastly, PD-linked astrocyte activation was significantly inhibited with 4PBA treatment. Altogether, our findings suggest that 4PBA exerts broad-spectrum neuroprotective effects in PD animal model.

COVID-19 and Parkinson's Disease: Possible Links in Pathology and Therapeutics.

The outbreak of SARs-CoV-2 with emerging new variants is leading to global health crisis and has brought a major concern for patients with comorbidities. Parkinson's disease (PD) is a motor neurodegenerative disease involving various metabolic and psychological ailments along with the common occurrence of hyposmia as observed in COVID-19 patients. In addition, the observed surplus inflammatory responses in both diseases are also alarming. Alongside, angiotensin-converting enzyme 2 (ACE2) receptor, essentially required by SARS-CoV-2 to enter the cell and dopamine decarboxylase (DDC), required for dopamine synthesis is known to co-regulate in the non-neuronal cells. Taken together, these conditions suggested the probable reciprocal pathological relation between COVID-19 and PD and also suggested that during comorbidities, the disease diagnosis and therapeutics are critical and may engender severe health complications. In this review, we discuss various events and mechanisms which may have implications for the exacerbation of PD conditions and must be taken into account during the treatment of patients.

Guanabenz mitigates the neuropathological alterations and cell death in Alzheimer's disease.

Alzheimer's disease (AD) pathology is characterized by cognitive impairment, increased acetylcholinesterase (AChE) activity, and impaired neuronal communication. Clinically, AChE inhibitors are being used to treat AD patients; however, these remain unable to prevent the disease progression. Therefore, further development of new therapeutic molecules is required having broad spectrum effects on AD-related various neurodegenerative events. Since repurposing is a quick mode to search the therapeutic molecules; henceforth, this study was conducted to evaluate the anti-Alzheimer activity of drug guanabenz which is already in use for the management of high blood pressure in clinics. The study was performed employing both cellular and rat models of AD along with donepezil as reference drug. Guanabenz treatment in both the experimental models showed significant protection against AD-specific behavioral and pathological indicators like AChE activity, tau phosphorylation, amyloid precursor protein, and memory retention. In conjunction, guanabenz also attenuated the AD-related oxidative stress, impaired mitochondrial functionality (MMP, cytochrome-c translocation, ATP level, and mitochondrial complex I activity), endoplasmic reticulum stress (GRP78, GADD153, cleaved caspase-12), neuronal apoptosis (Bcl-2, Bax, cleaved caspase-3), and DNA fragmentation. In conclusion, findings suggested the panoptic protective effect of guanabenz on disease-related multiple degenerative markers and signaling. Furthermore, clinical trial may shed light and expedite the availability of new therapeutic anti-Alzheimer's molecule for the wellbeing of AD patients.

Rivastigmine attenuates the Alzheimer's disease related protein degradation and apoptotic neuronal death signalling.

Rivastigmine is a clinical drug for patients of Alzheimer's disease (AD) exerting its inhibitory effect on acetylcholinesterase activity however, its effect on other disease-related pathological mechanisms are not yet known. This study was conducted to evaluate the effect of rivastigmine on protein aggregation and degradation related mechanisms employing streptozotocin (STZ) induced experimental rat model. The known inhibitory effect of rivastigmine on cognition and acetylcholinesterase activity was observed in both cortex and hippocampus and further its effect on tau level, amyloid aggregation, biochemical alterations, endoplasmic reticulum (ER) stress, calcium homeostasis, proteasome activity and apoptosis was estimated. STZ administration in rat brain caused significant cognitive impairment, augmented acetylcholinesterase activity, tau phosphorylation and amyloid aggregation which were significantly inhibited with rivastigmine treatment. STZ also caused significant biochemical alterations which were attenuated with rivastigmine treatment. Since AD pathology is related to protein aggregation and we have found disease-related amyloid aggregation, further the investigation was done to decipher the ER functionality and apoptotic signalling. STZ caused significantly altered level of ER stress related markers (GRP78, GADD153 and caspase-12) which were significantly inhibited with rivastigmine treatment. Furthermore, the effect of rivastigmine was estimated on proteasome activity in both regions. Rivastigmine treatment significantly enhances the proteasome activity and may contributes in removal of amyloid aggregation. In conclusion, findings suggested that along with inhibitory effect of rivastigmine on acetylcholinesterase activity and up to some extent on cognition, it has significant effect on disease-related biochemical alterations, ER functionality, protein degradation machinery and neuronal apoptosis.

Reciprocal Upshot of Nitric Oxide, Endoplasmic Reticulum Stress, and Ubiquitin Proteasome System in Parkinson's Disease Pathology.

Parkinson's disease (PD) pathology involves degeneration of nigrostriatal pathway, postulating symptoms associated with age, environment, and genetic anomalies, including nonlinear disease progression. Hallmark characteristics of PD include dopaminergic neuronal degeneration and death, which may also be exhibited by other neurological diseases, making the diagnosis of the disease intricate at early stage. Such obscure diagnosis of the disease, limited symptomatic improvements with available therapeutics, and their inability to modify the disease status instigate us to appraise the past research and formulate the colligating comprehensive insights. This review is accentuating on the role of nitric oxide, endoplasmic reticulum stress, and their association with the ubiquitin proteasome system (UPS) during PD pathology involving focus on ubiquitin ligases due to their regulatory functions. Meticulous understanding of these major disease-related pathological events and their functional alliance may render novel dimensions for better understanding of disease etiology, related mechanisms, as well as direction toward witnessing of new therapeutic targets for the management of Parkinson's patients.

Salubrinal attenuates nitric oxide mediated PERK:IRE1α: ATF-6 signaling and DNA damage in neuronal cells.

The present study was conducted to investigate the effect of salubrinal on nitric oxide mediated endoplasmic reticulum stress signaling and neuronal apoptosis. Rotenone treatment to neuro2a cells caused significantly decreased cell viability, increased cytotoxicity, augmented nitrite levels, increased nitrotyrosine level and augmented level of key ER stress markers (GRP-78, GADD153 and caspase-12). These augmented levels of ER stress markers could be attenuated with pretreatment of nitric oxide synthase inhibitor-aminoguanidine as well as with salubrinal. The rotenone treatment to neuro2a cells also triggered the ER stress induced up regulation of various signaling factors of unfolded protein response involving pPERK, ATF4, p-IRE1α, XBP-1 and ATF-6. Pretreatment of salubrinal significantly attenuated the activation of transmembrane kinases (PERK and IRE1) and ATF6 and restored the rotenone induced altered level of other UPR related signaling factors. Rotenone induced dephosphorylation of eIF2α was also inhibited with salubrinal treatment. Biochemically rotenone treatment to neuro2a cells caused the reactive oxygen species generation, depleted mitochondrial membrane potential and increased intra cellular calcium level which was attenuated with salubrinal treatment. Rotenone treatment to neuro2a cells also caused neuronal apoptosis, DNA fragmentation and chromatin condensation which were attenuated with salubrinal treatment. In conclusion, the findings suggested that rotenone causes the augmented level of nitric oxide which contributes in ER stress and could be inhibited by both aminoguanidine and/or salubrinal treatment. Further, salubrinal treatment attenuates the nitric oxide induced ER stress axis PERK:IRE1α:ATF-6 and inhibits the DNA damage and neuronal apoptosis.

Ulmosides A: Flavonoid 6-C-glycosides from Ulmus wallichiana attenuates lipopolysacchride induced oxidative stress, apoptosis and neuronal death.

Extract of Ulmus wallichiana is being used as traditional medicine used for the treatment of fractured bones however the effect of its individual flavonols is not known. The present study was conducted to investigate the effect of its novel flavonol, (2S, 3S)-(+)-30, 40, 5, 7-tetrahydroxydihydroflavonol-6-C-b-d-glucopyranoside named as Ulmoside A (UA), on lipopolysaccharides (LPS) treated neurons. LPS treatment to neuronal cells caused significant cytotoxicity, reactive oxygen species generation, depletion in glutathione and mitochondrial impairment which were significantly inhibited with UA treatment. LPS treatment also caused significant translocation of cytochrome-c, decreased level of Bcl2, increased level of Bax and cleaved caspase-3 in neuronal cells reflecting the involvement of intrinsic apoptotic pathway in neuronal death which was attenuated with UA treatment. Since LPS is a well known pro-inflammatory agent it also offered the significant increase in proinflammatory cytokines (tumor necrosis factors-α & interleukin 1-beta) however, UA treatment did not exhibit significant inhibition against LPS induced inflammatory response. LPS also caused the augmented level of inducible nitric oxide synthase (iNOS) which was also not inhibited with co treatment of UA. We have also observed the significant DNA fragmentation and augmented level of cleaved Poly (ADP-Ribose) polymerase 1 after LPS treatment which was significantly reverted with UA treatment. Findings suggested that UA acts through mitochondria and exhibited its anti-oxidative and anti-apoptotic activities in neuronal cells while no significant anti-inflammatory activity and effect on iNOS were observed.

New therapeutic activity of metabolic enhancer piracetam in treatment of neurodegenerative disease: Participation of caspase independent death factors, oxidative stress, inflammatory responses and apoptosis.

Piracetam, a nootropic drug that has been clinically used for decades but remains enigmatic due to no distinct understanding of its mechanism of action. The present study aimed to investigate the role of caspase independent pathway in piracetam mediated neuroprotection. LPS administration caused significant alterations in oxidative stress related parameters like glutathione, glutathione reductase and increased lipid peroxidation. LPS administration also caused augmented expression of inflammatory cytokines and astrocytes activation. Piracetam treatment offered significant protection against LPS induced oxidative and inflammatory parameters and inhibited astrocytes activation. LPS administration caused augmented level of reactive oxygen species and depleted mitochondrial membrane potential which were attenuated with piracetam treatment. This study for the first time demonstrates the role of caspase independent death factors in piracetam induced neuroprotective effects in rat brain. Translocation of mitochondrial resident apoptosis inducing factor and endonuclease G to nucleus through cytosol after LPS administration was significantly blocked with piracetam treatment. Further, LPS induced DNA fragmentation along with up regulated Poly [ADP-ribose] polymerase 1 (PARP1) levels were also inhibited with piracetam treatment. Apoptotic death was confirmed by the cleavage of caspase 3 as well as histological alteration in rat brain regions. LPS administration caused significantly increased level of cleaved caspase 3, altered neuronal morphology and decreased neuronal density which were restored with piracetam treatment. Collectively our findings indicate that piracetam offered protection against LPS induced inflammatory responses and cellular death including its antioxidative antiapoptotic activity with its attenuation against mitochondria mediated caspase independent pathway.

Involvement of glucose related energy crisis and endoplasmic reticulum stress: Insinuation of streptozotocin induced Alzheimer's like pathology.

The present study was conducted to correlate the cellular and molecular alterations in Alzheimer's pathology employing streptozotocin (STZ) induced experimental rat model. The STZ was administered in rat brain bilaterally by intracerebroventricular route using stereotaxic surgery followed by donepezil dosing. The Alzheimer's related pathological marker like acetylcholinesterase (AChE) activity, tau phosphorylation and amyloid aggregation were observed after STZ administration. STZ treatment showed decreased glucose and glucose transporters (GLUT) level along with augmented level of calcium in both cortical and hippocampal regions of rat brain. Increased calcium level may correlate with endoplasmic reticulum (ER) stress and significantly increased expression of ER stress markers like GRP78, GADD and caspase-12 were observed in STZ treated rat brain. Cellular communication was also affected by STZ administration as observed by increased expression connexin 43. With this view the activation of astrocytes and microglia was also assessed and observed by augmented GFAP and cd11b expression which were partially inhibited with donepezil treatment. The significantly increased level of degenerating neurons, caspase-3 and DNA fragmentation was also observed in rat brain regions which were not inhibited with donepezil treatment and validating the clinical observations. In conclusion, study indicated the STZ induced occurrence of Alzheimer's pathology. Further, STZ administration also caused depleted glucose level, inhibited mitochondrial activity, augmented calcium levels, ER stress, altered cellular communication and neuronal death which were partially attenuated with donepezil treatment.