Bach1 derepression is neuroprotective in a mouse model of Parkinson's disease.

Parkinson's disease (PD) is a progressive neurodegenerative movement disorder characterized by the loss of nigrostriatal dopaminergic neurons. Mounting evidence suggests that Nrf2 is a promising target for neuroprotective interventions in PD. However, electrophilic chemical properties of the canonical Nrf2-based drugs cause irreversible alkylation of cysteine residues on cellular proteins resulting in side effects. Bach1 is a known transcriptional repressor of the Nrf2 pathway. We report that Bach1 levels are up-regulated in PD postmortem brains and preclinical models. Bach1 knockout (KO) mice were protected against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurotoxicity and associated oxidative damage and neuroinflammation. Functional genomic analysis demonstrated that the neuroprotective effects in Bach1 KO mice was due to up-regulation of Bach1-targeted pathways that are associated with both Nrf2-dependent antioxidant response element (ARE) and Nrf2-independent non-ARE genes. Using a proprietary translational technology platform, a drug library screen identified a substituted benzimidazole as a Bach1 inhibitor that was validated as a nonelectrophile. Oral administration of the Bach1 inhibitor attenuated MPTP neurotoxicity in pre- and posttreatment paradigms. Bach1 inhibitor-induced neuroprotection was associated with the up-regulation of Bach1-targeted pathways in concurrence with the results from Bach1 KO mice. Our results suggest that genetic deletion as well as pharmacologic inhibition of Bach1 by a nonelectrophilic inhibitor is a promising therapeutic approach for PD.

Probable Mechanisms of Doxorubicin Antitumor Activity Enhancement by Ginsenoside Rh2.

Ginsenoside Rh2 increases the efficacy of doxorubicin (DOX) treatment in murine models of solid and ascites Ehrlich's adenocarcinoma. In a solid tumor model (treatment commencing 7 days after inoculation), DOX + Rh2 co-treatment was significantly more efficacious than DOX alone. If treatment was started 24 h after inoculation, the inhibition of tumor growth of a solid tumor for the DOX + Rh2 co-treatment group was complete. Furthermore, survival in the ascites model was dramatically higher for the DOX + Rh2 co-treatment group than for DOX alone. Mechanisms underlying the combined DOX and Rh2 effects were studied in primary Ehrlich's adenocarcinoma-derived cells and healthy mice's splenocytes. Despite the previously established Rh2 pro-oxidant activity, DOX + Rh2 co-treatment revealed no increase in ROS compared to DOX treatment alone. However, DOX + Rh2 treatment was more effective in suppressing Ehrlich adenocarcinoma cell adhesion than either treatment alone. We hypothesize that the benefits of DOX + Rh2 combination treatment are due to the suppression of tumor cell attachment/invasion that might be effective in preventing metastatic spread of tumor cells. Ginsenoside Rh2 was found to be a modest activator in a Neh2-luc reporter assay, suggesting that Rh2 can activate the Nrf2-driven antioxidant program. Rh2-induced direct activation of Nrf2 might provide additional benefits by minimizing DOX toxicity towards non-cancerous cells.

Benfotiamine treatment activates the Nrf2/ARE pathway and is neuroprotective in a transgenic mouse model of tauopathy.

Impaired glucose metabolism, decreased levels of thiamine and its phosphate esters, and reduced activity of thiamine-dependent enzymes, such as pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase and transketolase occur in Alzheimer's disease (AD). Thiamine deficiency exacerbates amyloid beta (Aß) deposition, tau hyperphosphorylation and oxidative stress. Benfotiamine (BFT) rescued cognitive deficits and reduced Aß burden in amyloid precursor protein (APP)/PS1 mice. In this study, we examined whether BFT confers neuroprotection against tau phosphorylation and the generation of neurofibrillary tangles (NFTs) in the P301S mouse model of tauopathy. Chronic dietary treatment with BFT increased lifespan, improved behavior, reduced glycated tau, decreased NFTs and prevented death of motor neurons. BFT administration significantly ameliorated mitochondrial dysfunction and attenuated oxidative damage and inflammation. We found that BFT and its metabolites (but not thiamine) trigger the expression of Nrf2/antioxidant response element (ARE)-dependent genes in mouse brain as well as in wild-type but not Nrf2-deficient fibroblasts. Active metabolites were more potent in activating the Nrf2 target genes than the parent molecule BFT. Docking studies showed that BFT and its metabolites (but not thiamine) bind to Keap1 with high affinity. These findings demonstrate that BFT activates the Nrf2/ARE pathway and is a promising therapeutic agent for the treatment of diseases with tau pathology, such as AD, frontotemporal dementia and progressive supranuclear palsy.

Crosstalk between Nrf2 signaling and mitochondrial function in Parkinson's disease.

Search for a definitive cure for neurodegenerative disorders like Parkinson's disease (PD) has met with little success. Mitochondrial dysfunction and elevated oxidative stress precede characteristic loss of dopamine-producing neurons from the midbrain in PD. The majority of PD cases are classified as sporadic (sPD) with an unknown etiology, whereas mutations in a handful of genes cause monogenic form called familial (fPD). Both sPD and fPD is characterized by proteinopathy and mitochondrial dysfunction leading to increased oxidative stress. These pathophysiological mechanisms create a vicious cycle feeding into each other, ultimately tipping the neurons to its demise. Effect of iron accumulation and dopamine oxidation adds an additional dimension to mitochondrial oxidative stress and apoptotic pathways affected. Nrf2 is a redox-sensitive transcription factor which regulates basal as well as inducible expression of antioxidant enzymes and proteins involved in xenobiotic detoxification. Recent advances, however, shows a multifaceted role for Nrf2 in the regulation of genes connected with inflammatory response, metabolic pathways, protein homeostasis, iron management, and mitochondrial bioenergetics. Here we review the role of mitochondria and oxidative stress in the PD etiology and the potential crosstalk between Nrf2 signaling and mitochondrial function in PD. We also make a case for the development of therapeutics that safely activates Nrf2 pathway in halting the progression of neurodegeneration in PD patients.

Distinct Nrf2 Signaling Mechanisms of Fumaric Acid Esters and Their Role in Neuroprotection against 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine-Induced Experimental Parkinson's-Like Disease.

UNLABELLED: A promising approach to neurotherapeutics involves activating the nuclear-factor-E2-related factor 2 (Nrf2)/antioxidant response element signaling, which regulates expression of antioxidant, anti-inflammatory, and cytoprotective genes. Tecfidera, a putative Nrf2 activator, is an oral formulation of dimethylfumarate (DMF) used to treat multiple sclerosis. We compared the effects of DMF and its bioactive metabolite monomethylfumarate (MMF) on Nrf2 signaling and their ability to block 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced experimental Parkinson's disease (PD). We show that in vitro DMF and MMF activate the Nrf2 pathway via S-alkylation of the Nrf2 inhibitor Keap1 and by causing nuclear exit of the Nrf2 repressor Bach1. Nrf2 activation by DMF but not MMF was associated with depletion of glutathione, decreased cell viability, and inhibition of mitochondrial oxygen consumption and glycolysis rates in a dose-dependent manner, whereas MMF increased these activities in vitro However, both DMF and MMF upregulated mitochondrial biogenesis in vitro in an Nrf2-dependent manner. Despite the in vitro differences, both DMF and MMF exerted similar neuroprotective effects and blocked MPTP neurotoxicity in wild-type but not in Nrf2 null mice. Our data suggest that DMF and MMF exhibit neuroprotective effects against MPTP neurotoxicity because of their distinct Nrf2-mediated antioxidant, anti-inflammatory, and mitochondrial functional/biogenetic effects, but MMF does so without depleting glutathione and inhibiting mitochondrial and glycolytic functions. Given that oxidative damage, neuroinflammation, and mitochondrial dysfunction are all implicated in PD pathogenesis, our results provide preclinical evidence for the development of MMF rather than DMF as a novel PD therapeutic. SIGNIFICANCE STATEMENT: Almost two centuries since its first description by James Parkinson, Parkinson's disease (PD) remains an incurable disease with limited symptomatic treatment. The current study provides preclinical evidence that a Food and Drug Administration-approved drug, dimethylfumarate (DMF), and its metabolite monomethylfumarate (MMF) can block nigrostriatal dopaminergic neurodegeneration in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of PD. We elucidated mechanisms by which DMF and its active metabolite MMF activates the redox-sensitive transcription factor nuclear-factor-E2-related factor 2 (Nrf2) to upregulate antioxidant, anti-inflammatory, mitochondrial biosynthetic and cytoprotective genes to render neuroprotection via distinct S-alkylating properties and depletion of glutathione. Our data suggest that targeting Nrf2-mediated gene transcription using MMF rather than DMF is a promising approach to block oxidative stress, neuroinflammation, and mitochondrial dysfunction for therapeutic intervention in PD while minimizing side effects.

Facet-specific ligand dynamics: scaling the descriptors of Cs2AgBiBr6 nanocrystals.

Facet control by primary amines can bolster the optoelectronic parameters of A2BIB'IIIX6 perovskite nanocrystals (NCs) with large indirect bandgaps. The 18-C amine competitively attaches to the (222) facet of Cs2AgBiBr6 (CABB) NCs, 16-C and 14-C bind to (400) and (440), and 12-C binds to (400). The NCs with only the (400) facet decrease the bandgap and exciton binding energy by 0.26 eV and 15 meV, respectively.

Pharmacokinetics of AAV9 Mediated Trastuzumab Expression in Rat Brain Following Systemic and Local Administration.

INTRODUCTION: Recombinant adeno-associated viruses(rAAVs) are an attractive tool to ensure long-term expression monoclonal antibody(mAb) in the central nervous system(CNS). It is still unclear whether systemic injection or local CNS administration of AAV9 is more beneficial for the exposure of the expressed mAb in the brain. Hence, we compared the biodistribution and transgene expression following AAV9-Trastuzumab administration through different routes. METHODS AND RESULT: In-house generated AAV9-Trastuzumab vectors were administered at 5E+11 Vgs/rat through intravenous(IV), intracerebroventricular(ICV), intra-cisterna magna(ICM) and intrastriatal(IST) routes. Vector and trastuzumab blood/plasma concentrations were assessed at different time points up to the terminal time point of 21 days. Different brain regions in addition to the spinal cord, cerebrospinal fluid(CSF) and interstitial fluid(ISF), were also analyzed at the terminal time point. Our results show that vector biodistribution and Trastuzumab expression in the brain could the ranked as follows: IST>ICM>ICV>IV. Rapid clearance of vector was observed after administration via the ICM and ICV routes. The ICV route produced similar expression levels across different brain regions, while the ICM route had better expression in the hindbrain and spinal cord region. The IST route had higher expression in the forebrain region compared to the hindbrain region. A sharp decline in trastuzumab plasma concentration was observed across all routes of administration due to anti-trastuzumab antibody response. CONCLUSION: In this study we have characterized vector biodistribution and transgene mAb expression after AAV9 vector administration through different routes in rats. IST and ICM represent the best administration routes to deliver antibody genes to the brain.

Selective inhibition of phosphodiesterase 5 enhances glutamatergic synaptic plasticity and memory in mice.

Phosphodiesterases (PDEs) belong to a family of proteins that control metabolism of cyclic nucleotides. Targeting PDE5, for enhancing cellular function, is one of the therapeutic strategies for male erectile dysfunction. We have investigated whether in vivo inhibition of PDE5, which is expressed in several brain regions, will enhance memory and synaptic transmission in the hippocampus of healthy mice. We have found that acute administration of sildenafil, a specific PDE5 inhibitor, enhanced hippocampus-dependent memory tasks. To elucidate the underlying mechanism in the memory enhancement, effects of sildenafil on long-term potentiation (LTP) were measured. The level of LTP was significantly elevated, with concomitant increases in basal synaptic transmission, in mice treated with sildenafil (1 mg/kg/day) for 15 days compared to control mice. These results suggest that moderate PDE5 inhibition enhances memory by increasing synaptic plasticity and transmission in the hippocampus.

Assessment of therapeutic potential of amantadine in methamphetamine induced neurotoxicity.

Methamphetamine epidemic has a broad impact on world's health care system. Its abusive potential and neurotoxic effects remain a challenge for the anti-addiction therapies. In addition to oxidative stress, mitochondrial dysfunction and apoptosis, excitotoxicity is also involved in methamphetamine induced neurotoxicity. The N-methyl-D-aspartate (NMDA) type of glutamate receptor is thought to be one of the predominant mediators of excitotoxicity. There is growing evidence that NMDA receptor antagonists could be one of the therapeutic options to manage excitotoxicity. Amantadine, a well-tolerated and modestly effective antiparkinsonian agent, was found to possess NMDA antagonistic properties and has shown to release dopamine from the nerve terminals. The current study aimed to evaluate the effect of amantadine pre-treatment against methamphetamine induced neurotoxicity. Results showed that methamphetamine treatment had depleted striatal dopamine, generated of reactive oxygen species and decreased activity of complex I in the mitochondria. Interestingly, amantadine, at high dose (10 mg/kg), did not prevent dopamine depletion moreover it exacerbated the behavioral manifestations of methamphetamine toxicity such as akinesia and catalepsy. Only lower dose of amantadine (1 mg/kg) produced significant scavenging of the reactive oxygen species induced by methamphetamine. Overall results from the present study suggest that amantadine should not be used concomitantly with methamphetamine as it may results in excessive neurotoxicity.

Investigate the chronic neurotoxic effects of diquat.

Chronic exposure to agricultural chemicals (pesticides/herbicides) has been shown to induce neurotoxic effects or results in accumulation of various toxic metabolic by-products. These substances have the relevant ability to cause or increase the risk for neurodegeneration. Diquat is an herbicide that has been extensively used in the United States of America and other parts of the world. Diquat is constantly released into the environment during its use as a contact herbicide. Diquat structurally resembles 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) and paraquat. Rotenone, paraquat, maneb and MPTP reproduce features of movement disorders in experimental animal models. Based on the structural similarity to other neurotoxins, chronic exposure of diquat can induce behavioral and neurochemical alterations associated with dopaminergic neurotoxicity. However, in the present study, diquat unlike other neurotoxins (rotenone, 6-hydroxydopamine, MPTP, paraquat and maneb) did not induce dopamine depletion in the mouse striatum. Although, notable exacerbation in motor impairment (swimming score, akinesia and open field) were evident that may be due to the decreased dopamine turnover and mild nigrostriatal neurodegeneration. These data indicate that, despite the apparent structural similarity to other dopaminergic neurotoxins, diquat did not exert severe deleterious effects on dopamine neurons in a manner that is unique to rotenone and MPTP.

Harnessing the Therapeutic Potential of the Nrf2/Bach1 Signaling Pathway in Parkinson's Disease.

Parkinson's disease (PD) is the second most common neurodegenerative movement disorder characterized by a progressive loss of dopaminergic neurons in the substantia nigra pars compacta. Although a complex interplay of multiple environmental and genetic factors has been implicated, the etiology of neuronal death in PD remains unresolved. Various mechanisms of neuronal degeneration in PD have been proposed, including oxidative stress, mitochondrial dysfunction, neuroinflammation, α-synuclein proteostasis, disruption of calcium homeostasis, and other cell death pathways. While many drugs individually targeting these pathways have shown promise in preclinical PD models, this promise has not yet translated into neuroprotective therapies in human PD. This has consequently spurred efforts to identify alternative targets with multipronged therapeutic approaches. A promising therapeutic target that could modulate multiple etiological pathways involves drug-induced activation of a coordinated genetic program regulated by the transcription factor, nuclear factor E2-related factor 2 (Nrf2). Nrf2 regulates the transcription of over 250 genes, creating a multifaceted network that integrates cellular activities by expressing cytoprotective genes, promoting the resolution of inflammation, restoring redox and protein homeostasis, stimulating energy metabolism, and facilitating repair. However, FDA-approved electrophilic Nrf2 activators cause irreversible alkylation of cysteine residues in various cellular proteins resulting in side effects. We propose that the transcriptional repressor of BTB and CNC homology 1 (Bach1), which antagonizes Nrf2, could serve as a promising complementary target for the activation of both Nrf2-dependent and Nrf2-independent neuroprotective pathways. This review presents the current knowledge on the Nrf2/Bach1 signaling pathway, its role in various cellular processes, and the benefits of simultaneously inhibiting Bach1 and stabilizing Nrf2 using non-electrophilic small molecules as a novel therapeutic approach for PD.

An Emerging Role of miRNAs in Neurodegenerative Diseases: Mechanisms and Perspectives on miR146a.

Significance: Advancements in and access to health care have led to unprecedented improvements in the quality of life and increased lifespan of human beings in the past century. However, aging is a significant risk factor for neurodegenerative diseases (NDs). Hence, improved life expectancy has led to an increased incidence of NDs. Despite intense research, effective treatments for NDs remain elusive. The future of neurotherapeutics development depends on effective disease modification strategies centered on carefully scrutinized targets. Recent Advances: As a promising new direction, recent evidence has demonstrated that epigenetic processes modify diverse biochemical pathways, including those related to NDs. Small non-coding RNAs, known as microRNAs (miRNAs), are components of the epigenetic system that alter the expression of target genes at the post-transcriptional level. Critical Issues: miRNAs are expressed abundantly in the central nervous system and are critical for the normal functioning and survival of neurons. Here, we review recent advances in elucidating miRNAs' roles in NDs and discuss their potential as therapeutic targets. In particular, neuroinflammation is a major pathological hallmark of NDs and miR146a is a crucial regulator of inflammation. Future Directions: Finally, we explore the possibilities of developing miR146a as a potential biomarker and therapeutic target where additional research may help facilitate the detection and amelioration of neuroinflammation in NDs. Antioxid. Redox Signal. 35, 580-594.

Challenges and Limitations of Targeting the Keap1-Nrf2 Pathway for Neurotherapeutics: Bach1 De-Repression to the Rescue.

The Keap1-Nrf2 signaling axis is a validated and promising target for cellular defense and survival pathways. This minireview discusses the potential off-target effects and their impact on future drug development originating from Keap1-targeting small molecules that function as displacement activators of the redox-sensitive transcription factor Nrf2. We argue that small-molecule displacement activators, similarly to electrophiles, will release both Nrf2 and other Keap1 client proteins from the ubiquitin ligase complex. This non-specificity is likely unavoidable and may result in off-target effects during Nrf2 activation by targeting Keap1. The small molecule displacement activators may also target Kelch domains in proteins other than Keap1, causing additional off-target effects unless designed to ensure specificity for the Kelch domain only in Keap1. A potentially promising and alternative therapeutic approach to overcome this non-specificity emerging from targeting Keap1 is to inhibit the Nrf2 repressor Bach1 for constitutive activation of the Nrf2 pathway and bypass the Keap1-Nrf2 complex.

Identification of a potent Nrf2 displacement activator among aspirin-containing prodrugs.

Aspirin is a desired leaving group in prodrugs aimed at treatment of neurodegeneration and other conditions. A library of aspirin derivatives of various scaffolds potentially activating Nrf2 has been tested in Neh2-luc reporter assay which screens for direct Nrf2 protein stabilizers working via disruption of Nrf2-Keap1 interaction. Most aspirin prodrugs had a pro-alkylating or pro-oxidant motif in the structure and, therefore, were toxic at high concentrations. However, among the active compounds, we identified a molecule resembling a well-known Nrf2 displacement activator, bis-1,4-(4-methoxybenzenesulfonamidyl) naphthalene (NMBSA). The direct comparison of the newly identified compound with NMBSA and its improved analog in the reporter assay showed no quenching with N-acetyl cysteine, thus pointing to Nrf2 stabilization mechanism without cysteine alkylation. The potency of the newly identified compound in the reporter assay was much stronger than NMBSA, despite its inhibitory action in the commercial fluorescence polarization assay was observed only in the millimolar range. Molecular docking predicted that mono-deacetylation of the novel prodrug should generate a potent displacement activator. The time-course of reporter activation with the novel prodrug had a pronounced lag-period pointing to a plausible intracellular transformation leading to an active product. Treatment of the novel prodrug with blood plasma or cell lysate demonstrated stepwise deacetylation as judge by liquid chromatography-mass spectrometry (LC-MS). Hence, the esterase-catalyzed hydrolysis of the prodrug liberates only acetyl groups from aspirin moiety and generates a potent Nrf2 activator. The discovered mechanism of prodrug activation makes the newly identified compound a promising lead for future optimization studies.

Protective effect of minocycline, a semi-synthetic second-generation tetracycline against 3-nitropropionic acid (3-NP)-induced neurotoxicity.

3-Nitropropionic acid (3-NP) is an irreversible inhibitor of the electron transport enzyme succinate dehydrogenase, a mitochondrial Complex II enzyme. Minocycline is a semi-synthetic second-generation tetracycline with neuroprotective activity and has the capability to effectively cross the blood-brain barrier. We investigated the effects of minocycline on behavioral, biochemical, inflammation related and neurochemical alterations induced by the sub-chronic administration of 3-nitropropionic acid to rats. Chronic pre-administration of minocycline (50 and 100mg/kg) dose dependently prevented 3-NP-induced dysfunction behavioral (hypoactivity, memory retention, locomotor and rota-rod activity). In addition, 3-NP produced a marked increase in lipid peroxidation levels whereas decreased the activities of catalase and succinate dehydrogenase. In contrast, pretreatment of 3-NP injected rats with minocycline resulted in the attenuation of all these alterations. A marked increase in an inflammatory cytokine TNF-alpha by 3-NP was also decreased by minocycline treatment. Neurochemically, the administration of 3-NP significantly decreased the levels of catecholamines in the brain homogenates (dopamine, norepinephrine and serotonin) which were reversed by pretreatment of minocycline. The present finding explains the neuroprotective effect of minocycline against 3-NP toxicity by virtue of its antioxidant and anti-inflammatory activity.

Inflammatory and neurochemical changes associated with 3-nitropropionic Acid neurotoxicity.

ABSTRACT Administration of 3-nitropropionic acid (3-NP), a hemotoxin of fungal origin, is associated with a rare neurodegenerative disease, Huntington disease. The present study was carried out to investigate the inflammatory and neurochemical changes associated with the subchronic administration of 3-nitropropionic acid in rats. The administration of 3-nitropropionic acid (20 mg/kg SC and 15 mg/kg IP for 7 days) resulted in a significant increase in inflammatory mediators such as tissue necrosis factor (TNF)-alpha, total nitric oxide, and nitrite levels in brain homogenate. It also resulted in a significant decrease in catecholamine neurotransmitters such as dopamine and serotonin (5-HT) and an increase in turnover of these neurotransmitters as compared to the control group, estimated by HPLC with ECD detector. Inflammatory and neurochemical changes were in accordance to the behavioral and biochemical changes induced by 3-NP. The present study suggests the involvement of inflammatory changes as an important causal factor for neurodegeneration associated with 3-NP.

Hsp90 Co-chaperone p23 contributes to dopaminergic mitochondrial stress via stabilization of PHD2: Implications for Parkinson's disease.

The heat shock factor 90 (hsp90) complex has long been associated with neuropathological phenotypes linked to Parkinson's disease (PD) and its inhibition is neuroprotective in disease models. Hsp90 is conventionally believed to act by suppressing induction of hsp70. Here, we report a novel hsp70-independent mechanism by which Hsp90 may also contribute to PD-associated neuropathology. We previously reported that inhibition of the enzyme prolyl hydroxylase domain 2 (PHD2) in conjunction with increases in hypoxia-inducible factor 1 alpha (HIF1α) results in protection of vulnerable dopaminergic substantia nigra pars compacta (DAergic SNpc) neurons in in vitro and in vivo models of PD. We discovered an increased interaction between PHD2 and the p23:Hsp90 chaperone complex in response to mitochondrial stress elicited by the mitochondrial neurotoxin 1-methyl-4-phenylpyridine (MPP+) within cultured DAergic cells. Genetic p23 knockdown was found to result in decreases in steady-state PHD2 protein and activity and reduced susceptibility to MPP+ neurotoxicity. Administration of the p23 inhibitor gedunin was also neuroprotective in these cells as well as in human induced pluripotent stem cell (iPSC)-derived neurons. Our data suggests that mitochondrial stress-mediated elevations in PHD2 interaction with the p23-hsp90 complex have detrimental effects on the survival of DAergic neurons, while p23 inhibition is neuroprotective. We propose that neurotoxic effects are tied to enhanced PHD2 stabilization by the hsp90-p23 chaperone complex that is abrogated by p23 inhibition. This demonstrates a novel connection between two independent pathways previously linked to PD, hsp90 and PHD2-HIF1α, which could have important implications for here-to-fore unexplored mechanisms underlying PD neuropathology.

L-ascorbic acid: A true substrate for HIF prolyl hydroxylase?

L-Ascorbate (L-Asc), but not D-isoascorbate (D-Asc) and N-acetylcysteine (NAC) suppress HIF1 ODD-luc reporter activation induced by various inhibitors of HIF prolyl hydroxylase (PHD). The efficiency of suppression by L-Asc was sensitive to the nature of HIF PHD inhibitor chosen for reporter activation. In particular, the inhibitors developed to compete with alpha-ketoglutarate (αKG), were less sensitive to suppression by the physiological range of L-Asc (40-100 µM) than those having a strong iron chelation motif. Challenging those HIF activators in the reporter system with D-Asc demonstrated that the D-isomer, despite exhibiting the same reducing potency with respect to ferric iron, had almost no effect compared to L-Asc. Similarly, no effect on reporter activation was observed with cell-permeable reducing agent NAC up to 1 mM. Docking of L-Asc and D-Asc acid into the HIF PHD2 crystal structure showed interference of Tyr310 with respect to D-Asc. This suggests that L-Asc is not merely a reducing agent preventing enzyme inactivation. Rather, the overall results identify L-Asc as a co-substrate of HIF PHD that may compete for the binding site of αKG in the enzyme active center. This conclusion is in agreement with the results obtained recently in cell-based systems for TET enzymes and jumonji histone demethylases, where L-Asc has been proposed to act as a co-substrate and not as a reducing agent preventing enzyme inactivation.

Activation of Nrf2 and Hypoxic Adaptive Response Contribute to Neuroprotection Elicited by Phenylhydroxamic Acid Selective HDAC6 Inhibitors.

Activation of HIF-1α and Nrf2 is a primary component of cellular response to oxidative stress, and activation of HIF-1α and Nrf2 provides neuroprotection in models of neurodegenerative disorders, including ischemic stroke, Alzheimer's and Parkinson's diseases. Screening a library of CNS-targeted drugs using novel reporters for HIF-1α and Nrf2 elevation in neuronal cells revealed histone deacetylase (HDAC) inhibitors as potential activators of these pathways. We report the identification of phenylhydroxamates as single agents exhibiting tripartite inhibition of HDAC6, inhibition of HIF-1 prolyl hydroxylase (PHD), and activation of Nrf2. Two superior tripartite agents, ING-6 and ING-66, showed neuroprotection against various cellular insults, associated with stabilization of both Nrf2 and HIF-1, and expression of their respective target genes in vitro and in vivo. Discovery of the innate ability of phenylhydroxamate HDAC inhibitors to activate Nrf2 and HIF provides a novel route to multifunctional neuroprotective agents and cautions against HDAC6 selective inhibitors as chemical probes of specific HDAC isoform function.

Immunological alteration & toxic molecular inductions leading to cognitive impairment & neurotoxicity in transgenic mouse model of Alzheimer's disease.

AIMS: Inflammation is considered to be one of the crucial pathological factors associated with the development of Alzheimer's disease, although supportive experimental evidence remains undiscovered. Therefore, the current study was carried out to better understand and establish the pathophysiological involvement of chronic inflammation in a double transgenic mouse model of Alzheimer's disease. MAIN METHODS: We analyzed amyloid-beta deposition, oxidative stress, biochemical, neurochemical and immunological markers in a 10month old (APΔE9) mouse model. Memory functions were assessed by behavioral testing followed by measurement of synaptic plasticity via extracellular field recordings. KEY FINDINGS: Substantial increases in amyloid-beta levels, beta-secretase activity, and oxidative stress, along with significant neurochemical alterations in glutamate and GABA levels were detected in the brain of APΔE9 mice. Interestingly, marked elevations of pro-inflammatory cytokines in whole brain lysate of APΔE9 mice were observed. Flow cytometric analysis revealed a higher frequency of CD4+ IL-17a and IFN-γ secreting T-cells in APΔE9 brain, indicating a robust T-cell infiltration and activation. Behavioral deficits in learning and memory tasks, along with impairment in long-term potentiation and associated biochemical changes in the expression of glutamatergic receptor subunits were evident. SIGNIFICANCE: Thus, this study establishes the role by which oxidative stress, alterations in glutamate and GABA levels and inflammation increases hippocampal and cortical neurotoxicity resulting in the cognitive deficits associated with Alzheimer's disease.