Search

Presents information on a study that examined the metabolism of alprazolam by rat liver and brain microsomes and compared it with the activity in microsomes prepared from human brain regions obtained at autopsy. Introduction to cytochromes P450; Results and discussion on the study; Methodology of the study.

Autophagy-lysosomal function is crucial for maintaining healthy lifespan and preventing age-related diseases. The transcription factor TFEB plays a key role in regulating this pathway. Decreased TFEB expression is associated with various age-related disorders, making it a promising therapeutic target. In this study, we screened a natural product library and discovered mitophagy-inducing coumarin (MIC), a benzocoumarin compound that enhances TFEB expression and lysosomal function. MIC robustly increases the lifespan of Caenorhabditis elegans in an HLH-30/TFEB-dependent and mitophagy-dependent manner involving DCT-1/BNIP3 while also preventing mitochondrial dysfunction in mammalian cells. Mechanistically, MIC acts by inhibiting ligand-induced activation of the nuclear hormone receptor DAF-12/FXR, which, in turn, induces mitophagy and extends lifespan. In conclusion, our study uncovers MIC as a promising drug-like molecule that enhances mitochondrial function and extends lifespan by targeting DAF-12/FXR. Furthermore, we discovered DAF-12/FXR as a previously unknown upstream regulator of HLH-30/TFEB and mitophagy., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Aging is associated with changes in circulating levels of various molecules, some of which remain undefined. We find that concentrations of circulating taurine decline with aging in mice, monkeys, and humans. A reversal of this decline through taurine supplementation increased the health span (the period of healthy living) and life span in mice and health span in monkeys. Mechanistically, taurine reduced cellular senescence, protected against telomerase deficiency, suppressed mitochondrial dysfunction, decreased DNA damage, and attenuated inflammaging. In humans, lower taurine concentrations correlated with several age-related diseases and taurine concentrations increased after acute endurance exercise. Thus, taurine deficiency may be a driver of aging because its reversal increases health span in worms, rodents, and primates and life span in worms and rodents. Clinical trials in humans seem warranted to test whether taurine deficiency might drive aging in humans.

Parkinson's disease (PD) is a chronic, neurodegenerative condition characterized by motor symptoms such as bradykinesia, rigidity, and tremor, alongside multiple nonmotor symptoms. The appearance of motor symptoms is linked to progressive dopaminergic neuron loss within the substantia nigra. PD incidence increases sharply with age, suggesting a strong association between mechanisms driving biological aging and the development and progression of PD. However, the role of aging in the pathogenesis of PD remains understudied. Numerous models of PD, including cell models, toxin-induced models, and genetic models in rodents and nonhuman primates (NHPs), reproduce different aspects of PD, but preclinical studies of PD rarely incorporate age as a factor. Studies using patient neurons derived from stem cells via reprogramming methods retain some aging features, but their characterization, particularly of aging markers and reproducibility of neuron type, is suboptimal. Investigation of age-related changes in PD using animal models indicates an association, but this is likely in conjunction with other disease drivers. The biggest barrier to drawing firm conclusions is that each model lacks full characterization and appropriate time-course assessments. There is a need to systematically investigate whether aging increases the susceptibility of mouse, rat, and NHP models to develop PD and understand the role of cell models. We propose that a significant investment in time and resources, together with the coordination and sharing of resources, knowledge, and data, is required to accelerate progress in understanding the role of biological aging in PD development and improve the reliability of models to test interventions., Competing Interests: Conflict of Interest statement All authors declare that they have no competing interests.

Cellular senescence is a potential tumor-suppressive mechanism that generally results in an irreversible cell cycle arrest. Senescent cells accumulate with age and actively secrete soluble factors, collectively termed the 'senescence-associated secretory phenotype' (SASP), which has both beneficial and detrimental effects. Although the contribution of senescent cells to age-related pathologies has been well-established outside the brain, emerging evidence indicates that brain cells also undergo cellular senescence and contribute to neuronal loss in the context of age-related neurodegenerative diseases. Contribution of senescent cells in the pathogenesis of neurological disorders has led to the possibility of eliminating senescence cells via pharmacological compounds called senolytics. Recently several senolytics have been demonstrated to elicit improved cognitive performance and healthspan in mouse models of neurodegeneration. However, their translation for use in the clinic still holds several potential challenges. This review summarizes available senolytics, their purported mode of action, and possible off-target effects. We also discuss possible alternative strategies that may help minimize potential side-effects associated with the senolytics approach., (Copyright © 2021 Elsevier B.V. All rights reserved.)

In the nematode Caenorhabditis elegans, signals derived from bacteria in the diet, the animal's major nutrient source, can modulate both behavior and healthspan. Here we describe a dual role for trimethylamine (TMA), a human gut flora metabolite, which acts as a nutrient signal and a neurotoxin. TMA and its associated metabolites are produced by the human gut microbiome and have been suggested to serve as risk biomarkers for diabetes and cardiovascular diseases. We demonstrate that the tyramine receptor TYRA-3, a conserved G protein-coupled receptor (GPCR), is required to sense TMA and mediate its responses. TMA activates guanylyl cyclase DAF-11 signaling through TYRA-3 in amphid neurons (ASK) and ciliated neurons (BAG) to mediate food-sensing behavior. Bacterial mutants deficient in TMA production enhance dauer formation, extend lifespan, and are less preferred as a food source. Increased levels of TMA lead to neural damage in models of Parkinson's disease and shorten lifespan. Our results reveal conserved signaling pathways modulated by TMA in C. elegans that are likely to be relevant for its effects in mammalian systems., (© 2021 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Mutations in the human ATP13A2 gene are associated with an early-onset form of Parkinson's disease (PD) known as Kufor Rakeb Syndrome (KRS). Patients with KRS show increased iron deposition in the basal ganglia, suggesting iron toxicity-induced neurodegeneration as a potential pathogenesis associated with the ATP13A2 mutation. Previously we demonstrated that functional losses of ATP13A2 disrupt the lysosomes ability to store excess iron, leading to reduce survival of dopaminergic neuronal cells. To understand the possible mechanisms involved, we studied a Caenorhabditis elegans mutant defective in catp-6 function, an ortholog of human ATP13A2 gene. Here we show that catp-6 mutant worms have defective autophagy and lysosomal function, demonstrate characteristic PD phenotypes including reduced motor function and dysregulated iron metabolism. Additionally, these mutants have defective mitochondrial health, which is rescuable via iron chelation or mitophagy induction., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Alzheimer's disease (AD) is a neurodegenerative disorder and the leading cause of dementia in aged populations worldwide. The deposition of toxic protein aggregates such as amyloid beta (Aβ) is a hallmark of AD, and there is growing awareness that a key driver of AD pathogenesis is the neuroinflammatory cascade triggered and sustained by these proteins. Consequently, interventions that suppress prolonged neuroinflammation represent viable therapeutic approaches for AD. In this context, we tested the natural product gedunin which is an anti-inflammatory molecule, found in the seeds of the neem tree (Azadirachta indica), whose mechanism of action remains to be fully elucidated. Using a mouse microglia cell line (IMG), we show that gedunin suppresses neuroinflammation arising from Aβ 1-42 oligomer exposure. Our results demonstrate that gedunin suppresses Aβ 1-42 -induced NF-κB activation and its targets, including nitric oxide (NO) and IL-1β, known proinflammatory molecules. Further, we show that gedunin inhibits neuroinflammation by activating nuclear factor 2 erythroid-related factor 2 (Nrf2) and its downstream targets γ-glutamylcysteine synthetase, heme oxygenase 1, and NADPH quinone dehydrogenase 1, which are involved in quenching reactive oxygen and nitrogen species (NO) generated by NF-κB activation. Nrf2 activation appears essential for the anti-inflammatory effect because when silenced, the proinflammatory effects of Aβ 1-42 are enhanced and the protective effect of gedunin against NO production is reduced. Additionally, using human neuronal cells (SH-SY5Y), we show that gedunin prevents neurotoxicity secondary to Aβ-induced microglial activation. In conclusion, our findings highlight a potential therapeutic role of gedunin in neurodegenerative diseases.

Several studies have suggested that increases in astrocytic monoamine oxidase B (MAO-B) levels in conjunction with Parkinson's disease (PD) may contribute to subsequent neuropathology associated with the disorder. MAO-B inhibitors are currently widely used as symptomatic therapeutics for PD and, although somewhat controversial, these drugs may also exhibit disease-modifying properties. To obtain a better understanding of the potential role of MAO-B in disease neuropathology, we created an inducible astrocyte-specific transgenic MAO-B mouse model. Here, we summarize findings associated with this model, including neuropathological PD features associated with it.

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., (Published by Elsevier B.V.)

Exposure to the herbicide paraquat (PQ) is associated with an increased risk of idiopathic Parkinson's disease (PD). Therapies based on PQ's presumed mechanisms of action have not, however, yielded effective disease therapies. Cellular senescence is an anticancer mechanism that arrests proliferation of replication-competent cells and results in a pro-inflammatory senescence-associated secretory phenotype (SASP) capable of damaging neighboring tissues. Here, we demonstrate that senescent cell markers are preferentially present within astrocytes in PD brain tissues. Additionally, PQ was found to induce astrocytic senescence and an SASP in vitro and in vivo, and senescent cell depletion in the latter protects against PQ-induced neuropathology. Our data suggest that exposure to certain environmental toxins promotes accumulation of senescent cells in the aging brain, which can contribute to dopaminergic neurodegeneration. Therapies that target senescent cells may constitute a strategy for treatment of sporadic PD, for which environmental exposure is a major risk factor., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Monoamine oxidase B (MAO-B) has been implicated in the pathogenesis of Alzheimer's disease (AD) and other neurodegenerative disorders. Increased MAO-B expression in astroglia has been observed adjacent to amyloid plaques in AD patient brains. This phenomenon is hypothesized to lead to increased production of hydrogen peroxide and reactive oxygen species (ROS), thereby contributing to AD pathology. Therefore, reduction of ROS-induced oxidative stress via inhibition of MAO-B activity may delay the progression of the disease. In the present study we report the pharmacological properties of sembragiline, a novel selective MAO-B inhibitor specifically developed for the treatment of AD, and on its effect on ROS-mediated neuronal injury and astrogliosis in MAO-B transgenic animals. Sembragiline showed potent and long-lasting MAO-B-selective inhibition and did not inhibit MAO-A at doses where full inhibition of MAO-B was observed. Such selectivity should translate into a favorable clinical safety profile. Indeed, sembragiline neither induced the serotonin syndrome when administered together with the serotonin precursor l-5-hydroxytryptophan in combination with antidepressants such as fluoxetine, nor potentiated the pressor effect of tyramine. Additionally, in experiments using a transgenic animal model conditionally overexpressing MAO-B in astroglia, sembragiline protected against neuronal loss and reduced both ROS formation and reactive astrogliosis. Taken together, these findings warrant further investigation of the potential therapeutic benefit of MAO-B inhibitors in patients with AD and other neurologic disorders., (Copyright © 2017 by The Author(s).)

Glial activation and subsequent release of neurotoxic proinflammatory factors are believed to play an important role in the pathogenesis of several neurological disorders including Parkinson's disease (PD). Inhibition of glial activation and inflammatory processes may represent a therapeutic target to alleviate neurodegeneration. Securinine, a major natural alkaloid product from the root of the plant Securinega suffruticosa , has been reported to have potent biological activity and is used in the treatment of neurological conditions such as amyotrophic lateral sclerosis, poliomyelitis, and multiple sclerosis. In this study, we explored the underlying mechanisms of neuroprotection elicited by securinine, particularly its anti-inflammatory effects in glial cells. Our results demonstrate that securinine significantly and dose-dependently suppressed the nitric oxide production in microglia and astrocytic cultures. In addition, securinine inhibited the activation of the inflammatory mediator NF- κ B, as well as mitogen-activated protein kinases in lipopolysaccharide- (LPS-) stimulated BV2 cells. Additionally, securinine also inhibited interferon- γ - (IFN- γ -) induced nitric oxide levels and iNOS mRNA expression. Furthermore, conditioned media (CM) from securinine pretreated BV2 cells significantly reduced mesencephalic dopaminergic neurotoxicity compared with CM from LPS stimulated microglia. These findings suggest that securinine may be a potential candidate for the treatment of neurodegenerative diseases related to neuroinflammation.

Loss of parkin E3 ligase activity as a result of parkin gene mutation in rare familial forms of Parkinson's disease (PD) has been shown to be detrimental to mitochondrial function and to contribute to ensuing neurodegeneration. This has been shown by ourselves and others to be in part due to reductions in parkin-mediated ubiquitination of the transcriptional repressor PARIS, limiting the protein's subsequent degradation by the proteasome. Subsequent elevations in PARIS protein levels result in reduced expression of the master mitochondrial regulator PGC-1α, impacting in turn on mitochondrial function. Here, we report that oxidatively-mediated reductions in parkin solubility and function in a mouse model of age-related sporadic PD coincides with increased PARIS levels and reduced PGC-1α signaling. Furthermore, restoration of PGC-1α expression was found to abrogate losses in mitochondrial function and degeneration of dopaminergic (DAergic) neurons within the substantia nigra pars compacta (SNpc) associated with this particular model. These findings suggest that the PGC-1α signaling pathway constitutes a viable therapeutic target for the treatment of not only familial PD, but also more common sporadic forms of the disorder., (Copyright © 2016 Elsevier Inc. All rights reserved.)

We previously reported that pharmacological inhibition of a class of enzymes known as prolyl hydroxylase domain proteins (PHDs) has neuroprotective effects in various in vitro and in vivo models of Parkinson's disease (PD). We hypothesized that this was due to inhibition of the PHD2 isoform, preventing it from hydroxylating the transcription factor hypoxia inducible factor 1 α (HIF1α), targeting it for eventual proteasomal degradation. HIF1α itself induces the transcription of various cellular stress genes, including several involved in iron metabolism. Although all three isoforms of PHD are expressed within vulnerable dopaminergic (DAergic) substantia nigra pars compacta neurons, only select downregulation of the PHD2 isoform was found to protect against in vivo neurodegenerative effects associated with the mitochondrial neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. These findings were corroborated in induced pluripotent stem cell-derived neurons, providing validation in a pertinent human cell model. PHD2 inhibition was found to result in increased expression of ATP13A2, mutation of which is responsible for a rare juvenile form of PD known as Kufor-Rakeb syndrome. Knockdown of ATP13A2 expression within human DAergic cells was found to abrogate restoration of cellular iron homeostasis and neuronal cell viability elicited by inhibition of PHD2 under conditions of mitochondrial stress, likely via effects on lysosomal iron storage. These data suggest that regulation of ATP13A2 by the PHD2-HIF1α signaling pathway affects cellular iron homeostasis and DAergic neuronal survival. This constitutes a heretofore unrecognized process associated with loss of ATP13A2 function that could have wide-ranging implications for it as a therapeutic target for PD and other related conditions., Significance Statement: Reductions in PHD2 activity within dopaminergic neurons in vivo and in cultured human induced pluripotent stem cell-derived neurons protects against mitochondrial stress-induced neurotoxicity. Protective effects are dependent on downstream HIF-1α expression. Knockdown of ATP13A2, a gene linked to a rare juvenile form of Parkinson's disease and recently identified as a novel HIF1α target, was found to abrogate maintenance of cellular iron homeostasis and neuronal viability elicited by PHD2 inhibition in vivo and in cultured dopaminergic cells under conditions of mitochondrial stress. Mechanistically, this was due to ATP13A2's role in maintaining lysosomal iron stores. This constitutes a novel mechanism by which alterations in ATP13A2 activity may be driving PD-related neuropathology., (Copyright © 2016 the authors 0270-6474/16/361086-10$15.00/0.)

Following its activation by PINK1, parkin is recruited to depolarized mitochondria where it ubiquitinates outer mitochondrial membrane proteins, initiating lysosomal-mediated degradation of these organelles. Mutations in the gene encoding parkin, PARK2, result in both familial and sporadic forms of Parkinson's disease (PD) in conjunction with reductions in removal of damaged mitochondria. In contrast to what has been reported for other PARK2 mutations, expression of the Q311X mutation in vivo in mice appears to involve a downstream step in the autophagic pathway at the level of lysosomal function. This coincides with increased PARIS expression and reduced expression of a reciprocal signaling pathway involving the master mitochondrial regulator peroxisome proliferator-activated receptor-gamma coactivator (PGC1α) and the lysosomal regulator transcription factor EB (TFEB). Treatment with rapamycin was found to independently restore PGC1α-TFEB signaling in a manner not requiring parkin activity and to abrogate impairment of mitochondrial quality control and neurodegenerative features associated with this in vivo model. Losses in PGC1α-TFEB signaling in cultured rat DAergic cells expressing the Q311X mutation associated with reduced mitochondrial function and cell viability were found to be PARIS-dependent and to be independently restored by rapamycin in a manner requiring TFEB. Studies in human iPSC-derived neurons demonstrate that TFEB induction can restore mitochondrial function and cell viability in a mitochondrially compromised human cell model. Based on these data, we propose that the parkin Q311X mutation impacts on mitochondrial quality control via PARIS-mediated regulation of PGC1α-TFEB signaling and that this can be independently restored via upregulation of TFEB function., Significance Statement: Mutations in PARK2 are generally associated with loss in ability to interact with PINK1, impacting on autophagic initiation. Our data suggest that, in the case of at least one parkin mutation, Q311X, detrimental effects are due to inhibition at the level of downstream lysosomal function. Mechanistically, this involves elevations in PARIS protein levels and subsequent effects on PGC1α-TFEB signaling that normally regulates mitochondrial quality control. Treatment with rapamycin independently restores PGC1α-TFEB signaling in a manner not requiring parkin activity and abrogates subsequent mitochondrial impairment and neuronal cell loss. Taken in total, our data suggest that the parkin Q311X mutation impacts on mitochondrial quality control via PARIS-mediated regulation of PGC1α-TFEB signaling and that this can be independently restored via rapamycin., (Copyright © 2015 the authors 0270-6474/15/3512833-12$15.00/0.)

Cellular senescence is a potent anti-cancer mechanism that arrests the proliferation of mitotically competent cells to prevent malignant transformation. Senescent cells accumulate with age in a variety of human and mouse tissues where they express a complex 'senescence-associated secretory phenotype' (SASP). The SASP includes many pro-inflammatory cytokines, chemokines, growth factors and proteases that have the potential to cause or exacerbate age-related pathology, both degenerative and hyperplastic. While cellular senescence in peripheral tissues has recently been linked to a number of age-related pathologies, its involvement in brain aging is just beginning to be explored. Recent data generated by several laboratories suggest that both aging and age-related neurodegenerative diseases are accompanied by an increase in SASP-expressing senescent cells of non-neuronal origin in the brain. Moreover, this increase correlates with neurodegeneration. Senescent cells in the brain could therefore constitute novel therapeutic targets for treating age-related neuropathologies., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Aims: Pharmacological activation of the adaptive response to hypoxia is a therapeutic strategy of growing interest for neurological conditions, including stroke, Huntington's disease, and Parkinson's disease. We screened a drug library with known safety in humans using a hippocampal neuroblast line expressing a reporter of hypoxia-inducible factor (HIF)-dependent transcription., Results: Our screen identified more than 40 compounds with the ability to induce hypoxia response element-driven luciferase activity as well or better than deferoxamine, a canonical activator of hypoxic adaptation. Among the chemical entities identified, the antihelminthic benzimidazoles represented one pharmacophore that appeared multiple times in our screen. Secondary assays confirmed that antihelminthics stabilized the transcriptional activator HIF-1α and induced expression of a known HIF target gene, p21(cip1/waf1), in post-mitotic cortical neurons. The on-target effect of these agents in stimulating hypoxic signaling was binding to free tubulin. Moreover, antihelminthic benzimidazoles also abrogated oxidative stress-induced death in vitro, and this on-target effect also involves binding to free tubulin., Innovation and Conclusions: These studies demonstrate that tubulin-binding drugs can activate a component of the hypoxic adaptive response, specifically the stabilization of HIF-1α and its downstream targets. Tubulin-binding drugs, including antihelminthic benzimidazoles, also abrogate oxidative neuronal death in primary neurons. Given their safety in humans and known ability to penetrate into the central nervous system, antihelminthic benzimidazoles may be considered viable candidates for treating diseases associated with oxidative neuronal death, including stroke.

Lithium has long been used as a treatment for the psychiatric disease bipolar disorder. However, previous studies suggest that lithium provides neuroprotective effects in neurodegenerative diseases such as Parkinson's disease (PD) and Alzheimer's disease. The exact mechanism by which lithium exerts these effects still remains unclear. In the present study, we evaluated the effects of low-dose lithium treatment in an aged mouse model expressing a parkin mutation within dopaminergic neurons. We found that low-dose lithium treatment prevented motor impairment as demonstrated by the open field test, pole test, and rearing behavior. Furthermore, lithium prevented dopaminergic striatal degeneration in parkin animals. We also found that parkin-induced striatal astrogliosis and microglial activation were prevented by lithium treatment. Our results further corroborate the use of this parkin mutant transgenic mouse line as a model for PD for testing novel therapeutics. The findings of the present study also provide further validation that lithium could be re-purposed as a therapy for PD and suggest that anti-inflammatory effects may contribute to its neuroprotective mechanisms., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Previously published data from our laboratory demonstrated that pharmacological inhibition of a family of enzymes known as prolyl hydroxylase domain proteins prevents neurotoxicity associated with the acute 1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine intoxication model of Parkinson's disease in young animals. In this study, we assessed whether prolyl hydroxylase domain inhibition was neuroprotective in an inducible genetic dopaminergic glutathione depletion model previously characterized by our laboratory that more closely recapitulates the age-related and progressive nature of the human disease. Pharmacological prolyl hydroxylase domain inhibition via 3,4-dihydroxybenzoate was found to significantly attenuate hallmark mitochondrial dysfunction and loss of dopaminergic substantia nigral pars compacta neurons associated with this model. These studies further validate the possibility that prolyl hydroxylase domain inhibition may constitute a viable therapy for Parkinson's disease.

Accumulation of mitochondrial DNA deletions is observed especially in dopaminergic neurons of the substantia nigra during ageing and even more in Parkinson's disease. The resulting mitochondrial dysfunction is suspected to play an important role in neurodegeneration. However, the molecular mechanisms involved in the preferential generation of mitochondrial DNA deletions in dopaminergic neurons are still unknown. To study this phenomenon, we developed novel polymerase chain reaction strategies to detect distinct mitochondrial DNA deletions and monitor their accumulation patterns. Applying these approaches in in vitro and in vivo models, we show that catecholamine metabolism drives the generation and accumulation of these mitochondrial DNA mutations. As in humans, age-related accumulation of mitochondrial DNA deletions is most prominent in dopaminergic areas of mouse brain and even higher in the catecholaminergic adrenal medulla. Dopamine treatment of terminally differentiated neuroblastoma cells, as well as stimulation of dopamine turnover in mice over-expressing monoamine oxidase B both induce multiple mitochondrial DNA deletions. Our results thus identify catecholamine metabolism as the driving force behind mitochondrial DNA deletions, probably being an important factor in the ageing-associated degeneration of dopaminergic neurons.

Exposure to environmental toxins is associated with a variety of age-related diseases including cancer and neurodegeneration. For example, in Parkinson's disease (PD), chronic environmental exposure to certain toxins has been linked to the age-related development of neuropathology. Neuronal damage is believed to involve the induction of neuroinflammatory events as a consequence of glial cell activation. Cellular senescence is a potent anti-cancer mechanism that occurs in a number of proliferative cell types and causes the arrest of proliferation of cells at risk of malignant transformation following exposure to potentially oncogenic stimuli. With age, senescent cells accumulate and express a senescence-associated secretory phenotype (SASP; that is the robust secretion of many inflammatory cytokines, growth factors and proteases). Whereas cell senescence in peripheral tissues has been causally linked to a number of age-related pathologies, little is known about the induction of cellular senescence and the SASP in the brain. On the basis of recently reported findings, we propose that environmental stressors associated with PD may act in part by eliciting senescence and the SASP within non neuronal glial cells in the ageing brain, thus contributing to the characteristic decline in neuronal integrity that occurs in this disorder., (© 2013 The Association for the Publication of the Journal of Internal Medicine.)

Microglial activation and subsequent release of toxic pro-inflammatory factors are believed to play an important role in neuronal cell death associated with Parkinson's disease (PD). Compounds that inhibit microglia activation and suppress pro-inflammatory factor release have been reported to have neuroprotective effects in animal models of PD. In this study, we tested whether diadzein, a natural isoflavone found in soybean, attenuated lipopolysaccharide (LPS)-induced release of inflammatory mediators in BV-2, a murine microglial cell line. Diadzein pretreatment was found to significantly suppress the production of the pro-inflammatory factors nitric oxide and IL-6 as well as their mRNA expression in conjunction with reductions in ROS production, p38 MAPK phosphorylation, and NF-κB activation. Furthermore, transfer of conditioned media (CM) from BV-2 cells pretreated with diadzein resulted in a significantly reduction in dopaminergic neurotoxicity compared with CM from microglia stimulated with LPS alone. Together, our results suggest that diadzein's neuroprotective properties may be due to its ability to dampen induction of microglial activation and the subsequent release of soluble pro-inflammatory factors. This appears to be via inhibition of oxidative induction of the p38 MAP kinase-NFκB pathway, resulting in reduced expression of pro-inflammatory genes and release of their corresponding gene products.

We have previously shown that increases in astrocytic monoamine oxidase-B (MAO-B) expression, mimicking that which occurs with aging and in neurodegenerative disease, in a doxycycline (dox)-inducible transgenic mouse model evokes neuropathological similarities to what is observed in the human parkinsonian brain. Additional behavioral and neuropathological studies could provide further validation for its usage as a model for Parkinson's disease (PD). In the present study, we utilized a battery of behavioral tests to evaluate age-related phenotype in this model. In the open field test, we found that dox-induction impaired motor ability with decreases in movement and ambulatory function as well as diminished stereotypical, repetitive movement episodes in both young and old mice. Older mice also showed decreased motor performance in the pole test when compared to younger mice. Furthermore, dox-induced older mice displayed severe hindlimb clasping and the most significant loss of dopamine (DA) in the striatum when compared to young and non-induced animals. Additionally, increased MAO-B activity significantly correlated with decreased expression of striatal DA. The results of our study further confirms that the dox-inducible astrocytic MAO-B transgenic mouse displays similar age-related behavioral and neuropathological features to other models of PD, and could serve as a useful tool to study PD pathophysiology and for the evaluation of therapeutic interventions.

Alpha-synuclein has been reported to be present in the nucleus and levels enhanced by oxidative stress. Herein, we sought to investigate the mechanistic role of nuclear alpha-synuclein. We found that alpha-synuclein nuclear localization coincided with enhanced chromatin binding both in an in vitro and a corresponding in vivo brain oxidative stress model previously characterized by our laboratory as well as in PD brain tissues. Genome-wide chromatin immunoprecipitation (ChIP)-on-chip analysis of alpha-synuclein:promoter binding in response to oxidative stress in vitro revealed that binding occurs at several promoters belonging to a range of functional categories including transcriptional regulation. Interestingly, given the important role of mitochondrial dysfunction in PD, this included binding to the promoter for the master mitochondrial transcription activator, PGC1alpha in vitro, in vivo, and in human brain tissue with age and PD. To test the possible mechanistic impact of alpha-synuclein PGC1alpha promotor binding, we assessed PGC1alpha promoter activity, mRNA, and protein levels and expression of candidate PGC1alpha target genes in our in vitro model. All were found to be reduced in conjunction with increased levels of aberrant mitochondrial morphology and impaired mitochondrial function. Exogenous PGC1alpha expression was found to attenuate alpha-synuclein-mediated mitochondrial dysfunction and subsequent neurotoxicity in vitro. Our data suggest that nuclear alpha-synuclein localization under conditions of oxidative stress may impact on mitochondrial function in part via the protein's capacity to act as a transcriptional modulator of PGC1alpha. This represents a novel role for alpha-synuclein as it relates to mitochondrial dysfunction in PD., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Parkinson's disease (PD) is an age-related neurodegenerative disorder characterized in part by the preferential loss of nigrostriatal dopaminergic neurons. Although the precise etiology of PD is unknown, accumulating evidence suggests that PD involves microglial activation that exerts neurotoxic effects through production of proinflammatory cytokines and increased oxidative and nitrosative stress. Thus, controlling microglial activation has been suggested as a therapeutic target for combating PD. Previously we demonstrated that pharmacological inhibition of a class of enzymes known as prolyl hydroxylases via 3,4-dihydroxybenzoate administration protected against MPTP-induced neurotoxicity, however the exact mechanisms involved were not elucidated. Here we show that this may be due to DHB's ability to inhibit microglial activation. DHB significantly attenuated LPS-mediated induction of nitric oxide synthase and pro-inflammatory cytokines in murine BV2 microglial cells in vitro in conjunction with reduced ROS production and activation of NFκB and MAPK pathways possibly due to up-regulation of HO-1 levels. HO-1 inhibition partially abrogates LPS-mediated NFκB activity and subsequent NO induction. In vivo, DHB pre-treatment suppresses microglial activation elicited by MPTP treatment. Our results suggest that DHB's neuroprotective properties could be due to its ability to dampen induction of microglial activation via induction of HO-1.

Impairment of the mitochondrial electron transport chain has been suggested to be a critical factor in the neuropathogenesis of Parkinson's disease (PD), as inhibition of mitochondrial complex I (CI) activity is consistently detected in PD patients as well as in mitochondrial toxin models of the disorder. Increased levels of various reactive oxygen and nitrogen species appear to contribute to CI inhibition and mitochondrial dysfunction in PD. Reactive nitrogen species (RNS) such as nitric oxide (NO) and its metabolite peroxynitrite (PN) may inhibit CI activity via several different mechanisms including S-nitrosylation, nitration, and protein thiol formation. Studies using various cell and animal PD models have demonstrated that selective mitochondrial CI inhibition in dopaminergic cells may be due to both NO-mediated S-nitrosylation and nitration of CI sub-units. Strategies to modulate mitochondrial NO levels will therefore likely be a promising approach to enhance mitochondrial function and protect dopaminergic neurons against oxidative or nitrosative insult.

Alpha-synuclein is the major protein component of Lewy bodies, a cardinal pathological feature of the degenerating Parkinsonian brain. Alpha-synuclein has been reported to be able to intercalate into membranes via formation of an alpha-helical structure at its N-terminal end. Recent in vitro studies from various laboratories have demonstrated that α-synuclein can physically associate with mitochondria and interfere with mitochondrial function. α-Syn predominantly associates with the inner mitochondrial membrane, where it can apparently interact with complex I resulting in reduced mitochondrial complex I activity and increased free radical production. However, the effect of in vivo α-synuclein accumulation within dopaminergic neurons on mitochondrial function has not been thoroughly studied. Examination of transgenic animals which overexpress the familial mutant A53T form of the protein selectively within dopaminergic neurons reveals that A53T localizes to the mitochondrial membranes as monomers and oligomers particularly under conditions of proteasomal inhibitory stress, and that this localization coincides with a selective age-related mitochondrial complex I inhibition and decreased substrate-specific respiration along with increases in mitochondrial autophagy (mitophagy)., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

We previously demonstrated that spare respiratory capacity of the TCA cycle enzyme alpha-ketoglutarate dehydrogenase (KGDH) was completely abolished upon increasing levels of MAO-B activity in a dopaminergic cell model system (Kumar et al., J Biol Chem 278:46432-46439, 2003). MAO-B mediated increases in H(2)O(2) also appeared to result in direct oxidative inhibition of both mitochondrial complex I and aconitase. In order to elucidate the contribution that each of these components exerts over metabolic respiratory control as well as the impact of MAO-B elevation on their spare respiratory capacities, we performed metabolic respiratory control analysis. In addition to KGDH, we assessed the activities and substrate-mediated respiration of complex I, pyruvate dehydrogenase (PDH), succinate dehydrogenase (SDH), and mitochondrial aconitase in the absence and presence of complex-specific inhibitors in specific and mixed substrate conditions in mitochondria from our MAO-B elevated cells versus controls. Data from this study indicates that Complex I and KGDH are the most sensitive to inhibition by MAO-B mediated H(2)O(2) generation, and could be instrumental in determining the fate of mitochondrial metabolism in this cellular PD model system.

Parkinson's disease (PD) is characterized by early glutathione depletion in the substantia nigra (SN). Among its various functions in the cell, glutathione acts as a substrate for the mitochondrial enzyme glutaredoxin 2 (Grx2). Grx2 is involved in glutathionylation of protein cysteine sulfhydryl residues in the mitochondria. Although monothiol glutathione-dependent oxidoreductases (Grxs) have previously been demonstrated to be involved in iron-sulfur (Fe-S) center biogenesis, including that in yeast, here we report data suggesting the involvement of mitochondrial Grx2, a dithiol Grx, in iron-sulfur biogenesis in a mammalian dopaminergic cell line. Given that mitochondrial dysfunction and increased cellular iron levels are two important hallmarks of PD, this suggests a novel potential mechanism by which glutathione depletion may affect these processes in dopaminergic neurons. We report that depletion of glutathione as substrate results in a dose-dependent Grx2 inhibition and decreased iron incorporation into a mitochondrial complex I (CI) and aconitase (m-aconitase). Mitochondrial Grx2 inhibition through siRNA results in a corresponding decrease in CI and m-aconitase activities. It also results in significant increases in iron-regulatory protein (IRP) binding, likely as a consequence of conversion of Fe-S-containing cellular aconitase to its non-Fe-S-containing IRP1 form. This is accompanied by increased transferrin receptor, decreased ferritin, and subsequent increases in mitochondrial iron levels. This suggests that glutathione depletion may affect important pathologic cellular events associated with PD through its effects on Grx2 activity and mitochondrial Fe-S biogenesis.

Resveratrol, a naturally occurring polyphenol, exhibits antioxidant, antiaging, and anticancer activity. Resveratrol has also been shown to inhibit tumor initiation, promotion, and progression in a variety of cell culture systems. Earlier, we showed that paraquat, a bipyridyl herbicide, triggers endoplasmic reticulum stress, cell dysfunction, and dopaminergic cell death. Due to its antioxidant activity, we assessed the ability of resveratrol to rescue cells from the toxic effects of paraquat. While resveratrol did not have any protective effect at low concentrations, it triggered endoplasmic reticulum (ER) stress-induced cell death at higher concentrations (50-250 microM). The present study was carried out to determine the mechanism by which resveratrol triggers ER stress and cell death in dopaminergic N27 cells. Our studies demonstrate that resveratrol triggers ER stress and cell dysfunction, caspase activation, p23 cleavage and inhibition of proteasomal activity in dopaminergic N27 cells. While over expression of uncleavable p23 was associated with decreased cell death, downregulation of p23 protein expression by siRNA resulted in enhancement of ER stress-induced cell death triggered by resveratrol indicating a protective role for the small co-chaperone p23 in dopaminergic cell death.

Apoptosis-inducing factor (AIF)-deficient harlequin (Hq) mice undergo neurodegeneration associated with a 40-50% reduction in complex I level and activity. We tested the hypothesis that AIF and complex I regulate reactive oxygen species (ROS) production by brain mitochondria. Isolated Hq brain mitochondria oxidizing complex I substrates displayed no difference compared to wild type (WT) in basal ROS production, H2O2 removal, or ROS production stimulated by complex I inhibitors rotenone or 1-methyl-4-phenylpyridinium. In contrast, ROS production caused by reverse electron transfer to complex I was attenuated by approximately 50% in Hq mitochondria oxidizing the complex II substrate succinate. Basal and rotenone-stimulated rates of H2O2 release from in situ mitochondria did not differ between Hq and WT synaptosomes metabolizing glucose, nor did the level of in vivo oxidative protein carbonyl modifications detected in synaptosomes, brain mitochondria, or homogenates. Our results suggest that AIF does not directly modulate ROS release from brain mitochondria. In addition, they demonstrate that in contrast to ROS produced by mitochondria oxidizing succinate, ROS release from in situ synaptosomal mitochondria or from isolated brain mitochondria oxidizing complex I substrates is not proportional to the amount of complex I. These findings raise the important possibility that complex I contributes less to physiological ROS production by brain mitochondria than previously suggested.

Dopaminergic neurodegeneration during Parkinson disease (PD) involves several pathways including proteasome inhibition, alpha-synuclein (alpha-syn) aggregation, mitochondrial dysfunction, and glutathione (GSH) depletion. We have utilized a systems biology approach and built a dynamic model to understand and link the various events related to PD pathophysiology. We have corroborated the modeling data by examining the effects of alpha-syn expression in the absence and presence of proteasome inhibition on GSH metabolism in dopaminergic neuronal cultures. We report here that the expression of the mutant A53T form of alpha-syn is neurotoxic and causes GSH depletion in cells after proteasome inhibition, compared to wild-type alpha-syn-expressing cells and vector control. Modeling data predicted that GSH depletion in these cells was due to ATP loss associated with mitochondrial dysfunction. ATP depletion elicited by combined A53T expression and proteasome inhibition results in decreased de novo synthesis of GSH via the rate-limiting enzyme gamma-glutamyl cysteine ligase. Based on these data and other recent reports, we propose a novel dynamic model to explain how the presence of mutated alpha-syn protein or proteasome inhibition may individually impact on mitochondrial function and in combination result in alterations in GSH metabolism via enhanced mitochondrial dysfunction.

Parkinson's disease (PD) is an adult-onset neurodegenerative disorder characterized by preferential loss of dopaminergic neurons in an area of the midbrain called the substantia nigra (SN) along with occurrence of intraneuronal inclusions called Lewy bodies. The majority of cases of PD are sporadic in nature with late onset (95% of patients); however a few PD cases (5%) are seen in familial clusters with generally earlier onset. Although PD has been heavily researched, so far the exact cause of the rather selective cell death is unknown. Multiple lines of evidence suggest an important role for oxidative stress. Dopaminergic neurons (DA) are particularly prone to oxidative stress due to DA metabolism and auto-oxidation combined with increased iron, decreased total glutathione levels and mitochondrial complex I inhibition-induced ROS production in the SN which can lead to cell death by exceeding the oxidative capacity of DA-containing cells in the region. Enhancing antioxidant capabilities and chelating labile iron pools in this region therefore constitutes a rational approach to prevent or slow ongoing damage of DA neurons. In this review, we summarize the various sources of reactive oxygen species that may cause redox imbalance in PD as well as potential therapeutic targets for attenuation of oxidative stress associated with PD.

Age-related increases in monoamine oxidase B (MAO-B) may contribute to neurodegeneration associated with Parkinson's disease (PD). The MAO-B inhibitor deprenyl, a long-standing antiparkinsonian therapy, is currently used clinically in concert with the dopamine precursor L-DOPA. Clinical studies suggesting that deprenyl treatment alone is not protective against PD associated mortality were targeted to symptomatic patients. However, dopamine loss is at least 60% by the time PD is symptomatically detectable, therefore lack of effect of MAO-B inhibition in these patients does not negate a role for MAO-B in pre-symptomatic dopaminergic loss. In order to directly evaluate the role of age-related elevations in astroglial MAO-B in the early initiation or progression of PD, we created genetically engineered transgenic mice in which MAO-B levels could be specifically induced within astroglia in adult animals. Elevated astrocytic MAO-B mimicking age related increase resulted in specific, selective and progressive loss of dopaminergic neurons in the substantia nigra (SN), the same subset of neurons primarily impacted in the human condition. This was accompanied by other PD-related alterations including selective decreases in mitochondrial complex I activity and increased mitochondrial oxidative stress. Along with a global astrogliosis, we observed local microglial activation within the SN. These pathologies correlated with decreased locomotor activity. Importantly, these events occurred even in the absence of the PD-inducing neurotoxin MPTP. Our data demonstrates that elevation of murine astrocytic MAO-B by itself can induce several phenotypes of PD, signifying that MAO-B could be directly involved in multiple aspects of disease neuropathology. Mechanistically this may involve increases in membrane permeant H(2)O(2) which can oxidize dopamine within dopaminergic neurons to dopaminochrome which, via interaction with mitochondrial complex I, can result in increased mitochondrial superoxide. Our inducible astrocytic MAO-B transgenic provides a novel model for exploring pathways involved in initiation and progression of several key features associated with PD pathology and for therapeutic drug testing.

Parkinson's disease (PD) features oxidative stress and accumulation of misfolded (unfolded, alternatively folded, or mutant) proteins with associated loss of dopaminergic neurons. Oxidative stress and the accumulated misfolded proteins elicit cellular responses that include an endoplasmic reticulum (ER) stress response that may protect cells against the toxic buildup of misfolded proteins. Chronic ER stress and accumulation of misfolded proteins in excessive amounts, however, overwhelm the cellular 'quality control' system and impair the protective mechanisms designed to promote correct folding and degrade faulty proteins, ultimately leading to organelle dysfunction and neuronal cell death. Paraquat belongs to a class of bipyridyl herbicides and triggers oxidative stress and dopaminergic cell death. Epidemiological studies suggest an increased risk for developing PD following chronic exposure to paraquat. The present study was carried out to determine the role of paraquat in triggering cellular stress particularly ER stress and to elucidate the pathways that couple ER stress to dopaminergic cell death. We demonstrate that paraquat triggers ER stress, cell dysfunction, and dopaminergic cell death. p23, a small co-chaperone protein, is cleaved during ER stress-induced cell death triggered by paraquat and blockage of the caspase cleavage site of p23 was associated with decreased cell death. Paraquat also inhibits proteasomal activity that may further trigger accumulation of misfolded proteins resulting in ER stress. Our results indicate a protective role for p23 in PD-related programmed cell death. The data also underscore the involvement of ER, caspases, and the proteasomal system in ER stress-induced cell death process.

Parkinson's disease is a neurodegenerative disorder characterized by the preferential loss of midbrain dopaminergic neurons in the substantia nigra (SN). One of the earliest detectable biochemical alterations that occurs in the Parkinsonian brain is a marked reduction in SN levels of total glutathione (glutathione plus glutathione disulfide), occurring before losses in mitochondrial complex I (CI) activity, striatal dopamine levels, or midbrain dopaminergic neurodegeneration associated with the disease. Previous in vitro data from our laboratory has suggested that prolonged depletion of dopaminergic glutathione results in selective impairment of mitochondrial complex I activity through a reversible thiol oxidation event. To address the effects of depletion in dopaminergic glutathione levels in vivo on the nigrostriatal system, we created genetically engineered transgenic mouse lines in which expression of gamma-glutamyl cysteine ligase, the rate-limiting enzyme in de novo glutathione synthesis, can be inducibly downregulated in catecholaminergic neurons, including those of the SN. A novel method for isolation of purified dopaminergic striatal synaptosomes was used to study the impact of dopaminergic glutathione depletion on mitochondrial events demonstrated previously to occur in vitro as a consequence of this alteration. Dopaminergic glutathione depletion was found to result in a selective reversible thiol-oxidation-dependent mitochondrial complex I inhibition, followed by an age-related nigrostriatal neurodegeneration. This suggests that depletion in glutathione within dopaminergic SN neurons has a direct impact on mitochondrial complex I activity via increased nitric oxide-related thiol oxidation and age-related dopaminergic SN cell loss.

Iron elevation is well-documented in the Parkinsonian midbrain but its cause and contribution to subsequent neurodegeneration remain unknown. Mice administered iron at doses equivalent to those found in iron-fortified human infant formula during a developmental period equivalent to the first human year of life display progressive midbrain neurodegeneration and enhanced vulnerability to toxic injury. This may have major implications for the impact of neonatal iron intake as a potential risk factor for later development of Parkinson's disease (PD).

The pathogenesis underlying the selective degeneration of nigral dopaminergic neurons in Parkinson's disease is not fully understood but several lines of evidence implicate the role of oxidative stress and mitochondrial dysfunction. Depletion in levels of the thiol reducing agent glutathione (GSH + GSSG) is the earliest reported biochemical event to occur in the Parkinsonian substantia nigra prior to selective loss of complex I (CI) activity associated with the disease believed to contribute to subsequent dopaminergic cell death. Recent studies from our laboratory have demonstrated that acute reduction in both cellular and mitochondrial glutathione levels results in increased oxidative stress and a decrease in mitochondrial function linked to a selective decrease in CI activity through an NO-mediated mechanism (Jha, N.; Jurma, O.; Lalli, G.; Liu, Y.; Pettus, E. H.; Greenamyre, J. T.; Liu, R. M.; Forman, H. J.; Andersen, J. K. Glutathione depletion in PC12 results in selective inhibition of mitochondrial complex I activity. Implications for Parkinson's disease J. Biol. Chem. 275: 26096-26101; 2000. Hsu, M.; Srinivas, B.; Kumar, J.; Subramanian, R.; Andersen, J. Glutathione depletion resulting in selective mitochondrial complex I inhibition in dopaminergic cells is via an NO-mediated pathway not involving peroxynitrite: implications for Parkinson's disease J. Neurochem. 92: 1091-1103.2005.). However, the effect of prolonged glutathione depletion on dopaminergic cells is not known. In this present study, using low concentrations of buthionine-S-sulfoximine, a chemical inhibitor of the de novo glutathione synthesizing enzyme glutamate cysteine ligase, we developed a chronic model in which glutathione depletion in dopaminergic N27 cells for a 7-day period was found to lead to inhibition of CI activity via a peroxynitrite-mediated event which is reversible by the thiol reducing agent, dithiothreitol, and coincides with increased S-nitrosation of mitochondrial proteins.

Glutathione is an abundant intracellular thiol antioxidant whose levels are reduced both in Parkinson's disease itself and in a widely used animal model of the disorder, systemic MPTP administration. Previous in vitro work from our laboratory has suggested that glutathione depletion may be directly responsible for mitochondrial dysfunction, which ultimately leads to dopaminergic cell death associated with the disease. Here, we demonstrate the ability of gamma-glutamylcysteine ethyl ester, a lipid permeable derivative of the major substrate for scavenger glutathione synthesis, to counteract glutathione loss and neurodegeneration associated with in vitro and in vivo administration of MPTP or its derivatives. This data suggests that prevention of glutathione depletion is a likely therapeutic target for the disease.

Up-regulation of activity of gamma-glutamyl transpeptidase (GGT) has been reported to occur in the Parkinsonian substantia nigra, the area of the brain affected by the disease. Increased GGT activity has been hypothesized to play a role in subsequent mitochondrial complex I (CI) inhibition by increasing cysteine as substrate for cellular uptake. Intracellular cysteine has been proposed to form toxic adducts with dopamine which can be metabolized to compounds which inhibit CI activity. We have demonstrated that in addition to CI inhibition, GGT activity is up-regulated in dopaminergic cells as a consequence of glutathione depletion. Inhibition of GGT rather than resulting in increased CI inhibition results in exacerbation of this inhibitory effect. This suggests that increased GGT activity is likely an adaptive response to the loss of glutathione to conserve intracellular glutathione content and results in a compensatory effect on CI activity rather than in its inhibition as has been previously widely hypothesized.

Cytochrome P-450 function as mono-oxygenases and metabolize xenobiotics. CYP1A1, a cytochrome P-450 enzyme, bioactivates polycyclic aromatic hydrocarbons to reactive metabolite(s) that bind to DNA and initiate carcinogenesis. Northern and immunoblot analyses revealed constitutive expression of Cyp1a1 and CYP1A1 in rat and human brain, respectively. CYP1A1 mRNA and protein were localized predominantly in neurons of cerebral cortex, Purkinje and granule cell layers of cerebellum and pyramidal neurons of CA1, CA2, and CA3 subfields of the hippocampus. RT-PCR analyses using RNA obtained from autopsy human brain samples demonstrated the presence of a splice variant having a deletion of 87 bp of exon 6. This splice variant was present in human brain, but not in the liver from the same individual, and was absent in rat brain and liver. Structural modeling indicated broadening of the substrate access channel in the brain variant. The study demonstrates the presence of a unique cytochrome P-450 enzyme in human brain that is generated by alternate splicing. The presence of distinct cytochrome P-450 enzymes in human brain that are different from well-characterized hepatic forms indicates that metabolism of xenobiotics including drugs could occur in brain by pathways different from those known to occur in liver.

Dopaminergic neurons of the midbrain are the main source of dopamine (DA) in the mammalian central nervous system. Their loss is associated with one of the most prominent human neurological disorders, Parkinson's disease (PD). Dopaminergic neurons are found in a 'harsh' region of the brain, the substantia nigra pars compacta, which is DA-rich and contains both redox available neuromelanin and a high iron content. Although their numbers are few, these dopaminergic neurons play an important role in the control of multiple brain functions including voluntary movement and a broad array of behavioral processes such as mood, reward, addiction, and stress. Studies into the developmental pathways which are involved in the generation of dopaminergic neurons in the brain have led to the identification of several specific transcription factors including Nurr1, Lmx1b and Pitx3, all shown to be important in the development of the mesencephalic dopaminergic system. The selective degeneration of these dopaminergic neurons in the substantia nigra pars compacta leads to PD but the exact cause for this nigral cell loss is still unknown.

Cytochromes P450 (P450), a family of heme-containing proteins, is involved in the oxidative metabolism of both foreign and endogenous compounds. Although liver is quantitatively the major organ involved in the metabolism of most xenobiotics, there is increasing evidence that these enzymes are present in extrahepatic tissues, such as lung, kidney, brain, etc and they may contribute to the in situ metabolism of xenobiotics in these organs. The possible relationship between genetic polymorphism seen in P4502D6 and incidence of neurodegenerative diseases, such as Parkinson's disease, has prompted the characterization of P4502D enzymes in rat brain. In the present study, we demonstrate that P4502D1 (the rat homologue of human P4502D6) is constitutively expressed in rat brain and the mRNA and protein are localized predominantly in neuronal cell population in the olfactory bulb, cortex, cerebellum, and hippocampus. An alternate spliced transcript of CYP2D1 having exon 3 deletion was detected in rat brain but not in liver. Deletion of exon 3 causes frame shift and generates a stop codon at 391 bp relative to the start codon ATG leading to premature termination of translation. Thus, Northern blotting and in situ hybridization represent contributions from functional transcripts and alternate spliced variants that do not translate into functional protein. Further, the splice variant having partial inclusion of intron 6 detected in human brain was not detected in rat brain indicating that alternate spliced gene products of P450 enzymes are generated in species-specific and tissue-specific manner.

A frameshift mutation 138delT generates an open reading frame in the pseudogene, cytochrome P4502D7 (CYP2D7), and an alternate spliced functional transcript of CYP2D7 containing partial inclusion of intron 6 was identified in human brain but not in liver or kidney from the same individual. mRNA and protein of the brain variant CYP2D7 were detected in 6 of 12 human autopsy brains. Genotyping revealed the presence of the frameshift mutation 138delT only in those human subjects who expressed the brain variant CYP2D7. Genomic DNA analysis in normal volunteers revealed the presence of functional CYP2D7 in 4 of 8 individuals. In liver, the major organ involved in drug metabolism, a minor metabolic pathway mediated by CYP2D6 metabolizes codeine (pro-drug) to morphine (active drug), whereas norcodeine is the major metabolite. In contrast, when expressed in Neuro2a cells, brain variant CYP2D7 metabolized codeine to morphine with greater efficiency compared with the corresponding activity in cells expressing CYP2D6. Morphine binds to micro-opioid receptors in certain regions of the central nervous system, such as periaqueductal gray, and produces pain relief. The brain variant CYP2D7 and micro-opioid receptor colocalize in neurons of the periaqueductal gray area in human brain, indicating that metabolism of codeine to morphine could occur at the site of opioid action. Histio-specific isoforms of P450 generated by alternate splicing, which mediate selective metabolism of pro-drugs within tissues, particularly the brain, to generate active drugs may play an important role in drug action and provide newer insights into the genetics of metabolism.

Cytochrome P4502D6, an important isoform of cytochrome P450, mediates the metabolism of several psychoactive drugs in liver. Quantitatively, liver is the major drug metabolizing organ, however metabolism of drugs in brain could modulate pharmacological and pharmacodynamic effects of psychoactive drugs at their site of action and explain some of the variation typically seen in patient population. We have measured cytochrome P450 content and examined constitutive expression of CYP2D mRNA and protein in human brain regions by reverse transcription polymerase chain reaction, Northern and immunoblotting and localized it by in situ hybridization and immunohistochemistry. CYP2D mRNA was expressed constitutively in neurons of cerebral cortex, Purkinje and granule cell layers of cerebellum, reticular neurons of midbrain and pyramidal neurons of CA1, CA2 and CA3 subfields of hippocampus. Immunoblot studies demonstrated the presence of cytochrome P4502D protein in cortex, cerebellum, midbrain, striatum and thalamus of human brain. Immunohistochemical localization showed the predominant presence of cytochrome P4502D not only in neuronal soma but also in dendrites of Purkinje and cortical neurons. These studies demonstrate constitutive expression of cytochrome P4502D in neuronal cell population in human brain, indicating its possible role in metabolism of psychoactive drugs directly at or near their site of action, in neurons, in human brain., (Copyright 2002 Elsevier Science B.V.)