Revisiting Proteasome Inhibitors: Molecular Underpinnings of Their Development, Mechanisms of Resistance and Strategies to Overcome Anti-Cancer Drug Resistance.

Proteasome inhibitors have shown relevant clinical activity in several hematological malignancies, namely in multiple myeloma and mantle cell lymphoma, improving patient outcomes such as survival and quality of life, when compared with other therapies. However, initial response to the therapy is a challenge as most patients show an innate resistance to proteasome inhibitors, and those that respond to the therapy usually develop late relapses suggesting the development of acquired resistance. The mechanisms of resistance to proteasome inhibition are still controversial and scarce in the literature. In this review, we discuss the development of proteasome inhibitors and the mechanisms of innate and acquired resistance to their activity-a major challenge in preclinical and clinical therapeutics. An improved understanding of these mechanisms is crucial to guiding the design of new and more effective drugs to tackle these devastating diseases. In addition, we provide a comprehensive overview of proteasome inhibitors used in combination with other chemotherapeutic agents, as this is a key strategy to combat resistance.

Neurotoxic effects of MPTP on mouse cerebral cortex: Modulation of neuroinflammation as a neuroprotective strategy.

Parkinson's disease (PD) is a progressive neurological disorder, mainly characterized by the progressive loss of dopaminergic neurons in the Substantia nigra pars compacta (SNpc) and by the presence of intracellular inclusions, known as Lewy bodies. Despite SNpc being considered the primary affected region in PD, the neuropathological features are confined solely to the nigro-striatal axis. With disease progression other brain regions are also affected, namely the cerebral cortex, although the spreading of the neurologic damage to this region is still not completely unraveled. Tauroursodeoxycholic acid (TUDCA) is an endogenous bile acid that has been shown to have antioxidant properties and to exhibit a neuroprotective effect in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mice model of PD. Moreover, TUDCA anti-inflammatory properties have been reported in glial cells, making it a prominent therapeutic agent in PD. Here, we used C57BL/6 mice injected with MPTP in a sub-acute paradigm aiming to investigate if the neurotoxic effects of MPTP could be extended to the cerebral cortex. In parallel, we evaluated the anti-oxidant, neuroprotective and anti-inflammatory effects of TUDCA. The anti-inflammatory mechanisms elicited by TUDCA were further dissected in microglia cells. Our results show that MPTP leads to a decrease of ATP and activated AMP-activated protein kinase levels in mice cortex, and to a transient increase in the expression of antioxidant downstream targets of nuclear factor erythroid 2 related factor 2 (Nrf-2), and parkin. Notably, MPTP increases pro-inflammatory markers, while down-regulating the expression of the anti-inflammatory protein Annexin-A1 (ANXA1). Importantly, we show that TUDCA treatment prevents the deleterious effects of MPTP, sustains increased levels of antioxidant enzymes and parkin, and most of all negatively modulates neuroinflammation and up-regulates ANXA1 expression. Additionally, results from cellular models using microglia corroborate TUDCA modulation of ANXA1 synthesis, linking inhibition of neuroinflammation and neuroprotection by TUDCA.

Novel insights into the antioxidant role of tauroursodeoxycholic acid in experimental models of Parkinson's disease.

Impaired mitochondrial function and generation of reactive oxygen species are deeply implicated in Parkinson's disease progression. Indeed, mutations in genes that affect mitochondrial function account for most of the familial cases of the disease, and post mortem studies in sporadic PD patients brains revealed increased signs of oxidative stress. Moreover, exposure to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a mitochondrial complex I inhibitor, leads to clinical symptoms similar to sporadic PD. The bile acid tauroursodeoxycholic acid (TUDCA) is an anti-apoptotic molecule shown to protect against MPTP-induced neurodegeneration in mice, but the mechanisms involved are still incompletely identified. Herein we used MPTP-treated mice, as well as primary cultures of mice cortical neurons and SH-SY5Y cells treated with MPP+ to investigate the modulation of mitochondrial dysfunction by TUDCA in PD models. We show that TUDCA exerts its neuroprotective role in a parkin-dependent manner. Overall, our results point to the pharmacological up-regulation of mitochondrial turnover by TUDCA as a novel neuroprotective mechanism of this molecule, and contribute to the validation of TUDCA clinical application in PD.

Altered expression of Sialyl Lewis X in experimental models of Parkinson's disease.

The mechanisms underlying neurodegeneration in Parkinson's disease (PD) are still not fully understood. Glycosylation is an important post-translational modification that affects protein function, cell-cell contacts and inflammation and can be modified in pathologic conditions. Although the involvement of aberrant glycosylation has been proposed for PD, the knowledge of the diversity of glycans and their role in PD is still minimal. Sialyl Lewis X (sLeX) is a sialylated and fucosylated tetrasaccharide with essential roles in cell-to-cell recognition processes. Pathological conditions and pro-inflammatory mediators can up-regulate sLeX expression on cell surfaces, which has important consequences in intracellular signalling and immune function. Here, we investigated the expression of this glycan using in vivo and in vitro models of PD. We show the activation of deleterious glycation-related pathways in mouse striatum upon treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a toxin-based model of PD. Importantly, our results show that MPTP triggers the presentation of more proteins decorated with sLeX in mouse cortex and striatum in a time-dependent manner, as well as increased mRNA expression of its rate-limiting enzyme fucosyltransferase 7. sLeX is expressed in neurons, including dopaminergic neurons, and microglia. Although the underlying mechanism that drives increased sLeX epitopes, the nature of the protein scaffolds and their functional importance in PD remain unknown, our data suggest for the first time that sLeX in the brain may have a role in neuronal signalling and immunomodulation in pathological conditions. KEY MESSAGES: MPTP triggers the presentation of proteins decorated with sLeX in mouse brain. MPTP triggers the expression of sLeX rate-limiting enzyme FUT 7 in striatum. sLeX is expressed in neurons, including dopaminergic neurons, and microglia. sLeX in the brain may have a role in neuronal signalling and immunomodulation.

NPC1-like phenotype, with intracellular cholesterol accumulation and altered mTORC1 signaling in models of Parkinson's disease.

Disruption of brain cholesterol homeostasis has been implicated in neurodegeneration. Nevertheless, the role of cholesterol in Parkinson's Disease (PD) remains unclear. We have used N2a mouse neuroblastoma cells and primary cultures of mouse neurons and 1-methyl-4-phenylpyridinium (MPP+), a known mitochondrial complex I inhibitor and the toxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), known to trigger a cascade of events associated with PD neuropathological features. Simultaneously, we utilized other mitochondrial toxins, including antimycin A, oligomycin, and carbonyl cyanide chlorophenylhydrazone. MPP+ treatment resulted in elevated levels of total cholesterol and in a Niemann Pick type C1 (NPC1)-like phenotype characterized by accumulation of cholesterol in lysosomes. Interestingly, NPC1 mRNA levels were specifically reduced by MPP+. The decrease in NPC1 levels was also seen in midbrain and striatum from MPTP-treated mice and in primary cultures of neurons treated with MPP+. Together with the MPP+-dependent increase in intracellular cholesterol levels in N2a cells, we observed an increase in 5' adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and a concomitant increase in the phosphorylated levels of mammalian target of rapamycin (mTOR). NPC1 knockout delayed cell death induced by acute mitochondrial damage, suggesting that transient cholesterol accumulation in lysosomes could be a protective mechanism against MPTP/MPP+ insult. Interestingly, we observed a negative correlation between NPC1 protein levels and disease stage, in human PD brain samples. In summary, MPP+ decreases NPC1 levels, elevates lysosomal cholesterol accumulation and alters mTOR signaling, adding to the existing notion that PD may rise from alterations in mitochondrial-lysosomal communication.

Cholesterol redistribution triggered by CYP46A1 gene therapy improves major hallmarks of Niemann-Pick type C disease but is not sufficient to halt neurodegeneration.

Cholesterol 24-hydroxylase (CYP46A1) is an exclusively neuronal cytochrome P450 enzyme responsible for converting cholesterol into 24S-hydroxycholesterol, which serves as the primary pathway for eliminating cholesterol in the brain. We and others have shown that increased activity of CYP46A1 leads to reduced levels of cholesterol and has a positive effect on cognition. Therefore, we hypothesized that CYP46A1 could be a potential therapeutic target in Niemann-Pick type C (NPC) disease, a rare and fatal neurodegenerative disorder, characterized by cholesterol accumulation in endolysosomal compartments. Herein, we show that CYP46A1 ectopic expression, in cellular models of NPC and in Npc1tm(I1061T) mice by adeno-associated virus-mediated gene therapy improved NPC disease phenotype. Amelioration in functional, biochemical, molecular and neuropathological hallmarks of NPC disease were characterized. In vivo, CYP46A1 expression partially prevented weight loss and hepatomegaly, corrected the expression levels of genes involved in cholesterol homeostasis, and promoted a redistribution of brain cholesterol accumulated in late endosomes/lysosomes. Moreover, concomitant with the amelioration of cholesterol metabolism dysregulation, CYP46A1 attenuated microgliosis and lysosomal dysfunction in mouse cerebellum, favoring a pro-resolving phenotype. In vivo CYP46A1 ectopic expression improves important features of NPC disease and may represent a valid therapeutic approach to be used concomitantly with other drugs. However, promoting cholesterol redistribution does not appear to be enough to prevent Purkinje neuronal death in the cerebellum. This indicates that cholesterol buildup in neurons might not be the main cause of neurodegeneration in this human lipidosis.

New Scaffolds of Proteasome Inhibitors: Boosting Anticancer Potential by Exploiting the Synergy of In Silico and In Vitro Methodologies.

Cancer is a complex multifactorial disease whose pathophysiology involves multiple metabolic pathways, including the ubiquitin-proteasome system, for which several proteasome inhibitors have already been approved for clinical use. However, the resistance to existing therapies and the occurrence of severe adverse effects is still a concern. The purpose of this study was the discovery of novel scaffolds of proteasome inhibitors with anticancer activity, aiming to overcome the limitations of the existing proteasome inhibitors. Thus, a structure-based virtual screening protocol was developed using the structure of the human 20S proteasome, and 246 compounds from virtual databases were selected for in vitro evaluation, namely proteasome inhibition assays and cell viability assays. Compound 4 (JHG58) was shortlisted as the best hit compound based on its potential in terms of proteasome inhibitory activity and its ability to induce cell death (both with IC50 values in the low micromolar range). Molecular docking studies revealed that compound 4 interacts with key residues, namely with the catalytic Thr1, Ala20, Thr21, Lys33, and Asp125 at the chymotrypsin-like catalytic active site. The hit compound is a good candidate for additional optimization through a hit-to-lead campaign.

Okadaic acid inhibits the trichostatin A-mediated increase of human CYP46A1 neuronal expression in a ERK1/2-Sp3-dependent pathway.

The CYP46A1 gene codes for the cholesterol 24-hydroxylase, a cytochrome P450 specifically expressed in neurons and responsible for the majority of cholesterol turnover in the central nervous system. Previously, we have demonstrated the critical participation of Sp transcription factors in the CYP46A1 response to histone deacetylase (HDAC) inhibitors, and in this study we investigated the involvement of intracellular signaling pathways in the trichostatin A (TSA) effect. Our results show that pretreatment of neuroblastoma cells with chemical inhibitors of mitogen-activated kinase kinase (MEK)1 significantly potentiates the TSA-dependent induction of cholesterol 24-hydroxylase, whereas inhibition of protein phosphatases by okadaic acid (OA) or overexpression of MEK1 partially impairs the TSA effect without affecting histone hyperacetylation at the promoter. Immunoblotting revealed that TSA treatment decreases ERK1/2 phosphorylation concomitantly with a decrease in Sp3 binding activity, which are both reversed by pretreatment with OA. Chromatin immunoprecipitation analysis demonstrated that TSA induces the release of p-ERK1/2 from the CYP46A1 proximal promoter, whereas pretreatment with OA restores the co-occupancy of Sp3-ERK1/2 in the same promoter fragments. We demonstrate for the first time the participation of MEK-ERK1/2 signaling pathway in HDAC inhibitor-dependent induction of cytochrome P450 gene expression, underlying the importance of this regulatory signaling mechanism in the control of brain cholesterol elimination.

Neuronal differentiation alters the ratio of Sp transcription factors recruited to the CYP46A1 promoter.

CYP46A1 is a neuron-specific cytochrome P450 that plays a pivotal role in maintaining cholesterol homeostasis in the CNS. However, the molecular mechanisms underlying human CYP46A1 expression are still poorly understood, partly because of the lack of a cellular model that expresses high levels of CYP46A1. Our previous studies demonstrated that specificity protein (Sp) transcription factors control CYP46A1 expression, and are probably responsible for cell-type specificity. Herein, we have differentiated Ntera2/cloneD1 cells into post-mitotic neurons and identified for the first time a human cell model that expresses high levels of CYP46A1 mRNA. Our results show a decrease in Sp1 protein levels, concomitant with the increase in CYP46A1 mRNA levels. This decrease was correlated with changes in the ratio of Sp proteins associated to the CYP46A1 proximal promoter. To examine if the increase in (Sp3+Sp4)/Sp1 ratio was observed in other Sp-regulated promoters, we have selected four genes--reelin, glutamate receptor subunit zeta-1, glutamate receptor subunit epsilon-1 and µ-opioid receptor--known to be expressed in the human brain and analyzed the Sp proteins binding pattern to the promoter of these genes, in undifferentiated and differentiated Ntera2/cloneD1. Our data indicate that the dissociation of Sp1 from promoter regions is a common feature amongst Sp-regulated genes that are up-regulated after neuronal differentiation.

Sp proteins play a critical role in histone deacetylase inhibitor-mediated derepression of CYP46A1 gene transcription.

We investigated whether the CYP46A1 gene, a neuronal-specific cytochrome P450, responsible for the majority of brain cholesterol turnover, is subject to transcriptional modulation through modifications in histone acetylation. We demonstrated that inhibition of histone deacetylase activity by trichostatin A (TSA), valproic acid and sodium butyrate caused a potent induction of both CYP46A1 promoter activity and endogenous expression. Silencing of Sp transcription factors through specific small interfering RNAs, or impairing Sp binding to the proximal promoter, by site-directed mutagenesis, led to a significant decrease in TSA-mediated induction of CYP46A1 expression/promoter activity. Electrophoretic mobility shift assay, DNA affinity precipitation assays and chromatin immunoprecipitation assays were used to determine the multiprotein complex recruited to the CYP46A1 promoter, upon TSA treatment. Our data showed that a decrease in Sp3 binding at particular responsive elements, can shift the Sp1/Sp3/Sp4 ratio, and favor the detachment of histone deacetylase (HDAC) 1 and HDAC2 and the recruitment of p300/CBP. Moreover, we observed a dynamic change in the chromatin structure upon TSA treatment, characterized by an increase in the local recruitment of euchromatic markers and RNA polymerase II. Our results show the critical participation of an epigenetic program in the control of CYP46A1 gene transcription, and suggest that brain cholesterol catabolism may be affected upon treatment with HDAC inhibitors.

GSTpi expression in MPTP-induced dopaminergic neurodegeneration of C57BL/6 mouse midbrain and striatum.

MPTP-induced dopaminergic neurotoxicity involves major biochemical processes such as oxidative stress and impaired energy metabolism, leading to a significant reduction in the number of nigrostriatal dopaminergic neurons. Glutathione S-transferase pi (GSTpi) is a phase II detoxifying enzyme that provides protection of cells from injury by toxic chemicals and products of oxidative stress. In humans, polymorphisms of GSTP1 affect substrate selectivity and stability increasing the susceptibility to parkinsonism-inducing effects of environmental toxins. Given the ability of MPTP to increase the levels of reactive oxygen species and the link between altered redox potential and the expression and activity of GSTpi, we investigated the effect of MPTP on GSTpi cellular concentration in an in vivo model of Parkinson's disease. The present study demonstrates that GSTpi is actively expressed in both substantia nigra pars compacta and striatum of C57BL/6 mice brain, mostly in oligodendrocytes and astrocytes. After systemic administration of MPTP, GSTpi expression is significantly increased in glial cells in the vicinity of dopaminergic neurons cell bodies and fibers. The results suggest that GSTpi expression may be part of the mechanism underlying the ability of glial cells to elicit protection against the mechanisms involved in MPTP-induced neuronal death.

Glutathione S-transferase pi regulates UV-induced JNK signaling in SH-SY5Y neuroblastoma cells.

Activation of c-Jun N-terminal kinase (JNK) signaling pathway is a key event in apoptosis. The cellular mechanisms underlying the control of JNK catalytic activity before and immediately after stress in neuronal cells are still not completely understood. Under resting conditions the basal activity of JNK is low, since JNK is kept inactive by the presence of one or more endogenous repressors, including glutathione S-transferase pi (GSTpi). The aim of this study was to investigate the control of JNK signaling by GSTpi. We examined the modifications of GSTpi protein expression and oligomerization after UV irradiation-induced stress in human SH-SY5Y neuroblastoma cells. In parallel, we investigated the effect of UV irradiation on JNK activation and c-Jun phosphorylation, and whether apoptosis represents a functional consequence triggered by this signaling pathway. We show that in SH-SY5Y cells JNK phosphorylation and activation precedes c-Jun phosphorylation and caspase-3 cleavage. Importantly, the increase of JNK enzymatic activity correlates with the dissociation of GSTpi-JNK complexes and the increased concentration of GSTpi multimer forms. Results presented herein show for the first time direct interaction between JNK and GSTpi in SH-SY5Y neuroblastoma cells, and suggest that in these cells GSTpi may serve as a regulator of JNK catalytic activity. This work contributes to further elucidate the mechanisms underlying the regulation of JNK activity under stress conditions.

Transcriptional regulation of the human CYP46A1 brain-specific expression by Sp transcription factors.

Brain defective cholesterol homeostasis has been associated with neurologic diseases, such as Alzheimer's and Huntington's disease. The elimination of cholesterol from the brain involves its conversion into 24(S)-hydroxycholesterol by CYP46A1, and the efflux of this oxysterol across the blood-brain barrier. Herein, we identified the regulatory elements and factors involved the human CYP46A1 expression. Functional 5'deletion analysis mapped a region spanning from nucleotides -236/-64 that is indispensable for basal expression of this TATA-less gene. Treatment of SH-SY5Y cells with mithramycin A resulted in a significant reduction of promoter activity, suggesting a role of Sp family of transcription factors in CYP46A1 regulation. Combination of Sp1, Sp3, and Sp4 over-expression studies in Drosophila SL-2 cells, and systematic promoter mutagenesis identified Sp3 and Sp4 binding to four GC-boxes as required and sufficient for high levels of promoter activity. Moreover, Sp3 and Sp4 were demonstrated to be the major components of the protein-DNA complexes observed in primary rat cortical extracts. Our results suggest that the cell-type specific expression of Sp transcription factors - substitution of Sp1 by Sp4 in neurons - is responsible for the basal expression of the CYP46A1 gene. This study delineates for the first time the mechanisms underlying the human CYP46A1 transcription and thereby elucidates potential pathways underlying cholesterol homeostasis in the brain.

Tauroursodeoxycholic Acid Improves Motor Symptoms in a Mouse Model of Parkinson's Disease.

Parkinson's disease (PD) is characterized by severe motor symptoms, and currently there is no treatment that retards disease progression or reverses damage prior to the time of clinical diagnosis. Tauroursodeoxycholic acid (TUDCA) is neuroprotective in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD; however, its effect in PD motor symptoms has never been addressed. In the present work, an extensive behavior analysis was performed to better characterize the MPTP model of PD and to evaluate the effects of TUDCA in the prevention/improvement of mice phenotype. MPTP induced significant alterations in general motor performance paradigms, including increased latency in the motor swimming, adhesive removal and pole tests, as well as altered gait, foot dragging, and tremors. TUDCA administration, either before or after MPTP, significantly reduced the swimming latency, improved gait quality, and decreased foot dragging. Importantly, TUDCA was also effective in the prevention of typical parkinsonian symptoms such as spontaneous activity, ability to initiate movement and tremors. Accordingly, TUDCA prevented MPTP-induced decrease of dopaminergic fibers and ATP levels, mitochondrial dysfunction and neuroinflammation. Overall, MPTP-injected mice presented motor symptoms that are aggravated throughout time, resembling human parkinsonism, whereas PD motor symptoms were absent or mild in TUDCA-treated animals, and no aggravation was observed in any parameter. The thorough demonstration of improvement of PD symptoms together with the demonstration of the pathways triggered by TUDCA supports a subsequent clinical trial in humans and future validation of the application of this bile acid in PD.

Oxidative Stress and Antioxidants in Neurological Diseases: Is There Still Hope?

Oxidative stress is a pathological feature common to a multitude of neurological diseases. The production of reactive oxygen species (ROS) is the main mechanism underlying this cellular redox imbalance. Antioxidants protect biological targets against ROS, therefore, they have been considered as attractive potential therapeutic agents to counteract ROS-mediated neuronal damage. However, despite encouraging in vitro and preclinical in vivo data, the clinical efficacy of antioxidant treatment strategies is marginal and most clinical trials using antioxidants as therapeutic agents in neurodegenerative diseases have yielded disappointing outcomes. This might in part be due to the need of adjustment in concentrations and time parameters between preclinical studies and clinical settings. Moreover new efficient delivery methods need to be investigated, particularly taking into account that a successful therapeutic agent for neurological diseases should readily cross the blood-brain barrier (BBB). In that sense, the use of compounds that cross the BBB and boost the endogenous antioxidant defense machinery, by activating for instance the Nrf2 pathway, or compounds that are able to modulate ROS production, such as NOX enzyme inhibitors, seems to represent a more promising approach to combat oxidative stress in the central nervous system (CNS). Here we present a brief overview of the main players in oxidative stress and outline evidences of their involvement in Parkinson's disease, Alzheimer's disease, Huntington's disease and multiple sclerosis. Finally, we review and critically discuss the potential of antioxidants as therapeutics for central nervous system disorders with a special focus on emerging novel therapeutic strategies.

Tauroursodeoxycholic Acid Protects Against Mitochondrial Dysfunction and Cell Death via Mitophagy in Human Neuroblastoma Cells.

Mitochondrial dysfunction has been deeply implicated in the pathogenesis of several neurodegenerative diseases. Thus, to keep a healthy mitochondrial population, a balanced mitochondrial turnover must be achieved. Tauroursodeoxycholic acid (TUDCA) is neuroprotective in various neurodegenerative disease models; however, the mechanisms involved are still incompletely characterized. In this study, we investigated the neuroprotective role of TUDCA against mitochondrial damage triggered by the mitochondrial uncoupler carbonyl cyanide m-chlorophelyhydrazone (CCCP). Herein, we show that TUDCA significantly prevents CCCP-induced cell death, ROS generation, and mitochondrial damage. Our results indicate that the neuroprotective role of TUDCA in this cell model is mediated by parkin and depends on mitophagy. The demonstration that pharmacological up-regulation of mitophagy by TUDCA prevents neurodegeneration provides new insights for the use of TUDCA as a modulator of mitochondrial activity and turnover, with implications in neurodegenerative diseases.

Nrf2 activation by tauroursodeoxycholic acid in experimental models of Parkinson's disease.

Parkinson's disease (PD) is a progressive neurological disorder, mainly characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta. Although the cause of PD remains elusive, mitochondrial dysfunction and severe oxidative stress are strongly implicated in the cell death that characterizes the disease. Under oxidative stress, the master regulator of cellular redox status, nuclear factor erythroid 2 related factor 2 (Nrf2), is responsible for activating the transcription of several cytoprotective enzymes, namely glutathione peroxidase (GPx) and heme oxygenase-1 (HO-1). Nrf2 is a promising target to limit reactive oxygen species (ROS)-mediated damage in PD. Here, we show that tauroursodeoxycholic acid (TUDCA) prevents both 1-methyl-4-phenylpyridinium (MPP+)- and α-synuclein-induced oxidative stress, through Nrf2 activation, in SH-SY5Y cells. Additionally, we used C57BL/6 male mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to elucidate the effect of TUDCA in this in vivo model of PD. In vivo, TUDCA treatment increases the expression of Nrf2, Nrf2 stabilizer DJ-1, and Nrf2 downstream target antioxidant enzymes HO-1 and GPx. Moreover, we found that TUDCA enhances GPx activity in the brain. Altogether, our results suggest that TUDCA is a promising agent to limit ROS-mediated damage, in different models of PD acting, at least in part, through modulation of the Nrf2 signaling pathway. Therefore, TUDCA should be considered a promising therapeutic agent to be implemented in PD.

S-Glutathionylation of Keap1: a new role for glutathione S-transferase pi in neuronal protection.

Oxidative stress is a key pathological feature of Parkinson's disease (PD). Glutathione S-transferase pi (GSTP) is a neuroprotective antioxidant enzyme regulated at the transcriptional level by the antioxidant master regulator nuclear factor-erythroid 2-related factor 2 (Nrf2). Here, we show for the first time that upon MPTP-induced oxidative stress, GSTP potentiates S-glutathionylation of Kelch-like ECH-associated protein 1 (Keap1), an endogenous repressor of Nrf2, in vivo. S-glutathionylation of Keap1 leads to Nrf2 activation and subsequently increases expression of GSTP. This positive feedback regulatory loop represents a novel mechanism by which GSTP elicits antioxidant protection in the brain.

Neuronal cholesterol metabolism increases dendritic outgrowth and synaptic markers via a concerted action of GGTase-I and Trk.

Cholesterol 24-hydroxylase (CYP46A1) is responsible for brain cholesterol elimination and therefore plays a crucial role in the control of brain cholesterol homeostasis. Altered CYP46A1 expression has been associated with several neurodegenerative diseases and changes in cognition. Since CYP46A1 activates small guanosine triphosphate-binding proteins (sGTPases), we hypothesized that CYP46A1 might be affecting neuronal development and function by activating tropomyosin-related kinase (Trk) receptors and promoting geranylgeranyl transferase-I (GGTase-I) prenylation activity. Our results show that CYP46A1 triggers an increase in neuronal dendritic outgrowth and dendritic protrusion density, and elicits an increase of synaptic proteins in the crude synaptosomal fraction. Strikingly, all of these effects are abolished by pharmacological inhibition of GGTase-I activity. Furthermore, CYP46A1 increases Trk phosphorylation, its interaction with GGTase-I, and the activity of GGTase-I, which is crucial for the enhanced dendritic outgrowth. Cholesterol supplementation studies indicate that cholesterol reduction by CYP46A1 is the necessary trigger for these effects. These results were confirmed in vivo, with a significant increase of p-Trk, pre- and postsynaptic proteins, Rac1, and decreased cholesterol levels, in crude synaptosomal fractions prepared from CYP46A1 transgenic mouse cortex. This work describes the molecular mechanisms by which neuronal cholesterol metabolism effectively modulates neuronal outgrowth and synaptic markers.