A sporadic Parkinson's disease model via silencing of the ubiquitin-proteasome/E3 ligase component, SKP1A.

Our and other's laboratory microarray-derived transcriptomic studies in human PD substantia nigra pars compacta (SNpc) samples have opened an avenue to concentrate on potential gene intersections or cross-talks along the dopaminergic (DAergic) neurodegenerative cascade in sporadic PD (SPD). One emerging gene candidate identified was SKP1A (p19, S-phase kinase-associated protein 1A), found significantly decreased in the SNpc as confirmed later at the protein level. SKP1 is part of the Skp1, Cullin 1, F-box protein (SCF) complex, the largest known class of sophisticated ubiquitin-proteasome/E3-ligases and was found to directly interact with FBXO7, a gene defective in PARK15-linked PD. This finding has led us to the hypothesis that a targeted site-specific reduction of Skp1 levels in DAergic neuronal cell culture and animal systems may result in a progressive loss of DAergic neurons and hopefully recreate motor disabilities in animals. The second premise considers the possibility that both intrinsic and extrinsic factors (e.g., manipulation of selected genes and mitochondria impairing toxins), alleged to play central roles in DAergic neurodegeneration in PD, may act in concert as modifiers of Skp1 deficiency-induced phenotype alterations ('dual-hit' hypothesis of neurodegeneration). To examine a possible role of Skp1 in DAergic phenotype, we have initially knocked down the expression of SKP1A gene in an embryonic mouse SN-derived cell line (SN4741) with short hairpin RNA (shRNA) lentiviruses (LVs). The deficiency of SKP1A closely recapitulated cardinal features of the DAergic pathology of human PD, such as decreased expression of DAergic phenotypic markers and cell cycle aberrations. Furthermore, the knocked down cells displayed a lethal phenotype when induced to differentiate exhibiting proteinaceous round inclusion structures, which were almost identical in composition to human Lewy bodies, a hallmark of PD. These findings support a role for Skp1 in neuronal phenotype, survival, and differentiation. The identification of Skp1 as a key player in DAergic neuron function suggested that a targeted site-specific reduction of Skp1 levels in mice SNpc may result in a progressive loss of DAergic neurons and terminal projections in the striatum. The injected LV SKP1shRNA to mouse SN resulted in decreased expression of Skp1 protein levels within DAergic neurons and loss of tyrosine hydroxylase immunoreactivity (TH-IR) in both SNpc and striatum that was accompanied by time-dependent motor disabilities. The reduction of the vertical movements, that is rearing, may be reminiscent of the early occurrence of hypokinesia and axial, postural instability in PD. According to the 'dual-hit' hypothesis of neurodegenerative diseases, it is predicted that gene-gene and/or gene-environmental factors would act in concert or sequentially to propagate the pathological process of PD. Our findings are compatible with this conjecture showing that the genetic vulnerability caused by knock down of SKP1A renders DAergic SN4741 cells especially sensitive to genetic reduction of Aldh1 and exposure to the external stressors MPP+ and DA, which have been implicated in PD pathology. Future consideration should be given in manipulation SKP1A expression as therapeutic window, via its induction genetically or pharmacological, to prevent degeneration of the nigra striatal dopamine neurons, since UPS is defective.

Lewy bodies, iron, inflammation and neuromelanin: pathological aspects underlying Parkinson's disease.

Since the description of some peculiar symptoms by James Parkinson in 1817, attempts have been made to define its cause or at least to enlighten the pathology of "Parkinson's disease (PD)." The vast majority of PD subtypes and most cases of sporadic PD share Lewy bodies (LBs) as a characteristic pathological hallmark. However, the processes underlying LBs generation and its causal triggers are still unknown. ɑ-Synuclein (ɑ-syn, encoded by the SNCA gene) is a major component of LBs, and SNCA missense mutations or duplications/triplications are causal for rare hereditary forms of PD. Thus, it is imperative to study ɑ-syn protein and its pathology, including oligomerization, fibril formation, aggregation, and spreading mechanisms. Furthermore, there are synergistic effects in the underlying pathogenic mechanisms of PD, and multiple factors-contributing with different ratios-appear to be causal pathological triggers and progression factors. For example, oxidative stress, reduced antioxidative capacity, mitochondrial dysfunction, and proteasomal disturbances have each been suggested to be causal for ɑ-syn fibril formation and aggregation and to contribute to neuroinflammation and neural cell death. Aging is also a major risk factor for PD. Iron, as well as neuromelanin (NM), show age-dependent increases, and iron is significantly increased in the Parkinsonian substantia nigra (SN). Iron-induced pathological mechanisms include changes of the molecular structure of ɑ-syn. However, more recent PD research demonstrates that (i) LBs are detected not only in dopaminergic neurons and glia but in various neurotransmitter systems, (ii) sympathetic nerve fibres degenerate first, and (iii) at least in "brain-first" cases dopaminergic deficiency is evident before pathology induced by iron and NM. These recent findings support that the ɑ-syn/LBs pathology as well as iron- and NM-induced pathology in "brain-first" cases are important facts of PD pathology and via their interaction potentiate the disease process in the SN. As such, multifactorial toxic processes posted on a personal genetic risk are assumed to be causal for the neurodegenerative processes underlying PD. Differences in ratios of multiple factors and their spatiotemporal development, and the fact that common triggers of PD are hard to identify, imply the existence of several phenotypical subtypes, which is supported by arguments from both the "bottom-up/dual-hit" and "brain-first" models. Therapeutic strategies are necessary to avoid single initiation triggers leading to PD.

Site-activated multi target iron chelators with acetylcholinesterase (AChE) and monoamine oxidase (MAO) inhibitory activities for Alzheimer's disease therapy.

The first class of site-activated chelators with dual inhibition of acetyl-cholinesterase (AChE) and monoamine oxidase (MAO), rationally designed for simultaneously targeting the multiple pathogenic processes in Alzheimer's disease (AD) without significantly disrupting healthy metal metabolism in the body are discussed. It is demonstrated that the novel prochelator 2 was a selective and potent MAO-A inhibitor in vitro (IC50: 0.0077 ± 0.0007 µM) with moderate inhibition of MAO-B (IC50: 7.90 ± 1.34 µM). In vitro prochelator 2 also selectively inhibited AChE in a time-dependent manner and reach maximum inhibition of AChE after 2 h preincubation (IC50: 0.52 ± 0.07 µM for AChE, versus 44.90 ± 6.10 µM for BuChE). Prochelator 2 showed little affinity for metal (Fe, Cu, and Zn) ions until it bound to and was activated by AChE that is located predominately in the brain, releasing an active iron chelator M30. M30 is an efficient chelator for metal (Fe, Cu, and Zn) ions with the capabilities to suppress oxidative stress, to selectively inhibit MAO-A and B in the brain, and to regulate cerebral biometals dyshomeostasis in vivo; M30 is also a neuroprotective-neurorestorative chelator with a broad spectrum of activities against ß-amyloid (Aß) generation, amyloid plaques and neurofibrillary tangles (NFT) formation, and Aß aggregation induced by metal (Cu and Zn) ions. Both M30 and prochelator 2 were not toxic to Human SH-SY5Y neuroblastoma cells at low concentrations, but prochelator 2 shows limited cytotoxicity, at high concentrations. Together, these data suggest that prochelator 2 is a promise lead for simultaneously modulating multiple targets in AD.

Selegiline: a molecule with innovative potential.

Monoamine oxidase B (MAO-B) inhibitors have an established role in the treatment of Parkinson's disease as monotherapy or adjuvant to levodopa. Two major recognitions were required for their introduction into this therapeutic field. The first was the elucidation of the novel pharmacological properties of selegiline as a selective MAO-B inhibitor by Knoll and Magyar and the original idea of Riederer and Youdim, supported by Birkmayer, to explore its effect in parkinsonian patients with on-off phases. In the 1960s, MAO inhibitors were mainly studied as potential antidepressants, but Birkmayer found that combined use of levodopa and various MAO inhibitors improved akinesia in Parkinson's disease. However, the serious side effects of the first non-selective MAO inhibitors prevented their further use. Later studies demonstrated that MAO-B, mainly located in glial cells, is important for dopamine metabolism in the brain. Recently, cell and molecular studies revealed interesting properties of selegiline opening new possibilities for neuroprotective mechanisms and a disease-modifying effect of MAO-B inhibitors.

Creation of a gene expression classifier for predicting Parkinson's disease rate of progression.

Parkinson's disease (PD) etiology is heterogeneous, genetic, and multi-factorial, resulting in a varied disease from a mild slow progression to a more severe rapid progression. Prognostic information on the nature of the patient's disease at diagnosis aids the physician in counseling patients on treatment options and life planning. In a cohort of PD patients from the PPMI study, the relative gene expression levels of SKP1A, UBE2K, ALDH1A1, PSMC4, HSPA8 and LAMB2 were measured in baseline blood samples by real-time quantitative PCR. At baseline PD patients were up to 2 years from diagnosis, H&Y scale ≤ 2 and PD treatment naïve. PD-Prediction algorithm comprised of ALDH1A1, LAMB2, UBE2K, SKP1A and age was created by logistic regression for predicting progression to ≤ 70% Modified Schwab and England Activities of Daily Living (S&E-ADL). In relation to patients negative for PD-Prediction (n = 180), patients positive (n = 30) for Cutoff-1 (at 82% specificity, 80.0% sensitivity) had positive hazard ratio (HR+) of 10.6 (95% CI, 2.2-50.1), and positive (n = 23) for Cutoff-2 (at 93% specificity, 47% sensitivity) had HR+ of 17.1 (95% CI, 3.2-89.9) to progress to ≤ 70% S&E-ADL within 3 years (P value < 0.0001). Likewise, patients positive for PD-Prediction Cutoff-1 (n = 49) had HR+ 4.3 (95% CI, 1.6-11.6) for faster time to H&Y 3 in relation to patients negative (n = 170) for PD-Prediction (P value = 0.0002). Our findings show an algorithm that seems to predict fast PD progression and may potentially be used as a tool to assist the physician in choosing an optimal treatment plan, improving the patient's quality of life and overall health outcome.

Monoamine oxidase inhibitors, and iron chelators in depressive illness and neurodegenerative diseases.

In early 1920s, tyramine oxidase was discovered that metabolized tyramine and in 1933 Blaschko demonstrated that this enzyme also metabolized adrenaline, noradrenaline and dopamine. Zeller gave it the name monoamine oxidase (MAO) to distinguish it from the enzyme that oxidatively deaminated diamines. MAO was recognized as an enzyme of crucial interest to pharmacologists because it catalyzed the major inactivation pathway for the catecholamines (and, later, 5-hydroxytryptamine, as well). Within the few decade, the inhibitors of MAO were discovered and introduced for the treatment of depressive illness which was established clinically. However, the first clinical use exposed serious side effects, pharmacological interest in, and investigation of, MAO continued, resulting in the characterization of two forms, distinct forms, MAO-A and -B, and selective inhibitors for them. Selective inhibitors of MAO-B (selegiline, rasagiline and safinamide) have found a therapeutic role in the treatment of Parkinson's disease and reversible inhibitors of MAO-A offered antidepressant activity without the serious side effects of the earlier nonselective MAO inhibitors. Subsequent molecular pharmacological have also generated the concept of neuroprotection, reflecting the possibility of slowing, halting and maybe reversing, neurodegeneration in Parkinson's or Alzheimer's diseases. Increased levels of oxidative stress through the accumulation of iron in the Parkinsonian and Alzheimer brains has been suggested to be critical for the initiation and progress of neurodegeneration. Selective inhibition of brain MAO could contribute importantly to lowering such stress, preventing the formation of hydrogen peroxide. Interaction of Iron with hydrogen peroxide and lead to Fenton reaction and production of the most reactive radical, namely hydroxyl radical. There are complex interactions between free iron levels in brain and MAO, and cascade of neurotoxic events may have practical outcomes for depressive disorders and neurodegenerative diseases. As consequence recent novel therapeutic drugs for neurodegenerative diseases has led to the development of multi target drugs, that possess selective brain MAO A and B inhibitory moiety, iron chelating and antioxidant activities and the ability to increase brain levels of endogenous neurotrophins, such as BDNF, GDNF VEGF and erythropoietin and induce mitochondrial biogenesis.

A promising drug candidate for the treatment of glaucoma based on a P2Y6-receptor agonist.

Extracellular nucleotides can regulate the production/drainage of the aqueous humor via activation of P2 receptors, thus affecting the intraocular pressure (IOP). We evaluated 5-OMe-UDP(α-B), 1A, a potent P2Y6-receptor agonist, for reducing IOP and treating glaucoma. Cell viability in the presence of 1A was measured using [3-(4, 5-dimethyl-thiazol-2-yl) 2, 5-diphenyl-tetrazolium bromide] (MTT) assay in rabbit NPE ciliary non-pigmented and corneal epithelial cells, human retinoblastoma, and liver Huh7 cells. The effect of 1A on IOP was determined in acute glaucomatous rabbit hyaluronate model and phenol-induced chronic glaucomatous rabbit model. The origin of activity of 1A was investigated by generation of a homology model of hP2Y6-R and docking studies. 1A did not exert cytotoxic effects up to 100 mM vs. trusopt and timolol in MTT assay in ocular and liver cells. In normotensive rabbits, 100 µM 1A vs. xalatan, trusopt, and pilocarpine reduced IOP by 45 vs. 20-30%, respectively. In the phenol animal model, 1A (100 µM) showed reduction of IOP by 40 and 20%, following early and late administration, respectively. Docking results suggest that the high activity and selectivity of 1A is due to intramolecular interaction between Pα-BH3 and C5-OMe which positions 1A in a most favorable site inside the receptor. P2Y6-receptor agonist 1A effectively and safely reduces IOP in normotense, acute, and chronic glaucomatous rabbits, and hence may be suggested as a novel approach for the treatment of glaucoma.

Neuroprotective and neurorestorative potential of propargylamine derivatives in ageing: focus on mitochondrial targets.

The mitochondrial theory of ageing proposes that accumulation of damage to mitochondrial function and DNA mutation lead to ageing of humans and animals. It has been suggested that mitochondria play dynamic roles in regulating synaptogenesis and morphological/functional responses of synaptic activity, and thus, deteriorating of mitochondrial function (e.g., deficits of the mitochondrial respiratory enzymes, reduced calcium influx, increased accumulation of mitochondrial DNA defects/apoptotic proteins and impairment of mitochondrial membrane potential) can lead to severe neuronal energy deficit, and in the long run, to modifications in neuronal synapses and neurodegeneration in the ageing brain. Hence, considering the mechanisms by which mitochondrial impairment can lead to neuronal death, the development of neuroprotective molecules that target various mitochondrial pathogenic processes can be effective in the treatment of ageing and age-related neurodegenerative diseases. This review addresses several aspects of the neuroprotective effects of propargylamine derivatives (e.g., the monoamine oxidase-B inhibitors, selegiline and rasagiline and the multifunctional drugs, ladostigil, M30 and VAR10303) in ageing with a special focus on mitochondrial molecular protective mechanisms.

Gel-based protease proteomics for identifying the novel calpain substrates in dopaminergic neuronal cell.

Calpains are a family of calcium-dependent cysteine proteases that are ubiquitously expressed in mammals and play critical roles in neuronal death by catalyzing substrate proteolysis. Here, we developed two-dimensional gel electrophoresis-based protease proteomics to identify putative calpain substrates. To accomplish this, cellular lysates from neuronal cells were first separated by pI, and the immobilized sample on a gel strip was incubated with a recombinant calpain and separated by molecular weight. Among 25 altered protein spots that were differentially expressed by at least 2-fold, we confirmed that arsenical pump-driving ATPase, optineurin, and peripherin were cleaved by calpain using in vitro and in vivo cleavage assays. Furthermore, we found that all of these substrates were cleaved in MN9D cells treated with either ionomycin or 1-methyl-4-phenylpyridinium, both of which cause a calcium-mediated calpain activation. Their cleavage was blocked by calcium chelator or calpain inhibitors. In addition, calpain-mediated cleavage of these substrates and its inhibition by calpeptin were confirmed in a middle cerebral artery occlusion model of cerebral ischemia, as well as a stereotaxic brain injection model of Parkinson disease. Transient overexpression of each protein was shown to attenuate 1-methyl-4-phenylpyridinium-induced cell death, indicating that these substrates may confer protection of varying magnitudes against dopaminergic injury. Taken together, the data indicate that our protease proteomic method has the potential to be applicable for identifying proteolytic substrates affected by diverse proteases. Moreover, the results described here will help us decipher the molecular mechanisms underlying the progression of neurodegenerative disorders where protease activation is critically involved.

DJ-1 deficiency triggers microglia sensitivity to dopamine toward a pro-inflammatory phenotype that is attenuated by rasagiline.

DJ-1 is an oxidative stress sensor that localizes to the mitochondria when the cell is exposed to oxidative stress. DJ-1 mutations that result in gene deficiency are linked to increased risk of Parkinson's disease (PD). Activation of microglial stress conditions that are linked to PD may result in neuronal death. We postulated that DJ-1 deficiency may increase microglial neurotoxicity. We found that down-regulation of DJ-1 in microglia using an shRNA approach increased cell sensitivity to dopamine as measured by secreted pro-inflammatory cytokines such as IL-1ß and IL-6. Furthermore, we discovered that DJ-1-deficient microglia had increased monoamine oxidase activity that resulted in elevation of intracellular reactive oxygen species and nitric oxide leading to increased dopaminergic neurotoxicity. Rasagaline, a monoamine oxidase inhibitor approved for treatment of PD, reduced the microglial pro-inflammatory phenotype and significantly reduced neurotoxicity. Moreover, we discovered that DJ-1-deficient microglia have reduced expression of triggering receptor expressed on myeloid cells 2 (TREM2), previously suggested as a risk factor for pro-inflammation in neurodegenerative diseases. Further studies of DJ-1-mediated cellular pathways in microglia may contribute useful insights into the development of PD providing future avenues for therapeutic intervention

Nigericin-induced impairment of autophagic flux in neuronal cells is inhibited by overexpression of Bak.

Bak is a prototypic pro-apoptotic Bcl-2 family protein expressed in a wide variety of tissues and cells. Recent studies have revealed that Bcl-2 family proteins regulate apoptosis as well as autophagy. To investigate whether and how Bak exerts a regulatory role on autophagy-related events, we treated independent cell lines, including MN9D neuronal cells, with nigericin, a K(+)/H(+) ionophore. Treatment of MN9D cells with nigericin led to an increase of LC3-II and p62 levels with concomitant activation of caspase. Ultrastructural examination revealed accumulation of autophagic vacuoles and swollen vacuoles in nigericin-treated cells. We further found that the LC3-II accumulated as a consequence of impaired autophagic flux and the disrupted degradation of LC3-II in nigericin-treated cells. In this cell death paradigm, both transient and stable overexpression of various forms of Bak exerted a protective role, whereas it did not inhibit the extent of nigericin-mediated activation of caspase-3. Subsequent biochemical and electron microscopic studies revealed that overexpressed Bak maintained autophagic flux and reduced the area occupied by swollen vacuoles in nigericin-treated cells. Similar results were obtained in nigericin-treated non-neuronal cells and another proton ionophore-induced cell death paradigm. Taken together, our study indicates that a protective role for Bak during ionophore-induced cell death may be closely associated with its regulatory effect on maintenance of autophagic flux and vacuole homeostasis.

Lithium's gene expression profile, relevance to neuroprotection A cDNA microarray study.

Lithium can prevent 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) dopaminergic neurotoxicity in mice. This is attributed to induced antioxidant and antiapoptotic state, which among other factors results from induction of Bcl-2 and reduction of Bax, however, cDNA microarray reveals that this represents only one cascade of lithium targets. From analyzing the gene expression profile of lithium, we are able to point out candidate genes that might be involved in the antioxidant and neuroprotective properties of lithium. Among these are, the cAMP response element binding (CREB) protein, extracellular signal-regulated kinase (ERK), both CREB and ERK-part of the mitogen-activated kinase pathway-were upregulated by lithium, downregulated by MPTP, and maintained in mice fed with lithium chloride (LiCl) supplemented diet and treated with MPTP. Our positive control included tyrosine hydroxylase which both its mRNA and protein levels were independently measured, in addition to Bcl-2 protein levels. Other important genes which were similarly regulated are plasma glutathione peroxidase precursor (GSHPX-P), protein kinase C alpha type, insulin-like growth factor binding protein 4 precursor, and interferon regulatory factor. In addition, some genes were oppositely regulated, i.e., downregulated by lithium, upregulated by MPTP, and maintained in mice fed with LiCl supplemented diet and treated with MPTP, among these genes were basic fibroblast growth factor receptor 1 precursor, inhibin alpha subunit, glutamate receptor subunit zeta 1 precursor (NMD-R1), postsynaptic density protein-95 which together with NMD-R1 can form an apoptotic promoting complex. The discussed targets represent part of genes altered by chronic lithium. In fact lithium affected the expressions of more than 50 genes among these were basic transcription factors, transcription activators, cell signaling proteins, cell adhesion proteins, oncogenes and tumor suppressors, intracellular transducers, survival and death genes, and cyclins, here we discuss the relevance of these changes to lithium's reported neuroprotective properties.

Effects of novel neuroprotective and neurorestorative multifunctional drugs on iron chelation and glucose metabolism.

Iron accumulation and iron-related oxidative stress are involved in several pathological conditions and provide a rationale for the development of iron chelators as novel promising therapeutic strategies. Thus, we have recently synthesized multifunctional non-toxic, brain permeable iron chelating compounds, M30 and HLA20, possessing the neuroprotective N-propargyl moiety of the anti-Parkinsonian drug, monoamine oxidase (MAO)-B inhibitor, rasagiline and the antioxidant-iron chelating moiety of an 8-hydroxyquinoline derivative of the iron chelator, VK28. Here, we examined the hepatic regulatory effects of these novel compounds using two experimental approaches: chelation activity and glucose metabolism parameters. The present study demonstrated that M30 and HLA20 significantly decreased intracellular iron content and reduced ferritin expression levels in iron-loaded hepatoma Hep3B cells. In electron microscopy analysis, M30 was shown to reduce the electron-dense deposits of siderosomes by ~30 %, as well as down-regulate cytosolic ferritin particles observed in iron-overloaded cells. In vivo studies demonstrated that M30 administration (1 mg/kg, P.O. three times a week) reduced hepatic ferritin levels; increased hepatic insulin receptor and glucose transporter-1 levels and improved glucose tolerance in C57BL/6 mice and in a mouse model of type-2 diabetes, the ob/ob (leptin(-/-)). The results clearly indicate that the novel multifunctional drugs, especially M30, display significant capacity of chelating intracellular iron and regulating glucose metabolism parameters. Such effects can have therapeutic significance in conditions with abnormal local or systemic iron metabolism, including neurological diseases.

The Neuroprotective Activities of the Novel Multi-Target Iron-Chelators in Models of Alzheimer's Disease, Amyotrophic Lateral Sclerosis and Aging.

The concept of chelation therapy as a valuable therapeutic approach in neurological disorders led us to develop multi-target, non-toxic, lipophilic, brain-permeable compounds with iron chelation and anti-apoptotic properties for neurodegenerative diseases, such as Parkinson's disease (PD), Alzheimer's disease (AD), age-related dementia and amyotrophic lateral sclerosis (ALS). Herein, we reviewed our two most effective such compounds, M30 and HLA20, based on a multimodal drug design paradigm. The compounds have been tested for their mechanisms of action using animal and cellular models such as APP/PS1 AD transgenic (Tg) mice, G93A-SOD1 mutant ALS Tg mice, C57BL/6 mice, Neuroblastoma × Spinal Cord-34 (NSC-34) hybrid cells, a battery of behavior tests, and various immunohistochemical and biochemical techniques. These novel iron chelators exhibit neuroprotective activities by attenuating relevant neurodegenerative pathology, promoting positive behavior changes, and up-regulating neuroprotective signaling pathways. Taken together, these results suggest that our multifunctional iron-chelating compounds can upregulate several neuroprotective-adaptive mechanisms and pro-survival signaling pathways in the brain and might function as ideal drugs for neurodegenerative disorders, such as PD, AD, ALS, and aging-related cognitive decline, in which oxidative stress and iron-mediated toxicity and dysregulation of iron homeostasis have been implicated.

Novel therapeutic approach for neurodegenerative pathologies: multitarget iron-chelating drugs regulating hypoxia-inducible factor 1 signal transduction pathway.

Our novel multimodal brain-permeable iron-chelating compounds M30 and HLA20 were demonstrated to possess neuroprotective/neurorescue activities in vitro and in vivo against several insults applicable to various neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. Neuroprotection by iron chelators has been widely recognized with respect to their ability to prevent reactive oxygen species generation in the Fenton reaction by sequestering redox-active iron. An additional neuroprotective mechanism of iron-chelating compounds is associated with their ability to regulate the transcriptional activator hypoxia-inducible factor 1 (HIF-1). HIF-1 is a 'master switch' being an important physiological response mechanism, likely enhancing neuroprotective compensatory pathways involved in many physiological processes within the brain. This mini-review will discuss the multifunctional mechanisms of action of the drugs, M30 and HLA20 in preclinical models of neurodegeneration with a specific emphasis on their ability to activate the HIF-1 signal transduction pathway.

Targeting SKP1, an ubiquitin E3 ligase component found decreased in sporadic Parkinson's disease.

Microarray-derived transcriptomic studies in human substantia nigra pars compacta (SNpc) samples from sporadic Parkinson's disease (SPD) cases have opened an avenue to concentrate on potential gene intersections or cross-talks along the dopaminergic (DAergic) neurodegenerative cascade in SPD. One emerging gene candidate identified by our group was SKP1A (p19, S-phase kinase-associated protein 1A), found significantly decreased in the SNpc. It is part of the SCF (Skp1, Cullin 1, F-box protein) complex, the largest class of sophisticated ubiquitin-proteasome/E3 ligases, and can directly interact with Fbxo7, a gene defective in PARK15-linked PD. In vitro target validation by viral-mediated RNA interference revealed that the deficiency of Skp1 in a mouse SN-derived DAergic neuronal cell line potentiated the damage caused by exogenous insults implicated in PD pathology and caused the death of neurons undergoing differentiation, which developed Lewy body-like, α-synuclein-positive inclusions preceding cell death. Furthermore, recent animal studies show that site-directed intranigral stereotaxic injections of lentiviruses targeting SKP1A induce pathological and behavioral deficits in mice, supporting a significant role of Skp1 in SN DAergic neuronal survival in SPD. Thus, strategies aimed at increasing the activity or content of Skp1 may represent a novel therapeutic approach that has the potential to treat PD.

The relevance of iron in the pathogenesis of Parkinson's disease.

Alterations of iron levels in the brain has been observed and documented in a number of neurodegenerative disorders including Parkinson's disease (PD). The elevated nigral iron levels observed in PD may reflect a dysfunction of brain iron homeostasis. Under normal physiological conditions excess iron can be sequestrated in ferritin and neuromelanin. Alternatively, the excess iron may represent a component of brain iron deposition associated with ageing. The aetiology of idiopathic PD largely remains an enigma. However, intensive investigations have provided a host of putative mechanisms that might contribute to the pathogenesis underlying the characteristic degeneration of the dopaminergic neurons in the substantia nigra (SN). The mechanisms proposed include oxidative (and nitrative) stress, inflammation, excitotoxicity, mitochondrial dysfunction, altered proteolysis and finally apoptotic induced cell death. Iron-mediated cellular destruction is mediated primarily via reactive oxygen or/and nitrogen species induced oxidative stress. Furthermore, these pathogenic mechanisms appear to be closely interlinked to the cascade of events leading to cellular death. There are conflicting reports about the stage during disease progression at which nigral iron change occurs in PD. Some have found that there are no changes in iron content SN in asymptomatic incidental Lewy body disease, suggesting it may represent a secondary event in the cascade of neuronal degeneration. In contrast, others have found an elevation of iron in SN in pre-clinical stages. These discrepancies may be attributed to the occurrence of different sub-groups of the disease. This concurs with the notion that PD represents a group of related diseases with a number of potential pathogenic pathways.

Translocation and oligomerization of Bax is regulated independently by activation of p38 MAPK and caspase-2 during MN9D dopaminergic neurodegeneration.

Bax is translocated into the mitochondrial membrane and oligomerized therein to initiate mitochondrial apoptotic signaling. Our previous study indicated that reactive oxygen species (ROS)-mediated activation of mitogen-activated protein kinase (MAPK) and caspase is critically involved in 6-hydroxydopamine (6-OHDA)-mediated neurodegeneration. Here, we specifically attempted to examine whether and how these death signaling pathways may be linked to Bax translocation and oligomerization. We found that 6-OHDA treatment triggered translocation and oligomerization of Bax onto the mitochondria in MN9D dopaminergic neuronal cells. These events preceded cytochrome c release into the cytosol. Cross-linking assay revealed that co-treatment with a ROS scavenger or a pan-caspase inhibitor inhibited 6-OHDA-induced Bax oligomerization. Among several candidates of ROS-activated MAPKs and caspases, we found that co-treatment with PD169316 or VDVAD specifically inhibited 6-OHDA-induced Bax oligomerization, suggesting critical involvement of p38 MAPK and caspase-2. Consequently, overexpression of a dominant negative form of p38 MAPK or a shRNA-mediated knockdown of caspase-2 indeed inhibited 6-OHDA-induced Bax oligomerization. However, activation of p38 MAPK and caspase-2 was independently linked to oligomerization of Bax. This specificity was largely confirmed with a Bax 6A7 antibody known to detect activated forms of Bax on the mitochondria. Taken together, our data suggest that there is an independent amplification loop of Bax translocation and oligomerization via caspase-2 and p38 MAPK during ROS-mediated dopaminergic neurodegeneration.