Systemic Administration of Proteasome Inhibitor Protects Against MPTP Neurotoxicity in Mice

Dysfunction of the proteasome has been suggested to contribute in the degeneration of nigrostriatal dopaminergic neurons. Here, we investigated to determine whether systematic administration of proteasome inhibitor, carbobenzoxy-l-γ-t-butyl-l-glutamyl-l-alanyl-l-leucinal (PSI) protects against MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) neurotoxicity in mice. Three administrations of MPTP at 1-h intervals to mice reduced significantly the concentration of dopamine, DOPAC (3,4-dihydroxyphenylacetic acid) and HVA (homovanillic acid) in the striatum after 5 days. In contrast, PSI (0.3 and 1.0 mg/kg) prevented a significant decrease in dopamine, DOPAC and HVA contents of the striatum 5 days after MPTP treatment. In our Western blot analysis study, PSI at a dose of 1.0 mg/kg prevented a significant decrease in TH (tyrosine hydroxylase) protein and a significant increase in glial fibrillary acidic protein 5 days after MPTP treatment. Furthermore, our immunohistochemical study showed that PSI at a dose of 1.0 mg/kg prevented a significant loss in TH immunopositive neurons in the striatum and substantia nigra 5 days after MPTP treatment. In contrast, PSI caused a significant increase in the number of intense ubiquitin immunopositive cells in the striatum and substantia nigra 5 days after MPTP treatment. These results indicate that proteasome inhibitors can protect against MPTP neurotoxicity in mice. The neuroprotective effect of PSI against dopaminergic cell damage may be mediated by the elevation of ubiquitination. Thus, our findings provide further valuable information for the pathogenesis of Parkinson’s disease. Takuya Oshikawa and Hayato Kuroiwa contributed equally to this work.     

Delayed Treatment with Arundic Acid Reduces the MPTP-induced Neurotoxicity in Mice

The authors investigated the protective effects of a novel astrocyte-modulating agent, arundic acid, in a 1-methyl-4-phenyl-1,2,3,6-tetrahyropyridine (MPTP) mouse model of Parkinson’s disease. Male mice received four intraperitoneal (i.p.) injections of MPTP (20 mg/kg) at 2 h intervals. The content of dopamine and its metabolites in the striatum was reduced markedly 7 days after MPTP treatment. The delayed treatment with arundic acid (30 mg/kg, i.p.) administered 3, 4, 5 and 6 days after MPTP treatment did not affect the depletion of dopamine and its metabolites in the striatum. Our immunohistochemical study with anti-tyrosine hydroxylase antibody, anti-neuronal nuclei antibody, anti-glial fibrillary acidic protein antibody, anti-S100β antibody and anti-nestin antibody showed that the delayed treatment with arundic acid had a protective effect against MPTP-induced neuronal damage in the striatum and the substantia nigra of mice. Furthermore, this agent ameliorated the severe reductions in number of isolectin reactive microglia in the striatum and the substantia nigra 7 days after MPTP treatment. These results demonstrate that the inhibition of S100β synthesis in astrocytes may be the major component of the beneficial effect of arundic acid. Thus, our present findings provide that the therapeutic strategies targeted to astrocytic modulation with arundic acid offers a great potential for restoring the functional capacity of the surviving dopaminergic neurons in individuals affected with Parkinson’s disease.     

Neurochemical Evidence for Agmatine Modulation of 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine (MPTP) Neurotoxicity

Agmatine treatment is known to exert neuroprotective effects in several models of neurotoxic and ischemic brain and spinal cord injuries. Here we sought to find out whether agmatine treatment would also prove to be neuroprotective in the mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson’s disease. Concomitant daily treatment (intraperitoneal injections) with agmatine (100 mg/kg for 5 days) and MPTP (40 mg/kg for 2 days) exacerbated MPTP-related toxicity as evidenced by a larger reduction in dopamine uptake into striatal synaptosomes (42.4% as compared to 58.3% of control, respectively). In contrast, agmatine treatment commencing after MPTP, produced partial protection (31%) against MPTP dopaminergic toxicity. The findings implicate agmatine in mechanisms regulating MPTP neurotoxicity, but underscore the characteristic neuroprotective efficacy of agmatine when applied after the insult.     

The Effects of Piroxicam in the Attenuation of MPP+/MPTP Toxicity In Vitro and In Vivo

Several lines of evidence support the neuroprotective action of cyclooxygenase-2 (COX-2) inhibitors in various models of Parkinson’s disease (PD). In the current study, we investigated the neuroprotective properties of several COX inhibitors against 1-methyl-4-phenylpyridinium (MPP+) in neuroblastoma Neuro 2A (N-2A) cells in vitro and the protection against degeneration of substantia nigra pars compacta (SNc) dopaminergic (DA) neurons after the administration of 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP) in C57/BL6 male mice. The data obtained demonstrate a lack of protective effects observed by COX 1-2 inhibitors ibuprofen and acetylsalicylic acid against MPP+ toxicity in N-2A, where piroxicam was protective in a dose dependent manner (MPP+ control: 15 ± 2% MPP+ piroxicam: 5 mM 89 ± 4%). The data also indicate a drop in mitochondrial oxygen (O₂) consumption and ATP during MPP+ toxicity with no restoration of mitochondrial function concurrent to a heightened concentration of somatic ATP during piroxicam rescue. These findings indicate that the neuroprotective effects of COX inhibitors against MPP+ are not consistent, but that piroxicam may work through an unique mechanism to propel anaerobic energy metabolism. On the other hand, using mice, piroxicam (20 mg/kg) was effective against MPTP-induced dopaminergic degeneration in the (SNc) and loss of locomotive function in mice. Administering a 3 day pre-treatment of piroxicam (20 mg/kg) was effective in antagonizing the losses in SNc tyrosine hydroxylase protein expression, SNc DA concentration and associated anomaly in ambulatory locomotor activity. It was concluded from these findings that piroxicam is unique among COX inhibitors in providing very significant neuroprotection against MPP+ in vitro and in vivo.     

Increased MPTP Neurotoxicity in Vesicular Monoamine Transporter 2 Heterozygote Knockout Mice

Abstract: The neurotoxic action of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been proposed to be attenuated by sequestration into intracellular vesicles by the vesicular monoamine transporter (VMAT2). The purpose of this study was to determine if mice with genetically reduced levels of VMAT2 (heterozygote knockout; VMAT2 +/−) were more vulnerable to MPTP. Striatal dopamine (DA) content, the levels of DA transporter (DAT) protein, and the expression of glial fibrillary acidic protein (GFAP) mRNA, a marker of gliosis, were assessed as markers of MPTP neurotoxicity. In all parameters measured VMAT2 +/− mice were more sensitive than their wild-type littermates (VMAT2 +/+). Administration of MPTP (7.5, 15, or 30 mg/kg, b.i.d.) resulted in dose-dependent reductions in striatal DA levels in both VMAT2 +/− and VMAT2 +/+ animals, but the neurotoxic potency of MPTP was approximately doubled in the VMAT2 +/− mice: 59 versus 23% DA loss 7 days after 7.5 mg/kg dose for VMAT2 +/− and VMAT2 +/+ mice, respectively. Dopaminergic nerve terminal integrity, as assessed by DAT protein expression, also revealed more drastic reductions in the VMAT2 +/− mice: 59 versus 35% loss at 7.5 mg/kg and 95 versus 58% loss at 15 mg/kg for VMAT2 +/− and VMAT2 +/+ mice, respectively. Expression of GFAP mRNA 2 days after MPTP was higher in the VMAT2 +/− mice than in the wild-type: 15.8- versus 7.8-fold increase at 7.5 mg/kg and 20.1- versus 9.6-fold at 15 mg/kg for VMAT2 +/− and VMAT2 +/+ mice, respectively. These observations clearly demonstrate that VMAT2 +/− mice are more susceptible to the neurotoxic effects of MPTP, suggesting that VMAT2-mediated sequestration of the neurotoxin into vesicles may play an important role in attenuating MPTP toxicity in vivo.     

From bench to bed: the potential of stem cells for the treatment of Parkinson’s disease

Parkinson’s disease (PD) is the most common movement disorder. The neuropathology is characterized by the loss of dopamine neurons in the substantia nigra pars compacta. Transplants of fetal/embryonic midbrain tissue have exhibited some beneficial clinical effects in open-label trials. Neural grafting has, however, not become a standard treatment for several reasons. First, the supply of donor cells is limited, and therefore, surgery is accompanied by difficult logistics. Second, the extent of beneficial effects has varied in a partly unpredictable manner. Third, some patients have exhibited graft-related side effects in the form of involuntary movements. Fourth, in two major double-blind placebo-controlled trials, there was no effect of the transplants on the primary endpoints. Nevertheless, neural transplantation continues to receive a great deal of interest, and now, attention is shifting to the idea of using stem cells as starting donor material. In the context of stem cell therapy for PD, stem cells can be divided into three categories: neural stem cells, embryonic stem cells, and other tissue-specific types of stem cells, e.g., bone marrow stem cells. Each type of stem cell is associated with advantages and disadvantages. In this article, we review recent advances of stem cell research of direct relevance to clinical application in PD and highlight the pros and cons of the different sources of cells. We draw special attention to some key problems that face the translation of stem cell technology into the clinical arena.     

Silencing α-Synuclein Gene Expression Enhances Tyrosine Hydroxylase Activity in MN9D Cells

alpha-Synuclein has been implicated in the pathogenesis of Parkinson's disease (PD). Previous studies have shown that alpha-synuclein is involved in the regulation of dopamine (DA) metabolism, possibly by down-regulating the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in DA biosynthesis. In this study, we constructed alpha-synuclein stably silenced MN9D/alpha-SYN(-) cells by vector mediated RNA interference and examined its effects on DA metabolism. We found that there were no significant differences in TH protein and mRNA levels between MN9D, MN9D/alpha-SYN(-) and MN9D/CON cells, suggesting that silencing alpha-synuclein expression does not affect TH gene expression. However, significant increases in phosphorylated TH, cytosolic 3, 4-dihydroxyphenylalanine (L-DOPA) and DA levels were observed in MN9D/alpha-SYN(-) cells. Our data show that TH activity and DA biosynthesis were enhanced by down-regulation of alpha-synuclein, suggesting that alpha-synuclein may act as a negative regulator of cytosolic DA. With respect to PD pathology, a loss of functional alpha-synuclein may result in increased DA levels in neurons that may lead to cell injury or even death.     

Adenosine A<Subscript>2A</Subscript> receptors in Parkinson’s disease treatment

Latest results on the action of adenosine A_2A receptor antagonists indicate their potential therapeutic usefulness in the treatment of Parkinson’s disease. Basal ganglia possess high levels of adenosine A_2A receptors, mainly on the external surfaces of neurons located at the indirect tracts between the striatum, globus pallidus, and substantia nigra. Experiments with animal models of Parkinson’s disease indicate that adenosine A_2A receptors are strongly involved in the regulation of the central nervous system. Co-localization of adenosine A_2A and dopaminergic D2 receptors in striatum creates a milieu for antagonistic interaction between adenosine and dopamine. The experimental data prove that the best improvement of mobility in patients with Parkinson’s disease could be achieved with simultaneous activation of dopaminergic D2 receptors and inhibition of adenosine A_2A receptors. In animal models of Parkinson’s disease, the use of selective antagonists of adenosine A_2A receptors, such as istradefylline, led to the reversibility of movement dysfunction. These compounds might improve mobility during both monotherapy and co-administration with L-DOPA and dopamine receptor agonists. The use of adenosine A_2A receptor antagonists in combination therapy enables the reduction of the L-DOPA doses, as well as a reduction of side effects. In combination therapy, the adenosine A_2A receptor antagonists might be used in both moderate and advanced stages of Parkinson’s disease. The long-lasting administration of adenosine A_2A receptor antagonists does not decrease the patient response and does not cause side effects typical of L-DOPA therapy. It was demonstrated in various animal models that inhibition of adenosine A_2A receptors not only decreases the movement disturbance, but also reveals a neuroprotective activity, which might impede or stop the progression of the disease. Recently, clinical trials were completed on the use of istradefylline (KW-6002), an inhibitor of adenosine A_2A receptors, as an anti-Parkinson drug.     

Roles of Glutathione (GSH) in Dopamine (DA) Oxidation Studied by Improved Tandem HPLC Plus ESI-MS

In this study, new procedure with improved tandem HPLC plus ESI-MS was utilized to decipher the protective role of glutathione (GSH) against dopamine (DA) oxidation. We demonstrated that auto-oxidation of DA could produce aminochrome (AM, a cyclized DA quinone), which could be effectively abrogated by reductants, especially by GSH. Furthermore GSH was demonstrated to be able to conjugate with AM to form various conjugates via condensation reactions without enzymatic catalysis. The GSH-AM conjugates tend to aggregate, possibly mediated by conjugated AM structures, but could be inhibited by GSH. We hypothesized that proteins conjugated by AM might facilitate Lewy body formation of Parkinson’s disease (PD) in dopaminergic neurons via similar polymerization. We proposed that GSH could protect dopaminergic neurons against DA-induced toxicity via various mechanisms. The imbalance between DA oxidation and GSH protective capacity could be a key factor contributing to PD. Strategies to use GSH analogues, GSH inducers or to control DA oxidation might work to control PD onset and development.     

Disturbance of Iron Metabolism as a Contributing Factor to SN Hyperechogenicity in Parkinson’s Disease: Implications for Idiopathic and Monogenetic Forms

A number of investigations have provided evidence for a central role of iron in the pathogenesis of Parkinson’s disease (PD). Recently it could be demonstrated that iron related changes of the substantia nigra may be one important factor contributing to the hyperechogenicity typicall visualized by transcranial sonography in idiopathic PD. Moreover, also patients with monogenetically caused PD show this hyperechogenicity, although to a lesser extent. According to numerous findings and experiments it seems plausible that iron also contributes to the pathophysiological cascades in the monogenetic forms of PD. Therefore, it is not only essential to acknowledge the pivotal role of iron for PD, but also to enhance the effort in finding therapeutic strategies to prevent the impact of iron on neurodegenerative processes. Moreover, early detection of subjects at risk is essential for the application of therapeutic strategies at a stage at which neuroprotection is still possible. Special issue dedicated to Dr. Moussa Youdim     

Modulation of α-Synuclein Aggregation by Dopamine: A Review

Parkinson’s disease (PD) is a progressive neurodegenerative disorder that is characterized by (1) the selective loss of dopaminergic neurons in the substantia nigra and (2) the deposition of misfolded α-synuclein (α-syn) as amyloid fibrils in the intracellular Lewy bodies in various region of the brain. Current thinking suggests that an interaction between α-syn and dopamine (DA) leads to the selective death of neuronal cells and the accumulation of misfolded α-syn. However, the exact mechanism by which this occurs is not fully defined. DA oxidation could play a key role is the pathogenesis of PD by causing oxidative stress, mitochondria dysfunction and impairment of protein metabolism. Here, we review the literature on the role of DA and its oxidative intermediates in modulating the aggregation pathways of α-syn.     

The Mechanistic Links Between Proteasome Activity,Aging and Age-related Diseases

Damaged and misfolded proteins accumulate during the aging process, impairing cell function and tissue homeostasis. These perturbations to protein homeostasis (proteostasis) are hallmarks of age-related neurodegenerative disorders such as Alzheimer’s, Parkinson’s or Huntington’s disease. Damaged proteins are degraded by cellular clearance mechanisms such as the proteasome, a key component of the proteostasis network. Proteasome activity declines during aging, and proteasomal dysfunction is associated with late-onset disorders. Modulation of proteasome activity extends lifespan and protects organisms from symptoms associated with proteostasis disorders. Here we review the links between proteasome activity, aging and neurodegeneration. Additionally, strategies to modulate proteasome activity and delay the onset of diseases associated to proteasomal dysfunction are discussed herein.     

Sequence conservation between porcine and human LRRK2

Leucine-rich repeat kinase 2 (LRRK2) is a member of the ROCO protein superfamily (Ras of complex proteins (Roc) with a C-terminal Roc domain). Mutations in the LRRK2 gene lead to autosomal dominant Parkinsonism. We have cloned the porcine LRRK2 cDNA in an attempt to characterize conserved and therefore likely functional domains. The LRRK2 cDNA contains an open reading frame of 7,578 bp. The predicted LRRK2 protein consists of 2,526 amino acids of 86–93% identity with its mammalian couterparts. The deduced amino acid sequence of encoded porcine LRRK2 protein displays extensive homology with its human counterpart, with greatest similarities in those regions that contain the kinase domain, the Roc domain and the COR motif. Expression of porcine LRRK2 mRNA in various organs and tissues is similar to its human counterpart and not limited to the brain. The obtained data show that the LRRK2 sequence and expression patterns are conserved across species. The porcine LRRK2 gene was mapped to chromosome 5q25. The results obtained suggest that the LRRK2 gene might be of particular interest in our attempt to generate a transgenic porcine model for Parkinson’s disease. The sequence of the porcine LRRK2 cDNA, encoding the LRRK2/dardarin protein, and the genomic sequence of LRRK2 have been submitted to DDBJ/EMBL/GenBank under the Accessions Numbers EU019992, and EU019994, respectively.     

Mitochondria and Neurodegeneration

Many lines of evidence suggest that mitochondria have a central role in ageing-related neurodegenerative diseases. However, despite the evidence of morphological, biochemical and molecular abnormalities in mitochondria in various tissues of patients with neurodegenerative disorders, the question “is mitochondrial dysfunction a necessary step in neurodegeneration?” is still unanswered. In this review, we highlight some of the major neurodegenerative disorders (Alzheimer’s disease, Parkinson’s disease, Amyotrophic lateral sclerosis and Huntington’s disease) and discuss the role of the mitochondria in the pathogenetic cascade leading to neurodegeneration.     

Long-Term L-DOPA Treatment Causes Indiscriminate Increase in Dopamine Levels at the Cost of Serotonin Synthesis in Discrete Brain Regions of Rats

(1) The treatment of choice for Parkinson’s disease (PD) is 3,4-dihydroxyphenylalanine (L-DOPA) with peripheral decarboxylase inhibitor, but long-term therapy leads to motor and psychiatric complications. In the present study we investigated 5-hydroxytryptamine (5-HT) and dopamine concentrations in serotonergic and dopaminergic nuclei following chronic administration of L-DOPA to find whether the neurotransmitter synthesis in these brain areas are compensated. (2) Rats were administered L-DOPA (250 mg/kg) and carbidopa (25 mg/kg) daily for 59 and 60 days, and killed on the 60th day, respectively at 24 h and 30 min after the last dose. L-DOPA, norepinephrine, 5-HT, 5-hydroxyindoleacetic acid (5-HIAA), dopamine, homovanillic acid (HVA), and 3,4-dihydroxyphenylacetic acid (DOPAC) were measured in striatum, nucleus raphe dorsalis (NRD), nucleus accumbens (NAc), substantia nigra, cerebellum, and cortex employing HPLC-electrochemical procedure. (3) Prolonged treatment of L-DOPA caused depression in the animals as revealed in a forced swim test. Serotonin content was significantly decreased in all brain regions studied 30 min after long-term L-DOPA, except in NAc. The cortex and striatum showed lowered levels of this indoleamine 24 h after 59 doses of L-DOPA. Dopamine, HVA, and DOPAC concentrations were significantly higher in all the regions studied after 30 min, and in the cerebellum after 24 h of L-DOPA. The levels of DOPAC were elevated in all the brain areas studied 24 h after prolonged L-DOPA treatment. (4) The present results suggest that long-term L-DOPA treatment results in significant loss of 5-HT in serotonergic and dopaminergic regions of the brain. Furthermore, while L-DOPA metabolism per se was uninfluenced, dopamine synthesis was severely impaired in all the regions. The imbalance of serotonin and dopamine formation may be the cause of overt cognitive, motor, and psychological functional aberrations seen in parkinsonian patients following prolonged L-DOPA treatment.     

Inhibition of Vesicular Monoamine Transporter-2 Activity in α-Synuclein Stably Transfected SH-SY5Y Cells

α-Synuclein plays a key role in the pathological neurodegeneration in Parkinson’s disease. Although its contribution to normal physiology remains elusive, the selective degeneration of α-synuclein-containing dopaminergic neurons in Parkinson’s disease may be linked to abnormal α-synuclein induced toxicity. In the present study, a complex of α-synuclein and vesicular monoamine transporter-2 was identified by GST-Pull Down experiment. In wild-type α-synuclein stably transfected SH-SY5Y cell lines, the activity of vesicular monoamine transporter-2 decreased by 31% as determined by [³H] dopamine uptake, and its expression also decreased in both protein and mRNA levels using western and northern blot analysis. Overexpression of wild-type α-synuclein did not induce cell death or apoptosis, but significantly enhanced the intracellular reactive oxygen species level as assayed by flow cytometry. These data suggest that Up-regulated α-synuclein expression inhibits the activity of vesicular monoamine transporter-2, thereby interrupting dopamine homeostasis and resulting in dopaminergic neuron injury in Parkinson’s disease.     

Voltage-gated Calcium Channels Provide an Alternate Route for Iron Uptake in Neuronal Cell Cultures

Recent studies suggest that iron enters cardiomyocytes via the L-type voltage-gated calcium channel (VGCC). The neuronal VGCC may also provide iron entry. As with calcium, extraneous iron is associated with the pathology and progression of neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease. VGCCs, ubiquitously expressed, may be an important route of excessive entry for both iron and calcium, contributing to cell toxicity or death. We evaluated the uptake of ⁴⁵Ca²⁺ and ⁵⁵Fe²⁺ into NGF-treated rat PC12, and murine N-2α cells. Iron not only competed with calcium for entry into these cells, but iron uptake (similar to calcium uptake) was inhibited by nimodipine, a specific L-type VGCC blocker, and enhanced by FPL 64176, an L-VGCC activator, in a dose-dependent manner. Taken together, these data suggest that voltage-gated calcium channels are an alternate route for iron entry into neuronal cells under conditions that promote cellular iron overload toxicity. Special issue dedicated to Dr. Moussa Youdim.     

Cell death pathways in Parkinson’s disease: proximal triggers, distal effectors, and final steps

Parkinson’s disease (PD) is a common neurodegenerative disorder. Neuronal cell death in PD is still poorly understood, despite a wealth of potential pathogenic mechanisms and pathways. Defects in several cellular systems have been implicated as early triggers that start cells down the road toward neuronal death. These include abnormal protein accumulation, particularly of alpha-synuclein; altered protein degradation via multiple pathways; mitochondrial dysfunction; oxidative stress; neuroinflammation; and dysregulated kinase signaling. As dysfunction in these systems mounts, pathways that are more explicitly involved in cell death become recruited. These include JNK signaling, p53 activation, cell cycle re-activation, and signaling through bcl-2 family proteins. Eventually, neurons become overwhelmed and degenerate; however, even the mechanism of final cell death in PD is still unsettled. In this review, we will discuss cell death triggers and effectors that are relevant to PD, highlighting important unresolved issues and implications for the development of neuroprotective therapies.