Mitochondrial alterations in Parkinson's disease: new clues

Mitochondrial dysfunction has long been associated with Parkinson's disease (PD). In particular, complex I impairment and subsequent oxidative stress have been widely demonstrated in experimental models of PD and in post-mortem PD samples. A recent wave of new studies is providing novel clues to the potential involvement of mitochondria in PD. In particular, (i) mitochondria-dependent programmed cell death pathways have been shown to be critical to PD-related dopaminergic neurodegeneration, (ii) many disease-causing proteins associated with familial forms of PD have been demonstrated to interact either directly or indirectly with mitochondria, (iii) aging-related mitochondrial changes, such as alterations in mitochondrial DNA, are increasingly being associated with PD, and (iv) anomalies in mitochondrial dynamics and intra-neuronal distribution are emerging as critical participants in the pathogenesis of PD. These new findings are revitalizing the field and reinforcing the potential role of mitochondria in the pathogenesis of PD. Whether a primary or secondary event, or part of a multi-factorial pathogenic process, mitochondrial dysfunction remains at the forefront of PD research and holds the promise as a potential molecular target for the development of new therapeutic strategies for this devastating, currently incurable, disease.     

Zonisamide reduces nigrostriatal dopaminergic neurodegeneration in a mouse genetic model of Parkinson's disease

Parkinson's disease (PD) is a chronic neurodegenerative disorder characterized by the loss of nigrostriatal dopaminergic neurons and consequent motor dysfunction. Zonisamide (1,2‐benzisoxazole‐3‐methanesulfonamide), which was originally developed as an antiepileptic drug, has been found to have therapeutic benefits for PD. However, the pharmacological mechanisms behind the beneficial actions of zonisamide in PD are not fully understood. Here, we investigated the neuroprotective effects of zonisamide on nigrostriatal dopaminergic neurons of the Engrailed mutant mouse, a genetic model of PD. Chronic administration of zonisamide in Engrailed mutant mice was shown to improve the survival of nigrostriatal dopaminergic neurons compared with that under saline treatment. In addition, dopaminergic terminals in the striatum and the motor function were improved in zonisamide‐treated Engrailed mutant mice to the levels of those in control mice. To clarify the mechanism behind the neuroprotective effects of zonisamide, the contents of neurotrophic factors were determined after chronic administration of zonisamide. Brain‐derived neurotrophic factor content was increased in the striatum and ventral midbrain of the zonisamide‐treated mice compared to saline‐treated mice. These findings imply that zonisamide reduces nigrostriatal dopaminergic cell death through brain‐derived neurotrophic factor signaling and may have similar beneficial effects in human parkinsonian patients as well.

     

Molecular determinants of the aggregation behavior of alpha- and beta-synuclein

Alpha- and beta-synuclein are closely related proteins, the first of which is associated with deposits formed in neurodegenerative conditions such as Parkinson's disease while the second appears to have no relationship to any such disorders. The aggregation behavior of alpha- and beta-synuclein as well as a series of chimeric variants were compared by exploring the structural transitions that occur in the presence of a widely used lipid mimetic, sodium dodecyl sulfate (SDS). We found that the aggregation rates of all these protein variants are significantly enhanced by low concentrations of SDS. In particular, we inserted the 11-residue sequence of mainly hydrophobic residues from the non-amyloid-beta-component (NAC) region of alpha-synuclein into beta-synuclein and show that the fibril formation rate of this chimeric protein is only weakly altered from that of beta-synuclein. These intrinsic propensities to aggregate are rationalized to a very high degree of accuracy by analysis of the sequences in terms of their associated physicochemical properties. The results begin to reveal that the differences in behavior are primarily associated with a delicate balance between the positions of a range of charged and hydrophobic residues rather than the commonly assumed presence or absence of the highly aggregation-prone region of the NAC region of alpha-synuclein. This conclusion provides new insights into the role of alpha-synuclein in disease and into the factors that regulate the balance between solubility and aggregation of a natively unfolded protein.     

Oxidative post-translational modifications of alpha-synuclein in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease

Structural and functional alterations of alpha-synuclein is a presumed culprit in the demise of dopaminergic neurons in Parkinson's disease (PD). Alpha-synuclein mutations are found in familial but not in sporadic PD, raising the hypothesis that effects similar to those of familial PD-linked alpha-synuclein mutations may be achieved by oxidative post-translational modifications. Here, we show that wild-type alpha-synuclein is a selective target for nitration following peroxynitrite exposure of stably transfected HEK293 cells. Nitration of alpha-synuclein also occurs in the mouse striatum and ventral midbrain following administration of the parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Conversely, beta-synuclein and synaptophysin were not nitrated in MPTP-intoxicated mice. Our data demonstrate that alpha-synuclein is a target for tyrosine nitration, which, by disrupting its biophysical properties, may be relevant to the putative role of alpha-synuclein in the neurodegeneration associated with MPTP toxicity and with PD.     

Structural transformation and aggregation of human alpha-synuclein in trifluoroethanol: non-amyloid component sequence is essential and beta-sheet formation is prerequisite to aggregation

Amyloid-like aggregation of alpha-synuclein and deposit in Lewy bodies are thought to be the major cause of Parkinson's disease. Here we describe the secondary structural transformation and aggregation of human alpha-synuclein and its C-terminus truncated fragments in trifluoroethanol. Proteins containing the NAC (non-amyloid component) segment undergo a three-state transition: from native random coil to beta-sheet and to alpha-helical structure, while the NAC deficient fragment and gamma-synuclein undergo a typical two-state coil-to-alpha transition. The beta-sheet form is highly hydrophobic that strongly binds to 1-anilinonaphthalene-8-sulfonic acid (ANS) and is prone to self-aggregation. The results suggest that the NAC sequence is essential to beta-sheet formation and the aggregation originates from the beta-sheet intermediate, which may be implicated in the pathogenesis of Parkinson's disease.     

Synergistic effects of pravastatin and pioglitazone in renal tubular epithelial cells induced by transforming growth factor-beta1

Recent studies suggest that treatment with PPAR-gamma agonists and statins have beneficial effects on renal disease. However, the combined effects of PPAR-gamma agonists and statins in human renal epithelial cells are unknown. Our present study revealed that there were synergistic effects of pravastatin and pioglitazone in the expression of alpha-smooth muscle actin (alpha-SMA), connective tissue growth factor (CTGF), fibronectin (FN), plasminogen activator inhibitor-1 (PAI-1) and collagen 1 in human renal proximal tubular epithelial cells induced by transforming growth factor-beta 1 (TGF-beta1). The beneficial effects of combined therapy against renal tubular epithelial cell injury are attributed, at least in part, to the inhibition of transdifferentiation, extracellular matrix deposition and cytokine production.     

Analysis of dopaminergic neuronal dysfunction in genetic and toxin‐induced models of Parkinson's disease in Drosophila

Drosophila melanogaster has contributed significantly to the understanding of disease mechanisms in Parkinson's disease (PD) as it is one of the very few PD model organisms that allow the study of age‐dependent behavioral defects, physiology and histology, and genetic interactions among different PD‐related genes. However, there have been contradictory results from a number of recent reports regarding the loss of dopaminergic neurons in different PD fly models. In an attempt to re‐evaluate and clarify this issue, we have examined three different genetic (α‐synuclein, Pink1, parkin) and two toxin‐based (rotenone and paraquat) models of the disease for neuronal cell loss. Our results showed no dopaminergic neuronal loss in all models tested. Despite this surprising result, we found additional phenotypes showing the dysfunctional status of the dopaminergic neurons in most of the models analyzed. A common feature found in most models is a quantifiable decrease in the fluorescence of a green‐fluorescent protein reporter gene in dopaminergic neurons that correlates well with other phenotypes found for these models and can be reliably used as a hallmark of the neurodegenerative process when modeling diseases affecting the dopaminergic system in Drosophila.

     

A new active vitamin D analog,ED-71, causes increase in bone mass with preferential effects on bone in osteoporotic patients

As a candidate for active vitamin D analogs that have selective effects on bone, 1alpha,25-dihydroxy-2beta-(3-hydroxypropoxy)vitamin D3 (ED-71) has been synthesized and is currently under clinical trials. In ovariectomized rat model for osteoporosis, ED-71 caused an increase bone mass at the lumbar vertebra to a greater extent than 1alpha-hydroxyvitamin D3 (alfacalcidol), while enhancing calcium absorption and decreasing serum parathyroid hormone levels to the same degree as alfacalcidol. ED-71 lowered the biochemical and histological parameters of bone resorption more potently than alfacalcidol, while maintaining bone formation markers.An early phase II clinical trial was conducted with 109 primary osteoporotic patients. The results indicate that oral daily administration of ED-71 (0.25, 0.5, 0.75, and 1.0 microgram) for 6 months increased lumbar bone mineral density in a dose-dependent manner without causing hypercalcemia and hypercalciuria. ED-71 also exhibited a dose-dependent suppression of urinary deoxypyridinoline with no significant reduction in serum osteocalcin. These results demonstrate that ED-71 has preferential effects on bone with diminished effects on intestinal calcium absorption. ED-71 offers potentially a new modality of therapy for osteoporosis with selective effects on bone.     

Regulation of protein kinase C Apl II by serotonin receptors in Aplysia

Serotonin (5-hydroxytryptamine, 5HT) is the neurotransmitter that mediates dishabituation in Aplysia. Serotonin mediates this behavioral change through the reversal of synaptic depression in sensory neurons (SNs). However, the 5HT receptors present in SNs and in particular, the receptor important for activation of protein kinase C (PKC) have not been fully identified. Using a recent genome assembly of Aplysia, we identified new receptors from the 5HT(2) , 5HT(4) , and 5HT(7) families. Using RT-PCR from isolated SNs, we found that three 5HT receptors, 5HT(1Apl(a)) , 5HT(2Apl) , and 5HT(7Apl) were expressed in SNs. These receptors were cloned and expressed in a heterologous system. In this system, 5HT(2Apl) could significantly translocate PKC Apl II in response to 5HT and this was blocked by pirenperone, a 5HT(2) receptor antagonist. Surprisingly, pirenperone did not block 5HT-mediated translocation of PKC Apl II in SNs, nor 5HT-mediated reversal of depression. Expression of 5HT(1Apl(a)) in SNs or genistein, an inhibitor of tyrosine kinases inhibited both PKC translocation and reversal of depression. These results suggest a non-canonical mechanism for the translocation of PKC Apl II in SNs.     

cdk5 modulates beta- and delta-catenin/Pin1 interactions in neuronal cells

The cdk5/p35 complex has been implicated in a variety of functions related to brain development, including axonal outgrown and neuronal migration. In this study, by co-immunoprecipitation and pull-down experiments, we have shown that the cdk5/p35 complex associates with and phosphorylates the neuronal delta-catenin. Immunocytochemical studies of delta-catenin and the cdk5-activator p35 in primary cortical neurons indicated that these proteins co-localize in the cell body of neuronal cells. In addition, cdk5 co-localized with beta-catenin in the cell-cell contacts and plasma membrane of undifferentiated and differentiated N2A cells. In this context, we identified Ser(191) and Ser(246) on beta-catenin structure as specific phosphorylation sites for cdk5/p35 complex. Moreover, Pin1, a peptidyl-prolyl isomerase (PPIase) directly bound to both, beta- and delta-catenin, once they have been phosphorylated by the cdk5/p35 complex. Studies indicate that the cdk5/p35 protein kinase system is directly involved in the regulatory mechanisms of neuronal beta- and delta-catenin.     

A metabolic shift induced by a PPAR panagonist markedly reduces the effects of pathogenic mitochondrial tRNA mutations

Mutations in mitochondrial DNA-encoded tRNA genes are associated with many human diseases. Activation of peroxisome proliferator-activated receptors (PPARs) by synthetic agonists stimulates oxidative metabolism, induces an increase in mitochondrial mass and partially compensates for oxidative phosphorylation system (OXPHOS) defects caused by single OXPHOS enzyme deficiencies in vitro and in vivo. Here, we analysed whether treatment with the PPAR panagonist bezafibrate in cybrids homoplasmic for different mitochondrial tRNA mutations could ameliorate the OXPHOS defect. We found that bezafibrate treatment increased mitochondrial mass, mitochondrial tRNA steady state levels and enhanced mitochondrial protein synthesis. This improvement resulted in increased OXPHOS activity and finally in enhanced mitochondrial ATP generating capacity. PPAR panagonists are known to increase the expression of PPAR gamma coactivator-1α (PGC-1α), a master regulator of mitochondrial biogenesis. Accordingly, we found that clones of a line harbouring a mutated mitochondrial tRNA gene mutation selected for the ability to grow in a medium selective for OXPHOS function had a 3-fold increase in PGC-1α expression, an increase that was similar to the one observed after bezafibrate treatment. These findings show that increasing mitochondrial mass and thereby boosting residual OXPHOS capacity can be beneficial to an important class of mitochondrial defects reinforcing the potential therapeutic use of approaches stimulating mitochondrial proliferation for mitochondrial disorders.     

Aquaporin‐4 deficiency reduces TGF‐β1 in mouse midbrains and exacerbates pathology in experimental Parkinson's disease

Aquaporin‐4 (AQP4), the main water‐selective membrane transport protein in the brain, is localized to the astrocyte plasma membrane. Following the establishment of a 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐induced Parkinson's disease (PD) model, AQP4‐deficient (AQP4^?/?) mice displayed significantly stronger microglial inflammatory responses and remarkably greater losses of tyrosine hydroxylase (TH⁺)‐positive neurons than did wild‐type AQP4 (AQP4^+/+) controls. Microglia are the most important immune cells that mediate immune inflammation in PD. However, recently, few studies have reported why AQP4 deficiency results in more severe hypermicrogliosis and neuronal damage after MPTP treatment. In this study, transforming growth factor‐β1 (TGF‐β1), a key suppressive cytokine in PD onset and development, failed to increase in the midbrain and peripheral blood of AQP4^?/? mice after MPTP treatment. Furthermore, the lower level of TGF‐β1 in AQP4^?/? mice partially resulted from impairment of its generation by astrocytes; reduced TGF‐β1 may partially contribute to the uncontrolled microglial inflammatory responses and subsequent severe loss of TH⁺ neurons in AQP4^?/? mice after MPTP treatment. Our study provides not only a better understanding of both aetiological and pathogenical factors implicated in the neurodegenerative mechanism of PD but also a possible approach to developing new treatments for PD via intervention in AQP4‐mediated immune regulation.     

Structural effects of Parkinson's disease linked DJ-1 mutations

Mutations in the protein DJ-1 are associated with familial forms of Parkinson's disease, indicating that DJ-1 may be involved in pathways related to the etiology of this disorder. Here we have used solution state NMR and circular dichroism spectroscopies to evaluate the extent of structural perturbations associated with five different Parkinson's disease linked DJ-1mutations: L166P, E64D, M26I, A104T, and D149A. Comparison of the data with those obtained for the wild-type protein shows that the L166P mutation leads to severe and global destabilization and unfolding of the protein structure, while the structure of the E64D mutation, as expected, is nearly unperturbed. Interestingly, the remaining three mutants all show different degrees of structural perturbation, which are accompanied by a reduction in the thermodynamic stability of the protein. The observed structural and thermodynamic differences are likely to underlie any functional variations between these mutants and the wild type, which in turn are likely responsible for the pathogenicity of these mutations.     

Long-term effects of tumor necrosis factor-alpha treatment on insulin signaling pathway in HepG2 cells and HepG2 cells overexpressing constitutively active Akt/PKB

Tumor necrosis factor-alpha (TNF-alpha) mediated attenuation of insulin signaling pathway is an important cause in several disorders like obesity, obesity linked diabetes mellitus. TNF-alpha actions vary depending upon concentration and time of exposure in various cells. In the present study, the effects of long-term TNF-alpha (1 ng/ml) exposure on the components of insulin signaling pathway in HepG2 and HepG2 cells overexpressing constitutively active Akt1/PKB-alpha (HepG2-CA-Akt/PKB) have been investigated. In parental HepG2 cells, TNF-alpha treatment for 24 h reduced the phosphorylation of Akt1/PKB-alpha and GSK-3beta and under these conditions cells also showed reduced insulin responsiveness in terms of Akt1/PKB-alpha and GSK-3beta phosphorylation. TNF-alpha pre-incubated HepG2-CA-Akt/PKB cells showed lower reduction in Akt1/PKB-alpha and GSK-3beta phosphorylation and insulin responsiveness after 24 h as compared to parental HepG2 cells. We report that the long-term TNF-alpha pre-incubation in both parental HepG2 and HepG2-CA-Akt/PKB-alpha cells leads to the reduction in the levels of IRS-1 without altering the levels of IRS-2. In order to understand the reason for the differential insulin resistance in both the cell types, the effect of long-term TNF-alpha treatment on the proteins upstream to Akt/PKB was investigated. TNF-alpha pre-incubation also showed reduced insulin-stimulated Tyr phosphorylation of insulin receptor (IR-beta) in both the cell types, moreover hyperphosphorylation of IRS-1 at Ser 312 residue was observed in TNF-alpha pre-incubated cells. As hyperphosphorylation of IRS-1 at Ser 312 can induce its degradation, it is possible that reduced insulin responsiveness after long-term TNF-alpha pre-incubation observed in this study is due to the decrease in IRS-1 levels.