BAX channel activity mediates lysosomal disruption linked to Parkinson disease

Lysosomal disruption is increasingly regarded as a major pathogenic event in Parkinson disease (PD). A reduced number of intraneuronal lysosomes, decreased levels of lysosomal-associated proteins and accumulation of undegraded autophagosomes (AP) are observed in PD-derived samples, including fibroblasts, induced pluripotent stem cell-derived dopaminergic neurons, and post-mortem brain tissue. Mechanistic studies in toxic and genetic rodent PD models attribute PD-related lysosomal breakdown to abnormal lysosomal membrane permeabilization (LMP). However, the molecular mechanisms underlying PD-linked LMP and subsequent lysosomal defects remain virtually unknown, thereby precluding their potential therapeutic targeting. Here we show that the pro-apoptotic protein BAX (BCL2-associated X protein), which permeabilizes mitochondrial membranes in PD models and is activated in PD patients, translocates and internalizes into lysosomal membranes early following treatment with the parkinsonian neurotoxin MPTP, both in vitro and in vivo, within a time-frame correlating with LMP, lysosomal disruption, and autophagosome accumulation and preceding mitochondrial permeabilization and dopaminergic neurodegeneration. Supporting a direct permeabilizing effect of BAX on lysosomal membranes, recombinant BAX is able to induce LMP in purified mouse brain lysosomes and the latter can be prevented by pharmacological blockade of BAX channel activity. Furthermore, pharmacological BAX channel inhibition is able to prevent LMP, restore lysosomal levels, reverse AP accumulation, and attenuate mitochondrial permeabilization and overall nigrostriatal degeneration caused by MPTP, both in vitro and in vivo. Overall, our results reveal that PD-linked lysosomal impairment relies on BAX-induced LMP, and point to small molecules able to block BAX channel activity as potentially beneficial to attenuate both lysosomal defects and neurodegeneration occurring in PD.     

Cdk5 suppression blocks SIRT1 degradation via the ubiquitin-proteasome pathway in Parkinson's disease models

The NAD⁺-dependent protein deacetylase sirtuin 1 (SIRT1), a member of the sirtuin family, may have a neuroprotective effect in multiple neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD) and Amyotrophic lateral sclerosis (ALS). Many studies have suggested that overexpression-induced or resveratrol-treated activation of SIRT1 could significantly ameliorate several neurodegenerative diseases in mouse models. However, the type of SIRT1, protein expression levels and underlying mechanisms remain unclear, especially in PD. In this study, the results demonstrated that SIRT1 knockout markedly worsened the movement function in MPTP-lesioned animal model of PD. SIRT1 expression was found to be markedly decreased not only in environmental factor PD models, neurotoxin MPP⁺-treated primary culture neurons and MPTP-induced mice but also in genetic factor PD models, overexpressed α-synuclein-A30PA53T SH-SY5Y stable cell line and hm²α-SYN-39 transgenic mouse strain. Importantly, the degradation of SIRT1 during MPP⁺ treatment was mediated by the ubiquitin-proteasome pathway. Furthermore, the results indicated that cyclin-dependent kinase 5 (Cdk5) was also involved in the decrease of SIRT1 expression, which could be efficiently blocked by the inhibition of Cdk5. In conclusion, our findings revealed that the Cdk5-dependent ubiquitin-proteasome pathway mediated degradation of SIRT1 plays a vital role in the progression of PD.     

Caffeine protects against MPTP-induced blood-brain barrier dysfunction in mouse striatum

The blood-brain barrier (BBB) is important physiologically. Pathologically, BBB disruption has been implicated in a wide spectrum of neurological disorders including Parkinson's disease (PD). Recent studies indicate that caffeine is protective against PD, but by poorly understood mechanisms. Using a MPTP neurotoxin model of PD we tested the hypothesis that the protective actions of caffeine were because of, at least in part, preventing MPTP-induced BBB dysfunction. FVB mice were pre-treated with caffeine (10 mg/kg, i.p.) or saline for 7 days prior to initiation of neurotoxin treatments; during the 7 days of neurotoxin treatment, caffeine or saline continued to be administered 10 min before each dose of MPTP (20 mg/kg, i.p.). Striatum (and for some studies hippocampus and cerebral cortex as well) were evaluated for BBB leakage, tight junction protein expression levels, integrity of dopaminergic neurons, and activation of astrocytes and microglia using immunostaining, immunoblotting and real-time PCR techniques. We found that caffeine blocked MPTP-induced decreases in numbers of tyrosine hydroxylase-positive dopaminergic neurons, increases in leakage of Evan's blue dye and FITC-albumin in striatum but not in cerebral cortex or hippocampus, decreases in levels of the tight junction proteins occludin and ZO-1, and increases in reactive gliosis. Our results suggest that caffeine might protect against PD and PD-like features in animal models, in part, by stabilizing the BBB.     

Attenuation of MPTP-induced neurotoxicity and locomotor dysfunction in Nucling-deficient mice via suppression of the apoptosome pathway

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity is one of the experimental models most commonly used to study the pathogenesis of Parkinson's disease (PD). Although the biochemical mechanisms underlying the cell death induced by MPTP remain to be clarified, it has been found that the mitochondrial apoptotic signaling pathway plays an important role in the neurotoxicity of MPTP. Nucling is a novel type of apoptosis-associated molecule, essential for cytochrome c, apoptosis protease activating factor 1 (Apaf-1), pro-caspase-9 apoptosome induction and caspase-9 activation following pro-apoptotic stress. Here we found that Nucling-deficient mice treated with MPTP did not exhibit locomotor dysfunction in an open-field test. The substantia nigra dopaminergic neurons of Nucling-deficient mice were resistant to the damaging effects of the neurotoxin MPTP. Up-regulated expression of apoptosome was attenuated in Nucling-deficient mice treated with MPTP. These results indicate an important role for Nucling in MPTP-induced neuronal degeneration and suggest that the suppression of Nucling would be of therapeutic benefit for the treatment of neurodegeneration in PD.     

Chronic L‐DOPA induces hyperactivity,normalization of gait and dyskinetic behavior in MitoPark mice

Dopamine (DA) replacement therapy continues to be the gold standard treatment for Parkinson's disease (PD), as it improves key motor symptoms including bradykinesia and gait disturbances. With time, treatment induces side effects in the majority of patients, known as L‐DOPA‐induced dyskinesia (LID), which are often studied in animals by the use of unilateral, toxin‐induced rodent models. In this study, we used the progressive, genetic PD model MitoPark to specifically evaluate bilateral changes in motor behavior following long‐term L‐DOPA treatment at three different stages of striatal DA depletion. Besides locomotor activity, we assessed changes in gait with two automated gait analysis systems and the development of dyskinetic behavior. Long‐term treatment with a moderate, clinically relevant dose of L‐DOPA (8 mg/kg) gradually produced age‐dependent hyperactivity in MitoPark mice. In voluntary and forced gait analyses, we show that MitoPark mice with severe DA depletion have distinct gait characteristics, which are normalized to control levels following long‐term L‐DOPA treatment. The cylinder test showed an age‐dependent and gradual development of bilateral LID. Significant increase in striatal FosB and prodynorphin expression was found to accompany the behavior changes. Taken together, we report that MitoPark mice model both behavioral and biochemical characteristics of long‐term L‐DOPA treatment in PD patients and provide a novel, consistent and progressive animal model of dyskinesia to aid in the discovery and evaluation of better treatment options to counteract LID.     

Proteomic profiling of the substantia nigra demonstrates CNDP2 overexpression in Parkinson's disease

Despite decades of intensive investigations, the precise sequence of molecular events and the specific proteins mediating the degenerative process underlying Parkinson's disease (PD) remain unraveled. Proteomic strategies may provide unbiased tools to identify novel candidates and explore original mechanisms involved in PD. Substantia nigra pars compacta (SN) tissue, whose degeneration is the hallmark of PD, was dissected from neuropathologically confirmed PD patients (n=3) and control subjects (n=3), before being submitted to a comparative 2-DE analysis. The present study revealed a subset of neuronal and/or glial proteins that appears to be deregulated in PD and likely to contribute to neurodegeneration. Observed alterations not only consolidate well accepted concepts surrounding PD pathogenesis such as oxidative stress and mitochondrial dysfunction but also point out to novel pathways. Among the latter, cytosolic non specific dipeptidase 2 (CNDP2), a relatively unknown protein not yet reported to be associated with PD pathogenesis, was shown to be increased in the SN of PD patients, as confirmed by Western blot. Immunohistochemical analyses demonstrated the presence of CNDP2 within the cytoplasm of SN dopaminergic neurons. Altogether, our findings support a key role of CNDP2 in PD neurodegeneration, by mechanisms that could involve oxidative stress, protein aggregation or inflammation. This article is part of a Special Issue entitled: Translational Proteomics.     

Identification of novel proteins associated with both alpha-synuclein and DJ-1

The molecular mechanisms leading to neurodegeneration in Parkinson disease (PD) remain elusive, although many lines of evidence have indicated that alpha-synuclein and DJ-1, two critical proteins in PD pathogenesis, interact with each other functionally. The investigation on whether alpha-synuclein directly interacts with DJ-1 has been controversial. In the current study, we analyzed proteins associated with alpha-synuclein and/or DJ-1 with a robust proteomics technique called stable isotope labeling by amino acids in cell culture (SILAC) in dopaminergic MES cells exposed to rotenone versus controls. We identified 324 and 306 proteins in the alpha-synuclein- and DJ-1-associated protein complexes, respectively. Among alpha-synuclein-associated proteins, 141 proteins displayed significant changes in the relative abundance (increase or decrease) after rotenone treatment; among DJ-1-associated proteins, 119 proteins displayed significant changes in the relative abundance after rotenone treatment. Although no direct interaction was observed between alpha-synuclein and DJ-1, whether analyzed by affinity purification followed by mass spectrometry or subsequent direct co-immunoprecipitation, 144 proteins were seen in association with both alpha-synuclein and DJ-1. Of those, 114 proteins displayed significant changes in the relative abundance in the complexes associated with alpha-synuclein, DJ-1, or both after rotenone treatment. A subset of these proteins (mortalin, nucleolin, grp94, calnexin, and clathrin) was further validated for their association with both alpha-synuclein and DJ-1 using confocal microscopy, Western blot, and/or immunoprecipitation. Thus, we not only confirmed that there was no direct interaction between alpha-synuclein and DJ-1 but also, for the first time, report these five novel proteins to be associating with both alpha-synuclein and DJ-1. Further characterization of these docking proteins will likely shed more light on the mechanisms by which DJ-1 modulates the function of alpha-synuclein, and vice versa, in the setting of PD.     

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson's Disease

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson''s disease (PD) has proved to be invaluable in advancing our understanding of the mechanisms underlying parkinsonian symptoms, since it recapitulates the changes in basal ganglia circuitry and pharmacology observed in parkinsonian patients^1-4. However, the precise cellular and molecular changes occurring at cortico-striatal synapses of the output pathways within the striatum, which is the major input region of the basal ganglia remain elusive, and this is believed to be site where pathological abnormalities underlying parkinsonian symptoms arise^3,5.In PD, understanding the mechanisms underlying changes in basal ganglia circuitry following degeneration of the nigro-striatal pathway has been greatly advanced by the development of bacterial artificial chromosome (BAC) mice over-expressing green fluorescent proteins driven by promoters specific for the two striatal output pathways (direct pathway: eGFP-D1; indirect pathway: eGFP-D2 and eGFP-A2a)⁸, allowing them to be studied in isolation. For example, recent studies have suggested that there are pathological changes in synaptic plasticity in parkinsonian mice^9,10. However, these studies utilised juvenile mice and acute models of parkinsonism. It is unclear whether the changes described in adult rats with stable 6-OHDA lesions also occur in these models. Other groups have attempted to generate a stable unilaterally-lesioned 6-OHDA adult mouse model of PD by lesioning the medial forebrain bundle (MFB), unfortunately, the mortality rate in this study was extremely high, with only 14% surviving the surgery for 21 days or longer¹¹. More recent studies have generated intra-nigral lesions with both a low mortality rate >80% loss of dopaminergic neurons, however expression of L-DOPA induced dyskinesia^11,12,13,14 was variable in these studies. Another well established mouse model of PD is the MPTP-lesioned mouse¹⁵. Whilst this model has proven useful in the assessment of potential neuroprotective agents¹⁶, it is less suitable for understanding mechanisms underlying symptoms of PD, as this model often fails to induce motor deficits, and shows a wide variability in the extent of lesion^{17, 18}.Here we have developed a stable unilateral 6-OHDA-lesioned mouse model of PD by direct administration of 6-OHDA into the MFB, which consistently causes >95% loss of striatal dopamine (as measured by HPLC), as well as producing the behavioural imbalances observed in the well characterised unilateral 6-OHDA-lesioned rat model of PD. This newly developed mouse model of PD will prove a valuable tool in understanding the mechanisms underlying generation of parkinsonian symptoms.     

Physicochemical and immunological characterization of the type E botulinum neurotoxin binding protein purified fromClostridium botulinum

Type E botulinum neurotoxin is produced byClostridium botulinum along with a neurotoxin binding protein which helps protect the neurotoxin from adversepH, temperature, and proteolytic conditions. The neurotoxin binding protein has been purified as a 118-kDa protein. Secondary structure content of the neurotoxin binding protein as revealed by far-UV circular dichroism spectroscopy was 19% α-helix, 50%β-sheets, 28% random coils, and 3%β-turns. This compared to 22% α-helix, 44%β-sheets, 34% random coils, and noβ-turns of the type E botulinum neurotoxin. The complex of the two proteins revealed 25%α-helix, 45%β-sheets, 27% random coils, and 3%β-turns, suggesting a significant alteration at least in theα-helical folding of the two proteins upon their interaction. Tyrosine topography is altered considerably (28%) when the neurotoxin and its binding protein are separated, indicating strong interaction between the two proteins. Gel filtration results suggested that type E neurotoxin binding protein clearly complexes with type E neurotoxin. The interaction is favored at lowpH as indicated by an initial binding rate of 8.4 min^?1 atpH 5.7 compared to 4.0 min^?1 atpH 7.5 as determined using a fiber optic-based biosensor. The neurotoxin and its binding protein apparently are of equivalent antigenicity, as both reacted equally on enzyme-linked immunosorbent assay to polyclonal antibodies raised against the toxoid of their complex.     

Chronic morphine alters the presynaptic protein profile: identification of novel molecular targets using proteomics and network analysis

Opiates produce significant and persistent changes in synaptic transmission; knowledge of the proteins involved in these changes may help to understand the molecular mechanisms underlying opiate dependence. Using an integrated quantitative proteomics and systems biology approach, we explored changes in the presynaptic protein profile following a paradigm of chronic morphine administration that leads to the development of dependence. For this, we isolated presynaptic fractions from the striata of rats treated with saline or escalating doses of morphine, and analyzed the proteins in these fractions using differential isotopic labeling. We identified 30 proteins that were significantly altered by morphine and integrated them into a protein-protein interaction (PPI) network representing potential morphine-regulated protein complexes. Graph theory-based analysis of this network revealed clusters of densely connected and functionally related morphine-regulated clusters of proteins. One of the clusters contained molecular chaperones thought to be involved in regulation of neurotransmission. Within this cluster, cysteine-string protein (CSP) and the heat shock protein Hsc70 were downregulated by morphine. Interestingly, Hsp90, a heat shock protein that normally interacts with CSP and Hsc70, was upregulated by morphine. Moreover, treatment with the selective Hsp90 inhibitor, geldanamycin, decreased the somatic signs of naloxone-precipitated morphine withdrawal, suggesting that Hsp90 upregulation at the presynapse plays a role in the expression of morphine dependence. Thus, integration of proteomics, network analysis, and behavioral studies has provided a greater understanding of morphine-induced alterations in synaptic composition, and identified a potential novel therapeutic target for opiate dependence.     

Binding preference of p62 towards LC3-ll during dopaminergic neurotoxin-induced impairment of autophagic flux

Accumulating evidence has revealed that autophagy may be beneficial for treatment of neurodegenerative diseases through removal of abnormal protein aggregates. However, the critical autophagic events during neurodegeneration remain to be elucidated. Here, we investigated whether prototypic autophagic events occur in the MN9D dopaminergic neuronal cell line upon exposure to N-methyl-4-phenylpyridinium (MPP (+) ), a well-known dopaminergic neurotoxin. MPP (+) treatment induced both morphological and biochemical characteristics of autophagy, such as accumulation of autophagic vacuoles and LC3-II form and decreased p62 levels. Further investigation revealed that these phenomena were largely the consequences of blocked autophagic flux. Following MPP (+) treatment, levels of LC3-II formed and p62 dramatically increased in the Triton X-100-insoluble fraction. Levels of ubiquitinated proteins also increased in this fraction. Further colocalization analyses revealed that the punctated spots positive for both p62 and LC3 were more intense following MPP (+) treatment, suggesting drug-induced enrichment of these two proteins in the insoluble fraction. Intriguingly, reciprocal immunoprecipitation analysis revealed that p62 mainly precipitated with LC3-II form following MPP (+) treatment. Transient transfection of the mutant form of Atg4B, Atg4B (C74A) , which inhibits LC3 processing, dramatically decreased binding between p62 and LC3-II form. Taken together, our results indicate that p62 can be efficiently localized to autophagic compartments via preferential binding with LC3-II form. This colocalization may assist in removal of detergent-insoluble forms of damaged cellular proteins during dopaminergic neurotoxin-induced impairment of autophagic flux.     

The effect of extracellular recombinant human heat shock protein 70 (Hsp70) on protein pattern observed after endotoxin-induced macrophage activation

The protein pattern of mouse macrophage strain (J774) has been investigated using 2D electrophoresis after combined action of bacterial endotoxins (LPS), heat shock treatment (HS) and administration of recombinant human Hsp70. The investigation demonstrated significant protective effect of HS and recombinant Hsp70 treatment applied before LPS introduction. This effect is apparently realized by means of several signal transduction systems. In the course of the investigation, we have identified eight proteins, which exhibited pronounced changes in their synthesis due to combined treatment. The data accumulated may shed light on molecular mechanisms underlying protective antiseptic action of HS and/or recombinant Hsp70 applied before LPS administration.