Lack of nigrostriatal pathology in a rat model of proteasome inhibition

Systemic administration of ubiquitin-proteasome system inhibitors to rodents has been reported to induce certain behavioral and neuropathological features of Parkinson's disease. The goal of this study was to replicate these observations by administering a proteasome inhibitor (PSI) to rats using McNaught and colleagues' protocol. No alterations in locomotor activity or striatal dopamine and its metabolites were observed. Differences in nigral dopaminergic cell number between proteasome inhibitor- and vehicle-treated rats and inclusion bodies were not found. Extending the time of survival after administration and using different solvents failed to alter results, indicating this proteasome inhibitor does not consistently produce the selective toxicity and pathological hallmarks characterizing Parkinson's disease.     

Systemic exposure to proteasome inhibitors causes a progressive model of Parkinson's disease

Environmental toxins have been implicated in the etiology of Parkinson's disease. Recent findings of defects in the ubiquitin-proteasome system in hereditary and sporadic forms of the illness suggest that environmental proteasome inhibitors are candidate PD-inducing toxins. Here, we systemically injected six doses of naturally occurring (epoxomicin) or synthetic (Z-lle-Glu(OtBu)-Ala-Leu-al [PSI]) proteasome inhibitors into adult rats over a period of 2 weeks. After a latency of 1 to 2 weeks, animals developed progressive parkinsonism with bradykinesia, rigidity, tremor, and an abnormal posture, which improved with apomorphine treatment. Positron emission tomography demonstrated reduced carbon-11-labeled 2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane (CFT) binding to dopaminergic nerve terminals in the striatum, indicative of degeneration of the nigrostriatal pathway. Postmortem analyses showed striatal dopamine depletion and dopaminergic cell death with apoptosis and inflammation in the substantia nigra pars compacta. In addition, neurodegeneration occurred in the locus coeruleus, dorsal motor nucleus of the vagus, and the nucleus basalis of Meynert. At neurodegenerative sites, intracytoplasmic, eosinophilic, alpha-synuclein/ubiquitin-containing, inclusions resembling Lewy bodies were present in some of the remaining neurons. This animal model induced by proteasome inhibitors closely recapitulates key features of PD and may be valuable in studying etiopathogenic mechanisms and putative neuroprotective therapies for the illness.     

Proteasomal inhibition causes loss of nigral tyrosine hydroxylase neurons

Dysfunction of the ubiquitin-proteasomal system (UPS) has been implicated in the pathogenesis of Parkinson's disease. The systemic administration of UPS inhibitors has been reported to induce nigrostriatal cell death and model Parkinson's disease pathology in rodents. We administered a synthetic, specific UPS inhibitor (PSI) subcutaneously to rats and quantified substantia nigral tyrosine hydroxylase-positive dopaminergic neurons by stereology. PSI caused a 15% decrease in UPS activity at 2 weeks and a 42% reduction in substantia nigra pars compacta tyrosine hydroxylase-positive neurons at 8 weeks. Systemic inhibition of the UPS warrants further evaluation as a means to model Parkinson's disease.     

Proteasome inhibition and Parkinson's disease modeling

Impaired proteasome function is a potential mechanism for dopaminergic neuron degeneration. To model this molecular defect, we administered systemically the reversible lipophilic proteasome inhibitor, carbobenzoxy-L-isoleucyl-gamma-t-butyl-L-glutamyl-L-alanyl-L-leucinal (PSI), to rodents. In contrast to a previous report, this approach failed to cause any detectable behavioral or neuropathological abnormality in either rats or mice. Although theoretically appealing, this specific model of Parkinson's disease appears to exhibit poor reproducibility.     

Proteasome inhibition leads to early loss of synaptic proteins in neuronal culture

A dysfunctional ubiquitin proteasome system may be a mediating factor of disease progression in Lewy body dementia (LBD). The effects of proteasome inhibition using lactacystin and epoxomicin in primary neuronal culture were studied to assess the validity of this model to reflect the cortical pathology of LBD. Treatment of primary cortical neurons with 5???M lactacystin for 24?h led to a 38?% reduction in the levels of ??-III-tubulin (p?<?0.05), a 48?% reduction in the levels of synaptophysin (p?<?0.05) and a 74?% reduction in the levels of drebrin (p?<?0.01), when compared to controls. Results for epoxomicin were similar. The loss of neuronal protein occurred prior to any loss of mitochondrial activity or cell death. The results are reflective of the loss of synapses and the synaptic changes observed in LBD, which may be an early event in the neurodegeneration of LBD. The similarities with the pathological changes in LBD highlight the possibility that this model can potentially provide a platform to test novel treatments.     

Brain proteasomal function in sporadic Parkinson's disease and related disorders

Because genetic defects relating to the ubiquitin-proteasome system were reported in familial parkinsonism, we evaluated proteasomal function in autopsied brains with sporadic Parkinson's disease. We found that proteasome peptidase activities in a fraction specific to the proteasome were preserved in five brain areas (including the striatum) of Parkinson's disease where neuronal loss is not observed. Striatal protein levels of two proteasome subunits were normal in Parkinson's disease but reduced mildly in disease controls (multiple system atrophy). Our brain data suggest that a systemic, global disturbance in the catalytic activity and degradation ability of the proteasome itself is unlikely to explain the cause of Parkinson's disease.     

Systemic proteasomal inhibitor exposure enhances dopamine turnover and decreases dopamine levels but does not affect MPTP-induced striatal dopamine depletion in mice

The validation of an in vivo proteasomal inhibitor (PSI) model to translate ubiquitin-proteasomal-system dysfunction involved in the pathogenesis of Parkinson's disease (PD) into a commonly accepted animal model is ongoing. Here we first report the effects of systemic administration of the proteasomal inhibitor Z-lle-Glu(OtBu)-Ala-Leu-CHO (3 mg/kg, s.c., six times over 2 weeks) alone to extend the rat model to mice. Second we investigate the consequences of PSI pretreatment 42 weeks before an acute treatment with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in C57bl/6 mice. HPLC postmortem neurochemistry showed a significant increase in dopamine turnover and decrease of striatal dopamine levels, only 14 weeks after PSI treatment, but no enhancement of dopamine turnover or differences in striatal dopamine levels when comparing MPTP with MPTP plus PSI treatment. Behavioral analysis (rotarod, open field activity) did not indicate that PSI affects this type of motor behavior. Systemic PSI administration in mice appears not to be a valid animal model under the experimental conditions used. Potential solutions are discussed.     

Effect of proteasome inhibitor on cultured mesencephalic dopaminergic neurons

Proteasomal dysfunction has been implicated in the pathogenesis of Parkinson's disease (PD). We examined the effect of a selective proteasomal inhibitor, epoxomicin, on primary cultured mesencephalic neurons. Exposing rat cultured mesencephalic neurons to epoxomicin for 24 h resulted in neurotoxicity in a dose-dependent manner. Epoxomicin caused mitochondrial dysfunction, reduction in reduced glutathione (GSH), and increased generation of free radicals. Neuronal damage was significantly blocked by antioxidative/GSH-augmenting agents. Epoxomicin also increased the expression of Bax and decreased that of Bcl-2, which may cause mitochondrial dysfunction and release of free radicals. Dopaminergic neurons were preferentially resistant to the toxicity of epoxomicin. Inhibiting the synthesis of tetrahydrobiopterin (BH(4)), which has been reported to have antioxidative function, increased the susceptibility of dopaminergic neurons, whereas increasing BH(4) levels protected non-dopaminergic neurons. These findings suggest that BH(4) is at least in part a contributing factor to grand the resistance to dopaminergic neurons against epoxomicin neurotoxicity. Our results suggest that proteasome inhibition causes the neurotoxicity in mesencephalic neurons, but that is not sufficient to reproduce the selective damage to dopaminergic neurons, such as that seen in PD.     

The pedunculopontine nucleus in Parkinson's disease

This study demonstrated a significant loss of neurons within the lateral part of the pedunculopontine nucleus pars compacta in individuals with idiopathic Parkinson's disease and in individuals with combined Parkinson's and Alzheimer's disease. We also examined the extent of neuronal loss within the substantia nigra pars compacta, locus ceruleus, dorsal raphe nucleus, and nucleus basalis of Meynert. The number of pedunculopontine nucleus pars compacta neurons in the patients with Parkinson's or Parkinson's and Alzheimer's disease was reduced (average, 40%) in comparison with the number in control subjects or patients with Alzheimer's disease (p less than 0.01). This finding correlated significantly with the extent of loss of substantia nigra pars compacta neurons (p less than 0.01).     

Comparison of the neurotoxic effects of proteasomal inhibitors in primary mesencephalic cultures

Impairment of the ubiquitin-proteasome system (UPS) has been implicated in the pathogenesis of Parkinson's disease (PD). Because the neurodegenerative process of PD results in a severe loss of dopaminergic cells, previous in vitro studies have investigated the possibility that these neurons may be particularly vulnerable to proteasomal inhibition. Results of this earlier work are difficult to compare, however, since they were obtained using different proteasomal inhibitors at various concentrations and under diverse culture conditions. Here, four UPS inhibitors, i.e., lactacystin, PSI, epoxomicin and MG-132, were directly evaluated in terms of their ability to damage dopaminergic and GABAergic neurons in primary rat mesencephalic cultures. Using a broad range of concentrations and different incubation lengths, we found that proteasomal inhibitors consistently killed both dopaminergic and GABAergic neurons. The degree of toxicity was slightly different, however, between the two neuronal populations. When measurements of neurotransmitter uptake were used as indicators of neuronal cell viability, the extent of reduction of dopamine uptake caused by proteasomal inhibitors was slightly greater than the decrease in GABA uptake. With PSI the difference in reduction of dopamine vs. GABA uptake was less than 10% and did not reach statistical significance. With the other three inhibitors, dopaminergic cells were up to 20% more affected than GABAergic neurons; this difference reached statistical significance only at specific concentrations and time points. Preincubation of cultures with alpha-methyl-p-tyrosine, an inhibitor of dopamine synthesis, reduced dopamine concentration by 65% but failed to significantly change lactacystin- and MG-132-induced damage to dopaminergic neurons. Data indicate a modest preferential toxicity of proteasomal inhibitors toward dopaminergic cells and thus only in part support the hypothesis that a selective vulnerability to UPS dysfunction underlies the pathogenesis of nigrostriatal degeneration in PD.     

Proteasome inhibitor model of Parkinson's disease in mice is confounded by neurotoxicity of the ethanol vehicle.

Defects in the ubiquitin-proteasome system have been implicated in Parkinson's Disease (PD). Recently, a rat model of PD was developed using a synthetic proteasome inhibitor (PSI), (Z-lle-Glu(OtBu)-Ala-Leu-al). We attempted to transfer this model to mouse studies, where genetics can be more readily investigated due to the availability of genetically modified mice. We treated C57BL/6 (B6) mice with six intraperitoneal injections of 6 mg/kg PSI in 50 mul of 70% ethanol over a 2-week-period. We found significant decreases in nigrostriatal dopamine in PSI-treated mice compared with saline-treated mice. However, we observed similar decreases in the ethanol-treated vehicle control group. Administration of ethanol alone led to significant long-term alterations in dopamine levels. Ethanol significantly eclipses the effects of PSI in the dopamine system, and therefore is a confounding vehicle for this model.     

α-Synuclein Transgenic Mice Reveal Compensatory Increases in Parkinson's Disease-Associated Proteins DJ-1 and Parkin and Have Enhanced α-Synuclein and PINK1 Levels After Rotenone Treatment

Parkinson's disease (PD) is a severe neurodegenerative disorder characterised by loss of dopaminergic neurons of the substantia nigra. The pathological hallmarks are cytoplasmic inclusions termed Lewy bodies consisting primarily of aggregated α-synuclein (αSN). Different lines of transgenic mice have been developed to model PD but have failed to recapitulate the hallmarks of this disease. Since treatment of rodents with the pesticide rotenone can reproduce nigrostriatal cell loss and other features of PD, we aimed to test chronic oral administration of rotenone to transgenic mice over-expressing human αSN with the A53T mutation. Initial assessment of this transgenic line for compensatory molecular changes indicated decreased brain β-synuclein expression and significantly increased levels of the PD-associated oxidative stress response protein, DJ-1, and the E3 ubiquitin ligase enzyme, Parkin. Rotenone treatment of 30 mg/kg for 25 doses over a 35-day period was tolerated in the transgenic mice and resulted in decreased spontaneous locomotor movement and increased cytoplasmic αSN expression. The mitochondrial Parkinson's-associated PTEN-induced kinase 1 protein levels were also increased in transgenic mouse brain after rotenone treatment; there was no change in brain dopamine levels or nigrostriatal cell loss. These hA53T αSN transgenic mice provide a useful model for presymptomatic Parkinson's features and are valuable for study of associated compensatory changes in early Parkinson's disease stages.     

Proteasome inhibitor-induced model of Parkinson's disease

We recently reported that systemic administration of a proteasome inhibitor induced a progressive levodopa-responsive, bradykinetic syndrome in rats with imaging, pathological, and biochemical features that strikingly resemble what is found in PD. This model has the potential to be a useful tool for studying the mechanism of cell death in Parkinson's disease and for testing putative neuroprotective agents. Since publication of these findings, several laboratories have sought to reproduce the model; some have been successful in replicating our findings, but others have not. The reason for this variability is not known, but resolution is critically important given the potential utility of this model. We have begun to examine various factors that alone or in combination might explain these differences, and we present in this article preliminary results from these studies.     

Proteasome inhibitor MG-132 induces dopaminergic degeneration in cell culture and animal models

Impairment in ubiquitin-proteasome system (UPS) has recently been implicated in Parkinson's disease, as demonstrated by reduced proteasomal activities, protein aggregation and mutation of several genes associated with UPS. However, experimental studies with proteasome inhibitors failed to yield consensus regarding the effect of proteasome inhibition on dopaminergic degeneration. In this study, we systematically examined the effect of the proteasome inhibitor MG-132 on dopaminergic degeneration in cell culture and animal models of Parkinson's disease. Exposure of immortalized dopaminergic neuronal cells (N27) to low doses of MG-132 (2-10 microM) resulted in dose- and time-dependent cytotoxicity. Further, exposure to MG-132 (5 microM) for 10 min led to dramatic reduction of proteasomal activity (>70%) accompanied by a rapid accumulation of ubiquitinated proteins in these cells. MG-132 treatment also induced increases in caspase-3 activity in a time-dependent manner, with significant activation occurring between 90 and 150 min. We also noted a 12-fold increase in DNA fragmentation in MG-132 treated N27 cells. Similarly, primary mesencephalic neurons exposed to 5 microM MG-132 also induced >60% loss of TH positive neurons but only a minimal loss of non-dopaminergic cells. Stereotaxic injection of MG-132 (0.4 microg in 4 microl) into the substantia nigra compacta (SNc) in C57 black mice resulted in significant depletion of ipisilateral striatal dopamine and DOPAC content as compared to the vehicle-injected contralateral control sides. Also, we observed a significant decrease in the number of TH positive neurons in the substantia nigra of MG-132-injected compared to the vehicle-injected sites. Collectively, these results demonstrate that the proteasomal inhibitor MG-132 induces dopamine depletion and nigral dopaminergic degeneration in both cell culture and animal models, and suggest that proteasomal dysfunction may promote nigral dopaminergic degeneration in Parkinson's disease.     

Disease-specific patterns of locus coeruleus cell loss.

Computer visualization techniques were used to map and to quantitatively reconstruct the entire locus coeruleus, including the nucleus subcoeruleus, to compare the topographic patterns of cell loss in postmortem brains from patients with Parkinson's disease, Alzheimer's disease, and Down syndrome. There was comparable cell loss in all three diseases (approximately 60%) compared with aged normal subjects, and there was a significant loss of nucleus subcoeruleus cells specifically in patients with Parkinson's disease (63%). There was a significant positive correlation between the magnitude of locus coeruleus cell loss and the duration of Alzheimer's disease, but no such correlation was found for Parkinson's disease. In patients with Parkinson's disease, there was comparable cell loss throughout the rostral-caudal extent of the nucleus; however, in patients with Alzheimer's disease and Down syndrome, the greatest cell loss always occurred within the rostral portion of the nucleus, with a relative sparing of caudal cells. These data are consistent with the hypothesis that cell loss in Parkinson's disease is the result of a pathological process that attacks the catecholaminergic cells of the locus coeruleus and the subcoeruleus in general; in Alzheimer's disease and Down syndrome, however, the pathological process only affects the rostral, cortical-projecting locus coeruleus cells and spares the caudal, noncortical-projecting cells.     

Reciprocal Effects of α-Synuclein Overexpression and Proteasome Inhibition in Neuronal Cells and Tissue

Defects in the 20S/26S proteasome and conformational changes in α-synuclein (α-syn) are implicated in the development of sporadic and familial cases of PD. The objective of this study was to evaluate whether α-syn affects proteolysis by the proteasome and, reciprocally, whether proteasome inhibition affects α-syn solubility and localization. Although α-syn directly inhibited purified 20S proteasomes reversibly in vitro, its overexpression in neuroblastoma (SH-SY5Y and SK-N-BE), embryonic kidney (HEK293) cells, or mouse brain did not affect proteasome activity. Proteasome inhibition with MG132 and epoxomicin in SH-SY5Y cells failed to induce α-syn aggregation, although it increased membrane bound forms of endogenous and overexpressed wild-type, but not mutant, α-syn. Concomitantly this treatment generated cytoplasmic α-syn inclusions devoid of polyubiquitin in a small percentage of cells. The combination of proteasome inhibition with serum deprivation, which induced oxidative stress and autophagy, caused the appearance of high molecular weight α-syn species, such as those found in Lewy bodies. Our data suggest that high concentrations of α-syn do not affect proteasome function in vivo, whereas proteasome inhibition can modify synuclein solubility, most prominently under conditions of cell stress which occur during aging. These results have implications for the convergence of age-related oxidative stress and impaired protein degradation in neurodegeneration.     

Dementia in Parkinson's disease: the problem of clinicopathological correlation

Four cases of Parkinson's disease with advanced dementia are described. Postmortem examination revealed cell loss in the substantia nigra, with Lewy bodies present, and loss of cells in the basal nucleus of Meynert. A few tangles were observed in the hippocampus, but no senile plaques or neurofibrillary tangles were found in the neocortex. The authors note that a dramatic dementia syndrome may occur with Parkinson's disease alone, without the associated cytoskeletal markers of Alzheimer's disease. Cases were characterized by disorientation, episodic confusion and hallucinations persisting off medication, disturbed behavior, and the absence of aphasia.     

Decrease in presynaptic inhibition on heteronymous monosynaptic Ia terminals in patients with Parkinson's disease.

PURPOSE: Heteronymous Ia facilitation from the quadriceps to the soleus was studied to clarify central motor control through presynaptic inhibition (PSI) on Ia terminals of spinal motoneurons in Parkinson's disease. METHODS: 17 patients with Parkinson's disease and 36 control subjects participated in the study. Because the early part of facilitation reflects the degree of PSI, the extent of facilitation was quantified as the slope, within 0.8 msec of onset. RESULTS: Heteronymous Ia facilitation in the patients was greater than in the age-matched control participants. PSI was negatively correlated with the walking speed of the patients. In the patients examined twice, lessening of bradykinesia was correlated to a decrease in PSI but not lessening of rigidity. CONCLUSION: These findings indicate that the degree of PSI is decreased and disturbance of the central control of PSI may relate to gait disturbance in Parkinson's disease.     

Modifications of local cerebral metabolic rates for glucose and motor behavior in rats with unilateral lesion of the subthalamic nucleus.

Inactivation of the subthalamic nucleus (STN) has attracted interest as a therapeutic tool in Parkinson's disease. The functional consequences of the inactivation, however, are uncertain. In this study definition of the pattern of changes of cerebral functional activity associated with lesion of the STN and dopaminergic stimulation, by using the [14C]deoxyglucose method, was sought. Six or 7 days following unilateral lesion of the STN, the animals were divided into two groups: One group (n = 10) was administered apomorphine (1 mg/kg) subcutaneously; the second group (n = 10) received saline. The [14C]deoxyglucose procedure was initiated 10 minutes following the drug or saline injection. The results show that systemic administration of apomorphine to rats with unilateral lesion of the STN causes ipsiversive rotational behavior and asymmetries of glucose utilization of defined brain areas, including the substantia nigra reticulata, globus pallidus, and entopeduncular nucleus. These nuclei are the main targets of the subthalamic excitatory projections. Lesion of the nucleus per se (without challenge with apomorphine) has no significant consequences on glucose utilization. The findings indicate that the STN is involved in the activation of the basal ganglia output nuclei induced by systemic dopaminergic stimulation.     

The basal nucleus of Meynert in idiopathic Parkinson's disease

Abstract– The basal nucleus of Meynert (bnM) was examined in 6 patients with idiopathic Parkinson's disease and in 5 age-matched controls. The histopathological study was followed by a quantitative analysis of the magnocellular population of the nucleus, with the determination of the number of neurons and their nucleolar volume. In Parkinson's disease, there was a neuronal loss ranging from 29.9% to 68.3% (mean 45.8%) and numerous surviving cells containing Lewy inclusion bodies. The percentage loss of cells did not display a significant correlation with the age of the patients, the duration of the illness, the mental state or the drug regime. On the other hand, no significant differences in nucleolar volume of bnM neurons were detected between patients and controls. The damage of the basa! nucleus of Meynert in Parkinson's disease is less severe than that generally observed in Alzheimer's disease, and is slightly more pronounced than that reported for progressive supranuclear palsy.