Patrick P. Michel

Levodopa in the treatment of Parkinson's disease: Current controversies

Levodopa is the most effective symptomatic agent in the treatment of Parkinsons disease (PD) and the “gold standard” against which new agents must be compared. However, there remain two areas of controversy: (1) whether levodopa is toxic, and (2) whether levodopa directly causes motor complications. Levodopa is toxic to cultured dopamine neurons, and this may be a problem in PD where there is evidence of oxidative stress in the nigra. However, there is little firm evidence to suggest that levodopa is toxic in vivo or in PD. Clinical trials have not clarified this situation. Levodopa is also associated with motor complications. Increasing evidence suggests that they are related, at least in part, to the short half‐life of the drug (and its potential to induce pulsatile stimulation of dopamine receptors) rather than to specific properties of the molecule. Treatment strategies that provide more continuous stimulation of dopamine receptors provide reduced motor complications in MPTP monkeys and PD patients. These studies raise the possibility that more continuous and physiological delivery of levodopa might reduce the risk of motor complications. Clinical trials to test this hypothesis are underway. We review current evidence relating to these areas of controversy.

Chronic systemic complex I inhibition induces a hypokinetic multisystem degeneration in rats

In Parkinsons disease, nigral dopaminergic neurones degenerate, whereas post‐synaptic striatal target neurones are spared. In some atypical parkinsonian syndromes, both nigral and striatal neurones degenerate. Reduced activity of complex I of the mitochondrial respiratory chain has been implicated in both conditions, but it remains unclear if this affects the whole organism or only the degenerating brain structures. We therefore investigated the differential vulnerability of various brain structures to generalized complex I inhibition. Male Lewis rats infused with rotenone, a lipophilic complex I inhibitor [2.5u2003mg/kg/day intraveneously (i.v.) for 28u2003days], were compared with vehicle‐infused controls. They showed reduced locomotor activity and loss of striatal dopaminergic fibres (54%), nigral dopaminergic neurones (28.5%), striatal serotoninergic fibres (34%), striatal DARPP‐32‐positive projection neurones (26.5%), striatal cholinergic interneurones (22.1%), cholinergic neurones in the pedunculopontine tegmental nucleus (23.7%) and noradrenergic neurones in the locus ceruleus (26.4%). Silver impregnation revealed pronounced degeneration in basal ganglia and brain stem nuclei, whereas the hippocampus, cerebellum and cerebral cortex were less affected. These data suggest that a generalized mitochondrial failure may be implicated in atypical parkinsonian syndromes but do not support the hypothesis that a generalized complex I inhibition results in the rather selective nigral lesion observed in Parkinsons disease.

Dysfunction of mitochondrial complex I and the proteasome: interactions between two biochemical deficits in a cellular model of Parkinson's disease

Two biochemical deficits have been described in the substantia nigra in Parkinsons disease, decreased activity of mitochondrial complex I and reduced proteasomal activity. We analysed interactions between these deficits in primary mesencephalic cultures. Proteasome inhibitors (epoxomicin, MG132) exacerbated the toxicity of complex I inhibitors [rotenone, 1‐methyl‐4‐phenylpyridinium (MPP+)] and of the toxic dopamine analogue 6‐hydroxydopamine, but not of inhibitors of mitochondrial complex II–V or excitotoxins [N‐methyl‐d‐aspartate (NMDA), kainate]. Rotenone and MPP+ increased free radicals and reduced proteasomal activity via adenosine triphosphate (ATP) depletion. 6‐hydroxydopamine also increased free radicals, but did not affect ATP levels and increased proteasomal activity, presumably in response to oxidative damage. Proteasome inhibition potentiated the toxicity of rotenone, MPP+ and 6‐hydroxydopamine at concentrations at which they increased free radical levels ≥ 40% above baseline, exceeding the cellular capacity to detoxify oxidized proteins reduced by proteasome inhibition, and also exacerbated ATP depletion caused by complex I inhibition. Consistently, both free radical scavenging and stimulation of ATP production by glucose supplementation protected against the synergistic toxicity. In summary, proteasome inhibition increases neuronal vulnerability to normally subtoxic levels of free radicals and amplifies energy depletion following complex I inhibition.

Ceramide Induces Apoptosis in Cultured Mesencephalic Neurons

Abstract: The death of dopaminergic and other neurons in primary cultures of the mesencephalon could be induced by treatment with ceramide, as in lymphocytes where it mediates activation by the cytokines tumor necrosis factor‐α and interleukin‐1β of a novel sphingomyelin‐dependent signaling pathway leading to apoptosis. The morphological hallmarks of this form of cell death—bleb formation, cell body shrinkage, nuclear chromatin condensation, and fragmentation—were observed in degenerating neurons. Internucleosomal DNA degradation could also be evidenced by gel electrophoresis. The C2 and C6 analogues as well as native ceramide, administered in a dodecane suspension, had a similar effect, whereas the closely related C2‐dihydroceramide, which lacks the 4–5 trans double bond in the sphingosine chain, failed to induce apoptosis. Neuronal death could be delayed by serum factors, dibutyryl cyclic AMP, and the protein synthesis inhibitor cycloheximide.

Annonacin, a lipophilic inhibitor of mitochondrial complex I, induces nigral and striatal neurodegeneration in rats: possible relevance for atypical parkinsonism in Guadeloupe

In Guadeloupe, epidemiological data have linked atypical parkinsonism with fruit and herbal teas from plants of the Annonaceae family, particularly Annona muricata. These plants contain a class of powerful, lipophilic complex I inhibitors, the annonaceous acetogenins. To determine the neurotoxic potential of these substances, we administered annonacin, the major acetogenin of A. muricata, to rats intravenously with Azlet osmotic minipumps (3.8 and 7.6u2003mg per kg per day for 28u2003days). Annonacin inhibited complex I in brain homogenates in a concentration‐dependent manner, and, when administered systemically, entered the brain parenchyma, where it was detected by matrix‐associated laser desorption ionization – time of flight mass spectrometry, and decreased brain ATP levels by 44%. In the absence of evident systemic toxicity, we observed neuropathological abnormalities in the basal ganglia and brainstem nuclei. Stereological cell counts showed significant loss of dopaminergic neurones in the substantia nigra (−u200331.7%), and cholinergic (−u200337.9%) and dopamine and cyclic AMP‐regulated phosphoprotein (DARPP‐32)‐immunoreactive GABAergic neurones (−u200339.3%) in the striatum, accompanied by a significant increase in the number of astrocytes (35.4%) and microglial cells (73.4%). The distribution of the lesions was similar to that in patients with atypical parkinsonism. These data are compatible with the theory that annonaceous acetogenins, such as annonacin, might be implicated in the aetiology of Guadeloupean parkinsonism and support the hypothesis that some forms of parkinsonism might be induced by environmental toxins.

The mitochondrial complex I inhibitor rotenone triggers a cerebral tauopathy

Reduced activity of the mitochondrial respiratory chain – particularly complexu2003I – may be implicated in the etiology of both Parkinsons disease and progressive supranuclear palsy, although these neurodegenerative diseases differ substantially as to their distinctive pattern of neuronal cell loss and the predominance of cerebral α‐synuclein or tau protein pathology. To determine experimentally whether chronic generalized complexu2003I inhibition has an effect on the distribution of α‐synuclein or tau, we infused rats systemically with the plant‐derived isoflavonoid rotenone. Rotenone‐treated rats with a pronounced metabolic impairment had reduced locomotor activity, dystonic limb posture and postural instability. They lost neurons in the substantia nigra and in the striatum. Spherical deposits of α‐synuclein were observed in a few cells, but cells with abnormal cytoplasmic accumulations of tau immunoreactivity were significantly more numerous in the striatum of severely lesioned rats. Abnormally high levels of tau immunoreactivity were found in the cytoplasm of neurons, oligodendrocytes and astrocytes. Ultrastructurally, tau‐immunoreactive material consisted of straight 15‐nm filaments decorated by antibodies against phosphorylated tau. Many tau+ cell bodies also stained positive for thioflavinu2003S, nitrotyrosine and ubiquitin. Some cells with abnormal tau immunoreactivity contained activated caspaseu20033. Our data suggest that chronic respiratory chain dysfunction might trigger a form of neurodegeneration in which accumulation of hyperphosphorylated tau protein predominates over deposits of α‐synuclein.

Is Bax a mitochondrial mediator in apoptotic death of dopaminergic neurons in Parkinson's disease?

Bax is a proapoptotic member of the Bcl‐2 family of proteins. It is believed to exert its action primarily by facilitating the release of cytochrome c from the mitochondrial intermembrane space into the cytosol, leading to caspase activation and cell death. Because alterations in mitochondrial respiratory function, caspase activation and cell death with morphologic features compatible with apoptosis have been observed post mortem in the brain of patients with Parkinsons disease, we tried to clarify the potential role of Bax in this process in an immunohistochemical study on normal and Parkinsons disease post‐mortem brain and primary mesencephalic cell cultures treated with MPP+. We found that Bax is expressed ubiquitously by dopaminergic (DA) neurons in post‐mortem brain of normal and Parkinsons disease subjects as well as in vitro. Using an antibody to Bax inserted into the outer mitochondrial membrane as an index of Bax activation, no significant differences were observed between control and Parkinsons disease subjects, regardless of the mesencephalic subregion analysed. However, in Parkinsons disease subjects, the percentage of Bax‐positive melanized SNpc neurons containing Lewy bodies, suggestive of DA neuronal suffering, was significantly higher than the overall percentage of Bax‐positive neurons among melanized neurons. Furthermore, all melanized SNpc neurons in Parkinsons disease subjects with activated caspase‐3 were also immunoreactive for Bax, suggesting that Bax anchored in the outer mitochondrial membrane of melanized SNpc neurons showing signs of neuronal suffering or apoptosis is increased compared with DA neurons that are apparently unaltered. Surprisingly, MPP+ treatment of tyrosine hydroxylase (TH)‐positive neurons in primary mesencephalic cultures did not cause redistribution of Bax, although cytochrome c was released from the mitochondria and nuclear condensation/fragmentation was induced. Taken together, these findings suggest that in the human pathology, Bax may be a cofactor in caspase activation, but our in vitro data fail to indicate a central role for Bax in apoptotic death of DA neurons in an experimental Parkinsons disease paradigm.

Differential expression of tyrosine hydroxylase and membrane dopamine transporter genes in subpopulations of dopaminergic neurons of the rat mesencephalon

Dopaminergic (DA) cells of the substantia nigra pars compacta (SNC) and the ventral tegmental area (VTA) display differences in their topography, biochemistry and susceptibility to pathological processes. Neuronal dopamine concentration is regulated in large part by tyrosine hydroxylase (TH), the rate-limiting enzyme of dopamine synthesis, and by the dopamine reuptake system. In the present study, TH protein, TH mRNA and dopamine membrane transporter (DAT) mRNA were quantified at cellular level in 4 arbitrary subregions of the rat ventral mesencephalon (lateral, middle, medial SNC and VTA), using in situ hybridization and immunoautoradiography. The distribution of labelling for TH protein and TH mRNA was almost superimposable and close to that of DAT mRNA in mesencephalic neurons. Lower values of cellular expression in TH protein, TH mRNA and DAT mRNA were observed in the lateral part of the SNC compared to the other subregions. TH and DAT expression were correlated in SNC but not in VTA. Indeed DA cells in this region expressed low levels of DAT mRNA in comparison to the middle and medial SNC. These results suggest a heterogeneity of DA metabolism among populations of mesencephalic cells. The relative lower expression of the DAT gene in VTA neurons suggests a less efficient dopamine reuptake capacity, which may partly account for the relative sparing of the mesolimbic system reported in Parkinsons disease and MPTP-treated animals.

The mitochondrial complex i inhibitor annonacin is toxic to mesencephalic dopaminergic neurons by impairment of energy metabolism

The death of dopaminergic neurons induced by systemic administration of mitochondrial respiratory chain complex I inhibitors such as 1-methyl-4-phenylpyridinium (MPP(+); given as the prodrug 1-methyl-1,2,3,6-tetrahydropyridine) or the pesticide rotenone have raised the question as to whether this family of compounds are the cause of some forms of Parkinsonism. We have examined the neurotoxic potential of another complex I inhibitor, annonacin, the major acetogenin of Annona muricata (soursop), a tropical plant suspected to be the cause of an atypical form of Parkinson disease in the French West Indies (Guadeloupe). When added to mesencephalic cultures for 24 h, annonacin was much more potent than MPP(+) (effective concentration [EC(50)]=0.018 versus 1.9 microM) and as effective as rotenone (EC(50)=0.034 microM) in killing dopaminergic neurons. The uptake of [(3)H]-dopamine used as an index of dopaminergic cell function was similarly reduced. Toxic effects were seen at lower concentrations when the incubation time was extended by several days whereas withdrawal of the toxin after a short-term exposure (<6 h) arrested cell demise. Unlike MPP(+) but similar to rotenone, the acetogenin also reduced the survival of non-dopaminergic neurons. Neuronal cell death was not excitotoxic and occurred independently of free radical production. Raising the concentrations of either glucose or mannose in the presence of annonacin restored to a large extent intracellular ATP synthesis and prevented neuronal cell demise. Deoxyglucose reversed the effects of both glucose and mannose. Other hexoses such as galactose and fructose were not protective. Attempts to restore oxidative phosphorylation with lactate or pyruvate failed to provide protection to dopaminergic neurons whereas idoacetate, an inhibitor of glycolysis, inhibited the survival promoting effects of glucose and mannose indicating that these two hexoses acted independently of mitochondria by stimulating glycolysis. In conclusion, our study demonstrates that annonacin promotes dopaminergic neuronal death by impairment of energy production. It also underlines the need to address its possible role in the etiology of some atypical forms of Parkinsonism in Guadeloupe.

Chronic activation of the cyclic AMP signaling pathway promotes development and long-term survival of mesencephalic dopaminergic neurons.

Abstract: Dibutyryl cyclic AMP (dbcAMP), a permeant analogue of cyclic AMP (cAMP), prevented, for at least 3 weeks, the death of tyrosine hydroxylase (TH)‐immunopositive dopaminergic neurons, which occurred spontaneously by apoptosis in mesencephalic cultures. Treatment with the cyclic nucleotide analogue also led to a significant increase in the uptake of [3H]dopamine, attesting that the rescued TH+ neurons were fully functional and differentiated. dbcAMP was most effective when added immediately after plating, but delayed treatment could still arrest the ongoing degenerative process. Trophic/survival effects were long‐lasting, declining only progressively after withdrawal of dbcAMP from the culture medium. They were independent of cell density and still detectable in the absence of serum proteins. The effects of dbcAMP were mimicked by depolarizing concentrations of potassium and by agents that increase endogenous production of cAMP, such as forskolin or 3‐isobutyl‐1‐methylxanthine, but not by native cAMP, which cannot cross cell membranes. Elimination of glial cells by arabinoside‐C did not reduce the activity of dbcAMP. GABAergic neurons, also present in these cultures, were much less dependent on the cyclic nucleotide analogue for their survival, and serotoninergic cells were not dependent at all. Therefore, cAMP‐dependent signaling may be particularly crucial for the maturation and long‐term survival of mesencephalic dopaminergic neurons.