Cerebrospinal fluid biomarkers in SARS-CoV-2 patients with acute neurological syndromes.

BACKGROUND AND PURPOSE: Mechanisms underlying acute brain injury in SARS-CoV-2 patients remain poorly understood. A better characterization of such mechanisms remains essential to preventing long-term neurological sequelae. Our present aim was to study a panel of biomarkers of neuroinflammation and neurodegeneration in the cerebrospinal fluid (CSF) of NeuroCOVID patients. METHODS: We retrospectively collected clinical and CSF biomarkers data from 24 NeuroCOVID adults seen at the University Hospital of Guadeloupe between March and June 2021. RESULTS: Among 24 NeuroCOVID patients, 71% had encephalopathy and 29% meningoencephalitis. A number of these patients also experienced de novo movement disorder (33%) or stroke (21%). The CSF analysis revealed intrathecal immunoglobulin synthesis in 54% of NeuroCOVID patients (two with a type 2 pattern and 11 with a type 3) and elevated neopterin levels in 75% of them (median 9.1nM, IQR 5.6-22.1). CSF neurofilament light chain (NfL) was also increased compared to a control group of non-COVID-19 patients with psychiatric illnesses (2905ng/L, IQR 1428-7124 versus 1222ng/L, IQR 1049-1566). Total-tau was elevated in the CSF of 24% of patients, whereas protein 14-3-3, generally undetectable, reached intermediate levels in two patients. Finally, CSF Aß1-42 was reduced in 52.4% of patients (median 536ng/L, IQR 432-904) with no change in the Aß1-42/Aß1-40 ratio (0.082, IQR 0.060-0.096). CONCLUSIONS: We showed an elevation of CSF biomarkers of neuroinflammation in NeuroCOVID patients and a rise of CSF NfL, evocative of neuronal damage. However, longitudinal studies are needed to determine whether NeuroCOVID could evolve into a chronic neurodegenerative condition.

Xenon-mediated neuroprotection in response to sustained, low-level excitotoxic stress.

Noble gases such as xenon and argon have been reported to provide neuroprotection against acute brain ischemic/anoxic injuries. Herein, we wished to evaluate the protective potential of these two gases under conditions relevant to the pathogenesis of chronic neurodegenerative disorders. For that, we established cultures of neurons typically affected in Alzheimer's disease (AD) pathology, that is, cortical neurons and basal forebrain cholinergic neurons and exposed them to L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) to generate sustained, low-level excitotoxic stress. Over a period of 4 days, PDC caused a progressive loss of cortical neurons which was prevented substantially when xenon replaced nitrogen in the cell culture atmosphere. Unlike xenon, argon remained inactive. Xenon acted downstream of the inhibitory and stimulatory effects elicited by PDC on glutamate uptake and efflux, respectively. Neuroprotection by xenon was mimicked by two noncompetitive antagonists of NMDA glutamate receptors, memantine and ketamine. Each of them potentiated xenon-mediated neuroprotection when used at concentrations providing suboptimal rescue to cortical neurons but most surprisingly, no rescue at all. The survival-promoting effects of xenon persisted when NMDA was used instead of PDC to trigger neuronal death, indicating that NMDA receptor antagonism was probably accountable for xenon's effects. An excess of glycine failed to reverse xenon neuroprotection, thus excluding a competitive interaction of xenon with the glycine-binding site of NMDA receptors. Noticeably, antioxidants such as Trolox and N-acetylcysteine reduced PDC-induced neuronal death but xenon itself lacked free radical-scavenging activity. Cholinergic neurons were also rescued efficaciously by xenon in basal forebrain cultures. Unexpectedly, however, xenon stimulated cholinergic traits and promoted the morphological differentiation of cholinergic neurons in these cultures. Memantine reproduced some of these neurotrophic effects, albeit with less efficacy than xenon. In conclusion, we demonstrate for the first time that xenon may have a therapeutic potential in AD.

Piperazine derivatives as iron chelators: a potential application in neurobiology.

Polysubstituted piperazine derivatives, designed as new iron chelators, were synthesized and fully characterized by nuclear magnetic resonance and mass spectroscopy. Their potential to prevent iron-induced neurotoxicity was assessed using a cellular model of Parkinson disease. We demonstrated their ability to provide sustained neuroprotection to dopaminergic neurons that are vulnerable in this pathology. The iron chelating properties of the new compounds were determined by spectrophotometric titration illustrating that high affinity for iron is not associated with important neuroprotective effects.

Is atypical parkinsonism in the Caribbean caused by the consumption of Annonacae?

An abnormally frequent atypical levodopa-unresponsive, akinetic-rigid syndrome with some similarity to PSP was identified in the Caribbean island Guadeloupe, and was associated with the consumption of plants of the Annonacea family, especially Annona muricata (corossol, soursop) suggesting a possible toxic etiology. Annonaceae contain two groups of potential toxins, alkaloids and acetogenins. Both alkaloids and annonacin, the most abundant acetogenin, were toxic in vitro to dopaminergic and other neurons. However we have focused our work on annonacin for two reasons: (1) annonacin was toxic in nanomolar concentrations, whereas micromolar concentrations of the alkaloids were needed, (2) acetogenins are potent mitochondrial poisons, like other parkinsonism-inducing compounds. We have also shown that high concentrations of annonacin are present in the fruit or aqueous extracts of the leaves of A. muricata, can cross the blood brain barrier since it was detected in brain parenchyma of rats treated chronically with the molecule, and induced neurodegeneration of basal ganglia in these animals, similar to that observed in atypical parkinsonism. These studies reinforce the concept that consumption of Annonaceae may contribute to the pathogenesis of atypical parkinsonism in Guadeloupe.

Levodopa in the treatment of Parkinson's disease: current controversies.

Levodopa is the most effective symptomatic agent in the treatment of Parkinson's 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.

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.

The role of glial reaction and inflammation in Parkinson's disease.

The glial reaction is generally considered to be a consequence of neuronal death in neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, and Parkinson's disease. In Parkinson's disease, postmortem examination reveals a loss of dopaminergic neurons in the substantia nigra associated with a massive astrogliosis and the presence of activated microglial cells. Recent evidence suggests that the disease may progress even when the initial cause of neuronal degeneration has disappeared, suggesting that toxic substances released by the glial cells may be involved in the propagation and perpetuation of neuronal degeneration. Glial cells can release deleterious compounds such as proinflammatory cytokines (TNF-alpha, Il-1beta, IFN-gamma), which may act by stimulating nitric oxide production in glial cells, or which may exert a more direct deleterious effect on dopaminergic neurons by activating receptors that contain intracytoplasmic death domains involved in apoptosis. In line with this possibility, an activation of proteases such as caspase-3 and caspase-8, which are known effectors of apoptosis, has been reported in Parkinson's disease. Yet, caspase inhibitors or invalidation of TNF-alpha receptors does not protect dopaminergic neurons against degeneration in experimental models of the disease, suggesting that manipulation of a single signaling pathway may not be sufficient to protect dopaminergic neurons. In contrast, the antiinflammatory drugs pioglitazone, a PPAR-gamma agonist, and the tetracycline derivative minocycline have been shown to reduce glial activation and protect the substantia nigra in an animal model of the disease. Inhibition of the glial reaction and the inflammatory processes may thus represent a therapeutic target to reduce neuronal degeneration in Parkinson's disease.

[Parkinson's disease: cell death mechanisms] / Maladie de Parkinson: mécanismes de la mort cellulaire.

Parkinson disease is a neurodegenerative disorder of aging characterized by a selective and progressive loss of dopaminergic neurons within the substantia nigra. The diagnosis of the disease is made when neuronal cell loss exceeds 50 p. cent indicating that the degenerative process started well before the onset of the first clinical symptoms. Three populations of dopaminergic neurons seem to coexist in the substantia nigra of parkinsonian patients; (1) senescent neurons that are still spared by the pathological process; (2) sick neurons exhibiting generally a preserved morphology but showing evidence of biochemical and metabolic abnormalities; (3) neurons which have entered into a final state of agony and exhibit the hallmarks of apoptosis, a controlled form of cell death that requires the activation of a particular type of proteases, caspases. In the inherited forms of the disease that are caused by mutations of genes encoding the Parkin, alpha-synuclein and UCHL-1 proteins, the degenerative process results from the dysfunction of an enzymatic complex of proteolysis, the proteasome. This probably leads to the intracellular accumulation of abnormal proteins that become deleterious for dopaminergic neurons. In the sporadic forms of the disease that are the most frequent, causes of the cell demise remain still unknown but neurodegeneration might also result from a decreased activity of the proteasome. A defect in the detoxification of reactive oxygen species or an energy failure caused by inhibition of the mitochondrial respiratory chain, at the complex I level, are other hypothesis that are frequently mentioned. Finally, activated glial cells (astrocytes and microglia) located around the degenerating dopaminergic neurons might also intervene in the mechanism of degeneration by perpetuating or even amplifying the primary neuronal insult. Proinflammatory cytokines acting on cell death membrane receptors and diffusable messengers such as nitric oxide could be part of this process.

[Parkinson disease: mechanisms of cell death]. / Maladie de Parkinson: mécanismes de la mort cellulaire.

Parkinson disease is a neurodegenerative disorder of aging characterized by a selective and progressive loss of dopaminergic neurons within the substantia nigra. The diagnosis of the disease is made when neuronal cell loss exceeds 50 p. 100 indicating that the degenerative process started well before the onset of the first clinical symptoms. Three populations of dopaminergic neurons seem to coexist in the substantia nigra of parkinsonian patients; (1) senescent neurons that are still spared by the pathological process; (2) sick neurons exhibiting generally a preserved morphology but showing evidence of biochemical and metabolic abnormalities; (3) neurons which have entered into a final state of agony and exhibit the hallmarks of apoptosis, a controlled form of cell death that requires the activation of a particular type of proteases, caspases. In the inherited forms of the disease that are caused by mutations of genes encoding the Parkin, alpha-synuclein and UCHL-1 proteins, the degenerative process results from the dysfunction of an enzymatic complex of proteolysis, the proteasome. This probably leads to the intracellular accumulation of abnormal proteins that become deleterious for dopaminergic neurons. In the sporadic forms of the disease that are the most frequent, causes of the cell demise remain still unknown but neurodegeneration might also result from a decreased activity of the proteasome. A defect in the detoxification of reactive oxygen species or an energy failure caused by inhibition of the mitochondrial respiratory chain, at the complex I level, are other hypothesis that are frequently mentioned. Finally, activated glial cells (astrocytes and microglia) located around the degenerating dopaminergic neurons might also intervene in the mechanism of degeneration by perpetuating or even amplifying the primary neuronal insult. Proinflammatory cytokines acting on cell death membrane receptors and diffusable messengers such as nitric oxide could be part of this process.

Noradrenaline provides long-term protection to dopaminergic neurons by reducing oxidative stress.

To better understand the neurotrophic function of the neurotransmitter noradrenaline, we have developed a model of mesencephalic cultures in which we find low concentrations (0.3-10 microM) of noradrenaline to be remarkably effective in promoting long-term survival and function of dopaminergic neurons. This protective action reproduced the effect of caspase inhibition. It was atypical in that it occurred independently of adrenoceptor activation and was mimicked by some antioxidants, redox metal chelators and the hydroxyl radical detoxifying enzyme catalase. Interestingly, intracellular reactive oxygen species (ROS) were drastically reduced by treatment with noradrenaline, indicating that the neurotransmitter itself acted as an antioxidant. Prevention of oxidative stress was, however, independent of the glutathione antioxidant defense system. Chemical analogues of noradrenaline bearing two free hydroxyl groups in the ortho position of the aromatic ring (o-catechols), as well as o-catechol itself, mimicked the survival promoting effects of the neurotransmitter, suggesting that this diphenolic structure was critical for both neuroprotection and reduction of ROS production. Paradoxically, the autoxidation of noradrenaline and the ensuing production of quinone metabolites may be required for both effects, as the neurotransmitter was spontaneously and rapidly degraded over time in the culture medium. These results support the concept that central noradrenergic mechanisms have a neuroprotective role, perhaps in part by reducing oxidative stress.

Survival promotion of mesencephalic dopaminergic neurons by depolarizing concentrations of K+ requires concurrent inactivation of NMDA or AMPA/kainate receptors.

The death of dopaminergic neurons that occurs spontaneously in mesencephalic cultures was prevented by depolarizing concentrations of K+ (20-50 mM). However, unlike that observed previously in other neuronal populations of the PNS or CNS, promotion of survival required concurrent blockade of either NMDA or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptors by the specific antagonists, MK-801 and GYKI-52466, respectively. Rescued neurons appeared to be healthy and functional because the same treatment also dramatically enhanced their capacity to accumulate dopamine. The effects on survival and uptake were rather specific to dopaminergic neurons, rapidly reversible and still observed when treatment was delayed after plating. Glutamate release increased substantially in the presence of elevated concentrations of K+, and chronic treatment with glutamate induced a loss of dopaminergic neurons that was prevented by MK-801 or GYKI-52466 suggesting that an excitotoxic process interfered with survival when only the depolarizing treatment was applied. The effects of the depolarizing stimulus in the presence of MK-801 were mimicked by BAY K-8644 and abolished by nifedipine, suggesting that neuroprotection resulted from Ca(2+) influx through L-type calcium channels. Measurement of intracellular calcium revealed that MK-801 or GYKI-52466 were required to maintain Ca(2+) levels within a trophic range, thus preventing K+-induced excitotoxic stress and Ca(2+) overload. Altogether, our results suggest that dopaminergic neurons may require a finely tuned interplay between glutamatergic receptors and calcium channels for their development and maturation.

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 Parkinson's disease, we tried to clarify the potential role of Bax in this process in an immunohistochemical study on normal and Parkinson's 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 Parkinson's 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 Parkinson's disease subjects, regardless of the mesencephalic subregion analysed. However, in Parkinson's 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 Parkinson's 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 Parkinson's disease paradigm.

The relationship between differentiation and survival in PC12 cells treated with cyclic adenosine monophosphate in the presence of epidermal growth factor or nerve growth factor.

We have asked whether treatment of PC12 cells with cyclic adenosine monophosphate (cAMP) and epidermal growth factor (EGF) results, like treatment with cAMP and nerve growth factor (NGF), in irreversible neuronal differentiation characterized by irreversible neurite extension, loss of serum-dependence, and death by apoptosis after trophic factor withdrawal. Although EGF alone, unlike NGF, did not cause morphological differentiation or prevent cell death, synergy between a cAMP-mediated signal transduction pathway and a pathway activated by the EGF receptor tyrosine kinase resulted in the same irreversible differentiation. EGF/cAMP-differentiated cells required cAMP to survive, but NGF, through a TrkA-dependent mechanism, could substitute for cAMP. The cyclin-dependent kinase inhibitors olomoucine and roscovitine also promoted survival of the irreversibly differentiated cells, by a mechanism that must be determined, since cell death was not associated with nuclear (3)H-thymidine accumulation, an index of mitotic activity.

Mitochondrial free calcium levels (Rhod-2 fluorescence) and ultrastructural alterations in neuronally differentiated PC12 cells during ceramide-dependent cell death.

Mitochondrial free calcium levels measured by Rhod-2 fluorescence and ultrastructure were examined during cell death in nerve growth factor (NGF)-differentiated PC12 cells that were 1) exposed to C2-ceramide, 2) deprived of serum to induce endogenous ceramide production, or 3) treated with calcium ionophore A23187. Rhod-2 fluorescence in mitochondria and also in the nucleolus increased to a maximum within 3 hours after C2-ceramide treatment or serum withdrawal. In A23187-treated cells, Rhod-2 fluorescence remained at baseline levels. In all three models, enlargement of the endoplasmic reticulum was the first ultrastructural alteration, followed by mitochondrial shrinkage in ionophore-treated cells, but by mitochondrial swelling in the ceramide-dependent models, in which rupture of the outer mitochondrial membrane and unfolding of the inner membrane were frequently seen. Dihydro-C2-ceramide, which did not cause cell death, had no effect on cellular ultrastructure. NGF, which inhibits ceramide-dependent cell death, prevented the effects of serum deprivation on mitochondrial ultrastructure but not on endoplasmic reticulum morphology or Rhod-2 fluorescence. Nuclear shrinkage with loss of nuclear membrane integrity, characterized by nuclear pores, free or surrounded by electron-dense filaments, was a late event in ceramide-dependent cell death. Chromatin condensation and other morphological features associated with apoptosis were seen in only a few atypical cells. Ceramide-mediated cell death, therefore, did not involve classical apoptosis but was mediated by a reproducible series of events beginning in the endoplasmic reticulum, followed by the mitochondria, and then the nucleus. NGF-dependent cell death inhibition intervenes at the mitochondrial level, not by blocking the increase in Rhod-2 fluorescence but by preventing the ultrastructural changes that follow.

Caspase-3: A vulnerability factor and final effector in apoptotic death of dopaminergic neurons in Parkinson's disease.

Caspase-3 is an effector of apoptosis in experimental models of Parkinson's disease (PD). However, its potential role in the human pathology remains to be demonstrated. Using caspase-3 immunohistochemistry on the postmortem human brain, we observed a positive correlation between the degree of neuronal loss in dopaminergic (DA) cell groups affected in the mesencephalon of PD patients and the percentage of caspase-3-positive neurons in these cell groups in control subjects and a significant decrease of caspase-3-positive pigmented neurons in the substantia nigra pars compacta of PD patients compared with controls that also could be observed in an animal model of PD. This suggests that neurons expressing caspase-3 are more sensitive to the pathological process than those that do not express the protein. In addition, using an antibody raised against activated caspase-3, the percentage of active caspase-3-positive neurons among DA neurons was significantly higher in PD patients than in controls. Finally, electron microscopy analysis in the human brain and in vitro data suggest that caspase-3 activation precedes and is not a consequence of apoptotic cell death in PD.

Neuropharmacologic aspects of apoptosis: significance for neurodegenerative diseases.

The cause of neuronal death in Parkinson's, Alzheimer's, and other neurodegenerative diseases is not known, except in some hereditary forms of these disorders in which a mutated gene has been identified. Even in these cases, the molecular mechanisms that underlie the loss of specific populations of neurons have not been determined, although it is highly probable that apoptosis is involved. Some of the biochemical events that occur during apoptosis have been elucidated. We focus in this review on the role played by the proapoptotic caspases, the antiapoptotic proteins of the Bcl-2 family, and the apoptosis associated signal transducers such as ceramide, calcium, and reactive nitrogen or oxygen species. The role of the mitochondria and the possible implication of cell cycle regulators will also be addressed. Of particular interest are the endogenous inhibitory mechanisms and the pharmacologic agents that can be used to block apoptosis signaling cascades, because they offer models for the development of therapeutic strategies designed to prevent the evolution of pathologic neurodegeneration.

Adenosine prevents the death of mesencephalic dopaminergic neurons by a mechanism that involves astrocytes.

The purinergic nucleoside adenosine effectively prevented the death of dopaminergic neurons that occurs spontaneously and progressively in cultures of rat mesencephalon. Adenosine also significantly increased dopamine uptake, attesting to the state of differentiation and functional integrity of the neurons that were rescued. The effects of adenosine were (a) specific to the dopaminergic neurons in these cultures, (b) long-lived, (c) still observed when the treatment was delayed after plating, (d) potentiated by inhibition of adenosine deaminase, and (e) abolished when this enzyme was added in excess to the culture medium. The action of adenosine was mimicked by 5'-(N-ethylcarboxamido)adenosine and dibutyryl-cyclic AMP, but not by CGS-21680, suggesting the possible involvement of A2B subtype purinergic receptors. ATP was also neuroprotective, but its action resulted principally from conversion to adenosine by ectonucleotidases. Several anticancer drugs, including cytosine arabinoside, have been shown previously to prevent apoptosis in cultured dopaminergic neurons by a mechanism that requires the inhibition of proliferating astrocytes. In the presence of adenosine, astrocytes were more differentiated, and their proliferation rate was significantly reduced, suggesting that the neurotrophic effect of the adenine nucleoside resulted also from the repression of the astroglial cells. We did not find evidence of a trophic intermediary in adenosine-treated cultures, however, leading to the hypothesis that limitation of astrocyte replication in itself and/or ensuing changes in the glial phenotype were crucial. Our results suggest that molecules that modulate adenine nucleotide/nucleoside release may be useful for the treatment of chronic neurodegenerative conditions affecting dopaminergic neurons, such as Parkinson's disease.