Sheela Vyas

Neuroinflammation in Parkinson's disease

Both epidemiological and genetic studies support a role of neuroinflammation in the pathophysiology of Parkinsons disease (PD). Furthermore, post mortem studies confirm the involvement of innate as well as adaptive immunity in the affected brain regions in patients with PD. Indeed, activated microglial cells and T lymphocytes have been detected in the substantia nigra of patients concomitantly with an increased expression of pro-inflammatory mediators. Preclinical investigations conducted in various animal models of PD indicate that inflammatory processes are instrumental in neuronal cell death even though they are unlikely to be a primary cause for neuronal loss. Neuroinflammatory processes in PD are rather involved in self-perpetuating deleterious events that lead to protracted neuronal degeneration. In line with this, recent data indicate that glucocorticoid receptors are important in curtailing microglial reactivity, and deregulation in their activity in PD could lead to sustained inflammation-mediated degeneration. Altogether, neuroinflammatory processes might represent a target for neuroprotection in PD.

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.

Transcriptional and post-transcriptional regulation of tyrosine hydroxylase gene by protein kinase C.

The role played by protein kinase C (PKC) in TH gene regulation was investigated at transcriptional and post‐transcriptional levels using PC12 cells. The cells were treated with the phorbol ester TPA, which not only activates PKC but also causes down‐regulation. PKC levels were monitored by [3H]PDBU binding assay and by using an anti‐PKC antibody that detected intact PKC (79 kd) as well as its catalytic and regulatory domains. The [3H]PDBU binding to the membrane‐associated PKC increased within 15‐30 min of TPA treatment; thereafter total cellular [3H]PDBU binding decreased to a minimum of 20% of the control at 8 h. The rate of decrease in binding was greater than the decrease in the intensity of the staining of PKC holo enzyme visualized by anti‐PKC antibody. TH mRNA levels, measured over the same time period, rose within 15 min of TPA treatment to peak at 4 h and subsequently declined below control level, paralleling the depletion of PKC. If cells depleted of PKC were reincubated in the normal medium, a recovery in PKC level was seen and, in parallel, TH mRNA levels increased to above control level. Furthermore, if down‐regulation of PKC was prevented by incubating the cells with the protease inhibitor leupeptin, a decrease beyond control level in TH mRNA was not observed. TPA rapidly induced TH gene transcription; a maximal increase of two‐fold was observed at 15 min, but the transcriptional rate then declined although it did not decrease beyond control values after 8 and 24 h of TPA treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term induction of tyrosine hydroxylase expression: compensatory response to partial degeneration of the dopaminergic nigrostriatal system in the rat brain.

Abstract: The present study was undertaken to examine the adaptive changes occurring 1 and 6 months after moderate or severe unilateral 6‐hydroxydopamine‐induced lesions confined to the lateral part of the rat substantia nigra pars compacta (SNC). The expression of tyrosine hydroxylase (TH) enzyme was analyzed in the remaining dopaminergic nigral cell bodies and in the corresponding striatal nerve endings. In the cell bodies of the lesioned SNC, TH mRNA content was increased (+20 to +30%) 6 months after the lesion without changes in cellular TH protein amounts. The depletion of TH protein in the nerve terminal area was less severe than the percentage of cell loss observed in the SNC at 1‐ and 6‐month postlesion intervals. Moreover, the decrease in TH protein in the ipsilateral striatum was less pronounced 6 months after lesion than 1 month after. That no corresponding change in TH protein content was observed in the cell bodies at a time when TH increased in nerve terminals suggests that the newly synthesized protein is probably rapidly transported to the striatal fibers. These results suggest the existence of a sequence of changes in TH expression between cell bodies and fibers, occurring spontaneously after partial denervation of the nigrostriatal pathway.

Expression of Bcl-2 in adult human brain regions with special reference to neurodegenerative disorders.

Abstract: The expression of the protooncogene bcl‐2, an inhibitor of apoptosis in various cells, was examined in the adult human brain. Several experimental criteria were used to verify its presence; mRNA was analyzed by northern blot with parallel experiments in mouse tissues, by RNase protection, and by in situ hybridization histochemistry. Bcl‐2 protein was detected by western blot analysis and immunohistochemistry. Two bcl‐2 mRNA species were identified in the human brain. The pattern of distribution of bcl‐2 mRNA at the cellular level showed labeling in neurons but not glia. The in situ hybridization signal was stronger in the pyramidal neurons of the cerebral cortex and in the cholinergic neurons of the nucleus basalis of Meynert than in the Purkinje neurons of the cerebellum. Both melanized and nonmelanized neurons were labeled in the substantia nigra. In the striatum, bcl‐2 mRNA was detected in some but not all neurons. In the regions examined for Bcl‐2 protein, the expression pattern correlated with the mRNA results. In patients with Alzheimers and Parkinsons diseases, quantification of bcl‐2 mRNA in the nucleus basalis of Meynert and substantia nigra, respectively, showed that the expression was unaltered compared with controls, raising the possibility that the expression of other components of apoptosis is modulated.

Toxic effects of iron for cultured mesencephalic dopaminergic neurons derived from rat embryonic brains

Iron, a transition metal possibly involved in the pathogenesis of Parkinsons disease, was tested for its toxic effects toward cultures of dissociated rat mesencephalic cells. When cultures were switched for 24 h to serum‐free conditions, the effective concentrations of ferrous iron (Fe2+) producing a loss of 50% of dopaminergic neurons, as quantified by tyrosine hydroxylase (TH) immunocytochem‐istry, TH mRNA in situ hybridization, and measurement of TH activity, were on the order of 200 μM. High‐affinity dopamine (DA) uptake, which reflects integrity and function of dopaminergic nerve terminals, was impaired at significantly lower concentrations (EC50= 67 μM). Toxic effects were not restricted to dopaminergic neurons inasmuch as trypan blue dye exclusion index and γ‐aminobutyric acid uptake, two parameters used to assess survival of other types of cells present in these cultures, were also affected. Protection against iron cytotoxicity was afforded by desferriox‐amine and apotransferrin, two ferric iron‐chelating agents. Normal supplementation of the culture medium by serum proteins during treatment was also effective, presumably vianonspecific sequestration. Potential interactions with DA were also investigated. Fe2+ at subtoxic concentrations and desferrioxamine in the absence of exogenous iron added to the cultures failed to potentiate or reduce DA cytotoxicity for mesencephalic cells, respectively. Transferrin, the glyco‐protein responsible for intracellular delivery of iron, was ineffective in initiating selective cytotoxic effects toward dopaminergic neurons preloaded with DA. Altogether, these results suggest (a) that ferrous iron is a potent neurotoxin for dopaminergic neurons as well as for other cell types in dissociated mesencephalic cultures, acting likely via autoxida‐tion into its ferric form, and (b) that the presence of intra‐and extracellular DA is not required for the observed toxic effects.

MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF THE RESPONSE OF DIFFERENTIATED PC12 CELLS TO CALCIUM STRESS

The mechanisms that lead ultimately to neuronal death in pathological ageing of the brain remain mostly unknown as in the case of Parkinsons disease where there is a progressive and selective loss of dopaminergic neurons within the substantia nigra. Dopamine‐expressing PC12 cells that were neuronally differentiated by nerve growth factor treatment were chosen as a culture model in which to study some of the changes that may occur during the course of the degenerative process. They were exposed to the calcium ionophore A23187 in order to produce a sustained rise in cytoplasmic calcium, a phenomenon related to various pathological conditions. The degenerative effects of the ionophore were dose‐ and time‐dependent. They were characterized by early fragmentation of the neurites followed ultimately by a loss in cell viability. Biochemical changes, such as a decrease in [3H]dopamine uptake and modulations of the tyrosine hydroxylase gene, were detected before macroscopic evidence of cell suffering (e.g. neurite fragmentation) could be observed. Although an ongoing degenerative process was occurring in cell somata, PC12 cells were able to recover upon ionophore withdrawal. Characteristics of apoptosis such as chromatin condensation and DNA fragmentation were detectable in a small population of dying cells. DNA fragmentation could be prevented by the endonuclease inhibitor aurintricarboxylic acid. New protein synthesis was not required, as cycloheximide failed to prevent degeneration. Taken together, these results suggest that differentiated PC12 cells react to calcium stress through a sequence of regulatory processes which appears to be independent of the apoptotic pathway.

A Ewing's Sarcoma Cell Line Showing Some, but Not All, of the Traits of a Cholinergic Neuron

Abstract: The Ewings sarcoma cell line ICB 112 was examined in detail for a cholinergic phenotype. Choline acetyltransferase activity (12.3 ± 2.9 nmol/h/mg of protein) was associated with the presence of multiple mRNA species labeled with a human choline acetyltransferase riboprobe. Choline was taken up by the cells by a high‐affinity, hemicholinium‐3‐sensitive transporter that was partially inhibited when lithium replaced sodium in the incubation medium; the choline taken up was quickly incorporated into both acetylcholine and phosphorylcholine. High‐affinity binding sites for vesamicol, an inhibitor of vesicular acetylcholine transport, were also present. The mRNAs for synaptotagmin (p65) and the 15‐kDa proteolipid were readily detected and were identical in size to those observed in cholinergic regions of the human brain. Cumulative acetylcholine efflux was increased by raising the extracellular potassium level or the addition of a calcium ionophore, but the time course of stimulated efflux was slow and persistent. These results show that this morphologically undifferentiated cell line is capable of acetylcholine synthesis and expresses markers for synaptic vesicles as well as proteins implicated in calcium‐dependent release but lacks an organized release mechanism.

Synergistic Differentiation by Chronic Exposure to Cyclic AMP and Nerve Growth Factor Renders Rat Phaeochromocytoma PC12 Cells Totally Dependent upon Trophic Support for Survival

Chronic dibutyryl cAMP (dbcAMP) treatment was observed not only to potentiate the differentiating actions of nerve growth factor (NGF) in PC12 cells, but to render them completely dependent on trophic support for survival even in the presence of serum proteins. When both NGF and dbcAMP were withdrawn from doubly differentiated PC12 cultures, degenerative events occurred after a lag period of 12–18 h, and by 48 h ≤ 5–10% of the cells remained viable. Reduction in [3H]dopamine uptake, an index of cell function and neurite integrity, paralleled cell demise. At the cellular level, ∼20–30% of the nuclei exhibited clear signs of chromatin fragmentation, as characterized by propidium iodide staining, suggesting that degeneration occurred by apoptosis. The cells could be rescued completely from degeneration by dbcAMP or by other cAMP analogues, whereas NGF and depolarization were also effective, but only partially. Phorbol 12‐myristate‐13‐acetate failed to afford protection. If deprivation was interrupted, cell demise could be stopped by restoration of initial culture conditions. Degenerative changes produced by deprivation and recovery processes were not inhibited by macromolecular synthesis inhibitors, e.g. cycloheximide and actinomycin‐D. However, chronic addition of cycloheximide prior to deprivation greatly impaired the differentiation of NGF/dbcAMP cells, allowing these cells to withstand trophic support withdrawal. Altogether our results indicate that the cAMP transduction pathway plays a crucial role not only in the differentiation but also in the survival of NGF/dbcAMP PC12 cells.

Induction of calbindin-D 28K gene and protein expression by physiological stimuli but not in calcium-mediated degeneration in rat PC12 pheochromocytoma cells

To understand the role of calbindin‐D 28K in neuronal degeneration, we examined its expression in differentiated PC12 cells in response to calcium intoxication, using the ionophore A23187 treatment, that results in cell degeneration and death. We first established that calbindin‐D 28K is expressed in PC12 cells. The amounts of calbindin‐D 28K mRNA and protein were increased by the differentiation factors, NGF and retinoic acid, but not by vitamin D3. Calbindin‐D 28K expression was also significantly up‐regulated by stimuli (depolarization, low concentrations of Ca2+ ionophore A23187) which increase intracellular calcium levels within the physiological range. In contrast, the calbindin‐D 28K mRNA and protein concentrations were not modulated by high concentrations of A23187, which resulted in cell degeneration and death. Experiments with the antisense oligonucleotides showed that, although the calbindin‐D 28K protein levels were decreased significantly, the progression of degenerative changes induced by calcium via A23187, was not altered.