Anat Shirvan

Immunosuppressive role of semaphorin‐3A on T cell proliferation is mediated by inhibition of actin cytoskeleton reorganization

Timely negative regulation of the immune system is critical to allow it to perform its duty while maintaining it under tight control to avoid overactivation. We previously reported that the neuronal receptor neuropilin‐1 (NP‐1) is expressed in human lymph nodes. However, the role of NP‐1 interaction with its physiological ligand semaphorin‐3A (Sema‐3A) on immune cells remains elusive. Here we show that Sema‐3A is expressed by activated DC and T cells, and that its secretion in DC/T cell cocultures is delayed. Sema‐3A/NP‐1 interaction down‐modulated T cell activation since addition of Sema‐3A in DC/T cell cocultures dramatically inhibited allogeneic T cell proliferation. More importantly, neutralization by blocking antibodies or by antagonist peptide of endogenous Sema‐3A produced by DC/T cell cocultures resulted in a 130% increase in T cell proliferation. Sema‐3A acted directly on T cells, since it could block anti‐CD3/CD28‐stimulated proliferation of T cells. Finally, immunomodulatory functions of Sema‐3A relied on the blockage of actin cytoskeleton reorganization, affecting TCR polarization and interfering with early TCR signal transduction events such as ZAP‐70 or focal adhesion kinase phosphorylation. Therefore, we propose that Sema‐3A secretion and the resulting NP‐1/Sema‐3A interaction are involved in a late negative feedback loop controlling DC‐induced T cell proliferation.

Anti-semaphorin 3A Antibodies Rescue Retinal Ganglion Cells from Cell Death following Optic Nerve Axotomy

Damage to the optic nerve in mammals induces retrograde degeneration and apoptosis of the retinal ganglion cell (RGC) bodies. The mechanisms that mediate the response of the neuronal cells to the axonal injury are still unknown. We have previously shown that semaphorins, axon guidance molecules with repulsive cues, are capable of mediating apoptosis in cultured neuronal cells (Shirvan, A., Ziv, I., Fleminger, G., Shina, R., He, Z., Brudo, I., Melamed, E., and Brazilai, A. (1999) J. Neurochem. 73, 961–971). In this study, we examined the involvement of semaphorins in an in vivo experimental animal model of complete axotomy of the rat optic nerve. We demonstrate that a marked induction of type III semaphorin proteins takes place in ipsilateral retinas at early stages following axotomy, well before any morphological signs of RGC apoptosis can be detected. Time course analysis revealed that a peak of expression occurred after 2–3 days and then declined. A small conserved peptide derived from semaphorin 3A that was previously shown to induce neuronal death in culture was capable of inducing RGC loss upon its intravitreous injection into the rat eye. Moreover, we demonstrate a marked inhibition of RGC loss when axotomized eyes were co-treated by intravitreous injection of function-blocking antibodies against the semaphorin 3A-derived peptide. Marked neuronal protection from degeneration was also observed when the antibodies were applied 24 h post-injury. We therefore suggest that semaphorins are key proteins that modulate the cell fate of axotomized RGC. Neutralization of the semaphorin repulsive function may serve as a promising new approach for treatment of traumatic injury in the adult mammalian central nervous system or of ophthalmologic diseases such as glaucoma and ischemic optic neuropathy that induce apoptotic RGC death.

Is there a rationale for neuroprotection against dopamine toxicity in Parkinson's disease?

Parkinsons disease is a progressive neurological disease caused by rather selective degeneration of the dopaminergic neurons in the substantia nigra. Though subject to intensive research, the etiology of this nigral loss is still undetermined and treatment is basically symptomatic. The current major hypothesis is that nigral neuronal death in PD is due to excessive oxidative stress generated by auto and enzymatic oxidation of the endogenous neurotransmitter dopamine (DA), the formation of neuromelanin (NM) and the presence of a high concentration of iron. In this review article although we concisely describe the effects of NM and iron on neuronal survival, we mainly focus on the molecular mechanisms of DA-induced apoptosis. DA exerts its toxic effects through its oxidative metabolites either in vitro or in vivo The oxidative metabolites then activate a very intricate web of signals, which culminate in cell death. The signal transduction pathways and genes, which are associated with DA toxicity are described in detail.

Two Waves of Cyclin B and Proliferating Cell Nuclear Antigen Expression During Dopamine‐Triggered Neuronal Apoptosis

Abstract: The neurotransmitter dopamine is capable of inducing apoptosis in postmitotic sympathetic neurons via its oxidative metabolites. To detect genes whose expression is transcriptionally regulated during the early stages of dopamine‐triggered apoptosis, we applied the differential display method to cultured sympathetic neurons. One of the up‐regulated genes was identified as cyclin B2, which exhibited two waves of induction and destruction, both at the mRNA and protein levels, resembling the sequential oscillations typical of two successive mitotic events in proliferating cells. The time window between the two waves was characterized by a change in expression of other cell‐cycle stage‐specific genes, and oscillations in proliferating cell nuclear antigen and alterations in cyclin A were observed. Cyclin D1 and cyclin‐dependent kinases were undetected and no sign of active DNA synthesis could be observed, indicating that activation of cell‐cycle components is incomplete. In comparison with a normal cell cycle, temporal expression profile of these mediators was unsynchronized. Whereas the first wave of cell‐cycle changes occurred prior to the commitment of the cells to the death process and could be tolerated by the cells, the second wave of changes coincided with the death commitment point. Our findings indicate that inappropriate and incomplete activation of some cell cycle‐related genes in postmitotic neurons occurs during dopamine‐triggered neuronal apoptosis.

Expression of cell cycle-related genes during neuronal apoptosis : Is there a distinct pattern?

An emerging hypothesis considers the process of neuronal apoptosis as a consequence of unscheduled and unsynchronized induction of cell cycle mediators. Induction of several cell cycle genes precedes neuronal apoptosis and may be involved in determination of cell fate. We have now characterized changes in expression of cell cycle genes during apoptosis induced by oxidative stress in chick post-mitotic sympathetic neurons. Induction of cyclin B occurred prior to the commitment of neurons to both dopamine- and peroxide-triggered apoptosis. Both the neuronal death and the rise in cyclin B were inhibited by antioxidant treatment, suggesting a functional role for cyclin B induction during neuronal apoptosis. Induction of the cyclin dependent kinase CDK5 protein coincided with the time point when neurons were irreversibly committed to die. Expression of other cell cycle mediators such as cyclin D1 and the cyclin dependent kinases CDC2 and CDK2 was undetected and not induced by exposure to oxidative stress. Comparative analysis of the profile of cell cycle mediators induced during neuronal apoptosis of different neuronal cell populations revealed no distinct pattern of events. There are no cell cycle stage-specific mediators that are ultimately stimulated during neuronal apoptosis, suggesting that multiple pathways of re-activating the dormant cell-cycle, converge to determine entry into apoptosis. Nevertheless, the existence of some cell cycle mediators, that were not reported so far to be induced in post mitotic neurons during oxidative stress, substantiate them as part of the strong differentiating forces.

Induction of neuronal apoptosis by Semaphorin3A-derived peptide

Collapsin-1/Semaphorin3A (Sema3A) belongs to the secreted type III semaphorins family of axon guidance molecules with chemorepulsive activity, and is suggested to play a major role in navigating axonal networks throughout development into their correct destinations. We have previously shown that semaphorins are mediators of neuronal apoptosis and can induce neuronal death in the absence of any other apoptotic trigger. We report here that exposure of neuronal cells to a small conserved peptide derived from Sema3A initiates an apoptotic death process. Administration of this peptide to cultured chick sympathetic and mouse cerebellar granule neurons caused a marked shrinkage of their axonal network and cell death, which was characterized as apoptotic, based on nuclear staining. Attenuation of neuronal cell death was obtained by treatment with antioxidants and by vascular endothelial growth factor. Survival of neurons exposed to this peptide increased by co-treatment with caspase inhibitors. Induction of apoptosis was specific to neuronal cells, similarly to that induced by the full-length Sema3A protein. Our findings therefore suggest active participation of this conserved Sema3A-derived peptide in semaphorin-induced neuronal death process.

The molecular mechanism of dopamine-induced apoptosis: identification and characterization of genes that mediate dopamine toxicity

Parkinsons disease (PD) is a progressive neurological disorder caused by rather selective degeneration of the dopaminergic (DA) neurons in the substantia nigra. Though subject to intensive research, the etiology of this nigral neuronal loss is still enigmatic and treatment is basically symptomatic. The current major hypothesis suggests that nigral neuronal death in PD is due to excessive oxidative stress generated by auto- and enzymatic oxidation of the endogenous neurotransmitter dopamine (DA), the formation of neuromelanin and presence of high concentrations of iron. We have found that DA toxicity is mediated through its oxidative metabolites. Whereas thiol-containing antioxidants provided marked protection against DA toxicity, ascorbic acid accelerated DA-induced death. Using the differential display approach, we sought to isolate and characterize genes whose expression is altered in response to DA toxicity. We found an upregulation of the collapsin response mediator protein (CRM) and TCP-1delta in sympathetic neurons, which undergo dopamine-induced apoptosis. The isolation of these genes led us to examine the expression and activity of CRM and TCP-1delta related genes. Indeed, we found a significant induction of mRNAs of the secreted collapsin-1 and the mitochondrial stress protein HSP60. Antibodies directed against collapsin-1 provided marked and prolonged protection of several neuronal cell types from dopamine-induced apoptosis. In a parallel study, using antisense technology, we found that inhibition of TCP-1delta expression significantly reduced DA-induced neuronal death. These findings suggest a functional role for collapsin-1 and TCP-1delta as positive mediators of DA-induced neuronal apoptosis.

Levodopa – an exotoxin or a therapeutic drug?

Abstract Auto-oxidation of levodopa generates toxic metabolites, such as free radicals, semiquinones and quinones. In vitro, levodopa is a powerful toxin that is lethal to cultures of neurones. This raises the concern that levodopa may also be toxic in vivo, and that chronic treatment with levodopa could induce further damage to nigrostriatal neurones in patients with Parkinson’s disease, accelerating the natural predetermined rate of disease progression. Although a few animal studies have shown that chronic levodopa may be toxic in vivo, most others report that it is not. The few available clinical studies also indicate that the course of Parkinson’s disease is not accelerated by chronic systemic treatment with levodopa.

Induction of mitosis-related genes during dopamine-triggered apoptosis in sympathetic neurons

It was suggested that neuronal degeneration in Parkinsons Disease (PD) is linked to dopamine (DA) toxicity. Dopamine has been shown to induce programmed cell death in both neuronal and non-neuronal cell types. We examined the molecular changes associated with dopamine-triggered apoptosis in sympathetic neurons using the differential display approach, and isolated 14 different DA responsive genes whose expression is altered during the early stages of the apoptotic process. Nine of these genes are upregulated and five are downregulated in response to DA exposure. Two of the upregulated genes were identified as cyclin B2 and a chicken homologue of chaperonin, a member of the heat shock protein family. Total increase in mRNA expression of both genes after 12 hours of exposure to DA was 40%. These two genes participate in cell cycle control and are specifically involved in determining entry of dividing cells into mitosis. Upregulation of mitosis-related genes in postmitotic sympathetic neurons undergoing apoptosis, may be indicative of an abortive attempt of these neurons to re-enter the cell cycle prior to their death. Possible implications to neuronal degeneration in PD are discussed.

Modulation of control mechanisms of dopamine-induced apoptosis - a future approach to the treatment of Parkinson's disease?

The cause for the progressive and selective degeneration of the dopaminergic (DA) nigrostriatal neurons in Parkinsons disease (PD) is still unknown. We suggest a novel approach, that links this neuronal degenerative process to inappropriate triggering of apoptosis, an active, controlled program of cellular self destruction, by excess oxidative stress mediated by DA metabolism. In support of this concept, we found that DA, the endogenous neurotransmitter, is capable of initiating apoptosis in cultured, postmitotic chick sympathetic neurons, an observation further extended to other cellular systems (PC-12 cells, cerebellar granular cells, thymocytes, splenocytes). In comparing the relative apoptosis-triggering potency of other mononamine neurotransmitters, DA was found to be the most active, whereas norepinephrine and serotonin had a moderate and a mild effects, respectively. This grading can be correlated with the relative involvement of the relevant neuronal systems (i.e., substantia nigra, locus ceruleus and raphe nuclei) in PD. We therefore hypothesize that neuronal degeneration in PD may be caused, at least in part, by a failure, either inherited or acquired, in cellular control systems of apoptosis, that may normally restrain the lethal potential of these endogenous neuro-transmitters and their potentially-toxic oxidation products. We therefore point at apoptosis-control systems as a critical scene of events, where the fate of nigrostriatal neurons is ultimately determined, and whose modulation may yield attenuation of the neuronal degenerative process. In support of this concept, we found that vector-driven stable expression of the proto-oncogene bcl-2, an inhibitor of apoptosis, can exert powerful cellular protection against DA toxicity in rat pheochromocytoma PC-12 cells. Furthermore, cell extracts from bcl-2-expressing cells were found to markedly inhibit in vitro oxidation of DA and production of DA-melanin. We also found that expression of bcl-2 can inhibit the decrease in intracellular reduced thiol (-SH) groups which we observed following exposure to DA. Research of the bcl-2 system and associated control mechanisms of apoptosis, possibly acting in association with intra-cellular anti-oxidant pathways, may therefore lead to novel therapeutic approaches for neuroprotection in PD.