Lalitha Madhavan

Apoptosis and glutamate-mediated excitotoxicity may play a role in the pathogenesis of many neurodegenerative disorders, including Parkinson's disease (PD). In the present study, we investigated whether stimulation of the 5-hydroxytryptamine 1A (5-HT1A) receptor attenuates N-methyl-D-aspartate- (NMDA) and 1-methyl-4-phenylpyridinium (MPP(+))-induced apoptotic cell death in cell culture models. A brief exposure (20 min) of M213-2O striatal cells to NMDA and glutamate produced a delayed increase in caspase-3 activity and DNA fragmentation in a dose- and time-dependent manner. NMDA-induced caspase-3 activity and DNA fragmentation were almost completely blocked by the 5-HT1A agonists 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) and (R)-5-fluoro-8 hydroxy-2-(dipropylamino)-tetralin (R-UH-301). Additionally, the protective effects of 8-OH-DPAT and R-UH-301 on NMDA-induced caspase-3 activation and apoptosis were reversed by pretreatment with the 5-HT1A antagonists N-[2-[4-(2-methoxyphenyl)-1-piperazinyl] ethyl]-N-(2-pyridinyl) cyclohexane carboxamide (WAY 100635) and S-UH-301, respectively. Similarly, dose- and time-dependent increases in caspase-3 activity and DNA fragmentation were observed in rat primary mesencephalic neurons after a brief exposure to NMDA and glutamate. Caspase-3 activation and DNA fragmentation in primary mesencephalic neurons were almost completely inhibited by 8-OH-DPAT. This neuroprotective effect of 8-OH-DPAT was reversed by WAY 100635. Additionally, 8-OH-DPAT blocked tyrosine hydroxylase (TH)-positive cell death after NMDA exposure and also almost completely attenuated the NMDA-induced Ca(2+) influx in primary mesencephalic cultures. Furthermore, 8-OH-DPAT and R-UH-301 blocked apoptotic cell death in the primary mesencephalic neurons that were exposed to the Parkinsonian toxin MPP(+). Together, these results suggest that 5-HT1A receptor stimulation may be a promising pharmacological approach in the development of neuroprotective agents for PD.

In addition to alleviating depression, trophic responses produced by antidepressants may regulate neural plasticity in the diseased brain, which not only provides symptomatic benefit but also potentially slows the rate of disease progression in Parkinson's disease (PD). Recent in vitro and in vivo data provide evidence that neurotrophic factors such as brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) may be key mediators of the therapeutic response to antidepressants. As such, we conducted a cross-sectional time-course study to determine whether antidepressant-mediated changes in neurotrophic factors occur in relevant brain regions in response to amitriptyline (AMI) treatment before and after intrastriatal 6-hydroxydopamine (6OHDA). Adult male Wistar rats were divided into seven cohorts and given daily injections (i.p.) of AMI (5mg/kg) or saline throughout the duration of the study. In parallel, various cohorts of intact or parkinsonian animals were sacrificed at specific time points to determine the impact of AMI treatment on trophic factor levels in the intact and degenerating nigrostriatal system. The left and right hemispheres of the substantia nigra, striatum, frontal cortex, piriform cortex, hippocampus, and anterior cingulate cortex were dissected, and BDNF and GDNF levels were measured with ELISA. Results show that chronic AMI treatment elicits effects in multiple brain regions and differentially regulates levels of BDNF and GDNF depending on the region. Additionally, AMI halts the progressive degeneration of dopamine (DA) neurons elicited by an intrastriatal 6-OHDA lesion. Taken together, these results suggest that AMI treatment elicits significant trophic changes important to DA neuron survival within both the intact and degenerating nigrostriatal system.

Neurotrophic factors are integrally involved in the development of the nigrostriatal system and in combination with gene therapy, possess great therapeutic potential for Parkinson's disease (PD). Pleiotrophin (PTN) is involved in the development, maintenance, and repair of the nigrostriatal dopamine (DA) system. The present study examined the ability of striatal PTN overexpression, delivered via psueudotyped recombinant adeno-associated virus type 2/1 (rAAV2/1), to provide neuroprotection and functional restoration from 6-hydroxydopamine (6-OHDA). Striatal PTN overexpression led to significant neuroprotection of tyrosine hydroxylase immunoreactive (THir) neurons in the substantia nigra pars compacta (SNpc) and THir neurite density in the striatum, with long-term PTN overexpression producing recovery from 6-OHDA-induced deficits in contralateral forelimb use. Transduced striatal PTN levels were increased threefold compared to adult striatal PTN expression and approximated peak endogenous developmental levels (P1). rAAV2/1 vector exclusively transduced neurons within the striatum and SNpc with approximately half the total striatal volume routinely transduced using our injection parameters. Our results indicate that striatal PTN overexpression can provide neuroprotection for the 6-OHDA lesioned nigrostriatal system based upon morphological and functional measures and that striatal PTN levels similar in magnitude to those expressed in the striatum during development are sufficient to provide neuroprotection from Parkinsonian insult.

Multiple laboratories have recently demonstrated that long-term dopaminergic transplants form Lewy bodies in patients with Parkinson's disease. Debate has arisen as to whether these Lewy bodies form from the transfer of α synuclein from the host to the graft or whether they form from intrinsic responses of the graft from being placed into what was, or became, an inflammatory focus. To test whether the former hypothesis was possible, we grafted fetal rat ventral mesencephalon into the dopamine depleted striatum of rats that had previously received 6-hydroxydopamine lesions. One month after the transplant, rats received viral over expression of human α synuclein (AAV2/6-α synuclein) or green fluorescent protein (AAV2/6-GFP) into the striatum rostral to the grafts. Care was taken to make sure that the AAV injections were sufficiently distal to the graft so no cells would be directly transfected. All rats were sacrificed five weeks after the virus injections. Double label immunohistochemistry combined with confocal microscopy revealed that a small number of grafted tyrosine hydroxylase (TH) neurons (5.7% ± 1.5% (mean ± SEM) of grafted dopamine cells) expressed host derived α synuclein but none of the grafted cells expressed host-derived GFP. The α synuclein in a few of these cells was misfolded and failed to be digested with proteinase K. These data indicate that it is possible for host derived α synuclein to transfer to grafted neurons supporting the concept that this is one possible mechanism by which grafted dopamine neurons form Lewy bodies in Parkinson's disease patients.

Recent studies indicate that transplanted neural stem/progenitor cells (NSPs) can interact with the environment of the central nervous system and stimulate protection and regeneration of host cells exposed to oxidative stress. Here, a set of animals grafted with NSPs and treated with 3-nitropropionic acid (3-NP) exhibited reduced behavioral symptoms and less severe damage of striatal cytoarchitecture than sham transplanted controls including better survival of neurons. Sites of tissue sparing correlated with the distribution pattern of donor cells in the host brain. To investigate the cellular and molecular bases of this phenomenon, we treated cocultures of NSPs and primary neural cell cultures with 3-NP to induce oxidative stress and to study NSP-dependent activation of antioxidant mechanisms and cell survival. Proactive presence of NSPs significantly improved cell viability by interfering with production of free radicals and increasing the expression of neuroprotective factors. This process was accompanied by elevated expression of ciliary neurotrophic factor (CNTF) and vascular endothelial growth factor (VEGF) in a network of NSPs and local astrocytes. Intriguingly, both in vitro and in vivo, enhanced growth factor secretion stimulated a robust upregulation of the antioxidant enzyme superoxide dismutase 2 (SOD2) in neurons and resulted in their improved survival. Our findings thus reveal a so far unrecognized mechanism of interaction between NSPs and surrounding cells accompanying neuroprotection: through mutual, NSP-triggered stimulation of growth factor production and activation of antioxidant mechanisms, cellular networks may shield the local environment from the arriving impact of oxidative stress.