Michelle Smeyne

A Long-Term Depression of AMPA Currents in Cultured Cerebellar Purkinje Neurons

Cerebellar long-term depression (LTD) is a model of synaptic plasticity in which conjunctive stimulation of parallel fiber and climbing fiber inputs to a Purkinje neuron induces a persistent depression of the parallel fiber-Purkinje neuron synapse. We report that an analogous phenomenon may be elicited in the cultured mouse Purkinje neuron when iontophoretic glutamate application and depolarization of the Purkinje neurons are substituted for parallel fiber and climbing fiber stimulation, respectively. The induction of LTD in these cerebellar cultures requires activation of both ionotropic (AMPA) and metabotropic quisqualate receptors, together with depolarization in the presence of external Ca2+. This postsynaptic alteration is manifest as a depression of glutamate or AMPA currents, but not aspartate or NMDA currents. These results strengthen the contention that the expression of cerebellar LTD is at least in part postsynaptic and provide evidence that activation of both ionotropic and metabotropic quisqualate receptors are necessary for LTD induction.

Glutathione metabolism and Parkinson's disease

It has been established that oxidative stress, defined as the condition in which the sum of free radicals in a cell exceeds the antioxidant capacity of the cell, contributes to the pathogenesis of Parkinson disease. Glutathione is a ubiquitous thiol tripeptide that acts alone or in concert with enzymes within cells to reduce superoxide radicals, hydroxyl radicals, and peroxynitrites. In this review, we examine the synthesis, metabolism, and functional interactions of glutathione and discuss how these relate to the protection of dopaminergic neurons from oxidative damage and its therapeutic potential in Parkinson disease.

Induction of cerebellar long-term depression in culture requires postsynaptic action of Sodium Ions

Cerebellar long-term depression (LTD) is a persistent attenuation of the parallel fiber-Purkinje neuron (PF-PN) synapse induced by conjunctive stimulation of PF and climbing fiber (CF) inputs. A similar phenomenon is seen in the voltage-clamped PN in tissue culture when iontophoretic quisqualate application and PN depolarization are substituted for PF and CF stimulation, respectively. In this model, LTD induction requires activation of both AMPA and metabotropic receptors, together with PN depolarization. We have sought to determine the role of the AMPA receptor in LTD induction. The AMPA receptor does not appear to exert its effect by directly gating Ca2+ influx. Replacement of external Na+ during quisqualate/depolarization conjunction with permeant ions caused a blockade of LTD induction, suggesting that Na+ influx through the AMPA-associated channel is necessary for this process. Similarly, pairing quisqualate pulses with depolarizing steps near ENa also failed to induce LTD. The present results indicate that postsynaptic Na+ influx is necessary for LTD induction. While a portion of the relevant Na+ influx is provided by voltage-gated channels, the AMPA-associated ion channel is most important in this regard.

GSTπ expression mediates dopaminergic neuron sensitivity in experimental parkinsonism

The cause of 95% of Parkinsons disease (PD) cases is unknown. It is hypothesized that PD arises from an interaction of free-radical-generating agents with an underlying genetic susceptibility to these compounds. Here we use the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of parkinsonism to examine the role of a dual function protein, GSTπ, in dopaminergic neuron death. GSTπ is the only GST family member expressed in substantia nigra neurons. GSTπ reduction by pharmacological blockade, RNA inhibition, and gene targeting increases sensitivity to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, suggesting that differential expression of GSTπ contributes to the sensitivity to xenobiotics in the substantia nigra and may influence the pathogenesis of reactive oxygen species-induced neurological disorders including PD.

Strain-dependent susceptibility to MPTP and MPP+-induced Parkinsonism is determined by glia

Parkinsons disease (PD) is a debilitating neurological disorder that strikes approximately 2% of people over age 50. Current hypotheses propose that the cause of PD is multifactorial, involving environmental agents and genetic predisposition. 1‐Methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) induces parkinsonism in many species, including humans and shows strain specificity in mice. The mechanism of strain specificity, however, remains unknown. Using novel chimeric murine substantia nigra cultures, we demonstrate that sensitivity to MPTP is conferred by glia and that it does not involve the MAO‐B conversion of MPTP to MPP+. C57Bl/6J dopaminergic neurons exposed to MPP+ demonstrated a 39% loss when cultured on C57Bl/6J glia compared with 17% neuron loss when cultured on resistant SWR/J glia. Similarly, SWR/J neurons exposed to MPP+ demonstrated a 4% loss when cultured on SWR/J glia, but a 14% loss when cultured on sensitive C57Bl/6J glia. The identification of glia as the critical cell type in the genesis of experimental Parkinsonism provides a target for the development of new anti‐parkinsonian therapies. GLIA 34:73–80, 2001.

Glia cell number modulates sensitivity to MPTP in mice

Free radical damage has been shown to play a significant role in the pathogenesis of a number of neurodegenerative diseases including Parkinsons disease. One model of experimental parkinsonism is the loss of substantia nigra cells following administration of MPTP. Previously, it has been shown that a number of inbred strains of mice have differential responses to this toxin, and this difference is dependent on glial cells. In this study, the number of glial cells in the substantia nigra pars compacta of C57Bl/6J (MPTP‐sensitive) and Swiss Webster (MPTP‐resistant) strains of mice was examined. The C57Bl/6J mice have an approximately 50% lower number of GFAP+ and S‐100β glial cells than the Swiss Webster mice. C57Bl/6J mice have a 25% increased number of resident nonactivated microglial cells. To determine whether this difference in cell number has functional significance, we used an in vitro SN culture system that allowed us to manipulate the number of glial cells. When C57Bl/6 neurons were grown on a glial mat plated with twice the number of cells, we were able to rescue the MPTP‐sensitive neurons from toxin‐induced cell death. This suggests that the number of glial cells in the SNpc may be an important factor in the survival of dopaminergic neurons following exposure to xenobiotics.

Inflammatory Effects of Highly Pathogenic H5N1 Influenza Virus Infection in the CNS of Mice

The A/VN/1203/04 strain of the H5N1 influenza virus is capable of infecting the CNS of mice and inducing a number of neurodegenerative pathologies. Here, we examined the effects of H5N1 on several pathological aspects affected in parkinsonism, including loss of the phenotype of dopaminergic neurons located in the substantia nigra pars compacta (SNpc), expression of monoamines and indolamines in brain, alterations in SNpc microglia number and morphology, and expression of cytokines, chemokines, and growth factors. We find that H5N1 induces a transient loss of the dopaminergic phenotype in SNpc and now report that this loss recovers by 90 d after infection. A similar pattern of loss and recovery was seen in monoamine levels of the basal ganglia. The inflammatory response in lung and different regions of the brain known to be targets of the H5N1 virus (brainstem, substantia nigra, striatum, and cortex) were examined at 3, 10, 21, 60, and 90 d after infection. In each of these brain regions, we found a significant increase in the number of activated microglia that lasted at least 90 d. We also quantified expression of IL-1α, IL-1β, IL-2, IL-6, IL-9, IL-10, IL-12(p70), IL-13, TNF-α, IFN-γ, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, macrophage colony-stimulating factor, eotaxin, interferon-inducible protein 10, cytokine-induced neutrophil chemoattractant, monocyte chemotactic protein-1, macrophage inflammatory protein (MIP) 1α, MIP-1β, and VEGF, and found that the pattern and levels of expression are dependent on both brain region and time after infection. We conclude that H5N1 infection in mice induces a long-lasting inflammatory response in brain and may play a contributing factor in the development of pathologies in neurodegenerative disorders.

Method for culturing postnatal substantia nigra as an in vitro model of experimental Parkinson's disease.

One of the most prevalent degenerative disorders of the nervous system is Parkinsons disease. The etiology of this disease is for the most part unknown, although it is posited to arise from an interaction of genetic and environmental factors. Although in vivo animal studies have been used to examine the effects of a number of Parkinson-inducing compounds, there is little information on reliable in vitro methodologies that can recapitulate the previously observed in vivo results. Here, we describe a method for generating mixed and chimeric neuron/glial cultures of postnatal substantia nigra (SN), independent of other monoaminergic nuclei in the ventral midbrain. Since many toxins do not affect regions of the midbrain except the SN, use of whole ventral midbrain from embryos can dilute any measurement of cell death. By specifically culturing ventrolateral midbrain containing the substantia nigra, one can more directly target the effects of dopaminergic toxins. In addition, this method can be used to test potential therapies for amelioration of Parkinsons disease.

HIF1α is necessary for exercise-induced neuroprotection while HIF2α is needed for dopaminergic neuron survival in the substantia nigra pars compacta.

Exercise reduces the risk of developing a number of neurological disorders and increases the efficiency of cellular energy production. However, overly strenuous exercise produces oxidative stress. Proper oxygenation is crucial for the health of all tissues, and tight regulation of cellular oxygen is critical to balance O2 levels and redox homeostasis in the brain. Hypoxia Inducible Factor (HIF)1α and HIF2α are transcription factors regulated by cellular oxygen concentration that initiate gene regulation of vascular development, redox homeostasis, and cell cycle control. HIF1α and HIF2α contribute to important adaptive mechanisms that occur when oxygen and ROS homeostasis become unbalanced. It has been shown that preconditioning by exposure to a stressor prior to a hypoxic event reduces damage that would otherwise occur. Previously we reported that 3 months of exercise protects SNpc dopaminergic (DA) neurons from toxicity caused by Complex I inhibition. Here, we identify the cells in the SNpc that express HIF1α and HIF2α and show that running exercise produces hypoxia in SNpc DA neurons, and alters the expression of HIF1α and HIF2α. In mice carrying a conditional knockout of Hif1α in postnatal neurons we observe that exercise alone produces SNpc TH+ DA neuron loss. Loss of HIF1α also abolishes exercise-induced neuroprotection. In mice lacking Hif2α in postnatal neurons, the number of TH+ DA neurons in the adult SNpc is diminished, but 3months of exercise rescues this loss. We conclude that HIF1α is necessary for exercise-induced neuroprotection and both HIF1α and HIF2α are necessary for the survival and function of adult SNpc DA neurons.

CHAPTER 6. Glutathione and Thiol Redox Signalling in Parkinson’s Disease

Glutathione (GSH) and thiol redox signalling are at the most basic level of cellular redox reactions and responses to oxidative stress. Oxidative stress plays a major role in the etiology of Parkinson’s disease. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) that cause this stress may be generated by a number of factors, including byproducts generated by oxidative phosphorylation in the mitochondrial production of ATP, exposure to toxic environmental agents, inflammation and secondary effects of genetic mutations. The known characteristics of dopaminergic (DA) neurons combined with the permissive cytoarchitecture of the substantia nigra pars compacta (SNpc) provide an environment that makes these cells particularly vulnerable in the event of oxidative stress. The antioxidant tripeptide GSH, cysteine residues, and the thiol redox reactions in which they participate, function to reduce oxidative stress and protect cells from their sequelae. In this chapter, we discuss the factors that contribute to oxidative stress in the SNpc and Parkinson’s disease, as well as mechanisms by which GSH synthesis, thiol-disulfide exchange and thiol-modulated enzymes can modulate gene transcription during oxidative stress.