Karen L. O'Malley

Metabotropic Glutamate Receptor 5 Is a Disulfide-linked Dimer

The sequences of the metabotropic glutamate receptors (mGluRs) show little homology with other members of the G protein-coupled receptor family and exhibit several distinctive features, including a large N-terminal extracellular domain with 17 cysteines in conserved positions. Here we demonstrate that mGluR5, as well as other mGluRs, behave as species approximately twice as large as expected from their sequence, but reducing conditions cause a decrease to the predicted molecular mass. Co-immunoprecipitation experiments using wild type and epitope-tagged receptors demonstrate that this is due to specific, disulfide-dependent dimerization of the receptor. The intermolecular disulfide that mediates dimerization occurs in the extracellular domain, within about 17 kDa from the N terminus.

Long-term behavioral recovery in Parkinsonian rats by an HSV vector expressing tyrosine hydroxylase

One therapeutic approach to treating Parkinsons disease is to convert endogenous striatal cells into levo-3,4-dihydroxyphenylalanine (L-dopa)-producing cells. A defective herpes simplex virus type 1 vector expressing human tyrosine hydroxylase was delivered into the partially denervated striatum of 6-hydroxydopamine-lesioned rats, used as a model of Parkinsons disease. Efficient behavioral and biochemical recovery was maintained for 1 year after gene transfer. Biochemical recovery included increases in both striatal tyrosine hydroxylase enzyme activity and in extracellular dopamine concentrations. Persistence of human tyrosine hydroxylase was revealed by expression of RNA and immunoreactivity.

Two distinct mechanisms are involved in 6-hydroxydopamine- and MPP+- induced dopaminergic neuronal cell death: Role of caspases, ROS, and JNK

In this study, we examined the possibility that MPTP and 6‐hydroxydopamine (6‐OHDA) act on distinct cell death pathways in a murine dopaminergic neuronal cell line, MN9D. First, we found that cells treated with 6‐OHDA accompanied ultrastructural changes typical of apoptosis, whereas MPP+ treatment induced necrotic manifestations. Proteolytic cleavage of poly(ADP‐ribose)polymerase by caspase was induced by 6‐OHDA, whereas it remained uncleaved up to 32 h after MPP+ treatment and subsequently disappeared. Accordingly, 6‐OHDA‐ but not MPP+‐induced cell death was significantly attenuated in the presence of a broad‐spectrum caspase inhibitor, N‐benzyloxy‐carbonyl‐Val‐Ala‐Asp‐fluomethylketone (Z‐VAD‐fmk). As measured by fluorometric probes, the level of reactive oxygen species (ROS) significantly increased after 6‐OHDA treatment. In contrast, the level of dihydroethidium‐sensitive ROS following MPP+ treatment remained unchanged while a slight increase in dichlorofluorescin‐sentive ROS was temporarily observed. As demonstrated by immunoblot analysis, the level of superoxide dismutase was down‐regulated following 6‐OHDA treatment, whereas it remained unchanged after MPP+ treatment. Cotreatment of cells with antioxidants such as N‐acetylcysteine or Mn(III)tetrakis(4‐benzoic acid)porphyrin chloride (MnTBAP, cell‐permeable superoxide dismutase mimetic) rescued 6‐OHDA‐ but not MPP+‐induced cell death, whereas inclusion of catalase or NG‐nitro‐l‐arginine had no effect in both cases. In addition, 6‐OHDA induced ROS‐mediated c‐Jun N‐terminal kinase (JNK) activation that was attenuated in the presence of N‐acetylcysteine or MnTBAP but not catalase or Z‐VAD‐fmk. In contrast, MPP+ has little effect on JNK activity, indicating that ROS and/or ROS‐induced cell death signaling pathway seems to play an essential role in 6‐OHDA–mediated apoptosis but not in MPP+‐induced necrosis in a mesencephalon‐derived, dopaminergic neuronal cell line. J. Neurosci. Res. 57:86–94, 1999.

Axon Degeneration in Parkinson's Disease

Parkinsons disease (PD) is the most common neurodegenerative disease of the basal ganglia. Like other adult-onset neurodegenerative disorders, it is without a treatment that forestalls its chronic progression. Efforts to develop disease-modifying therapies to date have largely focused on the prevention of degeneration of the neuron soma, with the tacit assumption that such approaches will forestall axon degeneration as well. We herein propose that future efforts to develop neuroprotection for PD may benefit from a shift in focus to the distinct mechanisms that underlie axon degeneration. We review evidence from human post-mortem studies, functional neuroimaging, genetic causes of the disease and neurotoxin models that axon degeneration may be the earliest feature of the disease, and it may therefore be the most appropriate target for early intervention. In addition, we present evidence that the molecular mechanisms of degeneration of axons are separate and distinct from those of neuron soma. Progress is being made in understanding these mechanisms, and they provide possible new targets for therapeutic intervention. We also suggest that the potential for axon re-growth in the adult central nervous system has perhaps been underestimated, and it offers new avenues for neurorestoration. In conclusion, we propose that a new focus on the neurobiology of axons, their molecular pathways of degeneration and growth, will offer novel opportunities for neuroprotection and restoration in the treatment of PD and other neurodegenerative diseases.

Enhanced early developmental expression of the metabotropic glutamate receptor mGluR5 in rat brain: Protein, mRNA splice variants, and regional distribution

Glutamate stimulates phosphatidyl inositol hydrolysis and mobilizes intracellular calcium through the mediation of metabotropic glutamate receptors (mGluRs), in particular the “Group I” receptors mGluRs, mGluR1, and mGluR5. This activity is markedly enhanced in developing brain relative to the adult. To determine whether this may be due to an increased amount of mGluR5 present in the developing brain, we examined mGluR5 expression using western blotting to measure mGluR5 protein, reverse transcription polymerase chain reaction (RT‐PCR) to measure mGluR5 mRNA, and immunocytochemistry to assess the regional distribution of mGluR5 morphologically. Western blotting revealed that in all brain regions examined there is more mGluR5 protein present in developing brain than in the adult. In most regions, the developmental decrease was over two‐fold. Total mGluR5 mRNA also decreased with development in most regions, but to a much lesser extent than the protein, suggesting that there is considerable post‐transcriptional regulation of the expression of this receptor. RT‐PCR analysis also demonstrated that in most regions the mGluR5a splice variant is most abundant in the young animals but mGluR5b predominates in the adult. Light microscopic immunocytochemistry indicated that expression is widespread in developing brain, and that the developmental decrease in receptor concentration is due to both an increased growth of receptor‐poor tissue regions and decreased expression within receptor‐rich regions.

Covalent and Noncovalent Interactions Mediate Metabotropic Glutamate Receptor mGlu5 Dimerization

Some, perhaps all, G protein-coupled receptors form homo- or heterodimers. We have shown that metabotropic glutamate receptors are covalent dimers, held together by one or more disulfide bonds near the N terminus. Here we report how mutating cysteines in this region affect dimerization and function. Covalent dimerization is preserved when cysteines 57, 93, or 99 are mutated but lost with replacement at 129. Coimmunoprecipitation under nondenaturing conditions indicates that the C[129]S mutant receptor remains a dimer, via noncovalent interactions. Both C[93]S and C[129]S bind [3H]quisqualate, whereas binding to C[57]S or C[99]S mutants is absent or greatly attenuated. The C[93]S and C[129]S receptors have activity similar to wild-type when assayed by fura-2 imaging of intracellular calcium in human embryonic kidney cells or electrophysiologically in Xenopus laevis oocytes. In contrast, C[57]S or C[99]S are less active in both assays but do respond with higher glutamate concentrations in the oocyte assay. These results demonstrate that 1) covalent dimerization is not critical for mGlu5 binding or function; 2) mGlu5 remains a noncovalent dimer even in the absence of covalent dimerization; and 3) high-affinity binding requires Cys-57 and Cys-99.

Developmental cell death in dopaminergic neurons of the substantia nigra of mice

Dopaminergic neurons in the substantia nigra pars compacta (SNpc) undergo natural cell death during development in rats. Controversy exists as to the occurrence of this phenomenon in SNpc dopaminergic neurons in the developing mouse. Herein, by using an array of morphologic techniques, we show that many SNpc neurons fulfill the criteria for apoptosis and that the number of apoptotic neurons in the SNpc vary in a time‐dependent manner from postnatal day 2 to 32. These dying neurons also show evidence of DNA fragmentation, of activated caspase‐3, and of cleavage of β‐actin. Some, but not all of the SNpc apoptotic neurons still express their phenotypic marker tyrosine hydroxylase, confirming their dopaminergic nature. Consistent with the importance of target‐derived trophic support in modulating developmental cell death, we demonstrate that destruction of intrinsic striatal neurons by a local injection of quinolinic acid (QA) dramatically enhances the magnitude of SNpc apoptosis and results in a lower number of adult SNpc dopaminergic neurons. Strengthening the apoptotic nature of the observed SNpc developmental cell death, we demonstrate that overexpression of the anti‐apoptotic protein Bcl‐2 attenuates both natural and QA‐induced SNpc apoptosis. The present study provides compelling evidence that developmental neuronal death with a morphology of apoptosis does occur in the SNpc of mice and that this process plays a critical role in regulating the adult number of dopaminergic neurons in the SNpc. J. Comp. Neurol. 424:476–488, 2000.

Oxidative stress-triggered unfolded protein response is upstream of intrinsic cell death evoked by parkinsonian mimetics

Oxidative stress is a key player in a variety of neurodegenerative disorders including Parkinsons disease. Widely used as a parkinsonian mimetic, 6‐hydroxydopamine (6‐OHDA) generates reactive oxygen species (ROS) as well as coordinated changes in gene transcription associated with the unfolded protein response (UPR) and apoptosis. Whether 6‐OHDA‐induced UPR activation is dependent on ROS has not yet been determined. The present study used molecular indicators of oxidative stress to place 6‐OHDA‐generated ROS upstream of the appearance of UPR markers such as activating transcription factor 3 (ATF3) and phosphorylated stress‐activated protein kinase (SAPK/JNK) signaling molecules. Antioxidants completely blocked 6‐OHDA‐mediated UPR activation and rescued cells from toxicity. Moreover, cytochrome c release from mitochondria was observed after the appearance of early UPR markers, suggesting that cellular stress pathways are responsible for its release. Mechanistically, the 6‐OHDA‐induced UPR was independent of intracellular calcium changes. Rather, evidence of protein oxidation was observed before the expression of UPR markers, suggesting that the rapid accumulation of damaged proteins triggered cell stress/UPR. Taken together, 6‐OHDA‐mediated cell death in dopaminergic cells proceeds via ROS‐dependent UPR up‐regulation which leads to an interaction with the intrinsic mitochondrial pathway and downstream caspase activation.

Characterization of the human dopamine D3 receptor expressed in transfected cell lines

A full-length cDNA clone of the human dopamine D3 receptor was obtained by the polymerase chain reaction (PCR) using reverse-transcribed RNA from human brain as the template. The cDNA was inserted into an expression vector which was then stably transfected into either Chinese hamster ovary (CHO), SK-N-MC human epithelioma or mouse CCL1.3 fibroblast cell lines. Post-transfection, the Bmax for D3 receptor expression was 1.9, 1.1 and 0.4 pmol/mg protein in the CHO-K1, SK-N-MC and CCL1.3 cell lines, respectively. The D3 receptor expressed in CHO-K1 and CCL1.3 cells exhibited similar radioligand binding profiles, especially for the D3-selective compound, 7-hydroxy-2-(di-n-propylamino)tetralin (7-OH-DPAT). Radioligand-binding competition curves of presumed D3 agonists were shifted to the right by the addition of guanine nucleotides and Na+ to the assay buffer. Presumed D3-receptor agonists had no effect on cAMP accumulation in any of the D3-transfected cell lines although cAMP accumulation was inhibited by dopamine D2 receptor activation in D2-transfected CHO and CCL1.3 cells and by activation of the exogenously expressed neuropeptide Y receptor in SK-N-MC cells. Also, D3 receptor activation neither potentiated ATP-stimulated arachidonic acid release from CHO cells nor stimulated inositol phosphate production in CCL1.3-cells although both of these responses were elicited by D2 agonists in D2-transfected cells. We conclude that the signalling properties of the D3 receptor differ from those of its closest homolog, the D2 receptor.

A Response Element for the Homeodomain Transcription Factor Ptx3 in the Tyrosine Hydroxylase Gene Promoter

Abstract: Tyrosine hydroxylase (TH) is the rate‐limiting enzyme in the biosynthesis of catecholamines, which takes place in different types of neuronal systems and nonneuronal tissues. The transcriptional regulation of the TH gene, which is complex and highly variable among different tissues, reflects this heterogeneity. We recently isolated a homeodomain transcription factor, named Ptx3, that is uniquely expressed in the dopaminergic neurons of the substantia nigra pars compacta and ventral tegmental area, which together form the mesencephalic dopaminergic system. This strict localization and its coinciding induction of expression with the TH gene during development suggested a possible role for this transcription factor in the control of the TH gene. We report here the presence of a responsive element for Ptx3 located at position ‐50 to ‐45 of the rat TH promoter. Transient transfections using TH promoter constructs and electrophoretic mobility shift assays using Ptx3‐containing nuclear extracts demonstrated that this region binds Ptx3 protein and confers a transcriptional effect on the TH gene. Depending on the cell type, the effect of Ptx3 was an eight‐ to 12‐fold enhancement of TH promoter activity in Neuro2A neuroblastoma cells, or a 60‐80% repression in nonneuronal human embryonic kidney 293 cells. Despite the close association of the Ptx3‐binding site and the major cyclic AMP‐response element in the TH gene, no interplay was found between Ptx3 and cyclic AMP‐modulating agents. In combination with the orphan nuclear receptor Nurr1, which is required for the induction of the TH gene in mesencephalic dopaminergic neurons, the TH promoter activity to Ptx3 was enhanced in Neuro2A cells. Nurr1 alone displayed only very weak activity on the TH promoter in this cell type. The results demonstrate that the homeodomain protein Ptx3 has the potential to act on the promoter of the TH gene in a markedly cell type‐dependent fashion. This suggests that Ptx3 contributes to the regulation of TH expression in mesencephalic dopaminergic neurons.