In Parkinson's disease(PD), conformational changes in the alpha-synuclein monomer precede formation of Lewy bodies. An examination of postmortem PD substantia nigra for evidence of pathological crosslinking of alpha-synuclein by tissue transglutaminase(tTG) suggested that both tTG and its substrate-characteristic N(epsilon)-(gamma-glutamyl)-lysine crosslink are increased in dopamine neurons. Unexpectedly, the isodipeptide crosslink was detected in the alpha-synuclein monomer as well as in higher molecular mass oligomers of alpha-synuclein. Crosslinked alpha-synuclein, formed at the expense of the total alpha-synuclein monomer, correlated with disease progression. The results indicate that most alpha-synuclein monomer in PD nigra is crosslinked by tTG and thus presumably functionally impaired. Related laboratory studies completed during the past year found that the differential vulnerability of striatal medium spiny neurons to excitotoxic injury correlates with basal levels of Bcl-2 and that prostaglandin PGA-1 protects these neurons against ischemic damage by inducing heat shock proteins. Clinically, as a prelude to Branch neuroprotective trials using a novel asset-sparing design, surrogate biomarker studies have been initiated. High priority pharmaceutical candidates are first evaluated for their ability, at safe and tolerable doses, to achieve CNS levels in the predicted therapeutic range and affect markers of relevant pharmacologic mechanisms using spinal fluid sampling and brain imaging techniques. For exmple, the ability of lithium and valproic acid to reduce GSK-3 mediated tau phosphorylation at epitopes implicated in the pathogenesis of the neurodegenerative process and of drugs such as memantine to normalize diminished brain BDNF are being studied in patients with Alzheimer?s and Huntington?s deseases. An assessment of the safety and initial neuroprotective efficacy of the chronic intralenticular infusion of recombinant-methionyl human glial cell line-derived neurotrophic factor(r-metHuGDNF)has been completed in one patient with progressive supranuclear palsy (PSP). She received GDNF under double-blind placebo-controlled conditions for 28 months at doses up to 50 ?g/day. After 22 months at peak dose, the patient succumbed to natural disease progression. There was no clinical, laboratory or radiological evidence of adverse events attributable to trophic factor administration. The rate of timed upper extremity motor task deterioration was substantially slowed, especially contralateral to the GDNF treated lenticular nucleus. These results suggest that chronic intraparenchymal infusion of GDNF at dose up to 50 ?g/day is safe and may delay symptom progression in PSP. Preclinical studies in rodent and primate models of PD completed during the past year have provided crucial new information regarding the palliative treatment of this disorder. One investigation evaluated a new approach to continuous dopaminomimetic delivery by subcutaneous polymeric implant and thus the Branch hypothesis concerning the deleterious consequences of the nonphysiologic stimulation of striatal dopaminergic receptors. As predicted, continuous stimulation of these receptors by the dopamine agonist apomorphine indefinitely postponed dyskinesias onset in MPTP-lesioned, levodopa-naive monkeys. A clinical evaluation of this strategy is now being implemented. Another study compared the individual and combined contribution of glutamatergic AMPA and NMDA receptor-mediated mechanisms in striatal spiny neurons to the pathogenesis of motor dysfunction attending dopaminergic treatment of PD. In both rodent and primate models, the reduction in levodopa-induced dyskinesia achieved by combined blockade of these ionotropic glutamatergic receptors far exceeded that obtained by blocking either receptor alone, suggesting that signaling from both receptors independently contributes to the pathogenesis of the mootor ccccomplication syndrome. Differential effects on D1 and D2 agonist-induced dyskinesias observed in this study support basal ganglia models in which D1 bearing spiny neurons giving rise to the direct striatonigral pathway express mainly AMPA receptors, while D2 bearing neurons projecting via the indirect striatopallidal pathway express mainly NMDA receptors. Clinical assessment of the potential benefits of combined NMDA and AMPA antagonist treatment is about to begin. A related study sought to determine whether activation of NR2B containing NMDA receptors contributes to the appearance of levodopa-induced response changes. The effects of selective NR2B antagonism (CP-101,606) on these response alterations in 6-hydroxydopamine lesioned rats indicated that activation of NR2B subunit containing NMDA receptors does participate in the development and maintenance of these motor response alterations through a mechanism involving increased phosphorylation of AMPA GluR1 receptor subunits. Other research, now in progress, has identified specific protein kinase C isoform changes occurring in striatal spiny neurons associated with parkinsonism and motor complications and discovered an effective pharmacologic approach to the normalization of these alterations. Improving the translation of recent findings from basic laboratory research to better therapies for neurologic disease constitutes a major challenge for the neurosciences as well as a critical goal for Branch research. ETB contributions to the development of adenosine A2A antagonist treatment for PD illustrate approaches to some of the relevant issues. Adenosine A2A receptors on striatal spiny neurons signal via kinases whose aberrant activation has been linked in earlier Branch studies to the appearance of parkinsonian signs after dopaminergic denervation and to the motor response complications associated with dopaminomimetic therapy. Our discovery of the ability of A2A receptor blockade to normalize certain of these kinases and thus benefit motor dysfunction in rodent and primate models rapidly guided the design of a clinical study using a highly selective antagonist (KW-6002) at these receptors in parkinsonian patients. The results confirmed that drugs capable of blocking. A2A receptors confer therapeutic benefit to parkinsonian patients and exemplify a strategy for successfully bridging a novel approach to PD therapy from an evolving research concept to pivotal clinical trials. Related investigator-initiated, proof-of-concept clinical studies completed during past year involving non-dopaminergic treatments that target selected striatal transmitter receptors also made important advances relevant to the treatment of PD. One trial,based on earlier Branch laboratory findings, evaluated the acute effects of serotonin 5HT1A autoreceptor stimulation on levodopa-induced motor response complications. Administration of the selective 5HT1A agonist(EMD-128130) substantially reduced dyskinesias and prolonged the antiparkinsonian response to levodopa, suggesting that 5HT1A receptor stimulation in levodopa-treated parkinsonian patients can modulate striatal dopaminergic function and that 5HT1A agonists may be useful as adjuvants in the treatment of this disorder. Another study assessed the contribution of alpha2 adrenergic receptor mediated mechanisms to the pathogenesis of motor dysfunction. Coadministration of a selective antagonist at these receptors(JP-1730), based on previous ETB preclinical findings, was found to provide considerable therapeutic benefit to patients with motor complications. Yet another clinical study found that a drug acting to reduce central glutamate release(riluzole)failed to ameliorate dyskinesias or other response complications in PD patients, suggesting that down regulation of glutamatergic mechanisms at metabotropic receptors or at non-striatal sites contributes to the pathogenesis of these disabling adverse effects
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