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L-dopa-induced dyskinesia (LID) is a common debilitating complication of dopamine replacement therapy in Parkinson's disease. Recent evidence suggests that LID may be linked causally to a hyperactivation of the Ras-ERK signaling cascade in the basal ganglia. We set out to determine whether specific targeting of Ras-guanine nucleotide-releasing factor 1 (Ras-GRF1), a brain-specific activator of the Ras-ERK pathway, may provide a therapy for LID. On the rodent abnormal involuntary movements scale, RasGRF1-deficient mice were significantly resistant to the development of dyskinesia during chronic L-dopa treatment. Furthermore, in a nonhuman primate model of LID, lentiviral vectors expressing dominant negative forms of Ras-GRF1 caused a dramatic reversion of dyskinesia severity leaving intact the therapeutic effect of L-dopa. These data reveal the central role of Ras-GRF1 in governing striatal adaptations to dopamine replacement therapy and validate a viable treatment for LID based on intracellular signaling modulation. doi: 10.10731pnas.1012071107

The inward L-dihydroxyphenylalanine (L-dopa) transport supplies renal proximal tubule cells (PTCs) with the precursor for dopamine synthesis. We have previously described insulin-induced stimulation of L-dopa uptake into PTCs. In the present paper we examined insulin-related signaling pathways involved in the increase of L-dopa transport into isolated rat PTCs. Insulin (50-500 [micro]U/ml) increased L-dopa uptake by PTCs, reaching the maximal increment (60% over the control) at 200 [micro]U/ml. At this concentration, insulin also increased insulin receptor tyrosine phosphorylation. Both effects were abrogated by the tyrosine kinase inhibitor genistein (5 [micro]M). In line, inhibition of the protein tyrosine phosphatase by pervanadate (0.2-100 [micro]M) caused a concentration-dependent increase in both the uptake of L-dopa (up to 400%) and protein tyrosine phosphorylation. A synergistic effect between pervanadate and insulin on L-dopa uptake was observed only when threshold (0.2 [micro]M), but not maximal (5 [micro]M), concentrations of pervanadate were assayed. Insulin-induced stimulation of L-dopa uptake was also abolished by inhibition of phosphatidylinositol 3-kinase (PI3K; 100 nM wortmannin, and 25 [micro]M LY-294002) and protein kinase C (PKC; 1 [micro]M RO-318220). Insulin-induced activation of PKC-[zeta] was confirmed in vitro by its translocation from the cytosol to the membrane fraction, and in vivo by immunohistochemistry studies. Insulin caused a wortmannin-sensitive increase in Akt/protein kinase B (Akt/PKB) phosphorylation and a dose-dependent translocation of Akt/PKB to the membrane fraction. Our findings suggest that insulin activates PKC-[zeta], and Akt/PKB downstream of PI3K, and that these pathways contribute to the insulin-induced increase of L-dopa uptake into PTCs. amino acid transport; second messengers; dopamine

Mussels adhere to a variety of surfaces by depositing a highly specific ensemble of 3,4-dihydroxyphenyl-L-alanine (DOPA) containing proteins. The adhesive properties of Mytilus edulis foot proteins mfp-1 and mfp-3 were directly measured at the nano-scale by using a surface forces apparatus (SFA). An adhesion energy of order W [approximately equal to] 3 x [10.sup.-4] J/[m.sup.2] was achieved when separating two smooth and chemically inert surfaces of mica (a common alumino-silicate clay mineral) bridged or 'glued' by mfp-3. This energy corresponds to an approximate force per plaque of [approximately equal to] 100 gm, more than enough to hold a mussel in place if no peeling occurs. In contrast, no adhesion was detected between mica surfaces bridged by mfp-1. AFM imaging and SFA experiments showed that mfp-1 can adhere well to one mica surface, but is unable to then link to another (unless sheared), even after prolonged contact time or increased load (pressure). Although mechanistic explanations for the different behaviors are not yet possible, the results are consistent with the apparent function of the proteins, i.e., mfp-1 is disposed as a 'protective' coating, and mfp-3 as the adhesive or 'glue' that binds mussels to surfaces. The results suggest that the adhesion on mica is due to weak physical interactions rather than chemical bonding, and that the strong adhesion forces of plaques arise as a consequence of their geometry (e.g., their inability to be peeled off) rather than a high intrinsic surface or adhesion energy, W. bioadhesion | Mytilus edulis

Mucosal changes in inflammatory bowel disease (IBD) are characterized by ulcerative lesions accompanied by a prominent infiltrate of inflammatory cells including lymphocytes, macrophages, and neutrophils and alterations in 5-hydroxytryptamine (5-HT)-producing enterochromaffin (EC) cells. Mechanisms involved in recruiting and activating these cells are thought to involve a complex interplay of inflammatory mediators. Studies in clinical and experimental IBD have shown the upregulation of various chemokines including monocyte chemottractant protein (MCP)-1 in mucosal tissues. However, precise information on the roles of this chemokine or the mechanisms by which it takes part in the pathogenesis of IBD are not clear. In this study, we investigated the role of MCP-1 in the development of hapten-induced experimental colitis in mice deficient in MCP-1. Our results showed a significant reduction in the severity of colitis both macroscopically and histologically along with a decrease in mortality in MCP-l-deficient mice compared with wild-type control mice. This was correlated with a downregulation of myeloperoxidase activity, IL-1[beta], IL-12p40, and IFN-[gamma]/production, and infiltration of CD[3.sup.+] T cells and macrophages in the colonic mucosa. In addition, we observed significantly lower numbers of 5-HT-expressing EC cells in the colon of MCP-1-deficient mice compared with those in wild-type mice after dinitrobenzenesulfonic acid. These results provide evidence for a critical role of MCP- 1 in the development of colonic inflammation in this model in the context of immune and enteric endocrine cells. monocyte chemottractant protein-1; dinitrobenzenesulfonic acid; CD3-positive cells; 5-hydroxytryptamine; serotonin

The long-term treatment of Parkinson's disease with L-dopa is often associated with the appearance of involuntary movements called L-dopa-induced dyskinesias. These debilitating side-effects are thought to result from an aberrant form of plasticity triggered by a combination of factors related to dopamine denervation and repeated L-dopa administration. In animal models of Parkinson's disease, dopamine denervation and repeated L-dopa administration are associated with an enhancement of opioid transmission in the basal ganglia. The exact role of this increased opioid activity is still under debate. It has been proposed that some of the changes in opioid transmission are directly involved in the genesis of L-dopa-induced dyskinesias. In this article, we suggest that changes in opioid transmission in the basal ganglia in response to denervation and repeated L-dopa therapy are, instead, part of compensatory mechanisms to prevent motor complications. Initially, these compensatory mechanisms might be sufficient to attenuate the parkinsonian syndrome and delay the appearance of involuntary movements. But with the progression of the disease and repeated exposure to L-dopa, these mechanisms eventually fail. These new insights could contribute to better understanding of the motor complications in Parkinson's disease and lead to the development or improvement of pharmacological strategies to prevent or reduce L-dopa-induced dyskinesias.

More than 50% of Parkinson's disease (PD) patients treated with L-dopa develop L-dopa-induced dyskinesias (LIDs) in the long term. Some patients exhibit severe dyskinesias soon after starting low doses of L-dopa, whereas other patients remain free of this disabling complication despite treatment with L-dopa. Avoiding or delaying the appearance of LIDs is one of the main objectives of the management of PD. Plasticity of the brain to adapt to a progressive disease, together with a non-physiological treatment strategy, might be the key physiopathological element that underlies LIDs. Neural plasticity varies among patients according to age and genetics. Thus, I propose that this variation explains the observed differences in the occurrence of LIDs in PD patients. Furthermore, I suggest that denervation and L-dopa treatment act as modulating and triggering factors of LIDs, respectively. In this article, the practical implications of these ideas and the role of pharmacogenetics in PD treatment are discussed. Treatment decisions are likely to rely on this information, challenging the relevance of current 'hot' debates about how to start treatment in PD.

The structure of a 3,4-dihydroxyphenyalanine(DOPA)-derived thiol self-assembled on a gold surface is investigated using X-ray photoelectron spectroscopy (XPS), infrared reflection-absorption spectroscopy (IRAS) and near-edge X-ray absorption fine structure spectroscopy (NEXAFS). Strong molecular binding of DOPA-PT on gold through the sulphur atom is achieved.

Historically, 3,4-dihydroxyphenylalanine (DOPA) has been considered to be an inert amino acid that alleviates the symptoms of Parkinson's disease by its conversion to dopamine via the enzyme aromatic L-amino acid decarboxylase. In contrast to this generally accepted idea, we propose that DOPA itself is a neurotransmitter and/or neuromodulator in addition to being a precursor of dopamine. Several criteria such as synthesis, metabolism, active transport, existence, physiological release, competitive antagonism and physiological or pharmacological responses must be satisfied before a compound is accepted as a neurotransmitter. Recent evidence suggests that DOPA fulfills these criteria in its involvement in baroreflex neurotransmission.

Intrastriatal delivery of the tyrosine hydroxylase gene by viral vectors is being explored as a tool for local delivery of L-dopa in animals with lesions of the nigrostriatal pathway. The functional effects reported using this approach have been disappointing, probably because the striatal L-dopa levels attained have been too low. In the present study, we have defined a critical threshold level of L-dopa, 1.5 pmol/mg of tissue, that has to be reached to induce any significant functional effects. Using new generation high-titer recombinant adeno-associated virus vectors, we show that levels of striatal L-dopa production exceeding this threshold can be obtained provided that tyrosine hydroxylase is coexpressed with the cofactor synthetic enzyme, GTP-cyclohydrolase-1. After striatal transduction with this combination of vectors, substantial functional improvement in both drug-induced and spontaneous behavior was observed in rats with either complete or partial 6-hydroxydopamine lesions of the nigrostriatal pathway. However, complete reversal of motor deficits occurred only in animals in which part of the striatal dopamine innervation was left intact. Spared nigrostriatal fibers thus may convert L-dopa to dopamine and store and release dopamine in a more physiologically relevant manner in the denervated striatum to mediate better striatal output-dependent motor function. We conclude that intrastriatal L-dopa delivery may be a viable strategy for treatment and control of adverse side effects associated with oral L-dopa therapy such as on-off fluctuations and drug-induced dyskinesias in patients with Parkinson's disease.

Byline: Ruth Djaldetti (1), Eldad Melamed (1) Keywords: Key words Parkinson's disease; Levodopa; Therapy Abstract: In the last decade there has been a surge of new therapeutic strategies for the treatment of Parkinson's disease along with a change of concepts about how the disease should be treated. The gold standard remains levodopa preparations, which have a rapid and dramatic symptomatic effect by replenishing the reduced dopamine levels in caudate and putamen nuclei. However, keeping in mind the complications that may emerge following long-term treatment, its initiation should possibly be delayed to the more advanced stages of the illness, especially in younger patients, in favour of dopamine agonists monotherapy. The adverse reactions that become prominent and disabling in late stages of the disease, i. e., dyskinesias, response fluctuations, and psychiatric side effects, can currently be managed by novel pharmacological as well as surgical strategies. Future therapies will focus on transplantation of dopaminergic embryonic tissue, gene therapy, and neuroprotective treatments. Author Affiliation: (1) Chairman, Department of Neurology, Rabin Medical Center, Beilinson Campus, Petah Tiqva 49100, Israel, and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel, Tel.: +972-3-937-6356, Fax: +972-3-922-3352, e-mail: emelamed@clalit.org.il, IL Article note: Received: 22 December 2000 / Accepted: 5 January 2001

Researchers have created a mutant form of the enzyme tyrosine phenol-lyase from Erwinia herbicola that does not require L-tyrosine. This bacterium is used to produce L-DOPA for the treatment of Parkinson's disease, but the wild enzyme requires tyrosine. The tyrosine must then be separated from L-DOPA.

Research was conducted to investigate the reaction of aromatic L-amino acid decarboxylase (AADC) with substrate dopa at various pH. Analysis of the reaction forming dopa-pyridoxal 5'-phosphate Schiff base indicates that dopa preferentially and directly binds to the unprotonated amino group of the enzyme.

Parkinson's disease (PD) is a progressive neurologic condition characterized by tremor, slowness, stiffness, and unstable posture. Degeneration of dopamine-producing neurons in the substantia nigra causes PD. Treatment with levodopa, a precursor of dopamine, initially ameliorates the clinical manifestations of PD. However, chronic levodopa treatment can produce severe involuntary movements (so-called dopa-induced dyskinesias or DID), limiting treatment. Pallidotomy, placement of a surgical lesion in the internal segment of the globus pallidus, reduces DID. Because this result is inconsistent with current theories of both basal ganglia function and DID, it prompted us to investigate the brain's response to levodopa. We measured regional cerebral blood flow response to levodopa with positron-emission tomography in 6 PD patients with DID, 10 chronically treated PD patients without DID, 17 dopa-naive PD patients, and 11 normals. The dose of levodopa was chosen to produce clinical benefit without inducing DID. This strategy allowed us to examine the brain response to levodopa across groups without the confounding effect of differences in motor behavior. We found that the DID group had a significantly greater response in ventrolateral thalamus than the other groups. This was associated with decreased activity in primary motor cortex. These findings are consistent with increased inhibitory output from the internal segment of the globus pallidus to thalamus after levodopa administration. They provide a physiological explanation for the clinical efficacy of pallidotomy and new insights into the physiology of the basal ganglia.

This communication describes a novel in vitro blood-brain barrier (BBB) model: organotypic slice cultures from the central nervous system were overlaid on endothelial cell monolayers grown on permeable membranes. Morphological, electrophysiological, and microdialysis approaches were carried out to characterize and validate this model. After 10 days in coculture, morphological studies reveal the presence of tight junctions. Electrophysiological recordings of neuronal activity performed on organotypic cultures with or without an endothelial cell monolayer show that amplitude of evoked responses were comparable, indicating good viability of cocultures after 2 weeks. Perfusion of known BBB permeable or nonpermeable molecules was used to test the coculture tightness in conjunction with electrophysiological or microdialysis approaches: application of glutamate (Glu), which doesn't easily cross the BBB, triggers off rhythmic activity only in control cultures, whereas epileptogenic activity was observed in both control cultures and cocultures during perfusions with picrotoxin, a molecule that can diffuse through the BBB. Finally, the microdialysis technique was used to determine the permeability of molecules coming from the perfusion chamber: L-dopa, dopamine, and Glu were employed to assess the selective permeability of the coculture model. Thus, these results indicate that the in vitro model described possesses characteristics similar to those of the BBB in situ and that cocultures of organotypic slices and endothelial cell monolayers have potential as a powerful tool for studying biochemical mechanisms regulating BBB function and drug delivery to the central nervous system.

Cultures of a cell line derived from pig proximal tubule epithelium, LLC-PK1, were investigated for their uptake of L-3,4-dihydroxyphenylalanine (L-dopa) and the decarboxylation of L-dopa to dopamine. The kinetics of absorption was determined for both the apical and basal membranes. The kinetics of dopamine formation was also determined based on the source of the substrate whether basal or apical. Dopamine secretion from this cell line was also investigated.