David G. Standaert

The Parkinson Progression Marker Initiative (PPMI)

The Parkinson Progression Marker Initiative (PPMI) is a comprehensive observational, international, multi-center study designed to identify PD progression biomarkers both to improve understanding of disease etiology and course and to provide crucial tools to enhance the likelihood of success of PD modifying therapeutic trials. The PPMI cohort will comprise 400 recently diagnosed PD and 200 healthy subjects followed longitudinally for clinical, imaging and biospecimen biomarker assessment using standardized data acquisition protocols at twenty-one clinical sites. All study data will be integrated in the PPMI study database and will be rapidly and publically available through the PPMI web site- www.ppmi-info.org. Biological samples including longitudinal collection of blood, cerebrospinal fluid (CSF) and urine will be available to scientists by application to an independent PPMI biospecimen review committee also through the PPMI web site. PPMI will rely on a partnership of government, PD foundations, industry and academics working cooperatively. This approach is crucial to enhance the potential for success of this ambitious strategy to develop PD progression biomarkers that will accelerate research in disease modifying therapeutics.

Molecular markers of early Parkinson's disease based on gene expression in blood

Parkinsons disease (PD) progresses relentlessly and affects five million people worldwide. Laboratory tests for PD are critically needed for developing treatments designed to slow or prevent progression of the disease. We performed a transcriptome-wide scan in 105 individuals to interrogate the molecular processes perturbed in cellular blood of patients with early-stage PD. The molecular multigene marker here identified is associated with risk of PD in 66 samples of the training set comprising healthy and disease controls [third tertile cross-validated odds ratio of 5.7 (P for trend 0.005)]. It is further validated in 39 independent test samples [third tertile odds ratio of 5.1 (P for trend 0.04)]. Insights into disease-linked processes detectable in peripheral blood are offered by 22 unique genes differentially expressed in patients with PD versus healthy individuals. These include the cochaperone ST13, which stabilizes heat-shock protein 70, a modifier of α-synuclein misfolding and toxicity. ST13 messenger RNA copies are lower in patients with PD (mean ± SE 0.59 ± 0.05) than in controls (0.96 ± 0.09) (P = 0.002) in two independent populations. Thus, gene expression signals measured in blood can facilitate the development of biomarkers for PD.

The pathophysiological basis of dystonias

Dystonias comprise a group of movement disorders that are characterized by involuntary movements and postures. Insight into the nature of neuronal dysfunction has been provided by the identification of genes responsible for primary dystonias, the characterization of animal models and functional evaluations and in vivo brain imaging of patients with dystonia. The data suggest that alterations in neuronal development and communication within the brain create a susceptible substratum for dystonia. Although there is no overt neurodegeneration in most forms of dystonia, there are functional and microstructural brain alterations. Dystonia offers a window into the mechanisms whereby subtle changes in neuronal function, particularly in sensorimotor circuits that are associated with motor learning and memory, can corrupt normal coordination and lead to a disabling motor disorder.

Atriopeptins as Cardiac Hormones

P Needleman, SP Adams, BR Cole, MG Currie, DM Geller, ML Michener, CB Saper, D Atriopeptins as cardiac hormones ISSN: 1524-4563 Copyright

IMMUNOHISTOCHEMICAL LOCALIZATION OF METABOTROPIC GLUTAMATE RECEPTORS MGLUR1A AND MGLUR2/3 IN THE RAT BASAL GANGLIA

Metabotropic glutamate receptors (mGluRs), which couple glutamate to second messengers, have important roles in the regulation of movement by the basal ganglia. We used two polyclonal antisera to mGluR1a and mGluR2/3 and confocal laser microscopy to investigate the localization of these receptors in the basal ganglia of the rat. The mGluRs were visualized in combination with an antibody to tyrosine hydroxylase (TH), an antibody to microtubule‐associated protein 2 (MAP2, a dendritic marker), or SV2 (an antibody to a protein associated with presynaptic terminals). In the neostriatum, punctate mGluR1a immunoreactivity (ir) was present in the neuropil. This staining did not colocalize with MAP2‐ir or SV2‐ir and was not altered by decortication or unilateral 6‐hydroxydopamine (6‐OHDA) lesions. In the globus pallidus and substantia nigra pars reticulata, however, mGluR1a‐ir was tightly clustered along large MAP2‐ir dendrites. In contrast to the variations in mGluR1a‐ir staining, similar punctate neuropil mGluR2/3‐ir staining was observed within all basal ganglia structures. In the neostriatum, these puncta were abundant; unlike mGluR1a, many mGluR2/3‐ir puncta colocalized with SV2‐ir, and striatal mGluR2/3‐ir puncta were markedly reduced in number after decortication. Neither mGluR1a‐ir nor mGluR2/3‐ir could be detected in TH‐ir soma within substantia nigra pars compacta, or in TH‐ir striatal terminals.

Continuous intrajejunal infusion of levodopa-carbidopa intestinal gel for patients with advanced Parkinson's disease: a randomised, controlled, double-blind, double-dummy study

BACKGROUND Levodopa is the most effective therapy for Parkinsons disease, but chronic treatment is associated with the development of potentially disabling motor complications. Experimental studies suggest that motor complications are due to non-physiological, intermittent administration of the drug, and can be reduced with continuous delivery. We aimed to assess efficacy and safety of levodopa-carbidopa intestinal gel delivered continuously through an intrajejunal percutaneous tube. METHODS In our 12-week, randomised, double-blind, double-dummy, double-titration trial, we enrolled adults (aged ≥ 30 years) with advanced Parkinsons disease and motor complications at 26 centres in Germany, New Zealand, and the USA. Eligible participants had jejunal placement of a percutaneous gastrojejunostomy tube, and were then randomly allocated (1:1) to treatment with immediate-release oral levodopa-carbidopa plus placebo intestinal gel infusion or levodopa-carbidopa intestinal gel infusion plus oral placebo. Randomisation was stratified by site, with a mixed block size of 2 or 4. The primary endpoint was change from baseline to final visit in motor off-time. We assessed change in motor on-time without troublesome dyskinesia as a prespecified key secondary outcome. We assessed efficacy in a full-analysis set of participants with data for baseline and at least one post-baseline assessment, and imputed missing data with the last observation carried forward approach. We assessed safety in randomly allocated patients who underwent the percutaneous gastrojejunostomy procedure. This study is registered with ClinicalTrials.gov, numbers NCT00660387 and NCT0357994. FINDINGS From baseline to 12 weeks in the full-analysis set, mean off-time decreased by 4.04 h (SE 0.65) for 35 patients allocated to the levodopa-carbidopa intestinal gel group compared with a decrease of 2.14 h (0.66) for 31 patients allocated to immediate-release oral levodopa-carbidopa (difference -1.91 h [95% CI -3.05 to -0.76]; p=0.0015). Mean on-time without troublesome dyskinesia increased by 4.11 h (SE 0.75) in the intestinal gel group and 2.24 h (0.76) in the immediate-release oral group (difference 1.86 [95% CI 0.56 to 3.17]; p=0.0059). In the safety analyses 35 (95%) of 37 patients allocated to the levodopa-carbidopa intestinal gel group had adverse events (five [14%] serious), as did 34 (100%) of 34 patients allocated to the immediate-release oral levodopa-carbidopa group (seven [21%] serious), mainly associated with the percutaneous gastrojejunostomy tube. INTERPRETATION Continuous delivery of levodopa-carbidopa with an intestinal gel offers a promising option for control of advanced Parkinsons disease with motor complications. Benefits noted with intestinal gel delivery were of a greater magnitude than were those obtained with medical therapies to date, and our study is, to our knowledge, the first demonstration of the benefit of continuous levodopa delivery in a double-blind controlled study. FUNDING AbbVie.

Metabotropic glutamate receptors are differentially regulated during development

The postnatal expression of metabotropic glutamate receptors was studied in rat brain by in situ hybridization and autoradiographic binding techniques. The messenger RNAs encoding five metabotropic glutamate receptor subtypes named mGluR1-5 had distinct regional and temporal expression profiles. mGluR1, mGluR2 and mGluR4 messenger RNA expression was low at birth and increased during postnatal development. In contrast, mGluR3 and mGluR5 were highly expressed at birth and decreased during maturation to adult levels of expression. [3H]Glutamate binding competition studies in developing brain disclosed the presence of two types of binding sites with the pharmacological properties of metabotropic glutamate receptors, having high (metabotropic type-1 binding sites; K1 = 8 nM) and low affinity (metabotropic type-2 binding sites; K1 = 50 microM) for quisqualic acid, as in adult rat brain. The densities of metabotropic binding sites changed during development in a complex, regionally specific fashion. Metabotropic type-1 binding sites were present at low levels at birth and gradually increased during the second postnatal week. In the striatum, globus pallidus and cerebellar granule layer, the increase in density of metabotropic type-1 binding sites was transient but persisted in the cerebellar molecular layer. In contrast, metabotropic type-2 binding sites were present at high densities in most regions in the first postnatal week and decreased during the second and third week, particularly in the thalamic reticular nucleus and globus pallidus. Only in the external cortex did both metabotropic type-1 and metabotropic type-2 binding sites increase during development. A striking correspondence between the temporal pattern of expression of specific metabotropic glutamate receptor transcripts and metabotropic binding sites was observed in the reticular nucleus of the thalamus (mGluR3; metabotropic type-2 binding sites) and cerebellum (mGluR1; metabotropic type-1 binding sites) suggesting early translation of these metabotropic glutamate receptor messenger RNAs into receptor proteins. In other regions the relationship between messenger RNA expression and binding sites was less direct: comparison between expression of metabotropic glutamate receptor messenger RNA and binding sites suggests both a pre- and postsynaptic location of some receptor subtypes. These data imply a functional role of mGluR3 and mGluR5 during synaptogenesis and maintenance of adult synapses and of mGluR1, mGluR2 and mGluR4 in mature synaptic transmission.

LRRK2 Inhibition Attenuates Microglial Inflammatory Responses

Missense mutations in leucine-rich repeat kinase 2 (LRRK2) cause late-onset Parkinsons disease (PD), and common genetic variation in LRRK2 modifies susceptibility to Crohns disease and leprosy. High levels of LRRK2 expression in peripheral monocytes and macrophages suggest a role for LRRK2 in these cells, yet little is known about LRRK2 expression and function in immune cells of the brain. Here, we demonstrate a role for LRRK2 in mediating microglial proinflammatory responses and morphology. In a murine model of neuroinflammation, we observe robust induction of LRRK2 in microglia. Experiments with toll-like receptor 4 (TLR4)-stimulated rat primary microglia show that inflammation increases LRRK2 activity and expression, while inhibition of LRRK2 kinase activity or knockdown of protein attenuates TNFα secretion and nitric oxide synthase (iNOS) induction. LRRK2 inhibition blocks TLR4 stimulated microglial process outgrowth and impairs ADP stimulated microglial chemotaxis. However, actin inhibitors that phenocopy inhibition of process outgrowth and chemotaxis fail to modify TLR4 stimulation of TNFα secretion and inducible iNOS induction, suggesting that LRRK2 acts upstream of cytoskeleton control as a stress-responsive kinase. These data demonstrate LRRK2 in regulating responses in immune cells of the brain and further implicate microglial involvement in late-onset PD.

Dopamine D1 Activation Potentiates Striatal NMDA Receptors by Tyrosine Phosphorylation-Dependent Subunit Trafficking

Interactions between dopaminergic and glutamatergic afferents in the striatum are essential for motor learning and the regulation of movement. An important mechanism for these interactions is the ability of dopamine, through D1 receptors, to potentiate NMDA glutamate receptor function. Here we show that, in striatal neurons, D1 receptor activation leads to rapid trafficking of NMDA receptor subunits, with increased NR1 and NR2B subunits in dendrites, enhanced coclustering of these subunits with the postsynaptic density scaffolding molecule postsynaptic density-95, and increased surface expression. The dopamine D1 receptor-mediated NMDA receptor trafficking is blocked by an inhibitor of tyrosine kinases. Blockers of tyrosine phosphatases also induce NMDA subunit trafficking, but this effect is nonselective and alters both NR2A- and NR2B-containing receptors. Furthermore, tyrosine phosphatase inhibition leads to the clustering of tyrosine-phosphorylated NR2B subunit along dendritic shafts. Our findings reveal that D1 receptor activation can potentiate striatal NMDA subunit function by directly promoting the surface insertion of the receptor complexes. This effect is regulated by the reciprocal actions of protein tyrosine phosphatases and tyrosine kinases. Modification of these pathways may be a useful therapeutic target for Parkinson’s disease and other basal ganglia disorders in which abnormal function of striatal NMDA receptors contributes to the symptoms of the diseases.

EXPRESSION OF GROUP ONE METABOTROPIC GLUTAMATE RECEPTOR SUBUNIT MRNAS IN NEUROCHEMICALLY IDENTIFIED NEURONS IN THE RAT NEOSTRIATUM, NEOCORTEX, AND HIPPOCAMPUS

Metabotropic glutamate receptors (mGluRs) can be divided into three groups based on sequence homology and pharmacology. We studied expression of group I mGluRs (mGluR1 and mGluR5) in identified neurons of the rat neostriatum, neocortex, and hippocampus using in situ hybridization. Tissue sections were hybridized with radiolabeled RNA probes for mGluR1 or mGluR5 and digoxygenin labeled RNA probes detecting somatostatin (SOM), preproenkephalin (ENK), preprotachykinin (SP), glutamic acid decarboxylase 67 (GAD67), parvalbumin (PARV), or choline acetyltransferase (ChAT) mRNA. In the striatum, mGluR1 hybridization signal was observed in all six neuronal populations. The strongest signal was found in SP-positive neurons, with a lower signal in ENK-positive neurons. All striatal interneurons were labeled less intensely than ENK- and SP-positive projection neurons. For striatal mGluR5 mRNA, both SP- and ENK-positive projection neurons were intensely labeled, but only GAD67-positive interneurons exhibited a significant signal. In the neocortex and hippocampus, mGluR1 and mGluR5 hybridization signals were studied in SOM-, GAD67-, and PARV-positive neurons. Hybridization signal for mGluR1 mRNA was intense in SOM-positive neurons of the cortex, CA1, CA3, and dentate gyrus, and weaker in GAD67-positive neurons of CA3 and dentate gyrus. MGluR5 signals were intensely labeled in SOM-, GAD67- and PARV-positive neuronal populations of the cortex and hippocampus. SOM-positive neurons were more intensely labeled in the hippocampus than cortex.