AV-101, a Pro-Drug Antagonist at the NMDA Receptor Glycine Site, Reduces L-Dopa Induced Dyskinesias in MPTP Monkeys

N-methyl-D-aspartate (NMDA) receptors have been implicated in L-Dopa-induced dyskinesias (LID) in Parkinson’s disease patients, but the use of antagonists that directly inhibit this receptor is associated with severe side effects. L-4-chlorokynurenine (4-Cl-KYN or AV-101) is a pro-drug of 7-chlorokynurenic acid (7-Cl-KYNA), a potent and specific antagonist of the glycine (GlyB) co-agonist site of NMDA receptors. The 7-Cl-KYNA has limited ability to cross the blood–brain barrier, whereas AV-101 readily accesses the brain. We investigated if AV-101 reduces LID in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned monkeys while maintaining the antiparkinsonian activity of L-Dopa. A first pilot study using three dyskinetic MPTP monkeys showed that acute AV-101 treatment (250 and 450 mg/kg) reduced LID and maintained the antiparkinsonian activity of L-Dopa. The main study using six additional dyskinetic MPTP monkeys showed that repeated AV-101 treatment (250 mg/kg, b.i.d. for 4 consecutive days) maintained their L-Dopa antiparkinsonian response. We measured significantly less LID when AV-101 was combined with L-Dopa treatment. AV-101 alone or with L-Dopa had no non-motor adverse effects in MPTP monkeys. Our study showed antidyskinetic activity of AV-101 in MPTP monkeys was comparable to amantadine tested previously in our laboratory in this model. We observed no adverse effects with AV-101, which is an improvement over amantadine, with its known side effects.</jats:p

Impact of prior JAK-inhibitor therapy with ruxolitinib on outcome after allogeneic hematopoietic stem cell transplantation for myelofibrosis: a study of the CMWP of EBMT.

JAK1/2 inhibitor ruxolitinib (RUX) is approved in patients with myelofibrosis but the impact of pretreatment with RUX on outcome after allogeneic hematopoietic stem cell transplantation (HSCT) remains to be determined. We evaluated the impact of RUX on outcome in 551 myelofibrosis patients who received HSCT without (n = 274) or with (n = 277) RUX pretreatment. The overall leukocyte engraftment on day 45 was 92% and significantly higher in RUX responsive patients than those who had no or lost response to RUX (94% vs. 85%, p = 0.05). The 1-year non-relapse mortality was 22% without significant difference between the arms. In a multivariate analysis (MVA) RUX pretreated patients with ongoing spleen response at transplant had a significantly lower risk of relapse (8.1% vs. 19.1%; p = 0.04)] and better 2-year event-free survival (68.9% vs. 53.7%; p = 0.02) in comparison to patients without RUX pretreatment. For overall survival the only significant factors were age > 58 years (p = 0.03) and HLA mismatch donor (p = 0.001). RUX prior to HSCT did not negatively impact outcome after transplantation and patients with ongoing spleen response at time of transplantation had best outcome

The number of striatal cholinergic interneurons expressing calretinin is increased in parkinsonian monkeys

The most abundant interneurons in the primate striatum are those expressing the calcium-binding protein calretinin (CR). The present immunohistochemical study provides detailed assessments of their morphological traits, number, and topographical distribution in normal monkeys (Macaca fascicularis) and in monkeys rendered parkinsonian (PD) by MPTP intoxication. In primates, the CR + striatal interneurons comprise small (8–12 μm), medium (12–20 μm) and large-sized (20–45 μm) neurons, each with distinctive morphologies. The small CR + neurons were 2–3 times more abundant than the medium-sized CR + neurons, which were 20–40 times more numerous than the large CR + neurons. In normal and PD monkeys, the density of small and medium-sized CR + neurons was twice as high in the caudate nucleus than in the putamen, whereas the inverse occurred for the large CR + neurons. Double immunostaining experiments revealed that only the large-sized CR + neurons expressed choline acetyltransferase (ChAT). The number of large CR + neurons was found to increase markedly (4–12 times) along the entire anteroposterior extent of both the caudate nucleus and putamen of PD monkeys compared to controls. Comparison of the number of large CR −/ChAT + and CR +/ChAT + neurons together with experiments involving the use of bromo-deoxyuridine (BrdU) as a marker of newly generated cells showed that it is the expression of CR by the large ChAT + striatal interneurons, and not their absolute number, that is increased in the dopamine-depleted striatum. These findings reveal the modulatory role of dopamine in the phenotypic expression of the large cholinergic striatal neurons, which are known to play a crucial role in PD pathophysiology

Membrane cholesterol removal and replenishment affect rat and monkey brain monoamine transporters

The dopamine transporter (DAT) is abundantly expressed in the striatum where it removes extracellular dopamine into the cytosol of presynaptic nerve terminals. It is the target of drugs of abuse and antidepressants. There is a loss of the DAT in Parkinson's disease affecting release of levodopa implicated in levodopa-induced dyskinesias. This study investigated the effect of cholesterol on DAT, serotonin transporter (SERT) and vesicular monoamine transporter 2 (VMAT2) in monkey and rat brains in vitro. DAT protein levels measured by Western blot remained unchanged with in vitro methyl-b-cyclodextrin (MCD) incubations to remove membrane cholesterol or with incubations to increase membrane cholesterol content. By contrast, striatal DAT specific binding labelled with [125I]RTI-121 or with [125I]RTI55 decreased with increasing concentrations of MCD and increased with cholesterol loading. Moreover, [125I]RTI-121 specific binding of striatal membranes depleted of cholesterol with MCD was restored to initial DAT content with addition of cholesterol showing its rapid and reversible effect. By contrast, striatal VMAT2 and SERT specific binding showed no or limited changes by cholesterol manipulations. Similar results were obtained for monkey caudate nucleus, putamen and nucleus accumbens. Membrane microviscosity was assessed by fluorescence polarization spectroscopy, using the probe 1,6-diphenyl1,3,5-hexatriene. DAT changes positively correlated with changes of membrane microviscosity in rat and monkey brain regions investigated and with membrane cholesterol contents. Similar findings were observed with desmosterol but to a lower extent than with cholesterol. These results show an important effect of cholesterol on the DAT associated with microviscosity changes that should be considered in drug therapies

Serotonin hyperinnervation of the striatum with high synaptic incidence in parkinsonian monkeys

The chronic use of L-Dopa for alleviating the motor symptoms of Parkinson’s disease often produces adverse effects such as dyskinesia. Unregulated release of dopamine by serotonin axons following L-Dopa administration is a major presynaptic determinant of these abnormal involuntary movements. The present study was designed to characterize the reorganization of serotonin striatal afferents following dopaminergic denervation in a primate model of Parkinson’s disease. Our sample comprised eight cynomolgus monkeys: four that were rendered parkinsonian following MPTP administration and four controls. The state of striatal serotonin and dopamine innervation was evaluated by means of immunohistochemistry with antibodies against serotonin transporter (SERT) and tyrosine hydroxylase. A detailed stereological investigation revealed a significant increase in the number of serotonin axon varicosities in the striatum of MPTP-intoxicated monkeys. This increase is particularly pronounced in the sensorimotor territory of the striatum, where the dopamine denervation is the most severe. Electron microscopic examinations indicate that, in contrast to the nucleus accumbens where the dopamine innervation is preserved, the SERT+ axon varicosities observed in the sensorimotor territory of the putamen establish twice as many synaptic contacts in MPTP-intoxicated monkeys than in controls. These findings demonstrate the highly plastic nature of the serotonin striatal afferent projections, a feature that becomes particularly obvious in the absence of striatal dopamine. Although the number of dorsal raphe serotonin neurons remains constant in parkinsonian monkeys, as shown in the present study, their ascending axonal projections undergo marked proliferative and synaptic adaptive changes that might play a significant role in the potential unregulated and ectopic release of dopamine by serotonin axons after L-Dopa treatment of Parkinson’s disease

Estrogen receptors and lesion-induced response of striatal dopamine receptors

Neuroprotection by 17β-estradiol and an estrogen receptor (ER) agonist against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) lesion were shown to implicate protein kinase B (Akt) signaling in mice. In order to evaluate the associated mechanisms, this study compared estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) intact or knockout (KO) and wild-type (WT) C57Bl/6 male mice following MPTP treatment of 7, 9, 11 mg/kg and/or 17β-estradiol. Striatal D1 and D2 dopamine (DA) receptors were measured by autoradiography with the specific ligands [3H]-SCH 23390 and [3H]-raclopride, respectively and signaling by Western blot for Akt, glycogen synthase kinase 3β (GSK3β) and extracellular-regulated signal kinases (ERK1 and ERK2). Control ERKOβ mice had lower striatal [3H]-SCH 23390 specific binding than WT and ERKOα mice; both KO mice had lower [3H]-raclopride specific binding. Striatal D1 receptors decreased with increasing doses of MPTP in correlation with striatal DA concentrations in ERKOα mice and remained unchanged in WT and ERKOβ mice. Striatal D2 receptors decreased with increasing doses of MPTP in correlation with striatal DA concentrations in WT and ERKOα mice and increased in ERKOβ mice. In MPTP-lesioned mice, 17β-estradiol treatment increased D1 receptors in ERKOα and ERKOβ mice and D2 receptors in WT and ERKOβ mice. MPTP did not affect striatal pAkt/Akt and pGSK3β/GSK3β levels in WT and ERKOα mice, while in vehicle-treated ERKOβ mice these levels were higher and increased with MPTP lesioning. Striatal pERK1/ERK1 and pERK2/ERK2 levels showed to a lesser extent a similar pattern. In conclusion, ERs affected the response of striatal DA receptors to a MPTP lesion and post receptor signaling

Prevention of L-Dopa-Induced Dyskinesias by MPEP Blockade of Metabotropic Glutamate Receptor 5 Is Associated with Reduced Inflammation in the Brain of Parkinsonian Monkeys

Proinflammatory markers were found in brains of Parkinson&rsquo;s disease (PD) patients. After years of L-Dopa symptomatic treatment, most PD patients develop dyskinesias. The relationship between inflammation and L-Dopa-induced dyskinesias (LID) is still unclear. We previously reported that MPEP (a metabotropic glutamate receptor 5 antagonist) reduced the development of LID in de novo MPTP-lesioned monkeys. We thus investigated if MPEP reduced the brain inflammatory response in these MPTP-lesioned monkeys and the relationship to LID. The panmacrophage/microglia marker Iba1, the phagocytosis-related receptor CD68, and the astroglial protein GFAP were measured by Western blots. The L-Dopa-treated dyskinetic MPTP monkeys had increased Iba1 content in the putamen, substantia nigra, and globus pallidus, which was prevented by MPEP cotreatment; similar findings were observed for CD68 contents in the putamen and globus pallidus. There was a strong positive correlation between dyskinesia scores and microglial markers in these regions. GFAP contents were elevated in MPTP + L-Dopa-treated monkeys among these brain regions and prevented by MPEP in the putamen and subthalamic nucleus. In conclusion, these results showed increased inflammatory markers in the basal ganglia associated with LID and revealed that MPEP inhibition of glutamate activity reduced LID and levels of inflammatory markers