Safinamide Modulates Striatal Glutamatergic Signaling in a Rat Model of Levodopa-Induced Dyskinesia.

Safinamide (Xadago) is a novel dual-mechanism drug that has been approved in the European Union and United States as add-on treatment to levodopa in Parkinson's disease therapy. In addition to its selective and reversible monoamine oxidase B inhibition, safinamide through use-dependent sodium channel blockade reduces overactive glutamatergic transmission in basal ganglia, which is believed to contribute to motor symptoms and complications including levodopa-induced dyskinesia (LID). The present study investigated the effects of safinamide on the development of LID in 6-hydroxydopamine (6-OHDA)-lesioned rats, evaluating behavioral, molecular, and neurochemical parameters associated with LID appearance. 6-OHDA-lesioned rats were treated with saline, levodopa (6 mg/kg), or levodopa plus safinamide (15 mg/kg) for 21 days. Abnormal involuntary movements, motor performance, molecular composition of the striatal glutamatergic synapse, glutamate, and GABA release were analyzed. In the striatum, safinamide prevented the rearrangement of the subunit composition of N-methyl-d-aspartate receptors and the levodopa-induced increase of glutamate release associated with dyskinesia without affecting the levodopa-stimulated motor performance and dyskinesia. Overall, these findings suggest that the striatal glutamate-modulating component of safinamide's activity may contribute to its clinical effects, where its long-term use as levodopa add-on therapy significantly improves motor function and "on" time without troublesome dyskinesia.

Intestinal permeability and Ménière's disease.

PURPOSE: Ménière disease (MD) is a multifactorial chronic disabling condition characterized by episodic vertigo, ear fullness, and hearing loss. MD patients often complain of aspecific gastrointestinal symptoms associated with autonomic dysregulation, frequently outweighed by the otological manifestations. Dietary modifications have been reported to improve the typical MD symptoms in some cases. Our purpose was to test the urinary levels of lactulose and mannitol (double sugar test) and the fecal calprotectin, both markers of altered intestinal permeability, in subjects with definite MD in an active and inactive stage. MATERIALS AND METHODS: Twenty-six with definite unilateral MD were studied: 14 patients were symptomatic for at least 3months with moderate to severe vertigo spells and a functional level ≥4; 12 patients had been asymptomatic (no vertigo spells) for at least 3months and had a functional level=1 at the time of testing. Twenty healthy volunteers were recruited as "control group". RESULTS: Lactulose and mannitol absorption was significantly increased in the symptomatic M patients compared to the asymptomatic group (p<0.02 and p<0.004, respectively) and to the controls. FC were also higher than normal only in the symptomatic group. (p<0.01). CONCLUSIONS: An altered intestinal permeability, according to the two assays, was found only in symptomatic MD patients. The rationale for a possible relationship between MD and intestinal permeability is forwarded. The double-sugar test and FC quantification might be implemented in the MD diagnostic workup.

CRISPR/Cas9-mediated knockout of Abcd1 and Abcd2 genes in BV-2 cells: novel microglial models for X-linked Adrenoleukodystrophy.

X-linked adrenoleukodystrophy (X-ALD), the most frequent peroxisomal disorder, is associated with mutation in the ABCD1 gene which encodes a peroxisomal ATP-binding cassette transporter for very long-chain fatty acids (VLCFA). The biochemical hallmark of the disease is the accumulation of VLCFA. Peroxisomal defect in microglia being now considered a priming event in the pathology, we have therefore generated murine microglial cells mutated in the Abcd1 gene and its closest homolog, the Abcd2 gene. Using CRISPR/Cas9 gene editing strategy, we obtained 3 cell clones with a single or double deficiency. As expected, only the combined absence of ABCD1 and ABCD2 proteins resulted in the accumulation of VLCFA. Ultrastructural analysis by electron microscopy revealed in the double mutant cells the presence of lipid inclusions similar to those observed in brain macrophages of patients. These observations are likely related to the increased level of cholesterol and the accumulation of neutral lipids that we noticed in mutant cells. A preliminary characterization of the impact of peroxisomal defects on the expression of key microglial genes such as Trem2 suggests profound changes in microglial functions related to inflammation and phagocytosis. The expression levels of presumed modifier genes have also been found modified in mutant cells, making these novel cell lines relevant for use as in vitro models to better understand the physiopathogenesis of X-ALD and to discover new therapeutic targets.

A microglial cell model for acyl-CoA oxidase 1 deficiency.

Acyl-CoA oxidase 1 (ACOX1) deficiency is a rare and severe peroxisomal leukodystrophy associated with a very long-chain fatty acid (VLCFA) ß-oxidation defect. This neurodegenerative disease lacks relevant cell models to further decipher the pathomechanisms in order to identify novel therapeutic targets. Since peroxisomal defects in microglia appear to be a key component of peroxisomal leukodystrophies, we targeted the Acox1 gene in the murine microglial BV-2 cell line. Using CRISPR/Cas9 gene editing, we generated an Acox1-deficient cell line and validated the allelic mutations, which lead to the absence of ACOX1 protein and enzymatic activity. The activity of catalase, the enzyme degrading H2O2, was increased, likely in response to the alteration of redox homeostasis. The mutant cell line grew more slowly than control cells without obvious morphological changes. However, ultrastructural analysis revealed an increased number of peroxisomes and mitochondria associated with size reduction of mitochondria. Changes in the distribution of lipid droplets containing neutral lipids have been observed in mutant cells; lipid analysis revealed the accumulation of saturated and monounsaturated VLCFA. Besides, expression levels of genes encoding interleukin-1 beta and 6 (IL-1ß and IL-6), as well as triggering receptor expressed on myeloid cells 2 (Trem2) were found modified in the mutant cells suggesting modification of microglial polarization and phagocytosis ability. In summary, this Acox1-deficient cell line presents the main biochemical characteristics of the human disease and will serve as a promising model to further investigate the consequences of a specific microglial peroxisomal ß-oxidation defect on oxidative stress, inflammation and cellular functions.

Increased production of 27-hydroxycholesterol in human colorectal cancer advanced stage: Possible contribution to cancer cell survival and infiltration.

So far, the investigation in cancer cell lines of the modulation of cancer growth and progression by oxysterols, in particular 27-hydroxycholesterol (27HC), has yielded controversial results. The primary aim of this study was the quantitative evaluation of possible changes in 27HC levels during the different steps of colorectal cancer (CRC) progression in humans. A consistent increase in this oxysterol in CRC mass compared to the tumor-adjacent tissue was indeed observed, but only in advanced stages of progression (TNM stage III), a phase in which cancer has spread to nearby sites. To investigate possible pro-tumor properties of 27HC, its effects were studied in vitro in differentiated CaCo-2 cells. Relatively high concentrations of this oxysterol markedly increased the release of pro-inflammatory interleukins 6 and 8, monocyte chemoattractant protein-1, vascular endothelial growth factor, as well as matrix metalloproteinases 2 and 9. The up-regulation of all these molecules, which are potentially able to favor cancer progression, appeared to be dependent upon a net stimulation of Akt signaling exerted by supra-physiological amounts of 27HC.

1-aryl-4-[(1-tetralinyl)alkyl]piperazines: alkylamido and alkylamino derivatives. Synthesis, 5-HT1A receptor affinity, and selectivity. 3.

The synthesis and binding profile on 5-HT1A, 5-HT2, D-1, D-2, alpha 1, and alpha 2 receptors of the N-4 long-chain arylpiperazines 22-40 are reported, where an amino or amido function is inserted into the intermediate chain linked to the alpha position of the tetralin nucleus. Unlike the buspirone analogues, for the amido derivatives studied in this paper, the terminal amide function of long-chain piperazines is not important for 5-HT1A receptor affinity binding, and its removal yields high-affinity 5-HT1A receptor agents.

Phenylimidazolidin-2-one derivatives as selective 5-HT3 receptor antagonists and refinement of the pharmacophore model for 5-HT3 receptor binding.

A possible bioisosterism between the benzamido and the phenylimidazolidin-2-one moieties has been suggested on the basis of the similarity between the molecular electrostatic potential (MEP) of metoclopramide, a D2 receptor antagonist with weak 5-HT3 receptor antagonist properties, and zetidoline, a D2 receptor antagonist. Starting from this premise, a series of phenylimidazolidin-2-one derivatives bearing a basic azabicycloalkyl or an imidazolylalkyl moiety were synthesized and evaluated for 5-HT3 receptor radioligand binding affinity ([3H]-GR 43,694). In vitro 5-HT3 receptor antagonist activity was tested in the guinea pig ileum assay (GPI). A number of high-affinity ligands were shown to be potent 5-HT3 receptor antagonists in vivo as determined by inhibition of the Bezold--Jarisch reflex in the anesthetized rat. In general, the imidazolylalkyl derivatives were found to be more active than azabicycloalkyls. 1-(3,5-Dichlorophenyl)-3-[(5-methyl-1H-imidazol-4-yl)methyl]imidazoli din-2-one (58), in particular, displayed very high affinity for the 5-HT3 receptor (Ki of 0.038 nM) with a Kb of 5.62 nM in the GPI assay, being more potent than the reference compounds (ondansetron, tropisetron, granisetron, and BRL 46,470) tested. 58 showed an ID50 comparable to that of ondansetron (2.2 micrograms/kg i.v.) in the Bezold--Jarisch reflex. A molecular modeling study based on this structurally novel series of compounds allowed the refinement of previously reported 5-HT3 receptor antagonist pharmacophore models.

Structure-activity relationship studies on the 5-HT(1A) receptor affinity of 1-phenyl-4-[omega-(alpha- or beta-tetralinyl)alkyl]piperazines. 4.

The synthesis of 1-phenylpiperazines, linked in the alpha or beta position of the tetralin moiety on the terminal part of the N-4 alkyl chain, and their radioligand binding affinities for 5-HT(1A), 5-HT(2A), D-1, D-2, alpha1, and alpha2 receptors along with SAR studies on the 5-HT(1A) receptor are reported. Several changes have been carried out on previous structures of type 2, by inserting the alkyl chain with variable length in the alpha or beta position of the tetralin moiety and by changing the position of the methoxy group on the aromatic ring of the tetralin nucleus. The highest affinity (IC50 = 0.50 nM) and selectivity for the 5-HT(1A) receptor were showed by 1-phenylpiperazine 2a with a three-membered alkyl chain bearing a 5-methoxytetralin-1-yl ring in the omega position.

Synthesis and in vitro and in vivo evaluation of dopaminergic ergoline derivatives.

A series of ergoline-amides was synthesised in the discovery of new dopaminomimetic agents. Several compounds exhibited in vivo high prolactin lowering activity (indirectly measured by the nidation test) in rats. For the most active, the potential anti-Parkinson activity was evaluated by observation of the contralateral turning behaviour in 6-OH-DA lesioned rats. The acute toxicity by oral route in mice was also studied.

Synthesis and antihypertensive activity of 2,4-dioxoimidazolidin-1-yl and perhydro-2,4-dioxopyrimidin-1-yl ergoline derivatives.

The synthesis and antihypertensive activity of a series of 2,4-dioxoimidazolidin-1-yl and perhydro-2,4-dioxopyrimidin-1-yl ergoline derivatives are reported. The oral antihypertensive activity was studied in spontaneously hypertensive rats (SHRs) by measuring systolic blood pressure by an indirect tail-cuff method at different times after treatment. The prolactin lowering activity (indirectly measured by the nidation test) in rats and the oral acute toxicity in mice were also studied. The results of this study revealed potent antihypertensive ergoline derivatives devoid of side-effects related to the dopaminergic stimulation and the importance of the delta 9,10 double bond for conferring high potency within these compounds.

Safinamide: from molecular targets to a new anti-Parkinson drug.

Ideal treatment in Parkinson's disease (PD) aims at relieving symptoms and slowing disease progression. Of all remedies, levodopa remains the most effective for symptomatic relief, but the medical need for neuroprotectant drugs is still unfulfilled. Safinamide, currently in phase III clinical trials for the treatment of PD, is a unique molecule with multiple mechanisms of action and a very high therapeutic index. It combines potent, selective, and reversible inhibition of MAO-B with blockade of voltage-dependent Na+ and Ca2+ channels and inhibition of glutamate release. Safinamide has neuroprotective and neurorescuing effects in MPTP-treated mice, in the rat kainic acid, and in the gerbil ischemia model. Safinamide potentiates levodopa-mediated increase of DA levels in DA-depleted mice and reverses the waning motor response after prolonged levodopa treatment in 6-OHDA-lesioned rats. Safinamide has excellent bioavailability, linear kinetics, and is suitable for once-a-day administration. Therefore, safinamide may be used in PD to reduce l-dopa dosage and also represents a valuable therapeutic drug to test disease-modifying potential.

Effect of ergolines on neurotransmitter systems in the rat brain.

The interaction of nicergoline with various monoaminergic receptors and its effects on monoamine turnover were studied in specific rat brain areas in comparison with those of its metabolites, 10-methoxy-1,6-dimethyl-ergoline-8 beta-methanol (MMDL) and 10-methoxy-6-methyl-ergoline-8 beta-methanol (MDL). Nicergoline showed marked in vitro affinity for alpha 1-noradrenergic receptors (IC50 = 0.2 nM), less for alpha 2, S1 and S2 (IC50 about 10(-7) M). Its strong interaction with alpha 1-receptors was confirmed in vivo. The affinity of the other ergolines for alpha 1-receptors was lower than that of nicergoline. The level of monoamine metabolites (HVA and DOPAC for dopamine, MOPEG-SO4 for noradrenaline and 5-HIAA for serotonin) was taken as a measure of their turnover in the rat brain. A single dose of nicergoline (20 mg/kg, s.c.) strongly enhanced noradrenaline turnover, less that of dopamine. These effects, probably due to the interaction with alpha-receptors, were more marked after parenteral than oral administration. MMDL shared nicergoline's effect on dopamine and MDL that on noradrenaline, but they were less active than the parent compound. The most interesting results were obtained after chronic treatment (6-7 weeks) with rather low doses: 5 mg/kg b.i.d. of nicergoline and equimolar doses of the metabolites. Nicergoline and MMDL both enhanced dopamine turnover, especially in mesolimbic areas. In conclusion, the present report confirms and extends previous results on nicergoline's effects on catecholamine turnover and shows that its metabolite MMDL shares its effects on dopamine. Finally, it is particularly interesting that both ergolines were more effective in old than in young rats.

Dopamine receptors status after unilateral nigral 6-OHDA lesion. Autoradiographic and in situ hybridization study in the rat brain.

The physiological effects of dopamine (DA) are mediated by several distinct receptor subtypes. The effects of unilateral nigral 6-hydroxydopamine (6-OHDA) lesions on DA receptors were investigated by receptor autoradiography using the D1 selective ligand [3H]SCH 23390 as well as the D2 ligand [3H]spiroperidol. mRNA distribution was studied by in situ hybridization. Lesioned rats were sacrificed at different time intervals. Receptor binding studies were performed on tissue sections using selective ligands. [35S]UTP labeled RNA probes were prepared from the different cDNA (D1, D2, D3) and used for in situ hybridization. A specific loss of receptor binding sites and mRNA hybridization was found in the lesioned substantia nigra pars compacta (SNc) at all times examined. Receptor binding studies revealed a different time-dependent increase in both D1 and D2 receptors. In situ hybridization showed that only D2 receptor mRNA increased in the caudate-putamen (CPu) of the lesioned side 15 d after 6-OHDA. No changes were observed in D1 and D3 receptor mRNA during the entire time-course.

Mechanism of the antihypertensive effect of FCE 22716, a new ergoline derivative, in the spontaneously hypertensive rat.

Both acute and chronic oral administration (1-20 mg/kg) of FCE 22716, a new ergoline derivative, resulted in a dose-related fall of arterial blood pressure lasting for more than 6 h. Tachycardia was observed only at high dosages. Yohimbine, propranolol and indometacin did not modify its antihypertensive effect; on the other hand pretreatment with prazosin, a selective alpha 1-adrenoceptor antagonist and pithing, almost completely neutralized its antihypertensive effect. Haloperidol, a dopamine antagonist that crosses the blood-brain barrier, also antagonized FCE 22716 activity. The lack of effects of domperidone (DA2-receptor antagonist selectively acting on the periphery) together with the finding that norepinephrine and epinephrine levels were unchanged after treatment with FCE 22716, seem to rule out an involvement of peripheral DA2-receptors. Both in vitro (isolated organs) and in vivo the compound antagonized responses mediated by stimulation of alpha 1-adrenoceptors and S2-receptors. Radioligand binding studies in different cerebral regions are in line with the above reported in vitro and in vivo results. These data suggest that FCE 22716 is endowed with a multitarget mechanism of action, mainly involving blockade of alpha 1-adrenoceptors and S2-receptors.

Recombinant human IL-2 is cytotoxic to oligodendrocytes after in vitro self aggregation.

Interleukin 2 (IL-2) is a cytokine produced by activated T cells that plays a crucial role in the immune response and exerts multiple functions in different tissues including the nervous system. The present study was carried out to investigate the effect of recombinant human IL-2 (rhIL-2) on the survival of differnet glial cells type and of cortical neurons in culture. The results demonstrate that rhIL-2 is selectively cytotoxic to oligodendrocytes only if preincubated in aqueous solution (1200 U/ml) for at least two days before being added to the culture. Other glial and neuronal cells were unaffected. The cytotoxic effect was temperature- and concentration-dependent occurring only when rhIL-2 was reincubated at 37 degrees C and was dose-dependently neutralized by antibodies raised against IL-2 indicating that an immunoreactive IL-2 like compound is the active principle. Polyacrilamide gel electrophoresis under native (PAGE) and denaturing (SDS-PAGE) conditions and gel filtration analysis of the rhIL-2 incubated solution suggest that rhIL-2 is cytotoxic to oligodendrocytes only after association into soluble high weight molecular aggregates.

Receptor adaptive responsiveness in disease models: 6-OHDA lesioned and spontaneously hypertensive rats.

We have examined the adaptive modifications of brain monoamine receptors in response to pathophysiological processes in animal disease models: 6-OHDA lesioned and spontaneously hypertensive rats (SHR). The two models share a similar increase in D-1 receptor densities, while noradrenergic receptors are affected in different way: alpha-1 and beta are supersensitive in 6-OHDA lesioned rats and only alpha-2 are increased in SHR. S-1 receptors too are up-regulated in SHR. We must notice that though receptor hypersensitivity in the 6-OHDA model is linked to massive decreases in neurotransmitter levels, this mechanism seems not to exist in the SHR model.