Genetics screening in an Italian cohort of patients with Amyotrophic Lateral Sclerosis: the importance of early testing and its implication.

INTRODUCTION: Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease with an elusive etiology. While environmental factors have been considered, familial ALS cases have raised the possibility of genetic involvement. This genetic connection is increasingly evident, even in patients with sporadic ALS. We allowed access to the genetic test to all patients attending our clinic to identify the prevalence and the role of genetic variants in the development of the disease and to identify patients with potentially treatable forms of the disease. MATERIALS AND METHODS: 194 patients with probable or definite ALS, were enrolled. A comprehensive genetic testing was performed, including sequencing all exons of the SOD1 gene and testing for hexanucleotide intronic repeat expansions (G4C2) in the C9orf72 gene using fluorescent repeat-primed PCR (RP-PCR). Whole Exome NGS Sequencing (WES) was performed, followed by an in silico multigene panel targeting neuromuscular diseases, spastic paraplegia, and motor distal neuropathies. We conducted statistical analyses to compare different patient groups. RESULTS: Clinically significant pathogenetic variants were detected in 14.43% of cases. The highest prevalence of pathogenetic variants was observed in fALS patients, but a substantial proportion of sALS patients also displayed at least one variant, either pathogenetic or of uncertain significance (VUS). The most observed pathogenetic variant was the expansion of the C9orf72 gene, which was associated with a shorter survival. SOD1 variants were found in 1.6% of fALS and 2.5% of sALS patients. DISCUSSION: The study reveals a significant number of ALS patients carrying pathogenic or likely pathogenic variants, with a higher prevalence in familial ALS cases. The expansion of the C9orf72 gene emerges as the most common genetic cause of ALS, affecting familial and sporadic cases. Additionally, SOD1 variants are detected at an unexpectedly higher rate, even in patients without a familial history of ALS, underscoring the crucial role of genetic testing in treatment decisions and potential participation in clinical trials. We also investigated variants in genes such as TARDBP, FUS, NEK1, TBK1, and DNAJC7, shedding light on their potential involvement in ALS. These findings underscore the complexity of interpreting variants of uncertain significance (VUS) and their ethical implications in patient communication and genetic counseling for patients' relatives. CONCLUSION: This study emphasizes the diverse genetic basis of ALS and advocates for integrating comprehensive genetic testing into diagnostic protocols. The evolving landscape of genetic therapies requires identifying all eligible patients transcending traditional familial boundaries. The presence of VUS highlights the multifaceted nature of ALS genetics, prompting further exploration of complex interactions among genetic variants, environmental factors, and disease development.

A novel CACNA1A R2201W variant in a woman with hemiplegic migraine.

INTRODUCTION: Familial hemiplegic migraine type 1 (FHM1) is a monogenic rare disease that is characterized by migraine attacks accompanied by unilateral weakness and is caused by mutations in the CACNA1A gene. We report the case of a patient with a clinical history consistent with hemiplegic migraine who underwent genetic testing that revealed a variant in the CACNA1A gene. CASE PRESENTATION: A 68-year-old woman was evaluated for progressive postural instability and subjective cognitive decline. She had suffered from recurrent migraine episodes accompanied by fully reversible unilateral weakness that had started around the age of thirty and had fully disappeared at the time of evaluation. Magnetic resonance imaging (MRI) showed an extensive leukoencephalopathy, with features suggestive of small vessel disease, significantly progressing over the years. Exome sequencing revealed the heterozygous variant c.6601C>T (p.Arg2201Trp) in the CACNA1A gene. This variant, located in a highly conserved region, causes the substitution of arginine with tryptophan at codon 2202 of exon 47, with a high likelihood of a damaging effect on protein activity and/or structure. DISCUSSION: This is the first report describing the missense mutation c.6601C>T (p.Arg2201Trp) in heterozygosity in the CACNA1A gene in a patient with clinical features of hemiplegic migraine. The presence of a diffuse leukoencephalopathy on MRI is not typical of hemiplegic migraine and may suggest a phenotypic variant related to this mutation or result from the combined effect of the patient's comorbidities.

Evaluating the contribution of the gene TARDBP in Italian patients with amyotrophic lateral sclerosis.

BACKGROUND AND OBJECTIVES: Genetic variants in the gene TARDBP, encoding TDP-43 protein, are associated with amyotrophic lateral sclerosis (ALS) in familial (fALS) and sporadic (sALS) cases. Objectives of this study were to assess the contribution of TARDBP in a large cohort of Italian ALS patients, to determine the TARDBP-associated clinical features and to look for genotype-phenotype correlation and penetrance of the mutations. METHODS: A total of 1992 Italian ALS patients (193 fALS and 1799 sALS) were enrolled in this study. Sanger sequencing of TARDBP gene was performed in patients and, when available, in patients' relatives. RESULTS: In total, 13 different rare variants were identified in 43 index cases (10 fALS and 33 sALS) with a cumulative mutational frequency of 2.2% (5.2% of fALS, 1.8% of sALS). The most prevalent variant was the p.A382T followed by the p.G294V. Cognitive impairment was detected in almost 30% of patients. While some variants, including the p.G294V and the p.G376D, were associated with restricted phenotypes, the p.A382T showed a marked clinical heterogeneity regarding age of onset, survival and association with cognitive impairment. Investigations in parents, when possible, showed that the variants were inherited from healthy carriers and never occurred de novo. CONCLUSIONS: In our cohort, TARDBP variants have a relevant frequency in Italian ALS patients and they are significantly associated with cognitive impairment. Clinical presentation is heterogeneous. Consistent genotype-phenotype correlations are limited to some mutations. A marked phenotypic variability characterizes the p.A382T variant, suggesting a multifactorial/oligogenic pathogenic mechanism.

A novel compound heterozygous mutation in GALC associated with adult-onset Krabbe disease: case report and literature review.

Krabbe disease (KD) is a rare autosomal recessive lipid storage leukodystrophy. It is caused by deficient enzyme activity resulting from mutations of the ß-galactocerebrosidase (GALC) gene. KD is distinguished into subtypes based on the age of onset; these are early infantile, late infantile, juvenile, and adult-onset. We report a case of a 47-year-old Caucasian man with a 2-year history of muscle atrophy and weakness in both hands associated with pyramidal signs and mild spasticity in the lower limbs. An extensive work-up led this motor neuron disease-like disorder to be diagnosed as adult-onset KD. The patient was found to be compound heterozygous for two GALC mutations (p.G286D and p.Y490N). These two rare missense mutations have previously been reported with other heterozygous mutations. However, their co-occurrence in a KD patient is novel. From the perspective of this case, we review the current literature on compound heterozygous mutations in adult-onset KD and their phenotypic variability.

Novel variants and cellular studies on patients' primary fibroblasts support a role for NEK1 missense variants in ALS pathogenesis.

In the last few years, NEK1 has been identified as a new gene related to amyotrophic lateral sclerosis (ALS). Loss-of-function variants have been mostly described, although several missense variants exist, which pathogenic relevance remains to be established. We attempted to determine the contribution of NEK1 gene in an Italian cohort of 531 sporadic and familial amyotrophic lateral sclerosis (ALS) patients applying massive parallel sequencing technologies. We filtered results of NEK1 gene and identified 20 NEK1 rare variants (MAF < 0.01) in 22 patients. In particular, we found two novel frameshift variants (p.Glu929Asnfs*12 and p.Val1030Ilefs*23), 18 missense variants, including the p.Arg261His in three patients, and a novel variant in the start codon, the p.Met1?, which most likely impairs translation initiation. To clarify the role of NEK1 missense variants we investigated NEK1 expression in primary fibroblast cultures. We obtained skin biopsies from four patients with NEK1 variants and we assessed NEK1 expression by western blot and immunofluorescence. We detected a decrease in NEK1 expression in fibroblasts from patients with NEK1 variants, suggesting that missense variants in NEK1 gene may have a pathogenic role. Moreover, we observed additional variants in ALS related genes in seven patients with NEK1 variants (32%), further supporting an oligogenic ALS model.

A novel exomal ATRX mutation with preferential transmission to offspring: A case report and review of the literature for transmission ratio distortion in ATRX families.

The present case report describes an Italian family with three affected probands, who exhibited serious mental disability, which has not been associated with other anomalies, except with slight facial dysmorphism. Molecular multigenic analysis for intellectual disability identified a previously unreported variant, p.Ile1765Met (c.5295C>G) in the SNF domain of the ATRX protein (in exon 24). The identified mutation was found in a hemizygous state in all three affected probands and in a heterozygous state in the asymptomatic mother and the female sibling. With respect to the phenotypic similarities found in the patients with those described in previous studies, the consistency in the mode of inheritance and segregation of the mutation, the variant reported in the present case report may be considered as 'likely pathogenic'. To investigate the hypothesis that the preferential transmission of the ATRX mutation observed in this family reflected a general trend, a meta­analysis into the segregation of ATRX mutations from published pedigrees, following allelic transmission from mothers who are heterozygous carriers to their offspring, was performed. A preferential transmission of the mutant allele to male offspring (58% of males inherited the mutant allele) was found; however, the bias was not statistically significant (P=0.29; χ2 test).

Non-syndromic isolated dominant optic atrophy caused by the p.R468C mutation in the AFG3 like matrix AAA peptidase subunit 2 gene.

Autosomal dominant optic atrophy (DOA) is the most frequent form of hereditary optic atrophy, a disease presenting with considerable inter- and intra-familial clinical variability. Although a number of mutations in different genes are now known to cause DOA, many cases remain undiagnosed. In an attempt to identify the underlying genetic defect, whole exome sequencing was performed in a 19-year-old male that had been affected by isolated DOA since childhood. The exome sequencing revealed a pathogenic mutation (p.R468C, c.1402C>T) in the AFG3 like matrix AAA peptidase subunit 2 (AFG3L2) gene, a gene known to be associated with spinocerebellar ataxia. The patient did not show any signs other than DOA. Thus, the result demonstrates the possibility that mutations in the AFG3L2 gene may be a cause of isolated autosomal DOA.

Long-term rearrangement of retinal structures in a novel mutation of X-linked retinoschisis.

The aim of the present study was to report a novel mutation in the retinoschisin 1 (RS1) gene in a Caucasian family affected by X-linked juvenile retinoschisis (XLRS) and to describe the long-term modification of retinal structure. Two brothers with an early onset maculopathy were diagnosed with XLRS. Fundus photography, fluorescein angiography, spectral domain optical coherence tomography and electroretinogram analyses were performed. Their sister was also examined. All subjects were screened for mutations in the RS1 gene. XLRS patients demonstrated a marked reduction of best-corrected visual acuity. SD-OCT scans reported a cystic degeneration primarily involving the inner nuclear layer, though some cysts were detected in the outer plexiform layer and in the ganglion cell layer. During the ten-year follow-up, a progressive retinal thickening and coalescence of the cysts was observed. Genetic testing revealed a novel mutation (p.Ile212Asn) in the RS1 gene in both XLRS patients, whereas their sister was not a genetic carrier. Several mutations of the RS1 gene were recognized to be responsible for XLRS. Although the correspondence between genotype and phenotype is still under debate, is reasonable that siblings affected by XLRS could share other genetic and/or epigenetic factors capable to influence clinical course of the disease.

Octopamine, unlike other trace amines, inhibits responses of astroglia-enriched cultures to lipopolysaccharide via a ß-adrenoreceptor-mediated mechanism.

Trace amines (TAs), i.e. ß-phenylethylamine, tyramine and octopamine, are generally regarded as sympathomimetic compounds with structural and functional analogy with catecholamines. Previous reports have shown particularly high levels of circulating TAs in migraine and cluster headache patients. However, no clues are yet available as to the pathophysiological significance of these alterations. The effect of different TAs on the release of nitric oxide was investigated in rat astroglial cells stimulated with lipopolysaccharide (LPS). Octopamine substantially inhibited the release of NO evoked by LPS. Tyramine and ß-PEA were ineffective. The inhibitory effect of octopamine was fully reverted by two selective antagonists of ß-adrenergic receptors, while α-adrenergic blockade was ineffective. These data, consistent with a role of octopamine as a modulator of NO release, uncover an interaction between octopamine and ß-adrenergic receptors in astroglial cells. These results may have an impact in understanding the mechanisms underlying migraine pathophysiology.

A new class of antitumor trans-amine-amidine-Pt(II) cationic complexes: influence of chemical structure and solvent on in vitro and in vivo tumor cell proliferation.

The reactions of cyclopropylamine, cyclopentylamine, and cyclohexylamine with trans-[PtCl2(NCMe)2] afforded the bis-cationic complexes trans-[Pt(amine)2(Z-amidine)2]2+[Cl-]2, 1-3. The solution behavior and biological activity have been studied in different solvents (DMSO, water, polyethylene glycol (PEG 400), and polyethylene glycol dimethyl ether (PEG-DME 500)). The biological activity was strongly influenced by the cycloaliphatic amine ring size, with trans-[Pt(NH2CH(CH2)4CH2)2{N(H) horizontal lineC(CH3)N(H)CH(CH2)4CH2}2]2+[Cl-]2 (3) being the most active compound. Complex 3 overcame both cisplatin and MDR resistance, inducing cancer cell death through p53-mediated apoptosis. Alkaline single-cell gel electrophoresis experiments indicated direct DNA damage, reasonably attributable to DNA adducts of trans-[PtCl(amine)(Z-amidine)2][Cl] species, which can evolve to produce disruptive and nonrepairable lesions on DNA, thus leading to the drug-induced programmed cancer cell death. Preliminary in vivo antitumor studies on C57BL mice bearing Lewis lung carcinoma highlighted that complex 3 promoted a significant and dose-dependent tumor growth inhibition without adverse side effects.

CHF5074, a novel gamma-secretase modulator, restores hippocampal neurogenesis potential and reverses contextual memory deficit in a transgenic mouse model of Alzheimer's disease.

The effects of compounds interfering with gamma-secretase, the enzymatic complex responsible of the formation of the amyloid-beta (Abeta) peptide from amyloid-beta protein precursor (AbetaPP), on plaque deposition in transgenic mouse models of Alzheimer's disease are known but scanty data are available on the effects of these drugs on brain plasticity. We evaluated the effects of long-term treatment with CHF5074, a new gamma-secretase modulator, on hippocampal neurogenesis, cortical synaptophysin levels, and contextual memory in transgenic mice carrying the double Swedish mutation of AbetaPP (Tg2576). Six-month old Tg2576 mice were treated with CHF5074 (375 ppm in the diet) up to 15 months of age. Age-matched control transgenic and wild-type mice received standard diet. Compared to wild-type animals, transgenic controls showed a significant decrease in the number of doublecortin-positive neuroblasts in dentate gyrus, synaptophysin intensity in the cortex, freezing to context in the contextual fear conditioning test. Compared to transgenic controls, CHF5074 treatment of Tg2576 mice resulted in a significant attenuation of the neurogenesis impairment in hippocampus (p=0.036), normalization of synaptophysin levels in cortex (p< 0.001), attenuation of plaque burden in the cortex (p=0.033), increases astroglial reaction around plaques (p=0.001), and attenuation of activated microglia (p=0.040). These effects were associated to a complete reversal of contextual memory deficit (p=0.006). Contextual memory significantly correlated with synaptophysin immunoreactivity in the cortex (r=0.548, p=0.0038).

In vitro antitumor activity of the water soluble copper(I) complexes bearing the tris(hydroxymethyl)phosphine ligand.

Monocationic hydrophilic complexes [Cu(thp)4](+) 3 and [Cu(bhpe)2](+) 4 were synthesized by ligand exchange reactions starting from the labile [Cu(CH3CN)4][PF6] precursor in the presence of an excess of the relevant hydrophilic phosphine. Complexes 3 and 4 were tested against a panel of several human tumor cell lines. Complex 3 has been shown to be about 1 order of magnitude more cytotoxic than cisplatin. Chemosensitivity tests performed on cisplatin and multidrug resistance phenotypes suggested that complex 3 acts via a different mechanism of action than the reference drug. Different short-term proliferation assays suggested that lysosomal damage is an early cellular event associated with complex 3 cytotoxicity, probably mediated by an increased production of reactive oxygen species. Cytological stains and flow cytometric analyses indicated that the phosphine copper(I) complex is able to inhibit the growth of tumor cells via G2/M cell cycle arrest and paraptosis accompanied with the loss of mitochondrial transmembrane potential.

1-(3',4'-Dichloro-2-fluoro[1,1'-biphenyl]-4-yl)-cyclopropanecarboxylic acid (CHF5074), a novel gamma-secretase modulator, reduces brain beta-amyloid pathology in a transgenic mouse model of Alzheimer's disease without causing peripheral toxicity.

Some nonsteroidal anti-inflammatory drugs has been shown to allosterically modulate the activity of gamma-secretase, the enzymatic complex responsible for the formation of beta-amyloid (Abeta). 1-(3',4'-Dichloro-2-fluoro[1,1'-biphenyl]-4-yl)-cyclopropanecarboxylic acid (CHF5074) is a new gamma-secretase modulator, devoid of anticyclooxygenase (COX) and Notch-interfering activities in vitro. We evaluated the effects of chronic CHF5074 treatment on brain Abeta pathology in Tg2576 transgenic mice. Twenty-eight animals of 9.5 to 10.5 months of age received CHF5074-medicated diet (375 ppm) or standard diet for 17 weeks. Compared with controls, CHF5074 treatment significantly reduced the area occupied by plaques and the number of plaques in cortex (-52.2 +/- 5.6%, p = 0.0003 and -48.9 +/- 6.6%, p = 0.0004, respectively) and hippocampus (-76.7 +/- 6.4%, p = 0.004 and -66.2 +/- 10.3%, p = 0.037, respectively). Biochemical analysis confirmed the histopathological measures, with CHF5074-treated animals showing reduced total brain Abeta40 (-49.2 +/- 9.2%, p = 0.017) and Abeta42 (-43.5 +/- 9.7%, p = 0.027) levels. In a human neuroglioma cell line expressing Swedish mutated form of amyloid precursor protein (H4swe), CHF5074 reduced Abeta42 and Abeta40 secretion, with an IC50 of 3.6 and 18.4 microM, respectively, values consistent with those measured in the brain of the CHF5074-treated Tg2576 mice (6.4 +/- 0.4 microM). At 5 microM, no effects were observed on Notch intracellular cleavage in human embryonic kidney 293swe cells. CHF5074 was well tolerated by Tg2576 mice. No abnormal findings were observed upon histopathological examination of the gastrointestinal tract, indicating the absence of COX-related toxicity. Semiquantitative histochemical evaluation of goblet cells in the ileum of vehicle- and CHF5074-treated animals yielded similar results, suggesting no effects on Notch pathway. CHF5074 is therefore a promising therapeutic agent for Alzheimer's disease.

Fifty Hertz electromagnetic field exposure stimulates secretion of beta-amyloid peptide in cultured human neuroglioma.

Recent epidemiological studies raise the possibility that individuals with occupational exposure to low frequency (50-60 Hz) electromagnetic fields (LF-EMF), are at increased risk of Alzheimer's disease (AD). However, the mechanisms through which LF-EMF may affect AD pathology are unknown. We here tested the hypothesis that the exposure to LF-EMF may affect amyloidogenic processes. We examined the effect of exposure to 3.1 mT 50 Hz LF-EMF on Abeta secretion in H4 neuroglioma cells stably overexpressing human mutant amyloid precursor protein. We found that overnight exposure to LF-EMF induces a significant increase of amyloid-beta peptide (Abeta) secretion, including the isoform Abeta 1-42, without affecting cell survival. These findings show for the first time that exposure to LF-EMF stimulates Abeta secretion in vitro, thus alluding to a potential link between LF-EMF exposure and APP processing in the brain.

In vitro and in vivo profiling of CHF5022 and CHF5074 Two beta-amyloid1-42 lowering agents.

Long-term use of non-steroidal anti-inflammatory drugs (NSAIDs) may delay or prevent the onset of Alzheimer's disease (AD). A subset of NSAIDs, including flurbiprofen, has been shown to selectively inhibit the production of beta-amyloid(1-42) (Abeta42), independently from their cyclooxygenase (COX) inhibiting activity. We evaluated the in vitro and in vivo profiles of CHF5022 and CHF5074, two flurbiprofen analogues. The in vitro Abeta inhibiting activity was evaluated in a human neuroglioma cell line (H4) carrying the double Swedish mutation (K595N/M596L) of the human amyloid precursor protein (APPsw). The in vitro anti-COX activity was evaluated using human recombinant enzymes isolated from transfected Sf-9 cells. The in vivo pharmacokinetic and pharmacodynamic profiles of the two compounds were evaluated in young APPsw transgenic mice (Tg2576) after oral gavage (100 or 300mgkg(-1) day(-1) for 4-5 days) and after medicated diet (375ppm for 4 weeks). R-Flurbiprofen was used as comparator. In vitro, CHF5022 and CHF5074 were found to be 3- and 7-fold more potent than R-flurbiprofen in inhibiting Abeta42 secretion (IC(50)s of 92, 40 and 268microM, respectively). Differently from R-flurbiprofen, CHF5022 and CHF5074 did not affect COX-1 (at 100microM) and COX-2 (at 300microM) activity. Similarly to R-flurbiprofen, no significant alteration in the expression profile of a subset of Notch intracellular domain-responsive genes was observed with either CHF5022 or CHF5074. In Tg2576 mice, CHF5022 was well tolerated when administered by oral gavage (100mgkg(-1) day(-1) for 5 days) or by medicated diet (56mg kg(-1) day(-1) for 4 weeks). R-Flurbiprofen was poorly tolerated in the diet (32mgkg(-1) day(-1)) with 55% of the animals dying during the first week of treatment. After 4-5 days of oral gavage, CHF5022 and CHF5074 plasma and brain levels at 3h were found to increase with the dose, leading to brain concentrations of about 10% and 5% of the corresponding plasma concentrations, respectively. In animals fed for 4 weeks with compound-supplemented diet, mean plasma (580microM) and brain (20microM) Cyrillic) concentrations of CHF5022 were 8 and 15 times higher than those of R-flurbiprofen. Plasma Abeta42 concentration was dose-dependently decreased by CHF5022 and CHF5074. Brain Abeta levels (formic acid-extractable) were not significantly affected by either compound, although Abeta42 levels tended to inversely correlate (P=0.105) with CHF5022 concentration in the brain. CHF5022 and CHF5074 thus appear to have a promising in vitro and in vivo profile. This warrants further evaluation of their long-term effects on Abeta brain pathology.

Longitudinal analysis of immune cell phenotypes in early stage multiple sclerosis: distinctive patterns characterize MRI-active patients.

To investigate whether peripheral immune abnormalities are associated with brain inflammation in multiple sclerosis, and whether differences in MRI activity are paralleled by changes in leukocyte composition, we conducted a prospective longitudinal study in patients at their clinical onset. Twenty patients presenting a first inflammatory event in the central nervous system suggestive of multiple sclerosis underwent, every 45 days for one year, immunophenotyping of 98 blood cell subsets together with brain MRI and clinical evaluation. Six patients showed intense MRI activity, six patients did not display MRI activity, while the remaining 8 patients had low (i.e. intermediate) MRI activity during the follow-up. Our results show that MRI-active and MRI-inactive patients display significant differences in ten lymphocyte subsets. Among these, there are both effector (CCR7-CD45RA-CD4+ alphabeta T cells, CCR5+ gammadelta T cells) and regulatory (DN CD28+ alphabeta T cells and CD25+CD8+ alphabeta T cells) lymphocytes pertaining to the innate and the acquired arms of the immune system. Moreover, these differences were, upon employment of a class prediction procedure based on "support vector machines" algorithm utilizing leave-one-out cross validation procedures, able to correctly assign patients to their respective MRI activity group. All 6 MRI-active and 6 MRI-inactive patients were correctly classified, and, upon application of a class prediction model in an unsupervised manner to the 8 patients with intermediate MRI activity, 6 were predicted as MRI-active and 2 as MRI-inactive patients. Also, when the mean values of the first three time points (T0, T1 and T2) were used for the prediction of all patients, the selected lymphocyte subsets correctly classified 90% of patients. Sensitivity was 91.7% and specificity was 87.5%. These results provide evidence showing that brain inflammation in multiple sclerosis is associated with distinct changes in peripheral lymphocyte subsets, and raise the possibility that the identified subsets may, after adequate validation, assist in the prediction of MRI activity in the early stages of multiple sclerosis.

Nimodipine selectively stimulates beta-amyloid 1-42 secretion by a mechanism independent of calcium influx blockage.

Several lines of evidence indicate that perturbed cellular Ca2+ homeostasis may play a prominent role in synaptic dysfunction and neuronal death in Alzheimer's disease (AD), suggesting a potential benefit of drugs capable to stabilize Ca2+ homeostasis. We here investigated the effects of a panel of L-type Ca2+ channel antagonists on the secretion of the amyloid beta-peptide (Abeta), which abnormally accumulates in the senile plaques of the brain of AD patients. We found that, in primary and immortalized neuronal cells in culture, nimodipine robustly stimulated secretion (up to about four-fold at 30 microM) of the highly amyloidogenic 42-residue isoform of Abeta (Abeta42), while leaving largely unaffected total Abeta secretion. An analogous effect was also observed in vivo, as the administration of a single dose of nimodipine (10 mg/kg i.p.) induced a significant rise of Abeta42 levels in plasma of Tg2576 mice. The effect of nimodipine was independent of blockage of L-type Ca2+ channels and capacitative calcium entry. Accordingly, nimodipine effect was largely Ca2+-independent, as neither depletion nor rise of extracellular Ca2+ abolished it. Hence, by showing that the effect of nimodipine on Abeta42 production is distinct from its ability to block Ca2+-influx pathways, we provide evidence for a previously uncharacterized effect of this long known molecule also used in clinical practice.

Synthesis and biological activity of flurbiprofen analogues as selective inhibitors of beta-amyloid(1)(-)(42) secretion.

Flurbiprofen, a nonsteroidal antiinflammatory drug (NSAID), has been recently described to selectively inhibit beta-amyloid(1)(-)(42) (Abeta42) secretion, the most toxic component of the senile plaques present in the brain of Alzheimer patients. The use of this NSAID in Alzheimer's disease (AD) is hampered by a significant gastrointestinal toxicity associated with cyclooxygenase (COX) inhibition. New flurbiprofen analogues were synthesized, with the aim of increasing Abeta42 inhibitory potency while removing anti-COX activity. In vitro ADME developability parameters were taken into account in order to identify optimized compounds at an early stage of the project. Appropriate substitution patterns at the alpha position of flurbiprofen allowed for the complete removal of anti-COX activity, while modifications at the terminal phenyl ring resulted in increased inhibitory potency on Abeta42 secretion. In rats, some of the compounds appeared to be well absorbed after oral administration and to penetrate into the central nervous system. Studies in a transgenic mice model of AD showed that selected compounds significantly decreased plasma Abeta42 concentrations. These new flurbiprofen analogues represent potential drug candidates to be developed for the treatment of AD.

Dopamine inhibits responses of astroglia-enriched cultures to lipopolysaccharide via a beta-adrenoreceptor-mediated mechanism.

We here investigated the effect of the catecholaminergic neurotransmitter dopamine (DA), on the release of two major inflammatory effectors, TNF-alpha and nitric oxide, in rat astroglia-enriched cultures stimulated with the bacterial endotoxin lipopolysaccharide (LPS). Upon LPS challenge, we observed a dramatic increase in the culture medium of the TNF-alpha protein, an effect thereafter followed by an increase of nitric oxide synthase type 2 (NOS2) mRNA and, at later times, of nitrite accumulation, an index of nitric oxide (NO) production. DA substantially inhibited the release of TNF-alpha and NO evoked by LPS, an effect not mimicked by selective agonists nor prevented by selective antagonists of the DA receptors. The inhibitory effects of DA were mimicked by noradrenalin and isoproterenol and fully reverted by propranolol, a selective antagonist of the beta-adrenergic receptors. In addition, selective antagonists of beta-adrenergic receptor type 1 (metoprolol) and type 2 (ICI-118,551) counteracted the inhibitory effects of DA on LPS-induced TNF-alpha and NO release. Accordingly, agents capable of elevating intracellular cyclic 3',5'-adenosine monophosphate (cAMP), such as forskolin and dibutyryl-cAMP, mimicked DA inhibitory effects on LPS-evoked accumulation of TNF-alpha and nitrite. These data, consistent with a role of DA as local modulator of glial inflammatory responses, uncover the existence of an interaction between DA and heterologous beta-adrenergic receptors in astroglial cells.