The neuroprotective and neuroplastic potential of glutamatergic therapeutic drugs in bipolar disorder.

The monoamine hypothesis has dominated research on the pathophysiology of mood disorders as well as the development of therapeutic drugs by over half a century. Nowadays a change of perspective is taking place. The glutamate system is increasingly implicated in the pathophysiology of mood disorders. The evidence spans from animal, post-mortem, imaging, pharmacological and genome-wide association studies. Bipolar disorder has been recently re-conceptualized as a synaptic plasticity-related disorder rather than simply as a result of deficits or excesses in individual neurotransmitters. A paradigm shift from a monoamine hypothesis to a neuroplasticity hypothesis focused on glutamate may represent a substantial advancement in the research for new drugs and therapies. In this review we summarize data from clinical and pre-clinical studies that have addressed glutamatergic alterations in bipolar disorder. Along with an in-depth discussion of glutamatergic alterations in bipolar disorder, we also report available data on the neuroprotective and neuroplastic potential of the classic mood stabilizers, ketamine, and psychedelics. The glutamatergic mechanisms underlying the efficacy of these drugs are described and discussed.

Observing and modeling long-term persistence of P. noctiluca in coupled complementary marine systems (Southern Tyrrhenian Sea and Messina Strait).

In the Mediterranean Sea, the Strait of Messina (MS) is a very peculiar area, connecting highly different regions and representing a privileged observatory for an early comprehension and assessment of ecosystems shifts. It is hypothesized that the outbreaks observed near the coast of many sites in the Mediterranean Sea may be the result of transport of permanent populations of P. noctiluca in pelagic waters to the coast, caused by specific hydrodynamic conditions. By both visual observations and numerical experiments our objective is twofold: (A) to help clarify whether the basin of the Aeolian Islands Archipelago (AIA), in the Southern Tyrrhenian Sea (STS), may be the site from which large populations of P. noctiluca are transported to the MS, and (B) to evaluate whether the upwelling turbulent system of the MS can be an energetic opportunity for this species. It should offer a rich habitat without jeopardizing the overall survival of the population, that is subject to stranding due to strong currents. Although very different, the two involved ecosystems (AIA and MS ) are complementary for the success of Pelagia noctiluca life cycle. Outputs obtained by coupling the 3D hydrodynamic model (SHYFEM) with a Lagrangian particle tracking model support the hypothesis of a connectivity between these two ecosystems, particularly in the first half of the year, indicating the coastal areas around the AIA as potential optimal source location for Pelagia larval stages. We support the very attractive hypothesis that two connected systems exist, the former one favours Pelagia's reproduction and acts as a nursery and the latter favours its growth due to higher productivity. We speculate that the reproductive population of the AIA is not permanent, but is renewed every year by individuals who have fed and quickly grown in the MS and who are passively transported by downwelling along canyon "corridors".

Hypermethioninemia in Campania: Results from 10 years of newborn screening.

In the last years tandem mass spectrometry (MS/MS) has become a leading technology used for neonatal screening purposes. Newborn screening by MS/MS on dried blood spot samples (DBS) has one of its items in methionine levels: the knowledge of this parameter allows the identification of infant affected by homocystinuria (cystathionine ß-synthase, CBS, deficiency) but can also lead, as side effect, to identify cases of methionine adenosyltransferase (MAT) type I/III deficiency. We started an expanded newborn screening for inborn errors of metabolism in Campania region in 2007. Here we report our ten years experience on expanded newborn screening in identifying patients affected by hypermethioninemia. During this period we screened approximately 77,000 infants and identified two cases: one case of classical homocystinuria and one patient affected by defect of MAT I/III. In this paper we describe these patients and their biochemical follow-up and review the literature concerning worldwide newborn screening reports on incidence of CBS and MAT deficiency.

Targeted metabolomics in the expanded newborn screening for inborn errors of metabolism.

Inborn errors of metabolism are genetic disorders due to impaired activity of enzymes, transporters, or cofactors resulting in accumulation of abnormal metabolites proximal to the metabolic block, lack of essential products or accumulation of by-products. Many of these disorders have serious clinical consequences for affected neonates, and an early diagnosis allows presymptomatic treatment which can prevent severe permanent sequelae and in some cases death. Expanded newborn screening for these diseases is a promising field of targeted metabolomics. Here we report the application, between 2007 and 2014, of this approach to the identification of newborns in southern Italy at risk of developing a potentially fatal disease. The analysis of amino acids and acylcarnitines in dried blood spots by tandem mass spectrometry revealed 24 affected newborns among 45,466 infants evaluated between 48 and 72 hours of life (overall incidence: 1 : 1894). Diagnoses of newborns with elevated metabolites were confirmed by gas chromatography-mass spectrometry, biochemical studies, and genetic analysis. Five infants were diagnosed with medium-chain acyl CoA dehydrogenase deficiency, 1 with methylmalonic acidemia with homocystinuria type CblC, 2 with isolated methylmalonic acidemia, 1 with propionic acidemia, 1 with isovaleric academia, 1 with isobutyryl-CoA dehydrogenase deficiency, 1 with beta ketothiolase deficiency, 1 with short branched chain amino acid deficiency, 1 with 3-methlycrotonyl-CoA carboxylase deficiency, 1 with formimino-transferase cyclodeaminase deficiency, and 1 with cystathionine-beta-synthase deficiency. Seven cases of maternal vitamin B12 deficiency and 1 case of maternal carnitine uptake deficiency were detected. This study supports the widespread application of metabolomic-based newborn screening for these genetic diseases.

Pharmacological treatments in alcohol use disorders: state of art and new perspectives.

The main focus of this narrative review is to present and discuss the most relevant clinical data about the pharmacological therapy for alcohol use disorders. By using PubMed we conducted a review of the clinical literature on drugs related to alcohol use disorders. All data are presented following the three phases of treatment: a) from withdrawal to abstinence; b) abstinence and relapse prevention; c) reduction of alcohol consumption. Historical evidence shows that in addition to the drugs already approved for the treatment of alcoholism, there are some off-label medications as effective as the approved ones which deserve therefore further study. The treatment of the alcoholic patient always requires a multidimensional and multidisciplinary approach, directed to the specific needs of each subject in order to achieve a correct care personalization. The study of the cognitive effects of each drug and pharmacogenetics will allow us to increasingly customize therapy for each individual patient.

Imaging studies on dopamine transporter and depression: a review of literature and suggestions for future research.

We review the conflicting results from imaging studies of dopamine transporter availability in depressed patients and also discuss the heterogeneity of the variables involved. Major depression includes diverse clinical manifestations and in recent years there has been an increasing interest in the identification of homogeneous phenotypes and different clinical subtypes of depression, e.g. anhedonic depression, retarded depression, etc. In addition, the use of different radioligands and imaging techniques, diverse rating scales, together with the lack of control of clinical variables (clinical course, recent or past use of substances of abuse, etc.) make it difficult to clearly identify neuronal regions or networks with consistently abnormal structures or functions in major depressive disorder. It is probably necessary to build a shared approach between clinicians and researchers in order to identify standardized procedures to better understand the role of the dopamine transporter in depression. We outline a list of major issues and also suggest some standardized procedures in collecting clinical and imaging data on major depressed patients. Our aim is to delineate a possible "modus operandi" that would be a proposal for neuroreceptor studies on major depression.

A novel GLA mutation in a Fabry family with glucose-6-phosphate dehydrogenase deficiency.

Fabry disease is an X-linked lysosomal disease caused by mutations of the alpha-galactosidase A (GLA) gene at chromosome subband Xq22.1. To date, more than 600 genetic mutations have been identified to determine the nature and frequency of the molecular lesions causing the classical and milder variant phenotypes and for precise carrier detection. We report here a Fabry family (mother, son and daughter) where the alpha-galactosidase A defect was associated with a glucose-6-phosphate dehydrogenase (G6PD) deficiency. Mutation analysis revealed for the GLA gene the presence of a new mutation, i.e., a small deletion (c.452delA) on exon 3 and for the G6PD gene the presence of 2 mutations, p.V68M (G6PD Asahi, G6PD A+) and p.N126D (G6PD A+) on exon 3 and exon 4, respectively.

Unfolded protein response is not activated in the mucopolysaccharidoses but protein disulfide isomerase 5 is deregulated.

Mucopolysaccharidoses (MPSs) are lysosomal storage diseases (LSDs) caused by defects in lysosomal enzymes involved in the catabolism of glycosaminoglycans. The pathogenesis of these disorders is still not completely known, although inflammation and oxidative stress appear to be common mechanisms, as in all LSDs. Recently, it was hypothesized that endoplasmic reticulum (ER) stress followed by an unfolded protein response (UPR) could be another common pathogenetic mechanism in LSDs. The aim of the present study was to verify if the UPR was elicited in the mucopolysaccharidoses and if the mechanism was MPS type- and mutation-dependent. To this end, we analyzed the UPR in vitro, in fibroblasts from patients with different types of mucopolysaccharidoses (MPS I, II, IIIA, IIIB, IVA) and in vivo, in the murine MPS IIIB model. In both cases we found no changes in mRNA levels of several UPR-related genes, such as the spliced or unspliced form of Xbp-1, Bip, Chop, Edem1, Edem2, Edem3. Therefore, we report here that the unfolded protein response of the ER is not triggered either in vitro or in vivo; accordingly, cytotoxicity assays indicated that affected fibroblasts are no more sensitive to apoptosis induction than normal cells. However, our results show that in most of the analyzed MPS fibroblasts the expression of a poorly known protein belonging to the family of the protein disulfide isomerases, namely Pdia5, is upregulated; here we discuss if its upregulation could be an early event of ER stress possibly related to the severity of the damage induced in the mutant proteins.

Major depressive disorder, anhedonia and agomelatine: an open-label study.

Despite a wide range of available antidepressants, the effect of the treatment is often suboptimal and there is a need for more effective and better tolerated drugs. Unlike other antidepressants, agomelatine represents a new approach to depression with an innovative mechanism of action. It is an agonist of melatoninergic receptors MT1 and MT2 and a selective antagonist of 5-HT2c receptors. In this open-label 8-week study we aimed to investigate the efficacy of agomelatine on depressive symptoms in patients with major depression. Secondary endpoints were the effect of agomelatine on anhedonia. Thirty major depressive patients received a flexible dose (25-50 mg; per os, daily) of agomelatine. Depressive (Hamilton Depression Scale) and anxious (Hamilton Anxiety Scale) symptoms, anhedonia (Snaith Hamilton Rating Scale), and sleep quality (Leeds Sleep Evaluation Questionnaire) were assessed. Twenty-four patients (80%) completed 8 weeks of treatment. Significant improvements were seen at all visits on the HAM-D (p<.05), HAM-A(p<.01), SHAPS (p<.05), LSEQ (p<.05). Nine subjects (30%) were responders and 5 (17%) remitters at week 1; 18 (60%) were remitters by the end of the trial. There was no serious adverse event. No aminotrasferase elevations were noted. In line with previous studies, in which agomelatine was associated with early clinical improvement, this study also provides evidence of an early response and the findings of improvements in depression scores. Moreover, this is the first study where agomelatine was effective in the treatment of anhedonia. Additional trials are needed to delineate the place of agomelatine in the contemporary pharmacotherapy for depressive disorders.

Large deletion involving exon 5 of the arylsulfatase B gene caused apparent homozygosity in a mucopolysaccharidosis type VI patient.

Apparent homozygosity for the mutation p.R315X present on exon 5 of the arylsulfatase B (ARSB) gene in a mucopolysaccharidosis type VI patient was solved in this study by further testing for a second mutation. Patient cDNA analysis revealed that the entire exon 5 of the ARSB gene was lacking; this new mutation was identified as c.899-1142del. As the genomic DNA sequencing excluded the presence of splicing mutations, polymerase chain reaction analysis was performed for polymorphisms listed in the NCBI SNP database for the ARSB gene. This allowed the mutation at the genomic DNA level to be identified as g.99367-102002del; this gross deletion, involving the entire exon 5 of the gene and parts of introns 4 and 5 led to a frameshift starting at amino acid 300 and resulting in a protein with 39% amino acids different from the normal enzyme. We stress that extensive DNA analysis needs to be performed in case of apparent homozygosity to avoid potential errors in genetic counseling.

Intracranial gene delivery of LV-NAGLU vector corrects neuropathology in murine MPS IIIB.

Mucopolysacccharidosis (MPS) IIIB is an inherited lysosomal storage disorder caused by the deficiency of alpha-N-acetylglucosaminidase (NAGLU). The disease is characterized by mild somatic features and severe neurological involvement with high mortality. Although several therapeutic approaches have been applied to the murine model of the disease, no effective therapy is available for patients. In this study, we used the lentiviral-NAGLU vector to deliver the functional human NAGLU gene into the brain of young adult MPS IIIB mice. We report the restoration of active enzyme with a sustained expression throughout a large portion of the brain, and a significantly improved behavioral performance of treated animals. Moreover, we analyzed the effect of therapy on the expression profile of some genes related to neurotrophic signaling molecules and inflammatory cytokines previously found altered in MPS IIIB mice. At 1 month from treatment, the level of cerebellin 1 (Cbln1) was decreased while the brain-derived neurotrophic factor (Bdnf) expression was increased, both reaching normal values. At 6 months from treatment a significant reduction in the expression of all the inflammation- and oxidative stress-related genes was observed, as well as the maintenance of the correction of the Bdnf gene expression. These results indicate that NAGLU delivery from intracerebral sources has the capacity to alleviate most disease manifestations in MPS IIIB mice; furthermore, Bdnf might be a response-to-therapy biomarker for MPS IIIB.

Mucopolysaccharidosis IIIB: oxidative damage and cytotoxic cell involvement in the neuronal pathogenesis.

Sanfilippo B syndrome (Mucopolysaccharidosis IIIB, MPS IIIB) is a lysosomal storage disease due to mutations in the gene encoding alpha-N-acetylglucosaminidase and is characterized by a severe neurological disorder. Although several studies have been reported for the murine model of the disease, the molecular basis and the sequence of events leading to neurodegeneration remain to be clarified. We previously suggested the possible involvement of the reactive oxygen species in the disease pathogenesis. In the present paper we extended the analysis of oxidative stress by evaluating the production of superoxide ions throughout the CNS and by evaluating the effect of the stress on the cellular macromolecules. These approaches applied to one-month-old, three-month-old and six-month-old mice revealed that oxidative stress is present in the affected cerebrum and cerebellum tissues from one month from birth, and that it results primarily in protein oxidation, both in the cerebrum and cerebellum, with lipid peroxidation, and especially DNA oxidation, appearing milder and restricted essentially to the cerebellum. We also identified additional genes possibly associated with the neuropathology of MPS IIIB disease. Real time RT-PCR analysis revealed an altered expression of the Sod1, Ret, Bmp4, Tgfb, Gzmb and Prf1 genes. Since Gzmb and Prf1 are proteins secreted by NK/cytotoxic T-cells, these data suggest the involvement of cytotoxic cells in the neuronal pathogenesis. Extending our previous study, findings reported in the present paper show that oxidative stress and all the analyzed stress-related pathological changes occur very early in the disease course, most likely before one month of age.

Molecular markers for the follow-up of enzyme-replacement therapy in mucopolysaccharidosis type VI disease.

MPS VI (mucopolysaccharidosis type VI) is a lysosomal storage disease in which deficient activity of the enzyme N-acetylgalactosamine 4-sulfatase [ASB (arylsulfatase B)] impairs the stepwise degradation of the GAG (glycosaminoglycan) dermatan sulfate. Clinical studies of ERT (enzyme replacement therapy) by using rhASB (recombinant human ASB) have been reported with promising results. The release of GAG into the urine is currently used as a biomarker of disease, reflecting in some cases disease severity and in all cases therapeutic responsiveness. Using RNA studies in four Italian patients undergoing ERT, we observed that TNFalpha (tumour necrosis factor alpha) might be a biomarker for MPS VI responsive to therapy. In addition to its role as a potential biomarker, TNFalpha expression could provide insights into the possible pathophysiological mechanisms underlying the mucopolysaccharidoses.

Cytokines, neurotrophins, and oxidative stress in brain disease from mucopolysaccharidosis IIIB.

Mucopolysaccharidosis IIIB (MPS IIIB; Sanfilippo syndrome type B) is characterized by profound neurological deterioration. Because a murine model of MPS IIIB disease is available, we focused on analysis of gene expression in the brain and cerebellum of 7-month-old MPS IIIB mice by pathway-specific filter microarrays designed to probe apoptotic-related, neurotrophic signalling molecules and inflammatory cytokines and receptors. Moreover, we extended the analysis with real-time PCR performed at 1, 3, 7 months after birth. Bdnf was down-regulated in the brain but up-regulated in the cerebellum at 7 months of age, both at RNA and at protein levels. Cbln1 presented a threefold increase in the oldest brains while remaining unaltered in the cerebellum. Ccl3, Casp11, gp91(phox), p67(phox), and p47(phox) showed an increased expression in both brain and cerebellum at each examined time point. Ccl3, in particular, exhibited in both organs and at all times tested approximately a tenfold increase in its expression. Insofar as p47(phox), p67(phox), and gp91(phox) are all components of the phagocyte NADPH oxidase, our results suggest the possible involvement of the reactive oxygen species in the genesis of neurodegeneration in MPS IIIB disease.

Gene therapy for a mucopolysaccharidosis type I murine model with lentiviral-IDUA vector.

Mucopolysaccharidosis type I is a lysosomal disease due to mutations in the IDUA gene, resulting in deficiency of alpha-L-iduronidase and accumulation of glycosaminoglycans (GAGs). Bone marrow transplantation and enzyme replacement are two therapies considered only moderately successful for affected patients, making the development of novel treatments necessary. We have previously shown the efficacy of lentivirus-mediated gene transfer to correct patient fibroblasts in vitro. Here we tested lentiviral-IDUA vector gene therapy in vivo on a murine MPS I model. Eight- to 10 week-old mice were injected with increasing lentiviral doses via the tail vein and analyzed 1 month after treatment. A single injection of lentiviral-IDUA vector resulted in transgene expression in several murine tissues, with the highest level reached in liver and spleen. Expression of 1% normal activity was sufficient in treated animals to normalize the GAG level in urine, liver, and spleen and was able to reduce the GAG level in kidney, heart, and lung. Polymerase chain reaction assays showed integration of the viral genome only in liver and spleen of treated animals, suggesting that the correction of the pathology in other tissues was due to secretion into the plasma by liver and spleen and uptake of corrective enzyme by distant tissues. Long-term (6 months) analysis showed the presence of enzyme-specific antibodies and the loss of enzyme activity and vector sequence in the target tissue, suggesting that the transgene-specific immune response interfered with long-term therapeutic correction and led to clearance of transduced cells. In conclusion, our results show the promising potential and the limitations of lentiviral-IDUA vector-mediated gene therapy in an in vivo model.

Treatment of the mouse model of mucopolysaccharidosis type IIIB with lentiviral-NAGLU vector.

The Sanfilippo syndrome type B (mucopolysaccharidosis IIIB) is an autosomal recessive disorder due to mutations in the gene encoding NAGLU (alpha-N-acetylglucosaminidase), one of the enzymes required for the degradation of the GAG (glycosaminoglycan) heparan sulphate. No therapy exists for affected patients. We have shown previously the efficacy of lentiviral-NAGLU-mediated gene transfer in correcting in vitro the defect on fibroblasts of patients. In the present study, we tested the therapy in vivo on a knockout mouse model using intravenous injections. Mice (8-10 weeks old) were injected with one of the lentiviral doses through the tail vein and analysed 1 month after treatment. A single injection of lentiviral-NAGLU vector resulted in transgene expression in liver, spleen, lung and heart of treated mice, with the highest level reached in liver and spleen. Expression of 1% normal NAGLU activity in liver resulted in a 77% decrease in the GAG content; more remarkably, an expression of 0.16% normal activity in lung was capable of decreasing the GAG level by 29%. Long-term (6 months) follow up of the gene therapy revealed that the viral genome integration persisted in the target tissues, although the real-time PCR analysis showed a decrease in the vector DNA content with time. Interestingly, the decrease in GAG levels was maintained in liver, spleen, lung and heart of treated mice. These results show the promising potential and the limitations of lentiviral-NAGLU vector to deliver the human NAGLU gene in vivo.

An adult Sanfilippo type A patient with homozygous mutation R206P in the sulfamidase gene.

The Sanfilippo type A syndrome, one of the most frequent forms of mucopolysaccharidosis III, is characterized by severe mental retardation, progressive neurological degeneration, and mild somatic changes. It is due to a deficiency of heparan-N-sulfatase (sulfamidase) activity and consequent excretion of heparan sulfate in the urine. The disease is transmitted through an autosomal recessive mechanism, and more than 60 gene mutations have been identified. Up to now, only 10 cases of attenuated form of Sanfilippo type A syndrome have been described, and the specific mutation has been identified only in two of them. We report here on a female patient, 20 years old, with Sanfilippo type A syndrome presenting with a mild clinical phenotype characterized essentially by a moderate nonevolving mental retardation. The genetic analysis demonstrated that the patient is homozygous for mutation R206P; presence of polymorphism R456H was also found. This study places R206P as a mild mutation underlying Sanfilippo type A disease.

In vitro gene therapy of mucopolysaccharidosis type I by lentiviral vectors.

Mucopolysaccharidosis type I (MPS I) results from a deficiency in the enzyme alpha-L-iduronidase (IDUA), and is characterized by skeletal abnormalities, hepatosplenomegaly and neurological dysfunction. In this study, we used a late generation lentiviral vector to evaluate the utility of this vector system for the transfer and expression of the human IDUA cDNA in MPS I fibroblasts. We observed that the level of enzyme expression in transduced cells was 1.5-fold the level found in normal cells; the expression persisted for at least two months. In addition, transduced MPS I fibroblasts were capable of clearing intracellular radiolabeled glycosaminoglycan (GAG). Pulse-chase experiments on transduced fibroblasts showed that the recombinant enzyme was synthesized as a 76-kDa precursor form and processed to a 66-kDa mature form; it was released from transduced cells and was endocytosed into a second population of untreated MPS I fibroblasts via a mannose 6-phosphate receptor. These results suggest that the lentiviral vector may be used for the delivery and expression of the IDUA gene to cells in vivo for treatment of MPS I.

Correction of mucopolysaccharidosis type IIIb fibroblasts by lentiviral vector-mediated gene transfer.

Mucopolysaccharidosis type IIIB (MPS IIIB; or Sanfilippo syndrome type B) is a lysosomal disease, due to glycosaminoglycan storage caused by mutations on the alpha-N-acetylglucosaminidase (NAGLU) gene. The disease is characterized by neurological dysfunction but relatively mild somatic manifestations. No effective treatment is available for affected patients. In the present study, we evaluated the role of a lentiviral vector as the transducing agent of NAGLU cDNA in MPS IIIB fibroblasts. The vector expressed high transduction efficiency and high levels of enzymic activity, 20-fold above normal levels, persisting for at least 2 months. PCR experiments confirmed the integration of the viral vector into the target genome. The NAGLU activity restored by virus infection was sufficient to normalize glycosaminoglycan accumulation, which is directly responsible for the disease phenotype. Metabolic labelling experiments on transduced fibroblasts exhibited, in the medium and in cellular lysates, polypeptide forms of 84 and 80 kDa respectively related to the precursor and mature forms of the enzyme. The enzyme secreted by transduced MPS IIIB fibroblasts was endocytosed in deficient cells by the mannose 6-phosphate system. Thus we show that lentiviral vectors may provide a therapeutic approach for the treatment of MPS IIIB disease.

[Analysis of decline in seminal fluid in the Italian population during the past 15 years]. / Analisi del declino qualitativo del liquido seminale in Italia negli ultimi 15 anni.

BACKGROUND AND AIMS: The authors wished to study whether there has been a real decline in the quality of semen over the past years, as reported in the literature by a number of studies. This paper analyses the basic parameters of semen from 1068 males who have acted as donors during the past 15 years (1981-1995) for our semen bank. METHODS: Tables have been drawn up showing the number of cases examined every year, the mean concentration of spermatozoa, the mean percentage of mobile sperm and the mean percentage of non-conforming elements. RESULTS: An analysis of the data reported here shows that the mean concentration of spermatozoa in semen has shrunk from 88 x 10(6)/ml in 1981 to 61 x 10(6)/ml in 1995, a reduction of 30.7%; mean total motility has diminished from 74 to 66%, whereas the mean percentage with typical morphology has fallen from 76 to 63%. No statistical analysis was performed since variance analysis could not be performed on the sample in question to isolate the various sources of error. CONCLUSIONS: In conclusion, there has been a marked fall in the quality of semen over the past few years in individuals who regard themselves as fertile and are selected as semen donors. In the discussion the authors examine the various hypotheses regarding the possible causes of this deterioration in quality, opting for the argument based on the increased use of pollutants during the period in question in urban and rural areas.