THE STUDY OF SNAP29 IN MITOSIS AND IN CEDNIK PATHOGENESIS.

Intracellular trafficking includes a series of regulated events that allow the transport of proteins and macromolecules. A key step of intracellular trafficking is the fusion between a containing-cargo vesicle and a target membrane, mediated by Soluble N-ethylmaleimide-sensitive fusion Attachment protein REceptor (SNARE) proteins. Snap29 is a cytosolic SNARE protein containing two SNARE domains required for fusion, whose specificity and activity is unclear. During the last few years, we and others have discovered that Snap29 is a key regulator of autophagy required for fusion of autophagosomes with lysosomes, the last trafficking step before cargo degradation. During the first part of my PhD, I contributed to uncover a novel function of Snap29 using Drosophila melanogaster, as a model system. We demonstrated that during mitosis Snap29 is repurposed as an outer kinetochore component, and that its localization depends on known kinetochore proteins, but does not require membranes or the autophagy process. Depletion of Snap29 in Drosophila S2 cells leads to cell division defects, such as failure to form a proper metaphase plate and segregate chromosomes correctly, or formation of aberrant mitotic spindles, ultimately leading to generation of micronuclei, aneuploidy and cell death. In addition, we observed that Snap29 is fundamental to determine correct tissue development and homeostasis in Drosophila, since its depletion or mutation determines disorganization and multilayering in the follicular epithelium, and tumor-like tissue alterations in eye imaginal discs. Since mutations affecting autophagy genes are not sufficient per se to induce such disruptions in the epithelial architecture, we hypothesize that these defects might be due to loss of Snap29 activity during mitosis. Mutations of SNAP29 human gene cause a rare neurocutaneous syndrome called CEDNIK (Cerebral Dysgenesis, Neuropathy, Ichthyosis and Keratoderma), which causes severe neurological and dermatological congenital manifestations associated with short life expectancy. So far, the most investigated aspects of this syndrome are dermatological alterations, likely caused by the impairment of SNAP29 activity during membrane trafficking. Other symptoms such as neonatal feeding impairment, muscle hypotonia, and neurological defects were never investigated neither in human patients nor in CEDNIK animal models. To study uncharacterized CEDNIK traits, in the second part of my PhD, we took advantage of an uncharacterized snap29 mutant in zebrafish. The presence of CEDNIK traits in homozygous mutant fish, such as keratoderma and microcephaly, indicated that snap29 zebrafish mutant could be a valid CEDNIK disease model. Importantly, by studying the homozygous fish, we found that they display trigeminal nerve formation and axon branching defects, suggesting the requirement of Snap29 for correct nervous system development. Such alterations correlate with mouth opening problems and swimming difficulties, as well as feeding impairment. In addition, we are currently characterizing defects in muscle fibers organization and angiogenesis and we are assessing whether Snap29 plays a role in autophagy and cell division in vivo. Overall, our findings demonstrate that Snap29 is a key regulator of cell division and shed light on uncharacterized aspects of CEDNIK syndrome, highlighting a pivotal role of Snap29 in nervous system development

A genetic model of CEDNIK syndrome in zebrafish highlights the role of the SNARE protein Snap29 in neuromotor and epidermal development

Homozygous mutations in SNAP29, encoding a SNARE protein mainly involved in membrane fusion, cause CEDNIK (Cerebral Dysgenesis, Neuropathy, Ichthyosis and Keratoderma), a rare congenital neurocutaneous syndrome associated with short life expectancy, whose pathogenesis is unclear. Here, we report the analysis of the first genetic model of CEDNIK in zebrafish. Strikingly, homozygous snap29 mutant larvae display CEDNIK-like features, such as microcephaly and skin defects. Consistent with Snap29 role in membrane fusion during autophagy, we observe accumulation of the autophagy markers p62 and LC3, and formation of aberrant multilamellar organelles and mitochondria. Importantly, we find high levels of apoptotic cell death during early development that might play a yet uncharacterized role in CEDNIK pathogenesis. Mutant larvae also display mouth opening problems, feeding impairment and swimming difficulties. These alterations correlate with defective trigeminal nerve formation and excess axonal branching. Since the paralog Snap25 is known to promote axonal branching, Snap29 might act in opposition with, or modulate Snap25 activity during neurodevelopment. Our vertebrate genetic model of CEDNIK extends the description in vivo of the multisystem defects due to loss of Snap29 and could provide the base to test compounds that might ameliorate traits of the disease

In vitro and in vivo nutraceutical characterization of two chickpea accessions: Differential effects on hepatic lipid over-accumulation

Dietary habits are crucially important to prevent the development of lifestyle-associated diseases. Diets supplemented with chickpeas have numerous benefits and are known to improve body fat composition. The present study was undertaken to characterize two genetically and phenotypically distinct accessions, MG_13 and PI358934, selected from a global chickpea collection. Rat hepatoma FaO cells treated with a mixture of free fatty acids (FFAs) (O/P) were used as an in vitro model of hepatic steatosis. In parallel, a high-fat diet (HFD) animal model was also established. In vitro and in vivo studies revealed that both chickpea accessions showed a significant antioxidant ability. However, only MG_13 reduced the lipid over-accumulation in steatotic FaO cells and in the liver of HFD fed mice. Moreover, mice fed with HFD + MG_13 displayed a lower level of glycemia and aspartate aminotransferase (AST) than HFD mice. Interestingly, exposure to MG_13 prevented the phosphorylation of the inflammatory nuclear factor kappa beta (NF-kB) which is upregulated during HFD and known to be linked to obesity. To conclude, the comparison of the two distinct chickpea accessions revealed a beneficial effect only for the MG_13. These findings highlight the importance of studies addressing the functional characterization of chickpea biodiversity and nutraceutical properties

CSRP3 mediates polyphenols-induced cardioprotection in hypertension

Berries contain bioactive polyphenols, whose capacity to prevent cardiovascular diseases has been established recently in animal models as well in human clinical trials. However, cellular processes and molecular targets of berries polyphenols remain to be identified. The capacity of a polyphenol-enriched diet (i.e., blueberries, blackberries, raspberries, strawberry tree fruits and Portuguese crowberries berries mixture) to promote animal survival and protect cardiovascular function from salt-induced hypertension was evaluated in a chronic salt-sensitive Dahl rat model. The daily consumption of berries improved survival of Dahl/salt-sensitive rats submitted to high-salt diet and normalized their body weight, renal function and blood pressure. In addition, a prophylactic effect was observed at the level of cardiac hypertrophy and dysfunction, tissue cohesion and cardiomyocyte hypertrophy. Berries also protected the aorta from fibrosis and modulated the expression of aquaporin-1, a channel involved in endothelial water and nitric oxide permeability. Left ventricle proteomics analysis led to the identification of berries and salt metabolites targets, including cystein and glycin-rich protein 3 (CSRP3), a protein involved in myocyte cytoarchitecture. In neonatal rat ventricular cardiomyocytes, CSRP3 was validated as a target of a berries-derived polyphenol metabolite, 4-methylcatechol sulfate, at micromolar concentrations, mimicking physiological conditions of human plasma circulation. Accordingly, siRNA silencing of CSRP3 and 4-methylcatechol sulfate pretreatment reversed cardiomyocyte hypertrophy and CSRP3 overexpression induced by phenylephrine. Our systemic study clearly supports the modulation of CSRP3 by a polyphenol-rich berries diet as an efficient cardioprotective strategy in hypertension-induced heart failure

Multiple functions of the SNARE protein Snap29 in autophagy, endocytic, and exocytic trafficking during epithelial formation in Drosophila

How autophagic degradation is linked to endosomal trafficking routes is little known. Here we screened a collection of uncharacterized Drosophila mutants affecting membrane transport to identify new genes that also have a role in autophagy. We isolated a loss of function mutant in Snap29 (Synaptosomal-associated protein 29 kDa), the gene encoding the Drosophila homolog of the human protein SNAP29 and have characterized its function in vivo. Snap29 contains 2 soluble NSF attachment protein receptor (SNARE) domains and a asparagine-proline-phenylalanine (NPF motif) at its N terminus and rescue experiments indicate that both SNARE domains are required for function, whereas the NPF motif is in part dispensable. We find that Snap29 interacts with SNARE proteins, localizes to multiple trafficking organelles, and is required for protein trafficking and for proper Golgi apparatus morphology. Developing tissue lacking Snap29 displays distinctive epithelial architecture defects and accumulates large amounts of autophagosomes, highlighting a major role of Snap29 in autophagy and secretion. Mutants for autophagy genes do not display epithelial architecture or secretion defects, suggesting that the these alterations of the Snap29 mutant are unlikely to be caused by the impairment of autophagy. In contrast, we fi nd evidence of elevated levels of hop-Stat92E (hopscotch-signal transducer and activator of transcription protein at 92E) ligand, receptor, and associated signaling, which might underlie the epithelial defects. In summary, our findings support a role of Snap29 at key steps of membrane trafficking, and predict that signaling defects may contribute to the pathogenesis of cerebral dysgenesis, neuropathy, ichthyosis, and palmoplantar keratoderma (CEDNIK), a human congenital syndrome due to loss of Snap29

Association between polymorphisms of TAS2R16 and susceptibility to colorectal cancer

Background: Genetics plays an important role in the susceptibility to sporadic colorectal cancer (CRC). In the last 10 years genome-wide association studies (GWAS) have identified over 40 independent low penetrance polymorphic variants. However, these loci only explain around 1‑4% of CRC heritability, highlighting the dire need of identifying novel risk loci. In this study, we focused our attention on the genetic variability of the TAS2R16 gene, encoding for one of the bitter taste receptors that selectively binds to salicin, a natural antipyretic that resembles aspirin. Given the importance of inflammation in CRC, we tested whether polymorphic variants in this gene could affect the risk of developing this neoplasia hypothesizing a role of TAS2R16 in modulating chronic inflammation within the gut. Methods: We performed an association study using 6 tagging SNPs, (rs860170, rs978739, rs1357949, rs1525489, rs6466849, rs10268496) that cover all TAS2R16 genetic variability. The study was carried out on 1902 CRC cases and 1532 control individuals from four European countries. Results: We did not find any statistically significant association between risk of developing CRC and selected SNPs. However, after stratification by histology (colon vs. rectum) we found that rs1525489 was associated with increased risk of rectal cancer with a (Ptrend of = 0.0071). Conclusions: Our data suggest that polymorphisms within TAS2R16 gene do not have a strong influence on colon cancer susceptibility, but a possible role in rectal cancer should be further evaluated in larger cohorts

[Occupational therapy in rheumatoid arthritis: short term prospective study in patients treated with anti-TNF-alpha drugs].

Objective: to assess the effect of occupational therapy (OT) in rheumatoid arthritis (RA) patients treated with anti- TNF-alpha drugs in a short-term open controlled prospective study. Methods: 31 RA subjects [(M/F=5/26; mean age= 56 (range=28-73) years; mean disease duration= 165 (range =15- 432) months], treated with anti- TNF-alpha drugs, were allocated to OT (n=15) or control (n=16) group. We evaluated at entry and 12 weeks the following outcome parameters including Health Assessment Questionnaire (HAQ), Short-Form Health Survey (SF-36), Global Health (GH), Ritchie index, number of swollen or tender joints, pain, patient and physician disease activity, Disease Activity Score (DAS28), erythrocyte sedimentation rate (ESR), C-reactive protein CRP) and the correct adherence to items regarding activity daily living (ADL). Results: at baseline, OT and control group had similar demographic and clinical features. After 12 weeks, the changes from baseline of main outcome parameters were not significantly different between the two groups. After 12 weeks, in 7 out of 11 items regarding ADL, the percentage of patients showing a correct adherence was significantly increased in OT group only. Moreover at the end of the study, the OT group showed a correct adherence to 8 out of 11 ADL items in an higher percentage of patients respect to the control group. Conclusion: our study sustains that OT improves self-management but not main parameters of disease activity or functional capacity. Nevertheless educational intervention should be considered as a useful tool in conjunction with pharmacological treatment

Plasma BDNF levels following transcranial direct current stimulation allow prediction of synaptic plasticity and memory deficits in 3 7Tg-AD mice.

Early diagnosis of Alzheimer\u2019s disease (AD) supposedly increases the effectiveness of therapeutic interventions. However, presently available diagnostic procedures are either invasive or require complex and expensive technologies, which cannot be applied at a larger scale to screen populations at risk of AD.We were looking for a biomarker allowing to unveil a dysfunction of molecular mechanisms, which underly synaptic plasticity and memory, before the AD phenotype is manifested and investigated the effects of transcranial direct current stimulation (tDCS) in 3 x Tg-AD mice, an experimental model of AD which does not exhibit any long-term potentiation (LTP) and memory deficits at the age of 3 months (3 x Tg-AD-3M). Our results demonstrated that tDCS differentially affected 3 x Tg-AD-3M and age-matched wild-type (WT) mice. While tDCS increased LTP at CA3-CA1 synapses and memory in WT mice, it failed to elicit these effects in 3 x Tg-AD-3M mice. Remarkably, 3 x Tg-AD-3M mice did not show the tDCS-dependent increases in pCREBSer133 and pCaMKIIThr286, which were found in WT mice. Of relevance, tDCS induced a significant increase of plasma BDNF levels in WT mice, which was not found in 3 x Tg-AD-3M mice. Collectively, our results showed that plasticity mechanisms are resistant to tDCS effects in the pre-AD stage. In particular, the lack of BDNF responsiveness to tDCS in 3 x Tg-AD-3M mice suggests that combining tDCS with dosages of plasma BDNF levels may provide an easy-todetect and low-cost biomarker of covert impairment of synaptic plasticity mechanisms underlying memory, which could be clinically applicable. Testing proposed here might be useful to identify AD in its preclinical stage, allowing timely and, hopefully, more effective disease-modifying interventions

Recurrent herpes simplex virus-1 infection induces hallmarks of neurodegeneration and cognitive deficits in mice

Herpes simplex virus type 1 (HSV-1) is a DNA neurotropic virus, usually establishing latent infections in the trigeminal ganglia followed by periodic reactivations. Although numerous findings suggested potential links between HSV-1 and Alzheimer's disease (AD), a causal relation has not been demonstrated yet. Hence, we set up a model of recurrent HSV-1 infection in mice undergoing repeated cycles of viral reactivation. By virological and molecular analyses we found: i) HSV-1 spreading and replication in different brain regions after thermal stress-induced virus reactivations; ii) accumulation of AD hallmarks including amyloid-\u3b2 protein, tau hyperphosphorylation, and neuroinflammation markers (astrogliosis, IL-1\u3b2 and IL-6). Remarkably, the progressive accumulation of AD molecular biomarkers in neocortex and hippocampus of HSV-1 infected mice, triggered by repeated virus reactivations, correlated with increasing cognitive deficits becoming irreversible after seven cycles of reactivation. Collectively, our findings provide evidence that mild and recurrent HSV-1 infections in the central nervous system produce an AD-like phenotype and suggest that they are a risk factor for AD