Possibile ruolo di contaminanti ambientali nella patogenesi del diabete mellito:studio degli effetti dell'esposizione di isole pancreatiche alla 2,3,7,8-tetraclorodibenzo-p-diossina.

La diossina è un noto contaminante ambientale appartenente alla classe dei cosidetti: ''persistent organic pollutants '' che si forma come prodotto secondario in processi industriali quali l'incenerimento dei rifiuti e la produzione di pesticidi. Essa, pertanto, può raggiungere tutti gli individui di una popolazione inducendo effetti tossici diffusi e persistenti. Numerosi studi epidemiologici hanno evidenziato una correlazione positiva fra esposizione alla diossina e insorgenza del diabete, malattia la cui incidenza è in continua crescita. Sulla base dei risultati ottenuti in precedenza su una linea cellulare β-pancreatica insulino-secernente, in questo lavoro di tesi, lo scopo è stato quello di studiare gli effetti tossici della diossina ed il relativo meccanismo di azione in un sistema sperimentale più vicino all'ambito fisiologico: le isole di Langerhans isolate dal pancreas del ratto. Le isole pancreatiche rappresentano, infatti, un vero e proprio organo endocrino in miniatura, al cui interno la funzione delle cellule β è regolata in modo più appropriato dagli stimoli fisiologici rispetto alla linea cellulare ed è anche influenzata dal continuo scambio di segnali chimici con gli altri tipi cellulari presenti all'interno dell'isola stessa. Inoltre, è stata studiata la possibilità di proteggere le isole dagli effetti della diossina (TCDD) utilizzando l' epigallocatechina-3-gallato che è capace di legarsi allo stesso recettore citoplasmatico (AhR) cui si lega la diossina. Le isole pancreatiche di ratto sono state isolate con il metodo della collagenasi, purificate su gradiente ed esposte per tempi diversi a diverse concentrazioni di 2,3,7,8-tetraclorodibenzo-p-diossina. Al termine del periodo di esposizione sono state valutate: la necrosi e l'apoptosi (mediante tecniche ELISA), le alterazioni ultrastrutturali (mediante microscopia elettronica) e le alterazioni della funzione secretoria stimolata da glucosio (mediante esperimenti di incubazione statica) e le alterazioni dell'espressione di geni funzionali e di geni pro- e anti-apoptotici (mediante tecniche di biologia molecolare). I risultati mostrano che quando le isole vengono incubate per 24h in presenza di varie concentrazioni di TCDD (1-50 nM), si ha un incremento dose-dipendente sia dell'apoptosi che della necrosi. Queste ultime vengono completamente prevenute in presenza dell' epigallocatechina-3-gallato. L'incubazione per 1 o 6 h con varie concentrazioni di TCDD determina una diminuzione significativa del rilascio di insulina stimolato da glucosio, mentre non risultano modificati il rilascio basale di insulina e il contenuto dell'ormone delle isole isolate. L'esposizione alla TCDD non influenza l'espressione del gene funzionale che codifica per l’insulina, mentre determina un aumento di geni pro-apoptotici quali PUMA, DP5, Bim. Concludendo, i risultati ottenuti mostrano che la diossina altera sia la funzione che la sopravvivenza delle isole di Langerhans isolate, fornendo un sostegno decisivo all'ipotesi, finora fondata solo su dati epidemiologici, che l'esposizione a contaminanti ambientali di tipo diossinico, anche a dosi molto basse, possa costituire un rilevante fattore di rischio nell'insorgenza del diabete di tipo 2

ERK1 and ERK2 mitogen-activated protein kinases affect Ras-dependent cell signaling differentially

BACKGROUND: The mitogen-activated protein (MAP) kinases p44(ERK1 )and p42(ERK2 )are crucial components of the regulatory machinery underlying normal and malignant cell proliferation. A currently accepted model maintains that ERK1 and ERK2 are regulated similarly and contribute to intracellular signaling by phosphorylating a largely common subset of substrates, both in the cytosol and in the nucleus. RESULTS: Here, we show that ablation of ERK1 in mouse embryo fibroblasts and NIH 3T3 cells by gene targeting and RNA interference results in an enhancement of ERK2-dependent signaling and in a significant growth advantage. By contrast, knockdown of ERK2 almost completely abolishes normal and Ras-dependent cell proliferation. Ectopic expression of ERK1 but not of ERK2 in NIH 3T3 cells inhibits oncogenic Ras-mediated proliferation and colony formation. These phenotypes are independent of the kinase activity of ERK1, as expression of a catalytically inactive form of ERK1 is equally effective. Finally, ectopic expression of ERK1 but not ERK2 is sufficient to attenuate Ras-dependent tumor formation in nude mice. CONCLUSION: These results reveal an unexpected interplay between ERK1 and ERK2 in transducing Ras-dependent cell signaling and proliferation. Whereas ERK2 seems to have a positive role in controlling normal and Ras-dependent cell proliferation, ERK1 probably affects the overall signaling output of the cell by antagonizing ERK2 activity

Naringenin Ameliorates Drosophila ReepA Hereditary Spastic Paraplegia-Linked Phenotypes

Defects in the endoplasmic reticulum (ER) membrane shaping and interaction with other organelles seem to be a crucial mechanism underlying Hereditary Spastic Paraplegia (HSP) neurodegeneration. REEP1, a transmembrane protein belonging to TB2/HVA22 family, is implicated in SPG31, an autosomal dominant form of HSP, and its interaction with Atlastin/SPG3A and Spastin/SPG4, the other two major HSP linked proteins, has been demonstrated to play a crucial role in modifying ER architecture. In addition, the Drosophila ortholog of REEP1, named ReepA, has been found to regulate the response to ER neuronal stress. Herein we investigated the role of ReepA in ER morphology and stress response. ReepA is upregulated under stress conditions and aging. Our data show that ReepA triggers a selective activation of Ire1 and Atf6 branches of Unfolded Protein Response (UPR) and modifies ER morphology. Drosophila lacking ReepA showed Atf6 and Ire1 activation, expansion of ER sheet-like structures, locomotor dysfunction and shortened lifespan. Furthermore, we found that naringenin, a flavonoid that possesses strong antioxidant and neuroprotective activity, can rescue the cellular phenotypes, the lifespan and locomotor disability associated with ReepA loss of function. Our data highlight the importance of ER homeostasis in nervous system functionality and HSP neurodegenerative mechanisms, opening new opportunities for HSP treatment

Further evidence supporting the role of GTDC1 in glycine metabolism and neurodevelopmental disorders

Copy number variants (CNVs) represent the genetic cause of about 15-20% of neurodevelopmental disorders (NDDs). We identified a similar to 67 kb de novo intragenic deletion on chromosome 2q22.3 in a female individual showing a developmental encephalopathy characterised by epilepsy, severe intellectual disability, speech delay, microcephaly, and thin corpus callosum with facial dysmorphisms. The microdeletion involved exons 5-6 of GTDC1, encoding a putative glycosyltransferase, whose expression is particularly enriched in the nervous system. In a previous study, a balanced de novo translocation encompassing GTDC1 was reported in a male child with global developmental delay and delayed speech and language development. Based on these premises, we explored the transcriptomic profile of our proband to evaluate the functional consequences of the novel GTDC1 de novo intragenic deletion in relation to the observed neurodevelopmental phenotype. RNA-seq on the proband's lymphoblastoid cell line (LCL) showed expression changes of glycine/serine and cytokine/chemokine signalling pathways, which are related to neurodevelopment and epileptogenesis. Subsequent analysis by ELISA (enzyme-linked immunosorbent assay) and HPLC (high-performance liquid chromatography) revealed increased levels of glycine in the proband's LCL and serum compared to matched controls. Given that an increased level of glycine has been observed in the plasma samples of individuals with Rett syndrome, a condition sharing epilepsy, microcephaly, and intellectual disability with our proband, we proposed that the GTDC1 downregulation is implicated in neurodevelopmental impairment by altering glycine metabolism. Furthermore, our findings expanded the phenotypic spectrum of the novel GTDC1-related condition, including microcephaly and epilepsy among relevant clinical features

The Fine Tuning of Drp1-Dependent Mitochondrial Remodeling and Autophagy Controls Neuronal Differentiation

Mitochondria play a critical role in neuronal function and neurodegenerative disorders, including Alzheimer’s, Parkinson’s and Huntington diseases and amyotrophic lateral sclerosis, that show mitochondrial dysfunctions associated with excessive fission and increased levels of the fission protein dynamin-related protein 1 (Drp1). Our data demonstrate that Drp1 regulates the transcriptional program induced by retinoic acid (RA), leading to neuronal differentiation. When Drp1 was overexpressed, mitochondria underwent remodeling but failed to elongate and this enhanced autophagy and apoptosis. When Drp1 was blocked during differentiation by overexpressing the dominant negative form or was silenced, mitochondria maintained the same elongated shape, without remodeling and this increased cell death. The enhanced apoptosis, observed with both fragmented or elongated mitochondria, was associated with increased induction of unfolded protein response (UPR) and ER-associated degradation (ERAD) processes that finally affect neuronal differentiation. These findings suggest that physiological fission and mitochondrial remodeling, associated with early autophagy induction are essential for neuronal differentiation. We thus reveal the importance of mitochondrial changes to generate viable neurons and highlight that, rather than multiple parallel events, mitochondrial changes, autophagy and apoptosis proceed in a stepwise fashion during neuronal differentiation affecting the nuclear transcriptional program

Drp1 overexpression induces desmin disassembling and drives kinesin-1 activation promoting mitochondrial trafficking in skeletal muscle

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Hereditary Spastic Paraplegia and Future Therapeutic Directions: Beneficial Effects of Small Compounds Acting on Cellular Stress

Hereditary spastic paraplegia (HSP) is a group of inherited neurodegenerative conditions that share a characteristic feature of degeneration of the longest axons within the corticospinal tract, which leads to progressive spasticity and weakness of the lower limbs. Mutations of over 70 genes produce defects in various biological pathways: axonal transport, lipid metabolism, endoplasmic reticulum (ER) shaping, mitochondrial function, and endosomal trafficking. HSPs suffer from an adequate therapeutic plan. Currently the treatments foreseen for patients affected by this pathology are physiotherapy, to maintain the outgoing tone, and muscle relaxant therapies for spasticity. Very few clinical studies have been conducted, and it's urgent to implement preclinical animal studies devoted to pharmacological test and screening, to expand the rose of compounds potentially attractive for clinical trials. Small animal models, such as Drosophila melanogaster and zebrafish, have been generated, analyzed, and used as preclinical model for screening of compounds and their effects. In this work, we briefly described the role of HSP-linked proteins in the organization of ER endomembrane system and in the regulation of ER homeostasis and stress as a common pathological mechanism for these HSP forms. We then focused our attention on the pharmacodynamic and pharmacokinetic features of some recently identified molecules with antioxidant property, such as salubrinal, guanabenz, N-acetyl cysteine, methylene blue, rapamycin, and naringenin, and on their potential use in future clinical studies. Expanding the models and the pharmacological screening for HSP disease is necessary to give an opportunity to patients and clinicians to test new molecules