Progressive retinal degeneration and glial activation in the Cln6nclf mouse model of neuronal ceroid lipofuscinosis : a beneficial effect of DHA and Curcumin supplementation

Neuronal ceroid lipofuscinosis (NCL) is a group of neurodegenerative lysosomal storage disorders characterized by vision loss, mental and motor deficits, and spontaneous seizures. Neuropathological analyses of autopsy material from NCL patients and animal models revealed brain atrophy closely associated with glial activity. Earlier reports also noticed loss of retinal cells and reactive gliosis in some forms of NCL. To study this phenomenon in detail, we analyzed the ocular phenotype of CLN6nclf mice, an established mouse model for variant-late infantile NCL. Retinal morphometry, immunohistochemistry, optokinetic tracking, electroretinography, and mRNA expression were used to characterize retinal morphology and function as well as the responses of Müller cells and microglia. Our histological data showed a severe and progressive degeneration in the CLN6nclf retina co-inciding with reactive Müller glia. Furthermore, a prominent phenotypic transformation of ramified microglia to phagocytic, bloated, and mislocalized microglial cells was identified in CLN6nclf retinas. These events overlapped with a rapid loss of visual perception and retinal function. Based on the strong microglia reactivity we hypothesized that dietary supplementation with immuno-regulatory compounds, curcumin and docosahexaenoic acid (DHA), could ameliorate microgliosis and reduce retinal degeneration. Our analyses showed that treatment of three-week-old CLN6nclf mice with either 5% DHA or 0.6% curcumin for 30 weeks resulted in a reduced number of amoeboid reactive microglia and partially improved retinal function. DHA-treatment also improved the morphology of CLN6nclf retinas with a preserved thickness of the photoreceptor layer in most regions of the retina. Our results suggest that microglial reactivity closely accompanies disease progression in the CLN6nclf retina and both processes can be attenuated with dietary supplemented immuno-modulating compounds

Relazione tecnica sulle attività della campagna oceanografica “Evatir 2015”

La campagna oceanografica “EVATIR 2015”, condotta a bordo della N/O “G. Dallaporta”, è la quinta campagna di valutazione acustica della biomassa pelagica nelle acque del Tirreno condotta dall’IAMC-CNR. La campagna è parte integrante del Progetto "Estensione della Campagna acustica Medias (Mediterranean International Acoustic Survey) nelle sub aree geografiche (GSA) 9 (Mar Ligure e Mar Tirreno settentrionale) e 10 (Mar Tirreno centrale e meridionale)" (CUP n. J52I15002440006), finanziato dal Mipaaf nell'ambito del Fondo Europeo per gli Affari Marittimi e la Pesca (FEAMP). Infatti, il FEAMP prevede un sostegno per le attività di raccolta e gestione e utilizzo dei dati, così come previsto all’art. 25, parr. 1 e 2, del Reg. (UE) 1380/2013 e ulteriormente specificato nel Reg. (CE) 199/2008. Le ricerche condotte in tale periodo sono state finalizzate principalmente alla valutazione della biomassa e della distribuzione spaziale delle popolazioni di piccoli pelagici ed allo studio delle relative condizioni ambientali. Le specie target del progetto sono l’acciuga europea (Engraulis encrasicolus) e la sardina (Sardina pilchardus), specie chiave sia a livello commerciale che ecologico. Le specie ittiche di piccoli pelagici, come sardine e acciughe, rappresentano uno dei prodotti sbarcati più importanti dalle marinerie del Mediterraneo e siciliane. Purtroppo, la gestione di queste risorse è abbastanza complicata poiché si tratta di specie a breve ciclo di vita, caratterizzate da ampie oscillazioni interannuali nella abbondanza dello stock dovuta principalmente al fallimento del reclutamento annuale. La variabilità nel reclutamento e le conseguenti oscillazioni di biomassa sono principalmente legate alla variabilità di habitat e in misura minore allo sforzo di pesca. L’obiettivo principale dei piani di monitoraggio di queste risorse si basa sulla possibilità di valutare di anno in anno le fluttuazioni di abbondanza dello stock e il conseguente livello di reclutamento, al fine di una della gestione sostenibile della pesca delle risorse stesse

Piattaforma per l’inserimento dei parametri di campionamento del Bongo e della Calvet sul Sistema Informativo di Capo Granitola

L’Istituto IAMC di Capo Granitola da più di quindici anni effettua delle campagne scientifiche a bordo di navi oceanografiche (Urania, G. Dallaporta e Minerva Uno). Nel corso dei campionamenti ittioplanctonici e zooplanctonici è previsto l’utilizzo di strumenti campionatori come retini Bongo 40, Bongo 60, Bongo 90 e Calvet. Ciò che differenzia i tre diversi strumenti “Bongo” è il diametro dei cilindri. L’obiettivo del campionamento è acquisire informazioni utili per la valutazione della distribuzione e abbondanza dei primi stadi di sviluppo delle popolazioni di piccoli pelagici e del plancton, nonché la valutazione mediante il Daily Egg Production Methods (DEPM) della biomassa dei riproduttori delle popolazioni pelagiche. Successivamente, sarà possibile correlare le stesse con dati oceanografici per la determinazione di possibili influenze derivate dalla struttura chimico- fisiche a mesoscala

Human serum Albumin-based nanoparticle-mediated in vitro gene delivery

The genetic treatment of neurodegenerative diseases still remains a challenging task since many approaches fail to deliver the therapeutic material in relevant concentrations into the brain. As viral vectors comprise the risk of immune and inflammatory responses, human serum albumin (HSA) nanoparticles were found to represent a safer and more convenient alternative. Their ability to cross the blood-brain barrier (BBB) and deliver drugs into the brain in order to enhance gene-based therapy has been previously demonstrated. The present study deals with the development of pGL3-PEI-coated HSA nanoparticles and subsequent in vitro testing in cerebellar granular and HeLa cells. The luciferase control vector pGL3 was chosen as reporter plasmid encoding for the firefly luciferase protein, linear polyethylenimine (22 kDa) as endosomolytic agent for enhancing the cells’ transfection. Studies on particle characteristics, their cellular uptake into aforementioned cell lines and on subcellular localisation, and transfection efficiency in the cerebellar cells proved the feasibility of nanoparticle-based gene delivery

Regulation of CRE-Dependent Transcriptional Activity in a Mouse Suprachiasmatic Nucleus Cell Line

We evaluated the signalling framework of immortalized cells from the hypothalamic suprachiasmatic nucleus (SCN) of the mouse. We selected a vasoactive intestinal peptide (VIP)-positive sub-clone of immortalized mouse SCN-cells stably expressing a cAMP-regulated-element (CRE)-luciferase construct named SCNCRE. We characterized these cells in terms of their status as neuronal cells, as well as for important components of the cAMP-dependent signal transduction pathway and compared them to SCN ex vivo. SCNCRE cells were treated with agents that modulate different intracellular signalling pathways to investigate their potency and timing for transcriptional CRE-dependent signalling. Several activating pathways modulate SCN neuronal signalling via the cAMP-regulated-element (CRE: TGACGCTA) and phosphorylation of transcription factors such as cAMP-regulated-element-binding protein (CREB). CRE-luciferase activity induced by different cAMP-signalling pathway-modulating agents displayed a variety of substance-specific dose and time-dependent profiles and interactions relevant to the regulation of SCN physiology. Moreover, the induction of the protein kinase C (PKC) pathway by phorbol ester application modulates the CRE-dependent signalling pathway as well. In conclusion, the cAMP/PKA- and the PKC-regulated pathways individually and in combination modulate the final CRE-dependent transcriptional output

<i>In-vitro</i> expression levels of luciferase reporter protein.

<p>Cb cells were transfected with pGL3-PEI-coated nanoparticles. The graph shows the percentage of positive wells showing luminescence on day 2, 3, 4 after washing the nanoparticles off. Well values higher than negative control average + 3x standard deviation, were considered positive. Results are shown as mean ± S.E.M. (n = 48).</p

Uptake of HSA nanoparticles and pGL3-PEI-coated HSA nanoparticles in cerebellar granule cells.

<p><b>A:</b> Flow cytometry was used to determine the autofluorescence of the HSA nanoparticles in Cb cells. Nanoparticle uptake into the cells increased with higher concentrations of used nanoparticles (0.1 mg/ml to 1.0 mg/ml) and with incubation time (18 h to 72 h). <b>B:</b> Temperature dependency of nanoparticles uptake at 4°C, 33°C and 39°C. <b>C:</b> Comparison between unmodified and pGL3-PEI-coated HSA nanoparticle uptake in Cb cells. Results are shown as mean ± S.E.M. (n = 3). <b>D:</b> Confocal microscopy images represent the uptake of nanoparticles in Cb cells at different concentrations. 0.1 mg/ml (1), 0.25 mg/ml (2), 0.5 mg/ml (3), 1 mg/ml (4). Scale bar 100 µm.</p

Degradation of pGL3-PEI-coated nanoparticles.

<p><b>A:</b> Dot blot immunoanalysis was used to evaluate the internalised HSA amount in Cb cells treated with pGL3-PEI-coated nanoparticles The HSA signal was related to the signal of the negative control (horizontal line). <b>B:</b> Similar conditions were used to evaluate the chemical degradation of pGL3-PEI-coated HSA nanoparticles with proteinase K. The HSA signal was related to the signal of non-degraded nanoparticles (horizontal line). Results are shown as mean ± S.E.M. (n = 3).</p

Particle characteristics of pGL3-PEI-coated nanoparticles, unmodified HSA nanoparticles, and pGL3-PEI complexes.

<p>Particle sizes [nm] (<b>A</b>) and surface charge [mV] (<b>B</b>) of unmodified HSA nanoparticles, pGL3-PEI complexes, and pGL3-PEI-coated HSA nanoparticles. Results are shown as mean ± S.E.M. (n = 3). (<b>C</b>) Scanning electron microscopy picture of pGL3-PEI coated HSA nanoparticles. Scale bar 700 nm.</p