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

Altered Expression of Ganglioside Metabolizing Enzymes Results in GM3 Ganglioside Accumulation in Cerebellar Cells of a Mouse Model of Juvenile Neuronal Ceroid Lipofuscinosis

Juvenile neuronal ceroid lipofuscinosis (JNCL) is caused by mutations in the CLN3 gene. Most JNCL patients exhibit a 1.02 kb genomic deletion removing exons 7 and 8 of this gene, which results in a truncated CLN3 protein carrying an aberrant C-terminus. A genetically accurate mouse model (Cln3Δex7/8 mice) for this deletion has been generated. Using cerebellar precursor cell lines generated from wildtype and Cln3Δex7/8 mice, we have here analyzed the consequences of the CLN3 deletion on levels of cellular gangliosides, particularly GM3, GM2, GM1a and GD1a. The levels of GM1a and GD1a were found to be significantly reduced by both biochemical and cytochemical methods. However, quantitative high-performance liquid chromatography analysis revealed a highly significant increase in GM3, suggesting a metabolic blockade in the conversion of GM3 to more complex gangliosides. Quantitative real-time PCR analysis revealed a significant reduction in the transcripts of the interconverting enzymes, especially of β-1,4-N-acetyl-galactosaminyl transferase 1 (GM2 synthase), which is the enzyme converting GM3 to GM2. Thus, our data suggest that the complex a-series gangliosides are reduced in Cln3Δex7/8 mouse cerebellar precursor cells due to impaired transcription of the genes responsible for their synthesis

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

FRET-assisted determination of CLN3 membrane topology

Juvenile neuronal ceroid lipofuscinosis (JNCL) is caused by mutations in the CLN3 gene, which encodes for a putative lysosomal transmembrane protein with thus far undescribed structure and function. Here we investigate the membrane topology of human CLN3 protein with a combination of advanced molecular cloning, spectroscopy, and in silico computation. Using the transposomics cloning method we first created a library of human CLN3 cDNA clones either with a randomly inserted eGFP, a myc-tag, or both. The functionality of the clones was evaluated by assessing their ability to revert a previously reported lysosomal phenotype in immortalized cerebellar granular cells derived from Cln3Δex7/8 mice (CbCln3Δex7/8). The double-tagged clones were expressed in HeLa cells, and FRET was measured between the donor eGFP and an acceptor DyLight547 coupled to a monoclonal α-myc antibody to assess their relative membrane orientation. The data were used together with previously reported experimental data to compile a constrained membrane topology model for hCLN3 using TOPCONS consensus membrane prediction algorithm. Our model with six transmembrane domains and cytosolic N- and C-termini largely agrees with those previously suggested but differs in terms of the transmembrane domain positions as well as in the size of the luminal loops. This finding improves understanding the function of the native hCLN3 protein

Flotillins Regulate Focal Adhesions by Interacting with α-Actinin and by Influencing the Activation of Focal Adhesion Kinase

Cell–matrix adhesion and cell migration are physiologically important processes that also play a major role in cancer spreading. In cultured cells, matrix adhesion depends on integrin-containing contacts such as focal adhesions. Flotillin-1 and flotillin-2 are frequently overexpressed in cancers and are associated with poor survival. Our previous studies have revealed a role for flotillin-2 in cell–matrix adhesion and in the regulation of the actin cytoskeleton. We here show that flotillins are important for cell migration in a wound healing assay and influence the morphology and dynamics of focal adhesions. Furthermore, anchorage-independent growth in soft agar is enhanced by flotillins. In the absence of flotillins, especially flotillin-2, phosphorylation of focal adhesion kinase and extracellularly regulated kinase is diminished. Flotillins interact with α-actinin, a major regulator of focal adhesion dynamics. These findings are important for understanding the molecular mechanisms of how flotillin overexpression in cancers may affect cell migration and, especially, enhance metastasis formation

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

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

<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

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

Illustration of the novel topology models of full-length and truncated forms of human CLN3.

<p>Myc-tag and eGFP insertion sites are illustrated on the new membrane topology model for the full-length human CLN3 (left) generated with Protter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102593#pone.0102593-Omasits1" target="_blank">[39]</a>. The corresponding topology of the most common truncated hCLN3 protein without insertion sites is shown on the right. The line indicates a common epitope used to generate antibodies used earlier <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102593#pone.0102593-Kyttala1" target="_blank">[17]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102593#pone.0102593-Ezaki1" target="_blank">[21]</a>.</p