Simonetta Sipione

Loss of normal huntingtin function: new developments in Huntington's disease research.

Huntingtons disease is characterized by a loss of brain striatal neurons that occurs as a consequence of an expansion of a CAG repeat in the huntingtin protein. The resulting extended polyglutamine stretch confers a deleterious gain-of-function to the protein. Analysis of the mutant protein has attracted most of the research activity in the field, however re-examination of earlier data and new results on the beneficial functions of normal huntingtin indicate that loss of the normal protein function might actually equally contribute to the pathology. Thus, complete elucidation of the physiological role(s) of huntingtin and its mode of action are essential and could lead to new therapeutic approaches.

Dysfunction of the cholesterol biosynthetic pathway in Huntington's disease.

The expansion of a polyglutamine tract in the ubiquitously expressed huntingtin protein causes Huntingtons disease (HD), a dominantly inherited neurodegenerative disease. We show that the activity of the cholesterol biosynthetic pathway is altered in HD. In particular, the transcription of key genes of the cholesterol biosynthetic pathway is severely affected in vivo in brain tissue from HD mice and in human postmortem striatal and cortical tissue; this molecular dysfunction is biologically relevant because cholesterol biosynthesis is reduced in cultured human HD cells, and total cholesterol mass is significantly decreased in the CNS of HD mice and in brain-derived ST14A cells in which the expression of mutant huntingtin has been turned on. The transcription of the genes of the cholesterol biosynthetic pathway is regulated via the activity of sterol regulatory element-binding proteins (SREBPs), and we found an ∼50% reduction in the amount of the active nuclear form of SREBP in HD cells and mouse brain tissue. As a consequence, mutant huntingtin reduces the transactivation of an SRE-luciferase construct even under conditions of SREBP overexpression or in the presence of an exogenous N-terminal active form of SREBP. Finally, the addition of exogenous cholesterol to striatal neurons expressing mutant huntingtin prevents their death in a dose-dependent manner. We conclude that the cholesterol biosynthetic pathway is impaired in HD cells, mice, and human subjects, and that the search for HD therapies should also consider cholesterol levels as both a potential target and disease biomarker.

Insulin expressing cells from differentiated embryonic stem cells are not beta cells.

Aim/hypothesisEmbryonic stem (ES) cells have been proposed as a potential source of tissue for transplantation for the treatment of Type 1 diabetes. However, studies showing differentiation of beta cells from ES cells are controversial. The aim of this study was to characterise the insulin-expressing cells differentiated in vitro from ES cells and to assess their suitability for the treatment of diabetes.MethodsES cell-derived insulin-expressing cells were characterised by means of immunocytochemistry, RT-PCR and functional analyses. Activation of the Insulin I promoter during ES-cell differentiation was assessed in ES-cell lines transfected with a reporter gene. ES cell-derived cultures were transplanted into STZ-treated SCID-beige mice and blood glucose concentrations of diabetic mice were monitored for 3 weeks.ResultsInsulin-stained cells differentiated from ES cells were devoid of typical beta-cell granules, rarely showed immunoreactivity for C-peptide and were mostly apoptotic. The main producers of proinsulin/insulin in these cultures were neurons and neuronal precursors and a reporter gene under the control of the insulin I promoter was activated in cells with a neuronal phenotype. Insulin was released into the incubation medium but the secretion was not glucose-dependent. When the cultures were transplanted in diabetic mice they formed teratomas and did not reverse the hyperglycaemic state.Conclusions/InterpretationOur studies show that insulin-positive cells in vitro-differentiated from ES cells are not beta cells and suggest that alternative protocols, based on enrichment of ES cell-derived cultures with cells of the endodermal lineage, should be developed to generate true beta cells for the treatment of diabetes.

Sphingolipids and gangliosides of the nervous system in membrane function and dysfunction

Simple sphingolipids such as ceramide and sphingomyelin (SM) as well as more complex glycosphingolipids play very important roles in cell function under physiological conditions and during disease development and progression. Sphingolipids are particularly abundant in the nervous system. Due to their amphiphilic nature they localize to cellular membranes and many of their roles in health and disease result from membrane reorganization and from lipid interaction with proteins within cellular membranes. In this review we discuss some of the functions of sphingolipids in processes that entail cellular membranes and their role in neurodegenerative diseases, with an emphasis on SM, ceramide and gangliosides.

Kinase inhibitors modulate huntingtin cell localization and toxicity.

Two serine residues within the first 17 amino acid residues of huntingtin (N17) are crucial for modulation of mutant huntingtin toxicity in cell and mouse genetic models of Huntingtons disease. Here we show that the stress-dependent phosphorylation of huntingtin at Ser13 and Ser16 affects N17 conformation and targets full-length huntingtin to chromatin-dependent subregions of the nucleus, the mitotic spindle and cleavage furrow during cell division. Polyglutamine-expanded mutant huntingtin is hypophosphorylated in N17 in both homozygous and heterozygous cell contexts. By high-content screening in live cells, we identified kinase inhibitors that modulated N17 phosphorylation and hence huntingtin subcellular localization. N17 phosphorylation was reduced by casein kinase-2 inhibitors. Paradoxically, IKKβ kinase inhibition increased N17 phosphorylation, affecting huntingtin nuclear and subnuclear localization. These data indicate that huntingtin phosphorylation at Ser13 and Ser16 can be modulated by small-molecule drugs, which may have therapeutic potential in Huntingtons disease.

Transplantation of prodrug-converting neural progenitor cells for brain tumor therapy

Since neural progenitor cells can engraft stably into brain tumors and differentiate along the neuronal and glial line, we tested the hypothesis that transplanted cytosine deaminase (CD)-expressing ST14A cells (an immortalized neural progenitor cell line) can convert locally 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU) and produce a regression of glioma tumors. ST14A, retrovirally transduced with the E. coli CD gene, showed a strong bystander effect on glioma cells as assessed by in vitro assay. Intracerebral injection of C6 glioma cells generated a rapidly growing tumoral mass. DiI prelabeled ST14A, coinjected into the rat brain with C6 glioma cells, survived in the tumoral mass up to 10 days and their number was not affected by in vivo 5-FC treatment. In contrast, a significant decrease of the glioma tumoral mass (−50%) was observed in 5-FC-treated rats. 5-FC had no effect on the tumor in the absence of CD-expressing ST14A cells. Our results support the feasibility of systems based on intratumoral transplantation of prodrug-converting cells for brain tumor therapy.

Shc signaling in differentiating neural progenitor cells

Previously we found that the availability of ShcA adapter is maximal in neural stem cells but that it is absent in mature neurons. Here we report that ShcC, unlike ShcA, is not present in neural stem/progenitor cells, but is expressed after cessation of their division and becomes selectively enriched in mature neurons. Analyses of its activity in differentiating neural stem/progenitor cells revealed that ShcC positively affects their viability and neuronal maturation via recruitment of the PI3K-Akt-Bad pathway and persistent activation of the MAPK pathway. We suggest that the switch from ShcA to ShcC modifies the responsiveness of neural stem/progenitor cells to extracellular stimuli, generating proliferation (with ShcA) or survival/differentiation (with ShcC).

The cloning and expression of a murine triacylglycerol hydrolase cDNA and the structure of its corresponding gene

A novel murine cDNA for triacylglycerol hydrolase (TGH), an enzyme that is involved in mobilization of triacylglycerol from storage pools in hepatocytes, has been cloned and expressed. The cDNA consists of 1962 bp with an open reading frame of 1695 bp that encodes a protein of 565 amino acids. Murine TGH is a member of the CES1A class of carboxylesterases and shows a significant degree of identity to other carboxylesterases from rat, monkey and human. Expression of the cDNA in McArdle RH7777 hepatoma cells showed a 3-fold increase in the hydrolysis of p-nitrophenyl laurate compared to vector-transfected cells. The highest expression of TGH was observed in the livers of mice, with lower expression in kidney, heart, adipose and intestinal (duodenum/jejunum) tissues. The murine gene that encodes TGH was cloned and exon-intron boundaries were determined. The gene spans approx. 35 kb and contains 14 exons. The results will permit future studies on the function of this gene via gene-targeting experiments and analysis of transcriptional regulation of the TGH gene.

Aberrant amplification of A2A receptor signaling in striatal cells expressing mutant huntingtin

Huntingtons disease (HD) is a neurodegenerative disorder caused by expansion of a CAG repeat in the gene encoding for Huntingtin (Htt), which results in progressive degeneration of the striatal GABAergic/enkephalin neurons. These neurons express both the A2A and D2 receptors, which stimulate and inhibit adenylyl cyclase, respectively. In this study we analyzed the possibility of an involvement of the A2A receptor and its signaling components in the pathogenesis of HD. We report here that striatal cells expressing mutant Htt exhibit increased binding affinities for the selective A2A receptor ligand 3H‐SCH‐58261. Furthermore, despite identical basal adenylyl cyclase activity in all cells, forskolin, a direct activator of this enzyme, significantly overstimulated cAMP production in mutant Htt cells with respect to parental or wild‐type Htt‐expressing cells. Michaelis‐Menten analysis of forskolin‐stimulated enzyme activity revealed a specific decrease of Km value in mutant Htt cells, indicating increased sensitivity for the substrate. Remarkably, coupling of the A2A receptor to adenylyl cyclase was also aberrantly increased. Nevertheless, in all clones, stimulation of cAMP production by 10−7 M NECA was fully counteracted by selective A2A receptor antagonists. Altogether, these data suggest that expression of mutant Htt induces an amplification of adenylyl cyclase‐transduced signals and an aberrant coupling of the A2A receptor to this transduction system. Given the involvement of adenylyl cyclase in key physiological functions, including cell growth and cell survival, we speculate that these changes may alter the susceptibility of striatal neurons to cell death and may contribute to the development of HD.

Modeling Huntington's disease in cells, flies, and mice.

A milestone in Huntington’s disease (HD) research is represented by the identification of the causative gene. With the genetics at hand, a series of transgenic cellular and animal models has been developed, which has greatly contributed to understanding of HD. All these models are described in this review, and are compared to each other, along with the information they have generated. Although the mechanism by which progressive loss of striatal neurons occurs in HD remains uncertain, hypotheses on mutant huntingtin toxicity involve impaired vescicular trafficking, transcriptional dysregulation, and/or activation of apoptotic pathways. The development of inducible HD mice has shown that neurodegeneration in HD may be at least partially blocked. Although traditionally considered a “gain-of-function” disease, the recent finding that normal huntingtin has an important role in neuronal survival suggests that loss of function of the normal protein might contribute to HD as well, also discloseing new perspectives on the therapeutical approach to the pathology.