Donato Goffredo

Huntingtin interacts with REST/NRSF to modulate the transcription of NRSE-controlled neuronal genes.

Huntingtin protein is mutated in Huntington disease. We previously reported that wild-type but not mutant huntingtin stimulates transcription of the gene encoding brain-derived neurotrophic factor (BDNF; ref. 2). Here we show that the neuron restrictive silencer element (NRSE) is the target of wild-type huntingtin activity on BDNF promoter II. Wild-type huntingtin inhibits the silencing activity of NRSE, increasing transcription of BDNF. We show that this effect occurs through cytoplasmic sequestering of repressor element-1 transcription factor/neuron restrictive silencer factor (REST/NRSF), the transcription factor that binds to NRSE. In contrast, aberrant accumulation of REST/NRSF in the nucleus is present in Huntington disease. We show that wild-type huntingtin coimmunoprecipitates with REST/NRSF and that less immunoprecipitated material is found in brain tissue with Huntington disease. We also report that wild-type huntingtin acts as a positive transcriptional regulator for other NRSE-containing genes involved in the maintenance of the neuronal phenotype. Consistently, loss of expression of NRSE-controlled neuronal genes is shown in cells, mice and human brain with Huntington disease. We conclude that wild-type huntingtin acts in the cytoplasm of neurons to regulate the availability of REST/NRSF to its nuclear NRSE-binding site and that this control is lost in the pathology of Huntington disease. These data identify a new mechanism by which mutation of huntingtin causes loss of transcription of neuronal genes.

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.

Calcium-dependent cleavage of endogenous wild-type huntingtin in primary cortical neurons.

Huntingtons disease (HD) is caused by a polyglutamine expansion in the amino-terminal region of huntingtin. Mutant huntingtin is proteolytically cleaved by caspases, generating amino-terminal aggregates that are toxic for cells. The addition of calpains to total brain homogenates also leads to cleavage of wild-type huntingtin, indicating that proteolysis of mutant and wild-type huntingtin may play a role in HD. Here we report that endogenous wild-type huntingtin is promptly cleaved by calpains in primary neurons. Exposure of primary neurons to glutamate or 3-nitropropionic acid increases intracellular calcium concentration, leading to loss of intact full-length wild-type huntingtin. This cleavage could be prevented by calcium chelators and calpain inhibitors. Degradation of wild-type huntingtin by calcium-dependent proteases thus occurs in HD neurons, leading to loss of wild-type huntingtin neuroprotective activity.

An optimized experimental strategy for efficient conversion of embryonic stem (ES)-derived mouse neural stem (NS) cells into a nearly homogeneous mature neuronal population

NS cells are a homogeneous population of neural stem cells which were previously derived from embryonic stem cells as well as from the fetal and adult brain. Our previous reports have described a 21 day long neuronal differentiation protocol able to reproducibly convert adult SVZ-derived NS (aNS) cells into a population composed of 65% mature neurons and 35% glial cells. Here we have developed a different procedure specifically applicable to ES-derived NS cells in order to fully explore their neurogenic capacity. Differently from the aNS differentiation procedure, optimized neuronal output from ES-derived NS cells requires replating of the cells on appropriate substrates followed by sequential exposure to modified media. In these conditions, ES-derived NS cells differentiate into neurons with a barely appreciable quota of astrocytes and occasional oligodendrocytes. In particular, 21 days after the beginning of the treatment, 85% of the cells has differentiated into molecularly and electrophysiologically mature neurons belonging to the GABAergic lineage. The procedure, which is applicable with no considerable differences to different ES-derived NS cell lines and to NS cells at different passages, opens to the possibility of molecular and biochemical studies on close-to-uniform stem cell derived neurons.

Differentiating embryonic stem-derived neural stem cells show a maturation-dependent pattern of voltage-gated sodium current expression and graded action potentials.

A population of mouse embryonic stem (ES)-derived neural stem cells (named NS cells) that exhibits traits reminiscent of radial glia-like cell population and that can be homogeneously expanded in monolayer while remaining stable and highly neurogenic over multiple passages has been recently discovered. This novel population has provided a unique in vitro system in which to investigate physiological events occurring as stem cells lose multipotency and terminally differentiate. Here we analysed the timing, quality and quantity of the appearance of the excitability properties of differentiating NS cells which have been long-term expanded in vitro. To this end, we studied the biophysical properties of voltage-dependent Na(+) currents as an electrophysiological readout for neuronal maturation stages of differentiating NS cells toward the generation of fully functional neurons, since the expression of neuronal voltage-gated Na(+) channels is an essential hallmark of neuronal differentiation and crucial for signal transmission in the nervous system. Using the whole cell and single-channel cell-attached variations of the patch-clamp technique we found that the Na(+) currents in NS cells showed substantial electrophysiological changes during in vitro neuronal differentiation, consisting mainly in an increase of Na(+) current density and in a shift of the steady-state activation and inactivation curves toward more negative and more positive potentials respectively. The changes in the Na(+) channel system were closely related with the ability of differentiating NS cells to generate action potentials, and could therefore be exploited as an appropriate electrophysiological marker of ES-derived NS cells undergoing functional neuronal maturation.

Setting the conditions for efficient, robust and reproducible generation of functionally active neurons from adult subventricular zone-derived neural stem cells.

Although new culture conditions enable homogeneous and long-term propagation of radial glia-like neural stem (NS) cells in monolayer and serum-free conditions, the efficiency of the conversion of NS cells into terminally differentiated, functionally mature neurons is relatively limited and poorly characterized. We demonstrate that NS cells derived from adult mouse subventricular zone robustly develop properties of mature neurons when exposed to an optimized neuronal differentiation protocol. A high degree of cell viability was preserved. At 22 days in vitro, most cells (65%) were microtubule-associated protein 2+ and coexpressed γ-aminobutyric acid (GABA), GAD67, calbindin and parvalbumin. Nearly all neurons exhibited sodium, potassium and calcium currents, and 70% of them fired action potentials. These neurons expressed functional GABAA receptors, whereas activable kainate, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartic acid receptors were present in approximately 80, 30 and 2% of cells, respectively. Antigenic and functional properties were efficiently and reliably reproduced across experiments and cell passages (up to 68). This is the first report showing a consistent and reproducible generation of large amounts of neurons from long-term passaged adult neural stem cells. Remarkably, the neuronal progeny carries a defined set of antigenic, biochemical and functional characteristics that make this system suitable for studies of NS cell biology as well as for genetic and chemical screenings.

p66ShcA adaptor molecule accelerates ES cell neural induction

SHC genes codify for a family of adaptor molecules comprising four genes. Previous data have implicated the Shc(s) molecules in stem cell division and differentiation. Specifically, the p66(ShcA) isoform has been found to contribute to longevity and resistance from oxidative stress. Here we report that p66(ShcA) is up-regulated during in vitro neural induction in embryonic stem cells. p66(ShcA) over-expression in ES cells reduces GSK-3beta kinase activation and increases beta-catenin stabilization and its transcriptional activity. p66(ShcA) over-expression results in ES cells undergoing an anticipated neural induction and accelerated neuronal differentiation. Similar effects are obtained in human ES cells over-expressing p66(ShcA). This study reveals a role for p66(ShcA) in the modulation of Wnt/beta-catenin pathway and in ES cell neuralization which is consistent between mouse and human.