Marzia Tartari

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.

Normal huntingtin function: an alternative approach to Huntington's disease

Several neurological diseases are characterized by the altered activity of one or a few ubiquitously expressed cell proteins, but it is not known how these normal proteins turn into harmful executors of selective neuronal cell death. We selected huntingtin in Huntingtons disease to explore this question because the dominant inheritance pattern of the disease seems to exclude the possibility that the wild-type protein has a role in the natural history of this condition. However, even in this extreme case, there is considerable evidence that normal huntingtin is important for neuronal function and that the activity of some of its downstream effectors, such as brain-derived neurotrophic factor, is reduced in Huntingtons disease.

Widespread Disruption of Repressor Element-1 Silencing Transcription Factor/Neuron-Restrictive Silencer Factor Occupancy at Its Target Genes in Huntington's Disease

Huntingtin is a protein that is mutated in Huntingtons disease (HD), a dominant inherited neurodegenerative disorder. We previously proposed that, in addition to the gained toxic activity of the mutant protein, selective molecular dysfunctions in HD may represent the consequences of the loss of wild-type protein activity. We first reported that wild-type huntingtin positively affects the transcription of the brain-derived neurotrophic factor (BDNF) gene, a cortically derived survival factor for the striatal neurons that are mainly affected in the disease. Mutation in huntingtin decreases BDNF gene transcription. One mechanism involves the activation of repressor element 1/neuron-restrictive silencer element (RE1/NRSE) located within the BDNF promoter. We now show that increased binding of the RE1 silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) repressor occurs at multiple genomic RE1/NRSE loci in HD cells, in animal models, and in postmortem brains, resulting in a decrease of RE1/NRSE-mediated gene transcription. The same molecular phenotype is produced in cells and brain tissue depleted of endogenous huntingtin, thereby directly validating the loss-of-function hypothesis of HD. Through a ChIP (chromatin immunoprecipitation)-on-chip approach, we examined occupancy of multiple REST/NRSF target genes in the postmortem HD brain, providing the first example of the application of this technology to neurodegenerative diseases. Finally, we show that attenuation of REST/NRSF binding restores BDNF levels, suggesting that relief of REST/NRSF mediated repression can restore aberrant neuronal gene transcription in HD.

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.

Systematic assessment of BDNF and its receptor levels in human cortices affected by Huntington's disease.

One cardinal feature of Huntingtons disease (HD) is the degeneration of striatal neurons, whose survival greatly depends on the binding of cortical brain‐derived neurotrophic factor (BDNF) with high‐affinity (TrkB) and low‐affinity neurotrophin receptors [p75 pan‐neurotrophin receptor (p75NTR)]. With a few exceptions, results obtained in HD mouse models demonstrate a reduction in cortical BDNF mRNA and protein, although autopsy data from a limited number of human HD cortices are conflicting. These studies indicate the presence of defects in cortical BDNF gene transcription and transport to striatum. We provide new evidence indicating a significant reduction in BDNF mRNA and protein in the cortex of 20 HD subjects in comparison with 17 controls, which supports the hypothesis of impaired BDNF production in human HD cortex. Analyses of the BDNF isoforms show that transcription from BDNF promoter II and IV is down‐regulated in human HD cortex from an early symptomatic stage. We also found that TrkB mRNA levels are reduced in caudate tissue but not in the cortex, whereas the mRNA levels of T‐Shc (a truncated TrkB isoform) and p75NTR are increased in the caudate. This indicates that, in addition to the reduction in BDNF mRNA, there is also unbalanced neurotrophic receptor signaling in HD.

Calcium Homeostasis and Mitochondrial Dysfunction in Striatal Neurons of Huntington Disease

Dysfunctions of Ca2+ homeostasis and of mitochondria have been studied in immortalized striatal cells from a commonly used Huntington disease mouse model. Transcriptional changes in the components of the phosphatidylinositol cycle and in the receptors for myo-inositol trisphosphate-linked agonists have been found in the cells and in the striatum of the parent Huntington disease mouse. The overall result of the changes is to delay myo-inositol trisphosphate production and to decrease basal Ca2+ in mutant cells. When tested directly, mitochondria in mutant cells behave nearly normally, but are unable to handle large Ca2+ loads. This appears to be due to the increased Ca2+ sensitivity of the permeability transition pore, which dissipates the membrane potential, prompting the release of accumulated Ca2+. Harmful reactive oxygen species, which are produced by defective mitochondria and may in turn stress them, increase in mutant cells, particularly if the damage to mitochondria is artificially exacerbated, for instance with complex II inhibitors. Mitochondria in mutant cells are thus peculiarly vulnerable to stresses induced by Ca2+ and reactive oxygen species. The observed decrease of cell Ca2+ could be a compensatory attempt to prevent the Ca2+ stress that would irreversibly damage mitochondria and eventually lead to cell death.

RESEARCH ARTICLE: Systematic Assessment of BDNF and Its Receptor Levels in Human Cortices Affected by Huntington's Disease

One cardinal feature of Huntingtons disease (HD) is the degeneration of striatal neurons, whose survival greatly depends on the binding of cortical brain‐derived neurotrophic factor (BDNF) with high‐affinity (TrkB) and low‐affinity neurotrophin receptors [p75 pan‐neurotrophin receptor (p75NTR)]. With a few exceptions, results obtained in HD mouse models demonstrate a reduction in cortical BDNF mRNA and protein, although autopsy data from a limited number of human HD cortices are conflicting. These studies indicate the presence of defects in cortical BDNF gene transcription and transport to striatum. We provide new evidence indicating a significant reduction in BDNF mRNA and protein in the cortex of 20 HD subjects in comparison with 17 controls, which supports the hypothesis of impaired BDNF production in human HD cortex. Analyses of the BDNF isoforms show that transcription from BDNF promoter II and IV is down‐regulated in human HD cortex from an early symptomatic stage. We also found that TrkB mRNA levels are reduced in caudate tissue but not in the cortex, whereas the mRNA levels of T‐Shc (a truncated TrkB isoform) and p75NTR are increased in the caudate. This indicates that, in addition to the reduction in BDNF mRNA, there is also unbalanced neurotrophic receptor signaling in HD.

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 evolutionary recent neuroepithelial cell adhesion function of huntingtin implicates ADAM10-Ncadherin

The Huntingtons disease gene product, huntingtin, is indispensable for neural tube formation, but its role is obscure. We studied neurulation in htt-null embryonic stem cells and htt-morpholino zebrafish embryos and found a previously unknown, evolutionarily recent function for this ancient protein. We found that htt was essential for homotypic interactions between neuroepithelial cells; it permitted neurulation and rosette formation by regulating metalloprotease ADAM10 activity and Ncadherin cleavage. This function was embedded in the N terminus of htt and was phenocopied by treatment of htt knockdown zebrafish with an ADAM10 inhibitor. Notably, in htt-null cells, reversion of the rosetteless phenotype occurred only with expression of evolutionarily recent htt heterologues from deuterostome organisms. Conversely, all of the heterologues that we tested, including htt from Drosophila melanogaster and Dictyostelium discoideum, exhibited anti-apoptotic activity. Thus, anti-apoptosis may have been one of htts ancestral function(s), but, in deuterostomes, htt evolved to acquire a unique regulatory activity for controlling neural adhesion via ADAM10-Ncadherin, with implications for brain evolution and development.

Co‐localization of brain‐derived neurotrophic factor (BDNF) and wild‐type huntingtin in normal and quinolinic acid‐lesioned rat brain

Loss of huntingtin‐mediated brain‐derived neurotrophic factor (BDNF) gene transcription has been described in Huntingtons disease (HD) [Zuccato et al. (2001) Science, 293, 493–498]. It has been shown that BDNF is synthesized in the pyramidal layer of cerebral cortex and released in the striatum [Altar et al. (1997) Nature, 389, 856–860; Conner et al. (1997) J. Neurosci., 17, 2295–2313]. Here we show the cellular localization of BDNF in huntingtin‐containing neurons in normal rat brain; our double‐label immunofluorescence study shows that huntingtin and BDNF are co‐contained in ≈99% of pyramidal neurons of motor cortex. In the striatum, huntingtin is expressed in 75% of neurons containing BDNF. In normal striatum we also show that BDNF is contained in cholinergic and in NOS‐containing interneurons, which are relatively resistant to HD degeneration. Furthermore, we show a reduction in huntingtin and in BDNF immunoreactivity in cortical neurons after striatal excitotoxic lesion. Our data are confirmed by an ELISA study of BDNF and by a Western blot analysis of huntingtin in cortex of quinolic acid (QUIN)‐lesioned hemispheres. In the lesioned striatum we describe that the striatal subpopulation of cholinergic neurons, surviving degeneration, contain BDNF. The finding that BDNF is contained in nearly all neurons that contain huntingtin in the normal cortex, along with the reduced expression of BDNF after QUIN injection of the striatum, shows that huntingtin may be required for BDNF production in cortex.