Scott Zeitlin

Increased apoptosis and early embryonic lethality in mice nullizygous for the Huntington's disease gene homologue.

The expansion of GAG triplet repeats in the translated region of the human HD gene, encoding a protein (huntingtin) of unknown function, is a dominant mutation leading to manifestation of Huntingtons disease. Targeted disruption of the homologous mouse gene (Hdh), to examine the normal role of huntingtin, shows that this protein is functionally indispensable, since nullizygous embryos become developmentally retarded and disorganized, and die between days 8.5 and 10.5 of gestation. Based on the observation that the level of the regionalized apoptotic cell death in the embryonic ectoderm, a layer expressing the Hdh gene, is much higher than normal in the null mutants, we propose that huntingtin is involved in processes counterbalancing the operation of an apoptotic pathway.

Inactivation of Hdh in the brain and testis results in progressive neurodegeneration and sterility in mice

Inactivation of the mouse homologue of the Huntington disease gene (Hdh) results in early embryonic lethality. To investigate the normal function of Hdh in the adult and to evaluate current models for Huntington disease (HD), we have used the Cre/loxP site-specific recombination strategy to inactivate Hdh expression in the forebrain and testis, resulting in a progressive degenerative neuronal phenotype and sterility. On the basis of these results, we propose that huntingtin is required for neuronal function and survival in the brain and that a loss-of-function mechanism may contribute to HD pathogenesis.

Mutant Huntingtin Impairs Axonal Trafficking in Mammalian Neurons In Vivo and In Vitro

ABSTRACT Recent data in invertebrates demonstrated that huntingtin (htt) is essential for fast axonal trafficking. Here, we provide direct and functional evidence that htt is involved in fast axonal trafficking in mammals. Moreover, expression of full-length mutant htt (mhtt) impairs vesicular and mitochondrial trafficking in mammalian neurons in vitro and in whole animals in vivo. Particularly, mitochondria become progressively immobilized and stop more frequently in neurons from transgenic animals. These defects occurred early in development prior to the onset of measurable neurological or mitochondrial abnormalities. Consistent with a progressive loss of function, wild-type htt, trafficking motors, and mitochondrial components were selectively sequestered by mhtt in human Huntingtons disease-affected brain. Data provide a model for how loss of htt function causes toxicity; mhtt-mediated aggregation sequesters htt and components of trafficking machinery leading to loss of mitochondrial motility and eventual mitochondrial dysfunction.

Time course of early motor and neuropathological anomalies in a knock-in mouse model of Huntington's disease with 140 CAG repeats.

Huntingtons disease (HD) is caused by an abnormal expansion of CAG repeats in the gene encoding huntingtin. The development of therapies for HD requires preclinical testing of drugs in animal models that reproduce the dysfunction and regionally specific pathology observed in HD. We have developed a new knock‐in mouse model of HD with a chimeric mouse/human exon 1 containing 140 CAG repeats inserted in the murine huntingtin gene. These mice displayed an increased locomotor activity and rearing at 1 month of age, followed by hypoactivity at 4 months and gait anomalies at 1 year. Behavioral symptoms preceded neuropathological anomalies, which became intense and widespread only at 4 months of age. These consisted of nuclear staining for huntingtin and huntingtin‐containing nuclear and neuropil aggregates that first appeared in the striatum, nucleus accumbens, and olfactory tubercle. Interestingly, regions with early pathology all receive dense dopaminergic inputs, supporting accumulating evidence for a role of dopamine in HD pathology. Nuclear staining and aggregates predominated in striatum and layer II/III and deep layer V of the cerebral cortex, whereas neuropil aggregates were found in the globus pallidus and layer IV/superficial layer V of the cerebral cortex. The olfactory system displayed early and marked aggregate accumulation, which may be relevant to the early deficit in odor discrimination observed in patients with HD. Because of their early behavioral anomalies and regionally specific pathology, these mice provide a powerful tool with which to evaluate the effectiveness of new therapies and to study the mechanisms involved in the neuropathology of HD. J. Comp. Neurol. 465:11–26, 2003.

Enhanced sensitivity to N-methyl-D-aspartate receptor activation in transgenic and knockin mouse models of Huntington's disease

We used two mouse models of Huntingtons disease (HD) to examine changes in glutamate receptor sensitivity and striatal electrophysiology. One model, a transgenic, consisted of mice expressing exon 1 of the human HD gene and carrying 141–157 CAG repeat sequences (R6/2 line). The second model, a CAG repeat “knockin,” consisted of mice with different lengths of CAG repeats (CAG71 and CAG94 repeats). The effects of glutamate receptor activation were examined by visualizing neurons in brain slices with infrared videomicroscopy and differential interference contrast optics to determine changes in somatic area (cell swelling). Striatal and cortical neurons in both models (R6/2 and CAG94) displayed more rapid and increased swelling to N‐methyl‐D‐aspartate (NMDA) than those in controls. This effect was specific as there were no consistent group differences after exposure to α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid (AMPA) or kainate (KA). Intracellular recordings revealed that resting membrane potentials (RMPs) in the R6/2 transgenics were significantly more depolarized than those in their respective controls. RMPs in CAG94 mice also were more depolarized than those in CAG71 mice or their controls in a subset of striatal neurons. Confirming previous results, R6/2 mice expressed behavioral abnormalities and nuclear inclusions. However, CAG71 and CAG94 knockins did not, suggesting that increased sensitivity to NMDA may occur early in the disease process. These findings imply that NMDA antagonists or compounds that alter sensitivity of NMDA receptors may be useful in the treatment of HD. J. Neurosci. Res. 58:515–532, 1999.

Presynaptic BDNF Required for a Presynaptic but Not Postsynaptic Component of LTP at Hippocampal CA1-CA3 Synapses

Brain-derived neurotrophic factor (BDNF) has been implicated in several forms of long-term potentiation (LTP) at different hippocampal synapses. Using two-photon imaging of FM 1-43, a fluorescent marker of synaptic vesicle cycling, we find that BDNF is selectively required for those forms of LTP at Schaffer collateral synapses that recruit a presynaptic component of expression. BDNF-dependent forms of LTP also require activation of L-type voltage-gated calcium channels. One form of LTP with presynaptic expression, theta burst LTP, is thought to be of particular behavioral importance. Using restricted genetic deletion to selectively disrupt BDNF production in either the entire forebrain (CA3 and CA1) or in only the postsynaptic CA1 neuron, we localize the source of BDNF required for LTP to presynaptic neurons. These results suggest that long-term synaptic plasticity has distinct presynaptic and postsynaptic modules. Release of BDNF from CA3 neurons is required to recruit the presynaptic, but not postsynaptic, module of plasticity.

RNA-mediated gene duplication: the rat preproinsulin I gene is a functional retroposon.

Rats and mice have two, equally expressed, nonallelic genes encoding preproinsulin (genes I and II). Cytological hybridization with metaphase chromosomes indicated that both genes reside on rat chromosome I but are approximately 100,000 kilobases apart. In mice the two genes reside on two different chromosomes. DNA sequence comparisons of the gene-flanking regions in rats and mice indicated that the preproinsulin gene I has lost one of the two introns present in gene II, is flanked by a long (41-base) direct repeat, and has a remnant of a polydeoxyadenylate acid tract preceding the downstream direct repeat. These structural features indicated that gene I was generated by an RNA-mediated duplication-transposition event involving a transcript of gene II which was initiated upstream from the normal capping site. Sequence divergence analysis indicated that the pair of the original gene and its retroposed, but functional, counterpart (which appeared about 35 million years ago) is maintained by strong negative selection operating primarily on the segments encoding the chains of the mature hormone, whereas the segments encoding the parts of the polypeptide that are eliminated during processing and also the introns and the flanking regions are evolving neutrally.

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.

Brain-Specific Knock-Out of Hypoxia-Inducible Factor-1α Reduces Rather Than Increases Hypoxic-Ischemic Damage

Hypoxia-inducible factor-1α (HIF-1α) plays an essential role in cellular and systemic O2 homeostasis by regulating the expression of genes important in glycolysis, erythropoiesis, angiogenesis, and catecholamine metabolism. It is also believed to be a key component of the cellular response to hypoxia and ischemia under pathophysiological conditions, such as stroke. To clarify the function of HIF-1α in the brain, we exposed adult mice with late-stage brain deletion of HIF-1α to hypoxic injuries. Contrary to expectations, the brains from the HIF-1α-deficient mice were protected from hypoxia-induced cell death. These surprising findings suggest that decreasing the level of HIF-1α can be neuroprotective. Gene chip expression analysis revealed that, contrary to expectations, the majority of hypoxia-dependent gene-expression changes were unaltered, whereas a specific downregulation of apoptotic genes was observed in the HIF-1α-deficient mice. Although the role of HIF-1α has been extensively characterized in vitro, in cancer models, and in chronic preconditioning paradigms, this is the first study to evaluate the role of HIF-1α in vivo in the brain in response to acute hypoxia/ischemia. Our data suggest, that in acute hypoxia, the neuroprotection found in the HIF-1α-deficient mice is mechanistically consistent with a predominant role of HIF-1α as proapoptotic and loss of function leads to neuroprotection. Furthermore, our data suggest that functional redundancy develops after excluding HIF-1α, leading to the preservation of gene expression regulating the majority of other previously characterized HIF-dependent genes.

Pre-mRNA splicing and the nuclear matrix.

We examined the relationship between pre-mRNA splicing and the nuclear matrix by using an in vivo system that we have developed. Plasmids containing the inducible herpesvirus tk gene promoter linked to an intron-containing segment of the rabbit beta-globin gene were transfected into HeLa cells, and then the promoter was transactivated by infection with a TK- virus. Northern analysis revealed that the globin pre-mRNA and all its splicing intermediates and products are associated with the nuclear matrix prepared from such transfected cells. When the nuclear matrix was incubated with a HeLa cell in vitro splicing extract in the presence of ATP, the amount of matrix-associated precursor progressively decreased without a temporal lag in the reaction, with a corresponding increase in free intron lariat. Thus, most of the events of the splicing process (endonucleolytic cuts and branching) occur in this in vitro complementation reaction. However, ligation of exons cannot be monitored in this system because of the abundance of preexisting mature mRNA. Since the matrix is not a self-splicing entity, whereas the in vitro splicing system cannot process efficiently deproteinized matrix RNA, we conclude from our in vitro complementation results (which can be reproduced by using micrococcal nuclease-treated splicing extract) that the nuclear matrix preparation retains parts of preassembled ribonucleoprotein complexes that have the potential to function when supplemented with soluble factors (presumably other than most of the small nuclear ribonucleoproteins known to participate in splicing) present in the HeLa cell extract.