Selective neuronal requirement for huntingtin in the developing zebrafish.

Huntington's disease shares a common molecular basis with eight other neurodegenerative diseases, expansion of an existing polyglutamine tract. In each case, this repeat tract occurs within otherwise unrelated proteins. These proteins show widespread and overlapping patterns of expression in the brain and yet the diseases are distinguished by neurodegeneration in a specific subset of neurons that are most sensitive to the mutation. It has therefore been proposed that expansion of the polyglutamine region in these genes may result in perturbation of the normal function of the respective proteins, and that this perturbation in some way contributes to the neuronal specificity of these diseases. The normal functions of these proteins have therefore become a focus for investigation as potential pathogenic pathways. We have used synthetic antisense morpholinos to inhibit the translation of huntingtin mRNA during early zebrafish development and have previously reported the effects of huntingtin reduction on iron transport and homeostasis. Here we report an analysis of the effects of huntingtin loss-of-function on the developing nervous system, observing distinct defects in morphology of neuromasts, olfactory placode and branchial arches. The potential common origins of these defects were explored, revealing impaired formation of the anterior-most region of the neural plate as indicated by reduced pre-placodal and telencephalic gene expression with no effect on mid- or hindbrain formation. These investigations demonstrate a specific 'rate-limiting' role for huntingtin in formation of the telencephalon and the pre-placodal region, and differing levels of requirement for huntingtin function in specific nerve cell types.

Robust homeostasis of Presenilin1 protein levels by transcript regulation.

The Presenilin proteins are essential facilitators of numerous developmental and cell signaling pathways. Point mutations in the human PRESENILIN genes (including mutations affecting splicing) have been linked to familial Alzheimer's disease. Zebrafish possess orthologues of the human PRESENILIN1 and PRESENILIN2 genes. We previously investigated forced aberrant splicing of zebrafish presenilin1 and discovered that high levels of incorporation into spliced transcripts of the intron cognate with human PRESENILIN1 intron 8 resulted in little or no change in Presenilin1 protein level and no identifiable embryonic phenotype. We now demonstrate that zebrafish embryos maintain relatively stable levels of normal Presenilin1 transcript and protein despite accumulating large amounts of aberrantly spliced presenilin1 transcript. We also show that increasing the levels of Presenilin1 protein decreases normal presenilin1 transcription. These two independent lines of evidence and the fact that blockage of Presenilin1 translation increases presenilin1 transcription support that regulation of presenilin1 transcript levels plays a major role in the homeostasis of Presenilin1 protein levels, presumably via a feedback mechanism that monitors the levels of Presenilin1 protein.

Huntingtin-deficient zebrafish exhibit defects in iron utilization and development.

Huntington's disease (HD) is one of nine neurodegenerative disorders caused by expansion of CAG repeats encoding polyglutamine in their respective, otherwise apparently unrelated proteins. Despite these proteins having widespread and overlapping expression patterns in the brain, a specific and unique subset of neurons exhibits particular vulnerability in each disease. It has been hypothesized that perturbation of normal protein function contributes to the specificity of neuronal vulnerability; however, the normal biological functions of many of these proteins including the HD gene product, Huntingtin (Htt), are unclear. To explore the roles of Htt, we have used antisense morpholino oligonucleotides to observe the effects of Htt deficiency in early zebrafish development. Knockdown of Htt expression resulted in a variety of developmental defects. Most notably, Htt-deficient zebrafish had hypochromic blood due to decreased hemoglobin production, despite the presence of iron within blood cells. Furthermore, transferrin receptor 1 transcripts were increased, suggesting cellular iron starvation. Provision of iron to the cytoplasm in a bio-available form restored hemoglobin production in Htt-deficient embryos. Since erythroid cells acquire iron via receptor-mediated endocytosis of transferrin, these results suggest a role for Htt in making endocytosed iron accessible for cellular utilization. Iron is required for oxidative energy production, and defects in iron homeostasis and energy metabolism are features of HD pathogenesis that are most pronounced in the major region of neurodegeneration. It is therefore plausible that perturbation of Htt's normal role in the iron pathway (by polyglutamine tract expansion) contributes to HD pathology, and particularly to its neuronal specificity.