Inactivation of the Huntington's disease gene (Hdh) impairs anterior streak formation and early patterning of the mouse embryo

BACKGROUND: Huntingtin, the HD gene encoded protein mutated by polyglutamine expansion in Huntington's disease, is required in extraembryonic tissues for proper gastrulation, implicating its activities in nutrition or patterning of the developing embryo. To test these possibilities, we have used whole mount in situ hybridization to examine embryonic patterning and morphogenesis in homozygous Hdh(ex4/5 )huntingtin deficient embryos. RESULTS: In the absence of huntingtin, expression of nutritive genes appears normal but E7.0–7.5 embryos exhibit a unique combination of patterning defects. Notable are a shortened primitive streak, absence of a proper node and diminished production of anterior streak derivatives. Reduced Wnt3a, Tbx6 and Dll1 expression signify decreased paraxial mesoderm and reduced Otx2 expression and lack of headfolds denote a failure of head development. In addition, genes initially broadly expressed are not properly restricted to the posterior, as evidenced by the ectopic expression of Nodal, Fgf8 and Gsc in the epiblast and T (Brachyury) and Evx1 in proximal mesoderm derivatives. Despite impaired posterior restriction and anterior streak deficits, overall anterior/posterior polarity is established. A single primitive streak forms and marker expression shows that the anterior epiblast and anterior visceral endoderm (AVE) are specified. CONCLUSION: Huntingtin is essential in the early patterning of the embryo for formation of the anterior region of the primitive streak, and for down-regulation of a subset of dynamic growth and transcription factor genes. These findings provide fundamental starting points for identifying the novel cellular and molecular activities of huntingtin in the extraembryonic tissues that govern normal anterior streak development. This knowledge may prove to be important for understanding the mechanism by which the dominant polyglutamine expansion in huntingtin determines the loss of neurons in Huntington's disease

Huntingtin: an iron-regulated protein essential for normal nuclear and perinuclear organelles

Huntington's disease (HD), with its selective neuronal cell loss, is caused by an elongated glutamine tract in the huntingtin protein. To discover the pathways that are candidates for the protein's normal and/or abnormal function, we surveyed 19 classes of organelle in Hdh(ex4/5)/Hdh(ex4/5) knock-out compared with wild-type embryonic stem cells to identify any that might be affected by huntingtin deficiency. Although the majority did not differ, dramatic changes in six classes revealed that huntingtin's function is essential for the normal nuclear (nucleoli, transcription factor-speckles) and perinuclear membrane (mitochondria, endoplasmic reticulum, Golgi and recycling endosomes) organelles and for proper regulation of the iron pathway. Moreover, upmodulation by deferoxamine mesylate implicates huntingtin as an iron-response protein. However, excess huntingtin produced abnormal organelles that resemble the deficiency phenotype, suggesting the importance of huntingtin level to the protein's normal pathway. Thus, organelles that require huntingtin to function suggest roles for the protein in RNA biogenesis, trafficking and iron homeostasis to be explored in HD pathogenesis

Dominant phenotypes produced by the HD mutation in STHdh(Q111) striatal cells

Lengthening a glutamine tract in huntingtin confers a dominant attribute that initiates degeneration of striatal neurons in Huntington's disease (HD). To identify pathways that are candidates for the mutant protein's abnormal function, we compared striatal cell lines established from wild-type and Hdh(Q111) knock-in embryos. Alternate versions of full-length huntingtin, distinguished by epitope accessibility, were localized to different sets of nuclear and perinuclear organelles involved in RNA biogenesis and membrane trafficking. However, mutant STHdh(Q111) cells also exhibited additional forms of the full-length mutant protein and displayed dominant phenotypes that did not mirror phenotypes caused by either huntingtin deficiency or excess. These phenotypes indicate a disruption of striatal cell homeostasis by the mutant protein, via a mechanism that is separate from its normal activity. They also support specific stress pathways, including elevated p53, endoplasmic reticulum stress response and hypoxia, as potential players in HD

The HD mutation does not alter neuronal death in the striatum of Hdh(Q92) knock-in mice after mild focal ischemia

Huntington's disease, with its dominant loss of striatal neurons, is triggered by an expanded glutamine tract in huntingtin. To investigate a proposed role for increased activation of the apoptotic cascade in mutant huntingtin's trigger mechanism, we examined huntingtin cleavage and lesion severity after mild ischemic injury in Hdh(Q92) mice. We found activation of calpain and caspase proteases and proteolysis of huntingtin in lesioned striatum. However, huntingtin fragments resembled products of calpain I, not caspase-3, cleavage and turnover was accompanied by augmented levels of full-length normal and mutant protein. By contrast, the number of apoptotic cells, total and striatal infarct size, and degree of neurologic deficit were similar in Hdh(Q92) and wild-type mice, indicating that the disease process neither strongly protected nor sensitized striatal neurons to apoptotic death. Thus, our findings do not support a role for increased apoptosis or caspase-3 cleavage in the mechanism by which mutant huntingtin triggers disease. However, they suggest that calpain activation and huntingtin regulation merit investigation as modifiers of disease progression in neurons injured by the harmful consequences of full-length mutant huntingtin

Analysis of oxidative events induced by expanded polyglutamine huntingtin exon 1 that are differentially restored by expression of heat shock proteins or treatment with an antioxidant

We recently reported that the transient expression of polyglutamine tracts of various size in exon 1 of the huntingtin polypeptide (httExl) generated abnormally high levels of intracellular reactive oxygen species that directly contributed to cell death. Here, we compared the protection generated by heat shock proteins to that provided by the antioxidant agent N-acetyl-L-cysteine. In cells expressing httExl with 72 glutamine repeats (httEx1-72Q), the overexpression of Hsp27 or Hsp70 plus Hdj-1(Hsp40) or treatment of the cells with N-acetyl-L-cysteine inhibited not only mitochondrial membrane potential disruption but also the increase in reactive oxygen species, nitric oxide and protein oxidation. However, only heat shock proteins and not N-acetyl-L-cysteine reduced the size of the inclusion bodies formed by httExl-72Q. In cells expressing httExl polypeptide with 103 glutamine repeats (httEx1-103Q), heat shock proteins neither decreased oxidative damage nor reduced the size of the inclusions. In contrast, N-acetyl-L-cysteine still efficiently decreased the oxidative damage induced by httExl-103Q polypeptide without altering the inclusions. N-Acetyl-L-cysteine was inactive with regard to proteasome inhibition, whereas heat shock proteins partially restored the caspase-like activity of this protease. These observations suggest some relationships between the presence of inclusion bodies and the oxidative damage induced by httExl-polyQ

Huntingtin Interacting Protein 1 Is a Clathrin Coat Binding Protein Required for Differentiation of late Spermatogenic Progenitors

Huntingtin-interacting protein 1 (HIP1) interacts with huntingtin, the protein whose gene is mutated in Huntington's disease. In addition, a fusion between HIP1 and platelet-derived growth factor β receptor causes chronic myelomonocytic leukemia. The HIP1 proteins, including HIP1 and HIP1-related (HIP1r), have an N-terminal polyphosphoinositide-interacting epsin N-terminal homology, domain, which is found in proteins involved in clathrin-mediated endocytosis. HIP1 and HIP1r also share a central leucine zipper and an actin binding TALIN homology domain. Here we show that HIP1, like HIP1r, colocalizes with clathrin coat components. We also show that HIP1 physically associates with clathrin and AP-2, the major components of the clathrin coat. To further understand the putative biological role(s) of HIP1, we have generated a targeted deletion of murine HIP1. HIP1(−/−) mice developed into adulthood, did not develop overt neurologic symptoms in the first year of life, and had normal peripheral blood counts. However, HIP1-deficient mice exhibited testicular degeneration with increased apoptosis of postmeiotic spermatids. Postmeiotic spermatids are the only cells of the seminiferous tubules that express HIP1. These findings indicate that HIP1 is required for differentiation, proliferation, and/or survival of spermatogenic progenitors. The association of HIP1 with clathrin coats and the requirement of HIP1 for progenitor survival suggest a role for HIP1 in the regulation of endocytosis

Huntingtin coordinates the dynein-mediated dynamic positioning of endosomes and lysosomes

We investigated the role of the membrane-associated scaffolding protein huntingtin (Htt) in the dynein-mediated transport of early, recycling, and late endosomes and lysosomes. Our observations support a model of Htt as a facilitator of dynein-mediated trafficking that can regulate the cytoskeletal association of dynamic organelles