Elucidating a normal function of huntingtin by functional and microarray analysis of huntingtin-null mouse embryonic fibroblasts

<p>Abstract</p> <p>Background</p> <p>The polyglutamine expansion in huntingtin (Htt) protein is a cause of Huntington's disease (HD). Htt is an essential gene as deletion of the mouse Htt gene homolog (<it>Hdh</it>) is embryonic lethal in mice. Therefore, in addition to elucidating the mechanisms responsible for polyQ-mediated pathology, it is also important to understand the normal function of Htt protein for both basic biology and for HD.</p> <p>Results</p> <p>To systematically search for a mouse Htt function, we took advantage of the <it>Hdh </it>+/- and <it>Hdh</it>-floxed mice and generated four mouse embryonic fibroblast (MEF) cells lines which contain a single copy of the <it>Hdh </it>gene (<it>Hdh</it>-HET) and four MEF lines in which the <it>Hdh </it>gene was deleted (<it>Hdh</it>-KO). The function of Htt in calcium (Ca²⁺) signaling was analyzed in Ca²⁺imaging experiments with generated cell lines. We found that the cytoplasmic Ca²⁺spikes resulting from the activation of inositol 1,4,5-trisphosphate receptor (InsP₃R) and the ensuing mitochondrial Ca²⁺signals were suppressed in the <it>Hdh</it>-KO cells when compared to <it>Hdh</it>-HET cells. Furthermore, in experiments with permeabilized cells we found that the InsP₃-sensitivity of Ca²⁺mobilization from endoplasmic reticulum was reduced in <it>Hdh</it>-KO cells. These results indicated that Htt plays an important role in modulating InsP₃R-mediated Ca²⁺signaling. To further evaluate function of Htt, we performed genome-wide transcription profiling of generated <it>Hdh</it>-HET and <it>Hdh</it>-KO cells by microarray. Our results revealed that 106 unique transcripts were downregulated by more than two-fold with p < 0.05 and 173 unique transcripts were upregulated at least two-fold with p < 0.05 in <it>Hdh</it>-KO cells when compared to <it>Hdh</it>-HET cells. The microarray results were confirmed by quantitative real-time PCR for a number of affected transcripts. Several signaling pathways affected by <it>Hdh </it>gene deletion were identified from annotation of the microarray results.</p> <p>Conclusion</p> <p>Functional analysis of generated Htt-null MEF cells revealed that Htt plays a direct role in Ca²⁺signaling by modulating InsP₃R sensitivity to InsP₃. The genome-wide transcriptional profiling of Htt-null cells yielded novel and unique information about the normal function of Htt in cells, which may contribute to our understanding and treatment of HD.</p

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

AbstractBrain-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

Deletion of Exon 20 of the Familial Dysautonomia Gene Ikbkap in Mice Causes Developmental Delay, Cardiovascular Defects, and Early Embryonic Lethality

Familial Dysautonomia (FD) is an autosomal recessive disorder that affects 1/3,600 live births in the Ashkenazi Jewish population, and leads to death before the age of 40. The disease is characterized by abnormal development and progressive degeneration of the sensory and autonomic nervous system. A single base pair substitution in intron 20 of the Ikbkap gene accounts for 98% of FD cases, and results in the expression of low levels of the full-length mRNA with simultaneous expression of an aberrantly spliced mRNA in which exon 20 is missing. To date, there is no animal model for the disease, and the essential cellular functions of IKAP - the protein encoded by Ikbkap - remain unknown. To better understand the normal function of IKAP and in an effort to generate a mouse model for FD, we have targeted the mouse Ikbkap gene by homologous recombination. We created two distinct alleles that result in either loss of Ikbkap expression, or expression of an mRNA lacking only exon 20. Homozygosity for either mutation leads to developmental delay, cardiovascular and brain malformations, accompanied with early embryonic lethality. Our analyses indicate that IKAP is essential for expression of specific genes involved in cardiac morphogenesis, and that cardiac failure is the likely cause of abnormal vascular development and embryonic lethality. Our results also indicate that deletion of exon 20 abolishes gene function. This implies that the truncated IKAP protein expressed in FD patients does not retain any significant biological function

Familial Dysautonomia: Mechanisms and Models

Abstract Hereditary Sensory and Autonomic Neuropathies (HSANs) compose a heterogeneous group of genetic disorders characterized by sensory and autonomic dysfunctions. Familial Dysautonomia (FD), also known as HSAN III, is an autosomal recessive disorder that affects 1/3,600 live births in the Ashkenazi Jewish population. The major features of the disease are already present at birth and are attributed to abnormal development and progressive degeneration of the sensory and autonomic nervous systems. Despite clinical interventions, the disease is inevitably fatal. FD is caused by a point mutation in intron 20 of the IKBKAP gene that results in severe reduction in expression of IKAP, its encoded protein. In vitro and in vivo studies have shown that IKAP is involved in multiple intracellular processes, and suggest that failed target innervation and/or impaired neurotrophic retrograde transport are the primary causes of neuronal cell death in FD. However, FD is far more complex, and appears to affect several other organs and systems in addition to the peripheral nervous system. With the recent generation of mouse models that recapitulate the molecular and pathological features of the disease, it is now possible to further investigate the mechanisms underlying different aspects of the disorder, and to test novel therapeutic strategies

Familial Dysautonomia: Mechanisms and Models

Abstract Hereditary Sensory and Autonomic Neuropathies (HSANs) compose a heterogeneous group of genetic disorders characterized by sensory and autonomic dysfunctions. Familial Dysautonomia (FD), also known as HSAN III, is an autosomal recessive disorder that affects 1/3,600 live births in the Ashkenazi Jewish population. The major features of the disease are already present at birth and are attributed to abnormal development and progressive degeneration of the sensory and autonomic nervous systems. Despite clinical interventions, the disease is inevitably fatal. FD is caused by a point mutation in intron 20 of the IKBKAP gene that results in severe reduction in expression of IKAP, its encoded protein. In vitro and in vivo studies have shown that IKAP is involved in multiple intracellular processes, and suggest that failed target innervation and/or impaired neurotrophic retrograde transport are the primary causes of neuronal cell death in FD. However, FD is far more complex, and appears to affect several other organs and systems in addition to the peripheral nervous system. With the recent generation of mouse models that recapitulate the molecular and pathological features of the disease, it is now possible to further investigate the mechanisms underlying different aspects of the disorder, and to test novel therapeutic strategies

Semi-quantitative RT-PCR analyses.

<p>Equal amount of total RNA from control (C) and mutant (M, Ikbkap^Δ20/Δ20) embryos at the indicated stages (E8.5-E10.5) was used for cDNA synthesis in the presence of reverse transcriptase (RT) and the resulting cDNA was used for amplification of the specified genes (see details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027015#s4" target="_blank">Materials and Methods</a>). Similar results were obtained for Ikbkap^3loxP/3loxP embryos (data not shown). For negative control (-), reverse transcription reaction was performed in the absence of RT using equal starting amounts of total RNA as in all other reactions. Amplification of the rRNA 18S (bottom panel) was used as internal control for normalization of cDNA template quantity in the PCR reactions. As marker 100 bp ladder (L) is also shown. Lack of Ikbkap amplification with oligos Ikbkap20for and Ikbkap23rev in the upper most panel reconfirms the genotyping results for the mutant (M) Ikbkap^Δ20/Δ20 embryos used for the depicted analyses.</p