A Series of N-terminal Epitope Tagged Hdh Knock-In Alleles Expressing Normal and Mutant Huntingtin: Their Application to Understanding the Effect of Increasing the Length of Normal Huntingtin’s Polyglutamine Stretch on CAG140 Mouse Model Pathogenesis

Background: Huntington’s disease (HD) is an autosomal dominant neurodegenerative disease that is caused by the expansion of a polyglutamine (polyQ) stretch within Huntingtin (htt), the protein product of the HD gene. Although studies in vitro have suggested that the mutant htt can act in a potentially dominant negative fashion by sequestering wild-type htt into insoluble protein aggregates, the role of the length of the normal htt polyQ stretch, and the adjacent proline-rich region (PRR) in modulating HD mouse model pathogenesis is currently unknown. Results: We describe the generation and characterization of a series of knock-in HD mouse models that express versions of the mouse HD gene (Hdh) encoding N-terminal hemaglutinin (HA) or 3xFlag epitope tagged full-length htt with different polyQ lengths (HA7Q-, 3xFlag7Q-, 3xFlag20Q-, and 3xFlag140Q-htt) and substitution of the adjacent mouse PRR with the human PRR (3xFlag20Q- and 3xFlag140Q-htt). Using co-immunoprecipitation and immunohistochemistry analyses, we detect no significant interaction between soluble full-length normal 7Q- htt and mutant (140Q) htt, but we do observe N-terminal fragments of epitope-tagged normal htt in mutant htt aggregates. When the sequences encoding normal mouse htt’s polyQ stretch and PRR are replaced with non-pathogenic human sequence in mice also expressing 140Q-htt, aggregation foci within the striatum, and the mean size of htt inclusions are increased, along with an increase in striatal lipofuscin and gliosis. Conclusion: In mice, soluble full-length normal and mutant htt are predominantly monomeric. In heterozygous knock-in HD mouse models, substituting the normal mouse polyQ and PRR with normal human sequence can exacerbate some neuropathological phenotypes

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

The matrix realization of affine Jacobi varieties and the extended Lotka-Volterra lattice

We study completely integrable Hamiltonian systems whose monodromy matrices are related to the representatives for the set of gauge equivalence classes $\boldsymbol{\mathcal{M}}_F$ of polynomial matrices. Let $X$ be the algebraic curve given by the common characteristic equation for $\boldsymbol{\mathcal{M}}_F$. We construct the isomorphism from the set of representatives to an affine part of the Jacobi variety of $X$. This variety corresponds to the invariant manifold of the system, where the Hamiltonian flow is linearized. As the application, we discuss the algebraic completely integrability of the extended Lotka-Volterra lattice with a periodic boundary condition.Comment: Revised version, 26 page

Improvement of neuropathology and transcriptional deficits in CAG 140 knock-in mice supports a beneficial effect of dietary curcumin in Huntington's disease

<p>Abstract</p> <p>Backgound</p> <p>No disease modifying treatment currently exists for Huntington's disease (HD), a fatal neurodegenerative disorder characterized by the formation of amyloid-like aggregates of the mutated huntingtin protein. Curcumin is a naturally occurring polyphenolic compound with Congo red-like amyloid binding properties and the ability to cross the blood brain barrier. CAG140 mice, a knock-in (KI) mouse model of HD, display abnormal aggregates of mutant huntingtin and striatal transcriptional deficits, as well as early motor, cognitive and affective abnormalities, many months prior to exhibiting spontaneous gait deficits, decreased striatal volume, and neuronal loss. We have examined the ability of life-long dietary curcumin to improve the early pathological phenotype of CAG140 mice.</p> <p>Results</p> <p>KI mice fed a curcumin-containing diet since conception showed decreased huntingtin aggregates and increased striatal DARPP-32 and D1 receptor mRNAs, as well as an amelioration of rearing deficits. However, similar to other antioxidants, curcumin impaired rotarod behavior in both WT and KI mice and climbing in WT mice. These behavioral effects were also noted in WT C57Bl/6 J mice exposed to the same curcumin regime as adults. However, neither locomotor function, behavioral despair, muscle strength or food utilization were affected by curcumin in this latter study. The clinical significance of curcumin's impairment of motor performance in mice remains unclear because curcumin has an excellent blood chemistry and adverse event safety profile, even in the elderly and in patients with Alzheimer's disease.</p> <p>Conclusion</p> <p>Together with this clinical experience, the improvement in several transgene-dependent parameters by curcumin in our study supports a net beneficial effect of dietary curcumin in HD.</p

Pharmacotherapy and Pregnancy: Highlights from the Third International Conference for Individualized Pharmacotherapy in Pregnancy

To address provider struggles to provide evidence-based, rational drug therapy to pregnant women, this third Conference was convened to highlight the current progress and research in the field. Speakers from academic centers, industry, and governmental institutions spoke about: the Food and Drug Administration’s role in pregnancy pharmacology and the new labeling initiative; drug registries in pregnancy; the pharmacist’s role in medication use in pregnancy; therapeutic areas such as preterm labor, gestational diabetes, nausea and vomiting in pregnancy, and hypertension; breast-feeding and medications; ethical challenges for consent in pregnancy drug studies; the potential for cord blood banks; and concerns about the fetus when studying drugs in pregnancy. The Conference highlighted several areas of collaboration within the current Obstetrics Pharmacology Research Units Network and hoped to educate providers, researchers, and agencies with the common goal to improve the ability to safely and effectively use individualized pharmacotherapy in pregnancy

Fiction Fix 11

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Potential function for the Huntingtin protein as a scaffold for selective autophagy

Although dominant gain-of-function triplet repeat expansions in the Huntingtin (HTT) gene are the underlying cause of Huntington disease (HD), understanding the normal functions of nonmutant HTT protein has remained a challenge. We report here findings that suggest that HTT plays a significant role in selective autophagy. Loss of HTT function in Drosophila disrupts starvation-induced autophagy in larvae and conditional knockout of HTT in the mouse CNS causes characteristic cellular hallmarks of disrupted autophagy, including an accumulation of striatal p62/SQSTM1 over time. We observe that specific domains of HTT have structural similarities to yeast Atg proteins that function in selective autophagy, and in particular that the C-terminal domain of HTT shares structural similarity to yeast Atg11, an autophagic scaffold protein. To explore possible functional similarity between HTT and Atg11, we investigated whether the C-terminal domain of HTT interacts with mammalian counterparts of yeast Atg11-interacting proteins. Strikingly, this domain of HTT coimmunoprecipitates with several key Atg11 interactors, including the Atg1/Unc-51–like autophagy activating kinase 1 kinase complex, autophagic receptor proteins, and mammalian Atg8 homologs. Mutation of a phylogenetically conserved WXXL domain in a C-terminal HTT fragment reduces coprecipitation with mammalian Atg8 homolog GABARAPL1, suggesting a direct interaction. Collectively, these data support a possible central role for HTT as an Atg11-like scaffold protein. These findings have relevance to both mechanisms of disease pathogenesis and to therapeutic intervention strategies that reduce levels of both mutant and normal HTT.Hereditary Disease Foundation (U.S.)Cure Huntington’s Disease Initiative, Inc.Fox Family Foundatio

IKK phosphorylates Huntingtin and targets it for degradation by the proteasome and lysosome

Expansion of the polyglutamine repeat within the protein Huntingtin (Htt) causes Huntington's disease, a neurodegenerative disease associated with aging and the accumulation of mutant Htt in diseased neurons. Understanding the mechanisms that influence Htt cellular degradation may target treatments designed to activate mutant Htt clearance pathways. We find that Htt is phosphorylated by the inflammatory kinase IKK, enhancing its normal clearance by the proteasome and lysosome. Phosphorylation of Htt regulates additional post-translational modifications, including Htt ubiquitination, SUMOylation, and acetylation, and increases Htt nuclear localization, cleavage, and clearance mediated by lysosomal-associated membrane protein 2A and Hsc70. We propose that IKK activates mutant Htt clearance until an age-related loss of proteasome/lysosome function promotes accumulation of toxic post-translationally modified mutant Htt. Thus, IKK activation may modulate mutant Htt neurotoxicity depending on the cell's ability to degrade the modified species

Networks of corporate power revisited

This paper examines developments through the quarter century since the publication of Stokman, Ziegler and Scott's (1985) iconic ten-nation study of the structure of interlocking directorships. The surprising decline of research in the area following the publication of Networks of Corporate Power is in part testimony to the rigour of the comparative methods used, raising the standard of evidence required for subsequent director interlock studies. But it also reflected a critical weakness in director interlock research to that point, the limited ability to answer what Mark Mizruchi has called the “So what?” question. While replicated studies found clear structures in director interlocks, varying from country to country, and there was some speculative fit with the distinctive political economies of these countries, there was little evidence of any effect of these structures on firm performance or activity. The more recent resurgence in director interlock research is in some ways rooted in a second generation of the original drivers; the ready availability of now large masses of data on firm governance and firm level performance and further advances in social network analytical techniques. Where Stokman and his colleagues manually compiled lists of directors scoured from company reports, these data are now routinely collected and compiled in accessible databases by government agencies and business information services in many countries. And there has been a gradual accumulation of advances in addressing the “so what” question