Genetic and pharmacological inhibition of calcineurin corrects the BDNF transport defect in Huntington's disease

<p>Abstract</p> <p>Background</p> <p>Huntington's disease (HD) is an inherited neurogenerative disease caused by an abnormal expansion of glutamine repeats in the huntingtin protein. There is currently no treatment to prevent the neurodegeneration caused by this devastating disorder. Huntingtin has been shown to be a positive regulator of vesicular transport, particularly for neurotrophins such as brain-derived neurotrophic factor (BDNF). This function is lost in patients with HD, resulting in a decrease in neurotrophic support and subsequent neuronal death. One promising line of treatment is therefore the restoration of huntingtin function in BDNF transport.</p> <p>Results</p> <p>The phosphorylation of huntingtin at serine 421 (S421) restores its function in axonal transport. We therefore investigated whether inhibition of calcineurin, the <it>bona fide </it>huntingtin S421 phosphatase, restored the transport defects observed in HD. We found that pharmacological inhibition of calcineurin by FK506 led to sustained phosphorylation of mutant huntingtin at S421. FK506 restored BDNF transport in two complementary models: rat primary neuronal cultures expressing mutant huntingtin and mouse cortical neurons from <it>Hdh</it>^Q111/Q111HD knock-in mice. This effect was the result of specific calcineurin inhibition, as calcineurin silencing restored both anterograde and retrograde transport in neurons from <it>Hdh</it>^Q111/Q111mice. We also observed a specific increase in calcineurin activity in the brain of <it>Hdh</it>^Q111/Q111mice potentially accounting for the selective loss of huntingtin phosphorylation and contributing to neuronal cell death in HD.</p> <p>Conclusion</p> <p>Our results validate calcineurin as a target for the treatment of HD and provide the first demonstration of the restoration of huntingtin function by an FDA-approved compound.</p

pARIS-htt: an optimised expression platform to study huntingtin reveals functional domains required for vesicular trafficking

<p>Abstract</p> <p>Background</p> <p>Huntingtin (htt) is a multi-domain protein of 350 kDa that is mutated in Huntington's disease (HD) but whose function is yet to be fully understood. This absence of information is due in part to the difficulty of manipulating large DNA fragments by using conventional molecular cloning techniques. Consequently, few studies have addressed the cellular function(s) of full-length htt and its dysfunction(s) associated with the disease.</p> <p>Results</p> <p>We describe a flexible synthetic vector encoding full-length htt called pARIS-htt (<b>A</b>daptable, <b>R</b>NAi <b>I</b>nsensitive &<b>S</b>ynthetic). It includes synthetic cDNA coding for full-length human htt modified so that: 1) it is improved for codon usage, 2) it is insensitive to four different siRNAs allowing gene replacement studies, 3) it contains unique restriction sites (URSs) dispersed throughout the entire sequence without modifying the translated amino acid sequence, 4) it contains multiple cloning sites at the N and C-ter ends and 5) it is Gateway compatible. These modifications facilitate mutagenesis, tagging and cloning into diverse expression plasmids. Htt regulates dynein/dynactin-dependent trafficking of vesicles, such as brain-derived neurotrophic factor (BDNF)-containing vesicles, and of organelles, including reforming and maintenance of the Golgi near the cell centre. We used tests of these trafficking functions to validate various pARIS-htt constructs. We demonstrated, after silencing of endogenous htt, that full-length htt expressed from pARIS-htt rescues Golgi apparatus reformation following reversible microtubule disruption. A mutant form of htt that contains a 100Q expansion and a htt form devoid of either HAP1 or dynein interaction domains are both unable to rescue loss of endogenous htt. These mutants have also an impaired capacity to promote BDNF vesicular trafficking in neuronal cells.</p> <p>Conclusion</p> <p>We report the validation of a synthetic gene encoding full-length htt protein that will facilitate analyses of its structure/function. This may help provide relevant information about the cellular dysfunctions operating during the disease. As proof of principle, we show that either polyQ expansion or deletion of key interacting domains within full-length htt protein impairs its function in transport indicating that HD mutation induces defects on intrinsic properties of the protein and further demonstrating the importance of studying htt in its full-length context.</p

Modulation of AMPA receptor surface diffusion restores hippocampal plasticity and memory in Huntington’s disease models

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Gpr158 mediates osteocalcin's regulation of cognition

That osteocalcin (OCN) is necessary for hippocampal-dependent memory and to prevent anxiety-like behaviors raises novel questions. One question is to determine whether OCN is also sufficient to improve these behaviors in wild-type mice, when circulating levels of OCN decline as they do with age. Here we show that the presence of OCN is necessary for the beneficial influence of plasma from young mice when injected into older mice on memory and that peripheral delivery of OCN is sufficient to improve memory and decrease anxiety-like behaviors in 16-mo-old mice. A second question is to identify a receptor transducing OCN signal in neurons. Genetic, electrophysiological, molecular, and behavioral assays identify Gpr158, an orphan G protein-coupled receptor expressed in neurons of the CA3 region of the hippocampus, as transducing OCN's regulation of hippocampal-dependent memory in part through inositol 1,4,5-trisphosphate and brain-derived neurotrophic factor. These results indicate that exogenous OCN can improve hippocampal-dependent memory in mice and identify molecular tools to harness this pathway for therapeutic purposes

Huntingtin proteolysis releases non-polyQ fragments that cause toxicity through dynamin 1 dysregulation

Cleavage of mutant huntingtin (HTT) is an essential process in Huntington's disease (HD), an inherited neurodegenerative disorder. Cleavage generates N-ter fragments that contain the polyQ stretch and whose nuclear toxicity is well established. However, the functional defects induced by cleavage of full-length HTT remain elusive. Moreover, the contribution of non-polyQ C-terminal fragments is unknown. Using time- and site-specific control of full-length HTT proteolysis, we show that specific cleavages are required to disrupt intramolecular interactions within HTT and to cause toxicity in cells and flies. Surprisingly, in addition to the canonical pathogenic N-ter fragments, the C-ter fragments generated, that do not contain the polyQ stretch, induced toxicity via dilation of the endoplasmic reticulum (ER) and increased ER stress. C-ter HTT bound to dynamin 1 and subsequently impaired its activity at ER membranes. Our findings support a role for HTT on dynamin 1 function and ER homoeostasis. Proteolysis-induced alteration of this function may be relevant to disease. Synopsis The development of a time and site-specifically controlled cleavage of the mutant huntingtin protein reveals a pathogenic mechanism induced by the non-polyQ-containing fragments that are generated upon proteolysis during disease progression. Huntingtin proteolysis generates N-ter fragments that contain the toxic polyQ stretch but also the corresponding C-ter fragments. N-ter to C-ter intramolecular interactions present in full-length huntingtin are abrogated by sequential cleavages. Whereas the N-ter polyQ fragments translocate into the nucleus, the non-polyQ C-ter huntingtin fragments remain in the cytoplasm and cause ER dilation, stress and cell death. C-ter huntingtin fragments bind and inactivate dynamin 1 at the ER thus causing ER dilation and toxicity. Site-specifically controlled cleavage of the mutant huntingtin protein reveals a pathogenic mechanism induced by non-polyQ-containing fragments that are generated upon proteolysis during disease progression.</p

Nemitin, a Novel Map8/Map1s Interacting Protein with Wd40 Repeats

In neurons, a highly regulated microtubule cytoskeleton is essential for many cellular functions. These include axonal transport, regional specialization and synaptic function. Given the critical roles of microtubule-associated proteins (MAPs) in maintaining and regulating microtubule stability and dynamics, we sought to understand how this regulation is achieved. Here, we identify a novel LisH/WD40 repeat protein, tentatively named nemitin (neuronal enriched MAP interacting protein), as a potential regulator of MAP8-associated microtubule function. Based on expression at both the mRNA and protein levels, nemitin is enriched in the nervous system. Its protein expression is detected as early as embryonic day 11 and continues through adulthood. Interestingly, when expressed in non-neuronal cells, nemitin displays a diffuse pattern with puncta, although at the ultrastructural level it localizes along the microtubule network in vivo in sciatic nerves. These results suggest that the association of nemitin to microtubules may require an intermediary protein. Indeed, co-expression of nemitin with microtubule-associated protein 8 (MAP8) results in nemitin losing its diffuse pattern, instead decorating microtubules uniformly along with MAP8. Together, these results imply that nemitin may play an important role in regulating the neuronal cytoskeleton through an interaction with MAP8

Brain energy rescue:an emerging therapeutic concept for neurodegenerative disorders of ageing

The brain requires a continuous supply of energy in the form of ATP, most of which is produced from glucose by oxidative phosphorylation in mitochondria, complemented by aerobic glycolysis in the cytoplasm. When glucose levels are limited, ketone bodies generated in the liver and lactate derived from exercising skeletal muscle can also become important energy substrates for the brain. In neurodegenerative disorders of ageing, brain glucose metabolism deteriorates in a progressive, region-specific and disease-specific manner — a problem that is best characterized in Alzheimer disease, where it begins presymptomatically. This Review discusses the status and prospects of therapeutic strategies for countering neurodegenerative disorders of ageing by improving, preserving or rescuing brain energetics. The approaches described include restoring oxidative phosphorylation and glycolysis, increasing insulin sensitivity, correcting mitochondrial dysfunction, ketone-based interventions, acting via hormones that modulate cerebral energetics, RNA therapeutics and complementary multimodal lifestyle changes

Maladie de Huntington : signalisation intracellulaire et mort neuronale

La présence d'une expansion anormalement importante de l'acide aminé glutamine (polyQ) dans la protéine huntingtine conduit à la maladie de Huntington (HD). Aucun traitement n'est disponible à l'heure actuelle pour retarder les symptômes de cette maladie neurodégénérative. L'étude de voies de signalisation régulant la mort neuronale dans HD a permis de montrer l'importance de la phosphorylation. La voie IGF-1/Akt/SGK inhibe la toxicité induite par la huntingtine-polyQ. L'effet anti-apoptotique de cette voie est médié par la phosphorylation directe de la sérine 421 de la huntingtine. De plus, des composants de cette voie sont altérés au cours de la pathologie. Quelle est la fonction de la huntingtine ? Plusieurs études montrent que la huntingtine est une protéine anti-apoptotique impliquée dans la dynamique intracellulaire. Nous avons récemment trouvé que la huntingtine stimule le transport des vésicules contenant un facteur neurotrophique, le BDNF (brain-derived neurotrophic factor). Ce mécanisme fait intervenir un complexe constitué de la huntingtin-associated protein- 1, HAP1, et de la sous-unité p150Glued de la dynactine. Cette fonction de la huntingtine dans le transport est perdue lorsque celle-ci contient une expansion anormale de glutamines. L'altération du transport de BDNF a pour conséquence une diminution du support trophique et conduit à la mort cellulaire

Maladie de Huntington (caractérisation du rôle neuroprotecteur de la voie de signalisation induite par IGF-1)

LE KREMLIN-B.- PARIS 11-BU Méd (940432101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF