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

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

Positive and negative viral associations in patients with acute respiratory tract infections in primary care: the ECOVIR study

IntroductionAcute respiratory infections (ARIs) are the most common viral infections encountered in primary care settings. The identification of causal viruses is still not available in routine practice. Although new strategies of prevention are being identified, knowledge of the relationships between respiratory viruses remains limited.Materials and methodsECOVIR was a multicentric prospective study in primary care, which took place during two pre-pandemic seasons (2018–2019 and 2019–2020). Patients presenting to their General practitioner (GP) with ARIs were included, without selecting for age or clinical conditions. Viruses were detected on nasal swab samples using a multiplex Polymerase Chain Reaction test focused on 17 viruses [Respiratory Syncytial Virus-A (RSV-A), RSV-B, Rhinovirus/Enterovirus (HRV), human Metapneumovirus (hMPV), Adenovirus (ADV), Coronaviruses (CoV) HKU1, NL63, 229E, OC43, Influenza virus (H1 and H3 subtypes), Influenza virus B, Para-Influenza viruses (PIVs) 1–4, and Bocavirus (BoV)].ResultsAmong the 668 analyzed samples, 66% were positive for at least one virus, of which 7.9% were viral codetections. The viral detection was negatively associated with the age of patients. BoV, ADV, and HRV occurred more significantly in younger patients than the other viruses (p &lt; 0.05). Codetections were significantly associated with RSV, HRV, BoV, hMPV, and ADV and not associated with influenza viruses, CoV, and PIVs. HRV and influenza viruses were negatively associated with all the viruses. Conversely, a positive association was found between ADV and BoV and between PIVs and BoV.ConclusionOur study provides additional information on the relationships between respiratory viruses, which remains limited in primary care

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

The Glasgow consensus on the delineation between pesticide emission inventory and impact assessment for LCA

Purpose: Pesticides are applied to agricultural fields in order to optimise crop yield and their global use is substantial. Their consideration in Life cycle assessment (LCA) is currently affected by important inconsistencies between the emission inventory and impact assessment phases of LCA. A clear definition of the delineation between the product system model (life cycle inventory, technosphere) and the natural environment (life cycle impact assessment, ecosphere) is currently missing and could be established via consensus building. Methods: A workshop held on the 11 May 2013, in Glasgow, UK, back to back with the 23rd SETAC Europe meeting had the goal of establishing consensus and creating clear guidelines where the boundary between the emission inventory and the impact characterisation model should be set in all three spatial dimensions and time when considering application of substances to an open agricultural field or in greenhouses, and consequent emissions to the natural environment and their potential impacts. More than 30 specialists in agrifood LCI, LCIA, risk assessment, and ecotoxicology, representing industry, government, and academia from 15 countries and four continents met to discuss and reach consensus. The resulting guidelines target LCA practitioners, data (base) and characterisation method developers, and decision makers. Results and discussion: Although, the initial goal was to define recommendations concerning boundaries between technosphere and ecosphere, it became clear that these strongly depend on goal and scope of an LCA study. Instead, the focus was on defining a clear interface between LCI and LCIA, capable of supporting any goal and scope requirements while avoiding double counting or exclusion of important emission flows and their potential impacts. Consensus was reached accordingly on distinct sets of recommendations for LCI and LCIA respectively, recommending for example that buffer zones should be considered as part of the crop production system and the change in yield per ha be considered. While the spatial dimensions of the field were not fixed, the temporal boundary between dynamic LCI fate modelling and steady-state LCIA fate modelling needs to be defined. Conclusions and recommendations: For pesticides application, the inventory should report: pesticide identification, crop, mass applied of each active ingredient, application method or formulation type, presence of buffer zones (y/n), location/country, application time in days before harvest and crop growth stage during application, adherence with Good Agricultural Practice (GAP), and whether the field is considered part of the technosphere or the ecosphere. Additionally, emission fractions to defined environmental media on-field and off-field should be reported. For LCIA, the directly concerned impact categories were identified as well as a list of relevant fate and exposure processes. Next steps and future work were identified: 1) establishing default emission fractions to environmental media for integration into LCI databases, and 2) interaction among impact model developers to extend current methods with new elements/processes mentioned in the recommendations, including targeted technical workshops on “how to” model specific processes.JRC.H.8-Sustainability Assessmen

A global database for metacommunity ecology, integrating species, traits, environment and space

The use of functional information in the form of species traits plays an important role in explaining biodiversity patterns and responses to environmental changes. Although relationships between species composition, their traits, and the environment have been extensively studied on a case-by-case basis, results are variable, and it remains unclear how generalizable these relationships are across ecosystems, taxa and spatial scales. To address this gap, we collated 80 datasets from trait-based studies into a global database for metaCommunity Ecology: Species, Traits, Environment and Space; “CESTES”. Each dataset includes four matrices: species community abundances or presences/absences across multiple sites, species trait information, environmental variables and spatial coordinates of the sampling sites. The CESTES database is a live database: it will be maintained and expanded in the future as new datasets become available. By its harmonized structure, and the diversity of ecosystem types, taxonomic groups, and spatial scales it covers, the CESTES database provides an important opportunity for synthetic trait-based research in community ecology

Is Huntington disease a developmental disorder?

The recently discovered roles of huntingtin in non-differentiated cells indicate that it is a key molecule in brain development. Humbert argues that the haploinsufficiency of wild-type huntingtin in Huntington disease might lead to various cellular alterations well before the onset of symptoms and ultimately cause disease