Generation and characterization of a murine model for Hereditary Spastic Paraplegia SPG11

Hereditary spastic paraplegias (HSPs) are clinically and genetically heterogenous group of movement disorders characterised mainly by a progressive spastic gait disorder and weakness of the legs, which results from a length-dependent axonopathy of corticospinal tract fibres. HSP type SPG11 is the most common recessive form and is caused by mutations in the SPG11 gene, which encodes the protein SPATACSIN. The pathophysiology of SPG11 is so far not well understood. In order to study the function of Spatacsin and to elucidate the key events leading to SPG11, Spg11 was disrupted in mice. Lack of Spatacsin in mice indeed causes a progressive gait disorder, which is paralleled by a progressive loss of cortical neurons and Purkinje cells. Degenerating neurons accumulate autofluorescent material. These deposits are associated with the lysosomal protein Lamp1, p62, a receptor for cargo destined to be degraded by autophagy, and with LC3, a marker for autophagosomes, suggesting that the deposits are related to autolysosomes. Distinct lysosomal functions, such as the processing of Cathepsin D and lysosomal pH do not differ between genotypes. Moreover, the levels of Lamp1 both in brain tissue and mouse embryonic fibroblast (MEF) lysates are not altered in knockout mice. Further supporting a defect in autophagic clearance, in knockout MEFs lipidated LC3 levels are increased and p62 levels are elevated in brain lysates. Consistently, the number of autolysosomes is increased while the number of lysosomes is decreased in KO MEFs. Upon starvation, the number of lysosomes decrease, but the lysosome number recovers upon sustained starvation only in wild-tpye MEFs. A depletion of lysosomes was also evident in vivo in Purkinje cells of 2 and 11-month-old knockout mice. These data suggest that owing to the decreased number of lysosomes, available for fusion with autophagosomes in knockout mice, autolysosomal clearance may be impaired which results in the accumulation of un-degraded material and finally causes death of particularly sensitive neurons like cortical motoneurons and Purkinje cells

Mouse models for hereditary spastic paraplegia uncover a role of PI4K2A in autophagic lysosome reformation

Hereditary spastic paraplegia (HSP) denotes genetically heterogeneous disorders characterized by leg spasticity due to degeneration of corticospinal axons. SPG11 and SPG15 have a similar clinical course and together are the most prevalent autosomal recessive HSP entity. The respective proteins play a role for macroautophagy/autophagy and autophagic lysosome reformation (ALR). Here, we report that spg11 and zfyve26 KO mice developed motor impairments within the same course of time. This correlated with enhanced accumulation of autofluorescent material in neurons and progressive neuron loss. In agreement with defective ALR, tubulation events were diminished in starved KO mouse embryonic fibroblasts (MEFs) and lysosomes decreased in neurons of KO brain sections. Confirming that both proteins act in the same molecular pathway, the pathologies were not aggravated upon simultaneous disruption of both. We further show that PI4K2A (phosphatidylinositol 4-kinase type 2 alpha), which phosphorylates phosphatidylinositol to phosphatidylinositol-4-phosphate (PtdIns4P), accumulated in autofluorescent deposits isolated from KO but not WT brains. Elevated PI4K2A abundance was already found at autolysosomes of neurons of presymptomatic KO mice. Immunolabelings further suggested higher levels of PtdIns4P at LAMP1-positive structures in starved KO MEFs. An increased association with LAMP1-positive structures was also observed for clathrin and DNM2/dynamin 2, which are important effectors of ALR recruited by phospholipids. Because PI4K2A overexpression impaired ALR, while its knockdown increased tubulation, we conclude that PI4K2A modulates phosphoinositide levels at autolysosomes and thus the recruitment of downstream effectors of ALR. Therefore, PI4K2A may play an important role in the pathogenesis of SPG11 and SPG15. Abbreviations: ALR: autophagic lysosome reformation; AP-5: adaptor protein complex 5; BFP: blue fluorescent protein; dKO: double knockout; EBSS: Earle’s balanced salt solution; FBA: foot base angle; GFP: green fluorescent protein; HSP: hereditary spastic paraplegia; KO: knockout; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; SQSTM1/p62: sequestosome 1; PI4K2A: phosphatidylinositol 4-kinase type 2 alpha; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns4P: phosphatidylinositol-4-phosphate; RFP: red fluorescent protein; SPG: spastic paraplegia gene; TGN: trans-Golgi network; WT: wild typ

Scoping review and evidence mapping of interventions aimed at improving reproducible and replicable science: Protocol

BACKGROUND: Many interventions, especially those linked to open science, have been proposed to improve reproducibility in science. To what extent these propositions are based on scientific evidence from empirical evaluations is not clear. AIMS: The primary objective is to identify Open Science interventions that have been formally investigated regarding their influence on reproducibility and replicability. A secondary objective is to list any facilitators or barriers reported and to identify gaps in the evidence. METHODS: We will search broadly by using electronic bibliographic databases, broad internet search, and contacting experts in the field of reproducibility, replicability, and open science. Any study investigating interventions for their influence on the reproducibility and replicability of research will be selected, including those studies additionally investigating drivers and barriers to the implementation and effectiveness of interventions. Studies will first be selected by title and abstract (if available) and then by reading the full text by at least two independent reviewers. We will analyze existing scientific evidence using scoping review and evidence gap mapping methodologies. RESULTS: The results will be presented in interactive evidence maps, summarized in a narrative synthesis, and serve as input for subsequent research. REVIEW REGISTRATION: This protocol has been pre-registered on OSF under doi https://doi.org/10.17605/OSF.IO/D65YS

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Do Not Trust the Pedigree: Reduced and Sex‐Dependent Penetrance at a Novel Mutation Hotspot in ATL1 Blurs Autosomal Dominant Inheritance of Spastic Paraplegia

ABSTRACT The hereditary spastic paraplegias (HSPs), a group of neurodegenerative movement disorders, are among the genetically most heterogeneous clinical conditions. Still, the more than 50 forms known so far apparently explain less than 80% of cases. The present study identified two large HSP families, which seemed to show an autosomal recessive and an X‐linked inheritance pattern. A set of genetic analyses including exome sequencing revealed plausible mutations only when assuming incomplete/sex‐dependent penetrance of adjacent alterations in the autosomal dominant HSP gene ATL1 (c.1243C>T and c.1244G>A, respectively). By screening of additional HSP patients for the presence of these alterations, we identified three more cases and obtained additional evidence for reduced penetrance. Bisulfate sequencing and haplotype analysis indicated that c.1243C and c.1244G constitute a mutational hotspot. Our findings suggest that misinterpretation of inheritance patterns and, consequently, misselection of candidate genes to be screened in gene‐focused approaches contribute to the apparently missing heritability in HSP and, potentially, in other genetically heterogeneous disorders. Inheritance patterns usually guide gene selection in mutational screening strategies. By whole exome sequencing of index cases with apparently novel forms of spastic paraplegia, we identify a mutational hotspot in the known dominant gene ATL1 and show that corresponding alterations are associated with a highly reduced and partially sex‐dependent risk of developing the disease. Our findings suggest that misleading family history may contribute to missing heritability in genetically heterogeneous disorders

Scoping review and evidence mapping of interventions aimed at improving reproducible and replicable science: Protocol

International audienceBackground: Many interventions, especially those linked to open science, have been proposed to improve reproducibility in science. To what extent these propositions are based on scientific evidence from empirical evaluations is not clear.Aims: The primary objective is to identify Open Science interventions that have been formally investigated regarding their influence on reproducibility and replicability. A secondary objective is to list any facilitators or barriers reported and to identify gaps in the evidence.Methods: We will search broadly by using electronic bibliographic databases, broad internet search, and contacting experts in the field of reproducibility, replicability, and open science. Any study investigating interventions for their influence on the reproducibility and replicability of research will be selected, including those studies additionally investigating drivers and barriers to the implementation and effectiveness of interventions. Studies will first be selected by title and abstract (if available) and then by reading the full text by at least two independent reviewers. We will analyze existing scientific evidence using scoping review and evidence gap mapping methodologies.Results: The results will be presented in interactive evidence maps, summarized in a narrative synthesis, and serve as input for subsequent research.Review registration: This protocol has been pre-registered on OSF under doi https://doi.org/10.17605/OSF.IO/D65YS

<i>In Vivo</i> Evidence for Lysosome Depletion and Impaired Autophagic Clearance in Hereditary Spastic Paraplegia Type SPG11

<div><p>Hereditary spastic paraplegia (HSP) is characterized by a dying back degeneration of corticospinal axons which leads to progressive weakness and spasticity of the legs. SPG11 is the most common autosomal-recessive form of HSPs and is caused by mutations in <i>SPG11</i>. A recent <i>in vitro</i> study suggested that Spatacsin, the respective gene product, is needed for the recycling of lysosomes from autolysosomes, a process known as autophagic lysosome reformation. The relevance of this observation for hereditary spastic paraplegia, however, has remained unclear. Here, we report that disruption of Spatacsin in mice indeed causes hereditary spastic paraplegia-like phenotypes with loss of cortical neurons and Purkinje cells. Degenerating neurons accumulate autofluorescent material, which stains for the lysosomal protein Lamp1 and for p62, a marker of substrate destined to be degraded by autophagy, and hence appears to be related to autolysosomes. Supporting a more generalized defect of autophagy, levels of lipidated LC3 are increased in Spatacsin knockout mouse embryonic fibrobasts (MEFs). Though distinct parameters of lysosomal function like processing of cathepsin D and lysosomal pH are preserved, lysosome numbers are reduced in knockout MEFs and the recovery of lysosomes during sustained starvation impaired consistent with a defect of autophagic lysosome reformation. Because lysosomes are reduced in cortical neurons and Purkinje cells <i>in vivo</i>, we propose that the decreased number of lysosomes available for fusion with autophagosomes impairs autolysosomal clearance, results in the accumulation of undegraded material and finally causes death of particularly sensitive neurons like cortical motoneurons and Purkinje cells in knockout mice.</p></div

Homozygous trapped mice represent Spatacsin knockout mice.

<p><b>(A)</b> Partial genomic structure of the targeted <i>Spg11</i> locus and the predicted mutant fusion protein as compared to wild-type Spatacsin; rectangles: exons, SA: splice acceptor, βgeo: β-galactosidase and neomycin fusion cassette, pA: polyadenylation site. The black arrowhead indicates the position of the epitope for antibody generation. CHC: clathrin heavy chain. <b>(B-H)</b> LacZ stainings of sections of the cortex (B-D), hippocampus (E), cerebellum (F), brain stem (G), and spinal cord (H) from 2-month-old heterozygous trapped mice shows that <i>Spg11</i> expression follows a neuronal pattern. DG: dentate gyrus; GL: granular layer, PCL: Purkinje cell layer, ML: molecular layer; SOC: superior olivary complex, IOC: inferior olivary complex. Scale bars: 100 μm. <b>(I)</b> Northern blot analysis of total brain RNA from wild-type (WT) mice shows a 7.6 kb WT-transcript which is absent in RNA isolated from homozygous trapped mice (KO). <i>Gapdh</i> served as a loading control. <b>(J)</b> Western Blot analysis with an affinity-purified monoclonal antibody directed against the deleted part of the Spatacsin protein (the position of the epitope is indicated in (A) by an arrowhead) detects a band of the predicted size in WT brain lysates, which is absent in brain lysates of homozygous trapped mice. Tubulin served as a loading control. (<b>K</b>) While Zfyve26 levels are diminished in brain lysates of Spatacsin KO mice, levels of the beta subunit of the AP-5 complex (Ap5b1) are not changed (n = 3; Student’s t-test: * indicates p<0.05; n.s.: not significant).</p