Collagen VI is a major extracellular matrix protein exerting a number of functions in different tissues, spanning from biomechanical to regulatory signals in the cell survival processes, and playing ...key roles in maintaining the stemness or determining the differentiation of several types of cells. In the last couple of years, emerging findings on collagen VI have led to increased interest in its role in the nervous system. The role of this protein in the peripheral nervous system was intensely studied and characterized in detail. Collagen VI acts as a regulator of Schwann cell differentiation and is required for preserving peripheral nerve myelination, function and structure, as well as for orchestrating nerve regeneration after injury. Although the role and distribution of collagen VI in the peripheral nervous system is now well established, the role of this distinctive extracellular matrix component in the central nervous system, along with its links to human neurological and neurodegenerative disorders, remains an open field of investigation. In this Review, we summarize and discuss a number of recent findings related to collagen VI in the central and peripheral nervous systems. We further link these findings to different aspects of the protein that are relevant to human diseases in these compartments in order to provide a comprehensive overview of the roles of this key matrix component in the nervous system.
The cardiac stroma contains multipotent mesenchymal progenitors. However, lineage relationships within cardiac stromal cells are poorly defined. Here, we identified heart-resident PDGFRa+ SCA-1+ ...cells as cardiac fibro/adipogenic progenitors (cFAPs) and show that they respond to ischemic damage by generating fibrogenic cells. Pharmacological blockade of this differentiation step with an anti-fibrotic tyrosine kinase inhibitor decreases post-myocardial infarction (post-MI) remodeling and leads to improvement in cardiac function. In the undamaged heart, activation of cFAPs through lineage-specific deletion of the gene encoding the quiescence-associated factor HIC1 reveals additional pathogenic potential, causing fibrofatty infiltration within the myocardium and driving major pathological features pathognomonic in arrhythmogenic cardiomyopathy (AC). In this regard, cFAPs contribute to multiple pathogenic cell types within cardiac tissue and therapeutic strategies aimed at modifying their activity are expected to have tremendous benefit for the treatment of diverse cardiac diseases.
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•Cardiac PDGFRa+ SCA-1+ cells (cFAPs) are multipotent mesenchymal progenitors in vivo•PDGFRa+ SCA-1+ cells differentiate into fibrogenic SCA-1− cells upon damage•Blockade of cFAP differentiation ameliorates cardiac dysfunction post-MI•Damage-free cFAP activation leads to arrhythmogenic cardiomyopathy
Soliman et al. identify a subset of cardiac fibroblasts as multipotent stromal progenitors and describe their dynamic response to damage. Beyond forming the scar, these cells have a pathogenic role in post-myocardial infarction remodeling, and their chronic activation by loss of quiescent factor Hic1 triggers fibrofatty infiltration and arrhythmogenic cardiomyopathy.
Dent disease (DD1) is a rare tubulopathy caused by mutations in the CLCN5 gene. Glomerulosclerosis was recently reported in DD1 patients and ClC-5 protein was shown to be expressed in human ...podocytes. Nephrin and actin cytoskeleton play a key role for podocyte functions and podocyte endocytosis seems to be crucial for slit diaphragm regulation. The aim of this study was to analyze whether ClC-5 loss in podocytes might be a direct consequence of the glomerular damage in DD1 patients. Three DD1 kidney biopsies presenting focal global glomerulosclerosis and four control biopsies were analyzed by immunofluorescence (IF) for nephrin and podocalyxin, and by immunohistochemistry (IHC) for ClC-5. ClC-5 resulted as down-regulated in DD1 vs. control (CTRL) biopsies in both tubular and glomerular compartments (p < 0.01). A significant down-regulation of nephrin (p < 0.01) in DD1 vs. CTRL was demonstrated. CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Caspase9) gene editing of CLCN5 in conditionally immortalized human podocytes was used to obtain clones with the stop codon mutation p.(R34Efs*14). We showed that ClC-5 and nephrin expression, analyzed by quantitative Reverse Transcription/Polymerase Chain Reaction (qRT/PCR) and In-Cell Western (ICW), was significantly downregulated in mutant clones compared to the wild type ones. In addition, F-actin staining with fluorescent phalloidin revealed actin derangements. Our results indicate that ClC-5 loss might alter podocyte function either through cytoskeleton disorganization or through impairment of nephrin recycling.
Collagen VI (ColVI) is an abundant and distinctive extracellular matrix protein secreted by fibroblasts in different tissues. Human diseases linked to mutations on ColVI genes are primarily affecting ...skeletal muscle due to non-cell autonomous myofiber defects. To date, it is not known whether and how fibroblast homeostasis is affected by ColVI deficiency, a critical missing information as this may strengthen the use of patients' fibroblasts for preclinical purposes. Here, we established primary and immortalized fibroblast cultures from ColVI null (
) mice, the animal model of ColVI-related diseases. We found that, under nutrient-stringent condition, lack of ColVI affects fibroblast survival, leading to increased apoptosis. Moreover,
fibroblasts display defects in the autophagy/lysosome machinery, with impaired clearance of autophagosomes and failure of Parkin-dependent mitophagy.
fibroblasts also show an increased activation of the Akt/mTOR pathway, compatible with the autophagy impairment, and adhesion onto purified ColVI elicits a major effect on the autophagic flux. Our findings reveal that ColVI ablation in fibroblasts impacts on autophagy regulation and cell survival, thus pointing at the new concept that this cell type may contribute to the pathological features of ColVI-related diseases.
Pericytes are a distinct type of cells interacting with endothelial cells in blood vessels and contributing to endothelial barrier integrity. Furthermore, pericytes show mesenchymal stem cell ...properties. Muscle-derived pericytes can demonstrate both angiogenic and myogenic capabilities. It is well known that regenerative abilities and muscle stem cell potential decline during aging, leading to sarcopenia. Therefore, this study aimed to investigate the potential of pericytes in supporting muscle differentiation and angiogenesis in elderly individuals and in patients affected by Ullrich congenital muscular dystrophy or by Bethlem myopathy, two inherited conditions caused by mutations in collagen VI genes and sharing similarities with the progressive skeletal muscle changes observed during aging. The study characterized pericytes from different age groups and from individuals with collagen VI deficiency by mass spectrometry-based proteomic and bioinformatic analyses. The findings revealed that aged pericytes display metabolic changes comparable to those seen in aging skeletal muscle, as well as a decline in their stem potential, reduced protein synthesis, and alterations in focal adhesion and contractility, pointing to a decrease in their ability to form blood vessels. Strikingly, pericytes from young patients with collagen VI deficiency showed similar characteristics to aged pericytes, but were found to still handle oxidative stress effectively together with an enhanced angiogenic capacity.
Mutations in the β-glucocerebrosidase (GBA1) gene do cause the lysosomal storage Gaucher disease (GD) and are among the most frequent genetic risk factors for Parkinson's disease (PD). So far, ...studies on both neuronopathic GD and PD primarily focused on neuronal manifestations, besides the evaluation of microglial and astrocyte implication. White matter alterations were described in the central nervous system of paediatric type 1 GD patients and were suggested to sustain or even play a role in the PD process, although the contribution of oligodendrocytes has been so far scarcely investigated.
We exploited a system to study the induction of central myelination in vitro, consisting of Oli-neu cells treated with dibutyryl-cAMP, in order to evaluate the expression levels and function of β-glucocerebrosidase during oligodendrocyte differentiation. Conduritol-B-epoxide, a β-glucocerebrosidase irreversible inhibitor was used to dissect the impact of β-glucocerebrosidase inactivation in the process of myelination, lysosomal degradation and α-synuclein accumulation in vitro. Moreover, to study the role of β-glucocerebrosidase in the white matter in vivo, we developed a novel mouse transgenic line in which β-glucocerebrosidase function is abolished in myelinating glia, by crossing the Cnp1-cre mouse line with a line bearing loxP sequences flanking Gba1 exons 9-11, encoding for β-glucocerebrosidase catalytic domain. Immunofluorescence, western blot and lipidomic analyses were performed in brain samples from wild-type and knockout animals in order to assess the impact of genetic inactivation of β-glucocerebrosidase on myelination and on the onset of early neurodegenerative hallmarks, together with differentiation analysis in primary oligodendrocyte cultures.
Here we show that β-glucocerebrosidase inactivation in oligodendrocytes induces lysosomal dysfunction and inhibits myelination in vitro. Moreover, oligodendrocyte-specific β-glucocerebrosidase loss-of-function was sufficient to induce in vivo demyelination and early neurodegenerative hallmarks, including axonal degeneration, α-synuclein accumulation and astrogliosis, together with brain lipid dyshomeostasis and functional impairment.
Our study sheds light on the contribution of oligodendrocytes in GBA1-related diseases and supports the need for better characterizing oligodendrocytes as actors playing a role in neurodegenerative diseases, also pointing at them as potential novel targets to set a brake to disease progression.
Background
Maintaining healthy mitochondria is mandatory for muscle viability and function. An essential surveillance mechanism targeting defective and harmful mitochondria to degradation is the ...selective form of autophagy called mitophagy. Ambra1 is a multifaceted protein with well‐known autophagic and mitophagic functions. However, the study of its role in adult tissues has been extremely limited due to the embryonic lethality caused by full‐body Ambra1 deficiency.
Methods
To establish the role of Ambra1 as a positive regulator of mitophagy, we exploited in vivo overexpression of a mitochondria‐targeted form of Ambra1 in skeletal muscle. To dissect the consequence of Ambra1 inactivation in skeletal muscle, we generated muscle‐specific Ambra1 knockout (Ambra1fl/fl:Mlc1f‐Cre) mice. Mitochondria‐enriched fractions were obtained from muscles of fed and starved animals to investigate the dynamics of the mitophagic flux.
Results
Our data show that Ambra1 has a critical role in the mitophagic flux of adult murine skeletal muscle and that its genetic inactivation leads to mitochondria alterations and myofibre remodelling. Ambra1 overexpression in wild‐type muscles is sufficient to enhance mitochondria clearance through the autophagy‐lysosome system. Consistently with this, Ambra1‐deficient muscles display an abnormal accumulation of the mitochondrial marker TOMM20 by +76% (n = 6–7; P < 0.05), a higher presence of myofibres with swollen mitochondria by +173% (n = 4; P < 0.05), and an alteration in the maintenance of the mitochondrial membrane potential and a 34% reduction in the mitochondrial respiratory complex I activity (n = 4; P < 0.05). Lack of Ambra1 in skeletal muscle leads to impaired mitophagic flux, without affecting the bulk autophagic process. This is due to a significantly decreased recruitment of DRP1 (n = 6–7 mice; P < 0.01) and Parkin (n = 6–7 mice; P < 0.05) to the mitochondrial compartment, when compared with controls. Ambra1‐deficient muscles also show a marked dysregulation of the endolysosome compartment, as the incidence of myofibres with lysosomal accumulation is 20 times higher than wild‐type muscles (n = 4; P < 0.05). Histologically, Ambra1‐deficient muscles of both 3‐ and 6‐month‐old animals display a significant decrease of myofibre cross‐sectional area and a 52% reduction in oxidative fibres (n = 6–7; P < 0.05), thus highlighting a role for Ambra1 in the proper structure and activity of skeletal muscle.
Conclusions
Our study indicates that Ambra1 is critical for skeletal muscle mitophagy and for the proper maintenance of functional mitochondria.
Wnt/β-catenin signaling plays key roles in several developmental and pathological processes. Domains of Wnt expression have been extensively investigated in the mouse, but the tissues receiving the ...signal remain largely unidentified. To define which cells respond to activated β-catenin during mammalian development, we generated the β-catenin-activated transgene driving expression of nuclear β-galactosidase reporter (BAT-gal) transgenic mice, expressing the lacZ gene under the control of β-catenin/T cell factor responsive elements. Reporter gene activity is found in known organizing centers, such as the midhindbrain border and the limb apical ectodermal ridge. Moreover, BAT-gal expression identifies novel sites of Wnt signaling, like notochord, endothelia, and areas of the adult brain, revealing an unsuspected dynamic pattern of β-catenin transcriptional activity. Expression of the transgene was analyzed in mutant backgrounds. In lipoprotein receptor-related protein 6-null homozygous mice, which lack a Wnt coreceptor, BAT-gal staining is absent in mutant tissues, indicating that BAT-gal mice are bona fide in vivo indicators of Wnt/β-catenin signaling. Analyses of BAT-gal expression in the adenomatous polyposis coli (multiple intestinal neoplasia/+) background revealed β-catenin transcriptional activity in intestinal adenomas but surprisingly not in normal crypt cells. In summary, BAT-gal mice unveil the entire complexity of Wnt/β-catenin signaling in mammals and have broad application potentials for the identification of Wnt-responsive cell populations in development and disease.
Ullrich congenital muscular dystrophy and Bethlem myopathy are skeletal muscle diseases that are due to mutations in the genes encoding collagen VI, an extracellular matrix protein forming a ...microfibrillar network that is particularly prominent in the endomysium of skeletal muscle. Myoblasts from patients affected by Ullrich congenital muscular dystrophy display functional and ultrastructural mitochondrial alterations and increased apoptosis due to inappropriate opening of the permeability transition pore, a mitochondrial inner membrane channel. These alterations could be normalized by treatment with cyclosporin A, a widely used immunosuppressant that desensitizes the permeability transition pore independently of calcineurin inhibition. Here, we report the results of an open pilot trial with cyclosporin A in five patients with collagen VI myopathies. Before treatment, all patients displayed mitochondrial dysfunction and increased frequency of apoptosis, as determined in muscle biopsies. Both of these pathologic signs were largely normalized after 1 month of oral cyclosporin A administration, which also increased muscle regeneration. These findings demonstrate that collagen VI myopathies can be effectively treated with drugs acting on the pathogenic mechanism downstream of the genetic lesion, and they represent an important proof of principle for the potential therapy of genetic diseases.
Dissection of mechanisms involved in the regulation of bone marrow microenvironment through cell-cell and cell-matrix contacts is essential for the detailed understanding of processes underlying bone ...marrow activities both under physiological conditions and in hematologic malignancies. Here we describe Emilin-2 as an abundant extracellular matrix component of bone marrow stroma.
Immunodetection of Emilin-2 was performed in bone marrow sections of mice from 30 days to 6 months of age. Emilin-2 expression was monitored in vitro in primary and mesenchymal stem cell lines under undifferentiated and adipogenic conditions. Hematopoietic stem cells and progenitors in bone marrow of 3- to 10-month-old wild-type and Emilin-2 null mice were analyzed by flow cytometry.
Emilin-2 is deposited in bone marrow extracellular matrix in an age-dependent manner, forming a meshwork that extends from compact bone boundaries to the central trabecular regions. Emilin-2 is expressed and secreted by both primary and immortalized bone marrow mesenchymal stem cells, exerting an inhibitory action in adipogenic differentiation. In vivo Emilin-2 deficiency impairs the frequency of hematopoietic stem/progenitor cells in bone marrow during aging.
Our data provide new insights in the contribution of bone marrow extracellular matrix microenvironment in the regulation of stem cell niches and hematopoietic progenitor differentiation.
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