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ABSTRACTEndoplasmic reticulum stress is an emerging significant player in the molecular pathology of connective tissue disorders. In response to endoplasmic reticulum stress, cells can upregulate macroautophagy/autophagy, a fundamental cellular homeostatic process used by cells to degrade and recycle proteins or remove damaged organelles. In these scenarios, autophagy activation can support cell survival. Here we demonstrated by in vitroand in vivoapproaches that megakaryocytes derived from col6a1−⁄−(collagen, type VI, alpha 1) null mice display increased intracellular retention of COL6 polypeptides, endoplasmic reticulum stress and apoptosis. The unfolded protein response is activated in col6a1−⁄−megakaryocytes, as evidenced by the upregulation of molecular chaperones, by the increased splicing of Xbp1mRNA and by the higher level of the pro-apoptotic regulator DDIT3/CHOP. Despite the endoplasmic reticulum stress, basal autophagy is impaired in col6a1−⁄−megakaryocytes, which show lower BECN1 levels and reduced autophagosome maturation. Starvation and rapamycin treatment rescue the autophagic flux in col6a1−⁄−megakaryocytes, leading to a decrease in intracellular COL6 polypeptide retention, endoplasmic reticulum stress and apoptosis. Furthermore, megakaryocytes cultured from peripheral blood hematopoietic progenitors of patients affected by Bethlem myopathy and Ullrich congenital muscular dystrophy, two COL6-related disorders, displayed increased apoptosis, endoplasmic reticulum stress and impaired autophagy. These data demonstrate that genetic disorders of collagens, endoplasmic reticulum stress and autophagy regulation in megakaryocytes may be interrelated.Abbreviations:7-AAD: 7-amino-actinomycin D; ATF: activating transcriptional factor; BAX: BCL2 associated X protein; BCL2: B cell leukemia/lymphoma 2; BCL2L1/Bcl-xL: BCL2-like 1; BM: bone marrow; COL6: collagen, type VI; col6a1−⁄−: mice that are null for Col6a1; DDIT3/CHOP/GADD153: DNA-damage inducible transcript 3; EGFP: enhanced green fluorescent protein; ER: endoplasmic reticulum; reticulophagy: endoplasmic reticulum-selective autophagy; HSPA5/Bip: heat shock protein 5; HSP90B1/GRP94: heat shock protein 90, beta (Grp94), member 1; LAMP2: lysosomal associated membrane protein 2; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; Mk: megakaryocytes; MTOR: mechanistic target of rapamycin kinase; NIMV: noninvasive mechanical ventilation; PI3K: phosphoinositide 3-kinase; PPP1R15A/GADD34: protein phosphatase 1, regulatory subunit 15A; RT-qPCR: reverse transcription-quantitative real-time PCR; ROS: reactive oxygen species; SERPINH1/HSP47: serine (or cysteine) peptidase inhibitor, clade H, member 1; sh-RNA: short hairpin RNA; SOCE: store operated calcium entry; UCMD: Ullrich congenital muscular dystrophy; UPR: unfolded protein response; WIPI2: WD repeat domain, phosphoinositide-interacting 2; WT: wild type; XBP1: X-box binding protein 1.

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. To establish the role of Ambra1 as a positive regulator of mitophagy, we exploited in vivooverexpression of a mitochondria‐targeted form of Ambra1 in skeletal muscle. To dissect the consequence of Ambra1 inactivation in skeletal muscle, we generated muscle‐specific Ambra1knockout (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. 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. Our study indicates that Ambra1 is critical for skeletal muscle mitophagy and for the proper maintenance of functional mitochondria.

Hemostatic abnormalities and impaired platelet function have been described in patients affected by connective tissue disorders. We observed a moderate bleeding tendency in patients affected by collagen VI–related disorders and investigated the defects in platelet functionality, whose mechanisms are unknown. We demonstrated that megakaryocytes express collagen VI that is involved in the regulation of functional platelet production. By exploiting a collagen VI–null mouse model (Col6a1−/−), we found that collagen VI–null platelets display significantly increased susceptibility to activation and intracellular calcium signaling. Col6a1−/−megakaryocytes and platelets showed increased expression of stromal interaction molecule 1 (STIM1) and ORAI1, the components of store-operated calcium entry (SOCE), and activation of the mammalian target of rapamycin (mTOR) signaling pathway. In vivo mTOR inhibition by rapamycin reduced STIM1 and ORAI1 expression and calcium flows, resulting in a normalization of platelet susceptibility to activation. These defects were cell autonomous, because transplantation of lineage-negative bone marrow cells from Col6a1−/−mice into lethally irradiated wild-type animals showed the same alteration in SOCE and platelet activation seen in Col6a1−/−mice. Peripheral blood platelets of patients affected by collagen VI–related diseases, Bethlem myopathy and Ullrich congenital muscular dystrophy, displayed increased expression of STIM1 and ORAI1 and were more prone to activation. Altogether, these data demonstrate the importance of collagen VI in the production of functional platelets by megakaryocytes in mouse models and in collagen VI–related diseases.

Skeletal muscle mitochondria readily accumulate Ca2+in response to SR store-releasing stimuli thanks to the activity of the mitochondrial calcium uniporter (MCU), the highly selective channel responsible for mitochondrial Ca2+uptake. MCU positively regulates myofiber size in physiological conditions and counteracts pathological loss of muscle mass. Here we show that skeletal muscle-specific MCU deletion inhibits myofiber mitochondrial Ca2+uptake, impairs muscle force and exercise performance, and determines a slow to fast switch in MHC expression. Mitochondrial Ca2+uptake is required for effective glucose oxidation, as demonstrated by the fact that in muscle-specific MCU−/−myofibers oxidative metabolism is impaired and glycolysis rate is increased. Although defective, mitochondrial activity is partially sustained by increased fatty acid (FA) oxidation. In MCU−/−myofibers, PDP2 overexpression drastically reduces FA dependency, demonstrating that decreased PDH activity is the main trigger of the metabolic rewiring of MCU−/−muscles. Accordingly, PDK4 overexpression in MCUfl/flmyofibers is sufficient to increase FA-dependent respiration. Finally, as a result of the muscle-specific MCU deletion, a systemic catabolic response impinging on both liver and adipose tissue metabolism occurs.

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EMILIN2 is an extracellular matrix constituent playing an important role in angiogenesis; however, the underlying mechanism is unknown. Here we show that EMILIN2 promotes angiogenesis by directly binding epidermal growth factor receptor (EGFR), which enhances interleukin-8 (IL-8) production. In turn, IL-8 stimulates the proliferation and migration of vascular endothelial cells. Emilin2null mice were generated and exhibited delayed retinal vascular development, which was rescued by the administration of the IL-8 murine ortholog MIP-2. Next, we assessed tumor growth and tumor-associated angiogenesis in these mice. Tumor cell growth in Emilin2null mice was impaired as well as the expression of MIP-2. The vascular density of the tumors developed in Emilin2null mice was prejudiced and vessels perfusion, as well as response to chemotherapy, decreased. Accordingly, human tumors expressing high levels of EMILIN2 were more responsive to chemotherapy. These results point at EMILIN2 as a key microenvironmental cue affecting vessel formation and unveil the possibility to develop new prognostic tools to predict chemotherapy efficacy.

Mutations of genes coding for collagen VI (COL6) cause muscle diseases, including Ullrich congenital muscular dystrophy and Bethlem myopathy. Although COL6 genetic variants were recently linked to brain pathologies, the impact of COL6 deficiency in brain function is still largely unknown. Here, a thorough behavioral characterization of COL6-null (Col6a1–/–) mice unexpectedly revealed that COL6 deficiency leads to a significant impairment in sensorimotor gating and memory/attention functions. In keeping with these behavioral abnormalities, Col6a1–/– mice displayed alterations in dopaminergic signaling, primarily in the prefrontal cortex. In vitro co-culture of SH-SY5Y neural cells with primary meningeal fibroblasts from wild-type and Col6a1–/– mice confirmed a direct link between COL6 ablation and defective dopaminergic activity, through a mechanism involving the inability of meningeal cells to sustain dopaminergic differentiation. Finally, patients affected by COL6-related myopathies were evaluated with an ad hoc neuropsychological protocol, revealing distinctive defects in attentional control abilities. Altogether, these findings point towards a previously undescribed role for COL6 in the proper maintenance of dopamine circuitry function and its related neurobehavioral features in both mice and humans. This article has an associated First Person interview with the first author of the paper.

Mutations in the collagen VI genes cause the Ullrich congenital muscular dystrophy (UCMD), with severe phenotype, and Bethlem myopathy (BM) with mild to moderate phenotype. Both, UCMD and BM patients show dystrophic features with degeneration/regeneration and replacement of muscle with fat and fibrous connective tissue. At molecular level, UCMD patients show autophagic impairment and increased PTP opening; these features are less severe in BM. To elucidate the biochemical mechanisms adopted by the muscle to adapt to collagen VI deficiency in BM and UCMD patients, a proteome analysis was carried out on human muscle biopsies. Qualitative and quantitative differences were assessed by 2D-DIGE coupled to MALDI-ToF/ToF MS. Proteomics results, coupled with immunoblotting, indicate changes in UPR, hexosamine pathway, and amino acid and fatty acid metabolism, suggesting an association of ER stress, metabolic dysregulation, autophagic impairment, and alteration in mechanotransduction signaling. Overall, these results indicate that despite the common downregulation of hexosamine pathway in UCMD and BM, in BM the protein quality control system is sustained by a metabolic adaptation supporting energy requirements for the maintenance of autophagy, counteracting ER misfolded protein overload. In UCMD, this multilayered system may be disrupted and worsened by the metabolic rewiring, which leads to lipotoxicity.

Less than half of the rural Guatemalan population has access to clean water, 98% of the country's water sources are contaminated and only 15 municipalities in the country have operational drinking water treatment systems. Guatemala also has among the world's worst health statistics for chronic malnutrition and substandard child growth attainment. Over the long term, chronic malnutrition and disease leads not only to stunted growth but also to the likelihood that children will not reach their full genetic and educational potential, thus reducing their productivity and earning power. Despite efforts to improve water systems over the past decade, improvements have not kept pace with population increases and migration. Investment in water systems has also declined because of the perceived high cost of centralized water treatment facilities. However, providing safe water does not have to be cost‐prohibitive. This article explores how the lack of safe water significantly contributes to the high incidence of diarrheal disease and chronic malnutrition and what can be done to provide clean drinking water to the Guatemalan population. Four factors, all of which can be accomplished at a relatively minor cost, are identified as key to providing clean drinking water to a household or small community.

A transgenic mouse line expressing the lacZ reporter under the control of a regulatory region of the col6a1gene has been used to investigate differentiation of Schwann cells. The data suggest that: (1) activation of col6a1gene transcription in the peripheral nervous system is part of the differentiation program of Schwann cells from neural crest cells stimulated by neuregulins; (2) once the Schwann cell precursors have acquired the competence of transcribing the col6a1gene, transcriptional regulation becomes independent from neuregulins and is modulated by different mechanisms, including cell cycle; (3) activation of transgene expression after birth in sciatic nerves corresponds to the time of withdrawal of immature Schwann cells from the cell cycle and the beginning of their differentiation into myelinating Schwann cells.

The region extending from −5.4 to −3.9 kilobase pairs from the transcription start site of the Col6a1gene has been previously shown to contain sequences activating tissue-specific transcription in articular cartilage, intervertebral disks, subepidermal, and vibrissae mesenchyme and peripheral nervous system (Braghetta, P., Fabbro, C., Piccolo, S., Marvulli, D., Bonaldo, P., Volpin, D., and Bressan, G. M. (1996) J. Cell Biol.135, 1163–1177). Analysis of expression of deletions of this region in transgenic mice has identified the 383-base pair fragment E–L as the most active sequence of the region. Linker-scanning mutagenesis analysis of segment E–J, which spans the 5′ 245 base pairs of E–L and is sufficient for high frequency expression in articular cartilage, showed that all the mutations reduced transcription considerably, suggesting that the integrity of the entire cluster of elements is necessary for enhancer activity. Electrophoretic mobility shift assays with nuclear extracts derived from various sources showed that fragment E–J binds numerous transcription factors (at least 22). These factors are present in most cells, expressing and nonexpressing α1(VI) collagen mRNA, but in different relative proportions, and none of them appears to be cell type-specific. Several lines of evidence indicate that sequence elements of the enhancer may have different functional roles in various cells. The data configure the −5.4/−3.9 region of theCol6a1gene as a new type of tissue-specific enhancer, characterized by a variety of tissues supporting its activation and by the dependence of its function only on ubiquitous transcription factors. This type of enhancer is postulated to be particularly important for genes such as those of the extracellular matrix, which are often expressed with broad tissue specificity.

Spiking studies underscore the important role filtration plays in removing protozoa from surface water. As part of a pilot‐scale examination of filtration media at the Cincinnati (Ohio) Water Works, heat‐inactivated Giardiacysts and Cryptosporidiumoocysts were spiked into surface water that had already undergone coagulation and sedimentation. This spiked water was then introduced into pilot‐scale filters containing sand only, typical dual anthracite–sand, and deep dual anthracite–sand media configurations. All of the media types used in this study demonstrated similar removal capabilities, with average cyst removal ranging from 2.7 to > 4.5 logs and average oocyst removal of 2.7–3.9 logs. Endospore and polystyrene bead removals were similar to those of the protozoa, but turbidity and particle counts demonstrated substantially less removal capability.

Analysis of the chromatin of different cell types has identified four DNase I-hypersensitive sites in the 5'-flanking region of the alpha1(VI) collagen gene, mapping at -4.6, -4.4, -2.5, and -0.1 kilobase (kb) from the RNA start site. The site at -2.5 kb was independent from, whereas the other three sites could be related to, alpha1(VI) mRNA expression. The site at -0.1 kb was present in cells expressing (NIH3T3 and C2C12) but absent in cells not expressing (EL4) the mRNA; the remaining two sites were apparently related with high levels of mRNA. DNase I footprinting and gel-shift assays with NIH3T3 and C2C12 nuclear extracts have located a binding site for transcription factor AP1 (activator protein 1) between nucleotides -104 and -73. When nuclear extracts from EL4 lymphocytes were used, the AP1 site-containing sequence was bound by proteins not related to AP1. The existence of the hypersensitive site at -0.1 kb may be related to the binding of AP1 and of additional factors to the core promoter (Piccolo, S., Bonaldo, P., Vitale, P., Volpin, D., and Bressan, G. M. (1995) J. Biol. Chem. 270, 19583-19590). The function of the AP1 binding site and of the core promoter in the transcriptional regulation of the Col6a1 gene was investigated by expressing several promoter-reporter gene constructs in transgenic mice and in cell cultures. The results indicate that regulation of transcription of the Col6a1 gene by different cis-acting elements (core promoter, AP1 binding site and enhancers) is not completely modular, but the final output depends on the specific interactions among the three elements in a defined cell type.

A pilot study suggests that DAF pretreatment helps reduce membrane fouling in hollow‐fiber MF. A pilot study investigated the use of dissolved air flotation (DAF) as a pretreatment to hollow‐fiber microfiltration (MF). Results showed that DAF‐pretreated feedwater achieved longer MF runs than raw feedwater. MF performances with DAF‐pretreated water were compared for fluxes of 70, 90, 110, and 135 gfd (120, 155, 190, and 230 L/m2/h). Performance results indicated that MF run lengths of more than 30 days could be achieved at flux conditions of 70 and 90 gfd (120 and 155 L/m2/h), with run lengths of 21 and 10 days for fluxes of 110 and 135 gfd (190 and 230 L/m2/h). Experimental results from this pilot study were also interpreted using a mathematical model to determine MF backwash efficiency at the different flux conditions. Both the experimental and modeling results suggest that membrane fouling by the DAF‐pretreated water was characterized by reduced pore blockage and lower cake compressibility as compared with treatment of raw feedwater.

A rat liver cDNA library, constructed in the plasmid expression vector pUEX, was immunoscreened using a rabbit polyclonal antiserum raised against rat liver Golgi membrane proteins. A sub-set of isolated clones were shown to encode the rat polymeric immunoglobulin receptor (pIgR). DNA sequence analysis of these clones provided the complete coding sequence of rat pIgR. Subsequent alignment of rat, rabbit and human predicted amino acid sequences demonstrated that the greatest degree of homology between the three pIgRs lies in their cytoplasmic tails; a region previously shown to be important for correct targetting and trancytosis of rabbit pIgR [(1984) Nature 308, 37–43].

Organelle-specific integral membrane proteins were identified by a novel strategy which gives rise to monospecific antibodies to these proteins as well as to the cDNA clones encoding them. A cDNA expression library was screened with a polyclonal antiserum raised against Triton X-114-extracted organelle proteins and clones were then grouped using antibodies affinity-purified on individual fusion proteins. The identification, molecular cloning and sequencing are described of a type 1 membrane protein (TGN38) which is located specifically in the trans-Golgi network.

A rat liver cDNA library, constructed in the plasmid expression vector pUEX, was immunoscreened using a rabbit polyclonal antiserum raised against rat liver Golgi membrane proteins. A sub‐set of isolated clones were shown to encode the rat polymeric immunoglobulin receptor (pIgR). DNA sequence analysis of these clones provided the complete coding sequence of rat pIgR. Subsequent alignment of rat, rabbit and human predicted amino acid sequences demonstrated that the greatest degree of homology between the three pIgRs lies in their cytoplasmic tails; a region previously shown to be important for correct targetting and trancytosis of rabbit pIgR [(1984) Nature 308, 37–43].

We have constructed a cDNA library in the plasmid expression vector pUEX enriched in sequences encoding membrane proteins. The procedure involved positive selection of sequences common to two different rat tissues (thus excluding tissue-specific mRNA) followed by positive selection between this material and RNA extracted from membrane bound polysomes (thus excluding cytoplasmic proteins). The resultant library prepared from rat kidney cDNA hybridized with rat liver poly(A)+ RNA, contained 30,000 clones and was shown to be enriched in cDNAs encoding membrane proteins. Seventeen clones selected because they encode large fusion proteins were shown to be single copy in the library, and not present in nucleotide data banks. Thus the strategy is particularly suitable for cloning low abundance cDNAs encoding membrane proteins.

Summary The ultrastructure of the cell wall and the distribution of alginate, cellulose and fucoidans were studied in the intertidal algaFucus virsoides. Microanalysis and precipitation with KPA revealed a cation localization in the wall, mainly corresponding with sulphated polysaccharide distribution. Thus, the adaptive resistance to changes in ionic environment seems to take place through a cation binding to the cell wall polysaccharides, principally at the thallus surface, employing an avoidance mechanism.

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.

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.

In Duchenne muscular dystrophy, the exon-skipping approach has obtained proof of concept in animal models, myogenic cell cultures, and following local and systemic administration in Duchenne patients. Indeed, we have previously demonstrated that low doses (7.5 mg/Kg/week) of 2′-O-methyl-phosphorothioate antisense oligoribonucleotides (AONs) adsorbed onto ZM2 nanoparticles provoke widespread dystrophin restoration 7 days after intraperitoneal treatment in mdx mice. In this study, we went on to test whether this dystrophin restoration was still measurable 90 days from the end of the same treatment. Interestingly, we found that both western blot and immunohistochemical analysis (up to 7% positive fibres) were still able to detect dystrophin protein in the skeletal muscles of ZM2-AON-treated mice at this time, and the level of exon-23 skipping could still be assessed by RT real-time PCR (up to 10% of skipping percentage). In contrast, the protein was undetectable by western blot analysis in the skeletal muscles of mdx mice treated with an identical dose of naked AON, and the percentage of dystrophin-positive fibres and exon-23 skipping were reminiscent of those of untreated mdx mice. Our data therefore demonstrate the long-term residual efficacy of this systemic low-dose treatment and confirm the protective effect nanoparticles exert on AON molecules.