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 STIM1 and ORAI1, the components of Store-Operated Calcium Entry (SOCE), and activation of the mTOR signaling pathway. In vivo mTOR inhibition by rapamycin reduced both STIM1 and ORAI1 expression and calcium flows, resulting in a normalization of platelet susceptibility to activation. These defects were cell-autonomous, as 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 as compared to 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.

Mutations of genes coding for Collagen VI (COL6) cause muscle diseases, including Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM). Although more recently COL6 genetic variants were 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 signalling, primarily in the prefrontal cortex (PFC). 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 at a novel role for COL6 in the proper maintenance of dopamine circuitry function and its related neurobehavioral features in both mice and humans.

Background: Congenital muscular dystrophies (CMD) are a clinically and genetically heterogeneous group of neuromuscular disorders characterized by muscle weakness. The two most prevalent forms of CMD, collagen VI-related myopathies (COL6RM) and laminin a2 deficient CMD type 1A (MDC1A), are both caused by deficiency or dysfunction of extracellular matrix proteins. Previously, we showed that an intramuscular transplantation of human adipose-derived stem cells (ADSC) into the muscle of the Col6a1-/- mice results in efficient stem cell engraftment, migration, long-term survival, and continuous production of the collagen VI protein, suggesting the feasibility of the systemic cellular therapy for COL6RM. In order for this therapeutic approach to work however, stem cells must be efficiently targeted to the entire body musculature. Thus, the main goal of this study is to test whether muscle homing of systemically transplanted ADSC can be enhanced by employing muscle-specific chemotactic signals originating from CMD-affected muscle tissue. Methods: Proteomic screens of chemotactic molecules were conducted in the skeletal muscles of COL6RM- and MDC1A-affected patients and CMD mouse models to define the inflammatory and immune activities, thus, providing potential markers of disease activity or treatment effect. Also using a pre-clinical animal model, recapitulating mild Ullrich congenital muscular dystrophy (UCMD), the therapeutic relevance of identified chemotactic pathways was investigated in vivo, providing a basis for future clinical investigations. Results: Comprehensive proteomic screens evaluating relevant human and mouse skeletal muscle biopsies offered chemotactic axes to enhance directional migration of systemically transplanted cells into CMD-affected muscles, including CCL5-CCR1/3/5, CCL2-CCR2, CXCL1/2-CXCR1,2 and CXCL7-CXCR2. Also, the specific populations of ADSC selected with an affinity for the chemokines being released by damaged muscle showed efficient migration to injured site and presented their therapeutic effect.Conclusions: Collectively, identified molecules provided insight into the mechanisms governing directional migration and intra-muscular trafficking of systemically infused stem cells, thus, permitting broad and effective application of the therapeutic adult stem cells for CMD treatment.

Background: Congenital muscular dystrophies (CMD) are a clinically and genetically heterogeneous group of neuromuscular disorders characterized by muscle weakness. The two most prevalent forms of CMD, collagen VI-related myopathies (COL6RM) and laminin a2 deficient CMD type 1A (MDC1A), are both caused by deficiency or dysfunction of extracellular matrix proteins. Previously, we showed that an intramuscular transplantation of human adipose-derived stem cells (ADSC) into the muscle of the Col6a1-/- mice results in efficient stem cell engraftment, migration, long-term survival, and continuous production of the collagen VI protein, suggesting the feasibility of the systemic cellular therapy for COL6RM. In order for this therapeutic approach to work however, stem cells must be efficiently targeted to the entire body musculature. Thus, the main goal of this study is to test whether muscle homing of systemically transplanted ADSC can be enhanced by employing muscle-specific chemotactic signals originating from CMD-affected muscle tissue. Methods: Proteomic screens of chemotactic molecules were conducted in the skeletal muscles of COL6RM- and MDC1A-affected patients and CMD mouse models to define the inflammatory and immune activities, thus, providing potential markers of disease activity or treatment effect. Also using a pre-clinical animal model, recapitulating mild Ullrich congenital muscular dystrophy (UCMD), the therapeutic relevance of identified chemotactic pathways was investigated in vivo, providing a basis for future clinical investigations.Results: Comprehensive proteomic screens evaluating relevant human and mouse skeletal muscle biopsies offered chemotactic axes to enhance directional migration of systemically transplanted cells into CMD-affected muscles, including CCL5-CCR1/3/5, CCL2-CCR2, CXCL1/2-CXCR1,2 and CXCL7-CXCR2. Also, the specific populations of ADSC selected with an affinity for the chemokines being released by damaged muscle showed efficient migration to injured site and presented their therapeutic effect.Conclusions: Collectively, identified molecules provided insight into the mechanisms governing directional migration and intra-muscular trafficking of systemically infused stem cells, thus, permitting broad and effective application of the therapeutic adult stem cells for CMD treatment.

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