Collagen XIII occurs as both a transmembrane-bound and a shed extracellular protein and is able to regulate the formation and function of neuromuscular synapses. Its absence results in myasthenia: presynaptic and postsynaptic defects at the neuromuscular junction (NMJ), leading to destabilization of the motor nerves, muscle regeneration and atrophy. Mutations in have recently been found to cause congenital myasthenic syndrome, characterized by fatigue and chronic muscle weakness, which may be lethal. We show here that muscle defects in collagen XIII-deficient mice stabilize in adulthood, so that the disease is not progressive until very late. Sciatic nerve crush was performed to examine how the lack of collagen XIII or forced expression of its transmembrane form affects the neuromuscular synapse regeneration and functional recovery following injury. We show that collagen XIII-deficient male mice are unable to achieve complete NMJ regeneration and functional recovery. This is mainly attributable to presynaptic defects that already existed in the absence of collagen XIII before injury. Shedding of the ectodomain is not required, as the transmembrane form of collagen XIII alone fully rescues the phenotype. Thus, collagen XIII could serve as a therapeutic agent in cases of injury-induced PNS regeneration and functional recovery. We conclude that intrinsic alterations at the NMJ in mice contribute to impaired and incomplete NMJ regeneration and functional recovery after peripheral nerve injury. However, such alterations do not progress once they have stabilized in early adulthood, emphasizing the role of collagen XIII in NMJ maturation. Collagen XIII is required for gaining and maintaining the normal size, complexity, and functional capacity of neuromuscular synapses. Loss-of-function mutations in cause congenital myasthenic syndrome 19, characterized by postnatally progressive muscle fatigue, which compromises patients' functional capacity. We show here in collagen XIII-deficient mice that the disease stabilizes in adulthood once the NMJs have matured. This study also describes a relevant contribution of the altered NMJ morphology and function to neuromuscular synapses, and PNS regeneration and functional recovery in collagen XIII-deficient mice after peripheral nerve injury. Correlating the animal model data on collagen XIII-associated congenital myasthenic syndrome, it can be speculated that neuromuscular connections in congenital myasthenic syndrome patients are not able to fully regenerate and restore normal functionality if exposed to peripheral nerve injury.

Alongside playing structural roles, the extracellular matrix (ECM) acts as an interaction platform for cellular homeostasis, organ development, and maintenance. The necessity of the ECM is highlighted by the diverse, sometimes very serious diseases that stem from defects in its components. The neuromuscular junction (NMJ) is a large peripheral motor synapse differing from its central counterparts through the ECM included at the synaptic cleft. Such synaptic basal lamina (BL) is specialized to support NMJ establishment, differentiation, maturation, stabilization, and function and diverges in molecular composition from the extrasynaptic ECM. Mutations, toxins, and autoantibodies may compromise NMJ integrity and function, thereby leading to congenital myasthenic syndromes (CMSs), poisoning, and autoimmune diseases, respectively, and all these conditions may involve synaptic ECM molecules. With neurotransmission degraded or blocked, muscle function is impaired or even prevented. At worst, this can be fatal. The article reviews the synaptic BL composition required for assembly and function of the NMJ molecular machinery through the lens of studies primarily with mouse models but also with human patients. In‐depth focus is given to collagen XIII, a postsynaptic‐membrane‐spanning but also shed ECM protein that in recent years has been revealed to be a significant component for the NMJ. Its deficiency in humans causes CMS, and autoantibodies against it have been recognized in autoimmune myasthenia gravis. Mouse models have exposed numerous details that appear to recapitulate human NMJ phenotypes relatively faithfully and thereby can be readily used to generate information necessary for understanding and ultimately treating human diseases. Anat Rec, 2019. © 2019 Wiley Periodicals, Inc.

Collagen XVIII (COL18A1) is an abundant heparan sulfate proteoglycan in vascular basement membranes. Here, we asked (i) if the loss of collagen XVIII would result in blood-brain barrier (BBB) breakdown, pathological alterations of small arteries and capillaries and neuroinflammation as found in cerebral small vessel disease (CSVD) and (ii) if such changes may be associated with remodeling of synapses and neural extracellular matrix (ECM). We found that 5-month-old Col18a1-/- mice had elevated BBB permeability for mouse IgG in the deep gray matter, and intravascular erythrocyte accumulations were observed in capillaries and arterioles in the cortex, hippocampus, and deep gray matter, with a lesser accumulation in the white matter. The permeability of the BBB increased with age in Col18a1-/- mice and affected cortical regions and the hippocampus in mice aged 12 months. However, none of the Col18a1-/- mice displayed hallmarks typical of more advanced stages of CSVD, such as perivascular space enlargement or large bleeds or infarcts. Collagen XVIII deficiency-induced BBB leakage was accompanied by activation of microglia and astrocytes, a loss of aggrecan in the ECM of perineuronal nets associated with fast-spiking inhibitory interneurons and accumulation of the perisynaptic ECM proteoglycan brevican and the microglial complement protein C1q at excitatory synapses. As the pathway underlying these regulations, we found increased signaling through the TGF-ß1/Smad3/TIMP-3 cascade. Thus, collagen XVIII proved to be crucial for the structural integrity of small vessels and its absence leads to pathological changes typical of early stages of CSVD. Furthermore, this study highlights an association between the alterations of perivascular ECM, extracellular proteolysis, and perineuronal/perisynaptic ECM, as a possible substrate of synaptic and cognitive alterations in CSVD.

BackgroundThe pathological alterations of small arteries and capillaries defined as cerebral small vessel disease (CSVD) are characterized by age-dependent blood-brain barrier (BBB) breakdown and vessel wall remodeling manifesting in small vessel occlusions and perivascular bleeds. As collagen XVIII (Col18a1) is an abundant heparan sulfate proteoglycan in vascular basement membranes, we focused on studying the role of collagen XVIII in vascular integrity and neurovascular unit maintenance in the context of CSVD.MethodsIn this study, we examined BBB breakdown, neuroinflammation and (peri)synaptic protein alterations using immunohistochemistry in 5- and 12-month-old Col18a1-/- and Col18a1+/+ mice. To further characterize molecular alterations, we used qPCR and compared the expression levels of major genes encoding tight junction and basement membrane proteins, inflammatory markers, neural extracellular matrix (ECM) proteins and proteinases.ResultsOur immunohistochemical analysis revealed progression of small vessel leakage in Col18a1-/- mice between 5 and 12 months. A large fraction (60%) of the small vessel area became mouse IgG-immunopositive in the hippocampus and retrosplenial cortex of 12-month-old Col18a1-/- mice. However, none of the Col18a1-/- mice displayed hallmarks typical of more advanced stages of CSVD, such as perivascular or large bleeds or infarcts. Collagen XVIII deficiency-induced BBB leakage was accompanied by activation of microglia and astrocytes, leading to perivascular ECM remodeling, upregulation of Timp3 and accumulation of perisynaptic ECM proteoglycan brevican and complement protein C1q, which may underlie impaired synaptic plasticity and loss of synapses.ConclusionOur findings highlight that Col18a1-/- mice represent a valuable model of early CSVD and call for more mechanistic analysis of underlying mechanisms and cognitive dysfunction in this model.

Idiopathic pulmonary fibrosis (IPF) is a severe lung disease with a poor prognosis and few treatment options. In the most widely used experimental model for this disease, bleomycin is administered into the lungs of mice, causing a reaction of inflammation and consequent fibrosis that resembles the progression of human IPF. The inflammation and fibrosis together induce changes in gene expression that can be analyzed with reverse transcription quantitative real-time PCR (RT-qPCR), in which accurate normalization with a set of stably expressed reference genes is critical for obtaining reliable results. This work compares ten commonly used candidate reference genes in the late, fibrotic phase of bleomycin-induced pulmonary fibrosis and ranks them from the most to the least stable using NormFinder and geNorm. Sdha, Polr2a and Hprt were identified as the best performing and least variable reference genes when alternating between normal and fibrotic conditions. In order to validate the findings, we investigated the expression of Tnf and Col1a1, representing the hallmarks of inflammation and fibrotic changes, respectively. With the best three genes as references, both were found to be upregulated relative to untreated controls, unlike the situation when analyzed solely with Gapdh, a commonly used reference gene. We therefore recommend Sdha, Polr2a and Hprt as reference genes for RT-qPCR in the 4-week bleomycin challenge that represents the late fibrotic phase.