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The interaction of sclerostin (Scl) with the low‐density lipoprotein receptor‐related protein 4 (LRP4) leads to a marked reduction in bone formation by inhibiting the Wnt/β‐catenin pathway. To characterize the Scl–LRP4 binding interface, we sorted a combinatorial library of Scl variants and isolated variants with reduced affinity to LRP4. We identified Scl single‐mutation variants enriched during the sorting process and verified their reduction in affinity toward LRP4—a reduction that was not a result of changes in the variants' secondary structure or stability. We found that Scl positions K75 (loop 1) and V136 (loop 3) are critical hotspots for binding to LRP4. Our findings establish the foundation for targeting these hotspots for developing novel therapeutic strategies to promote bone formation. [ABSTRACT FROM AUTHOR]

Synaptic development requires multiple signaling pathways to ensure successful connections. Transmembrane receptors are optimally positioned to connect the synapse and the rest of the neuron, often acting as synaptic organizers to synchronize downstream events. One such organizer, the LDL receptor-related protein LRP4, is a cell surface receptor that has been most well-studied postsynaptically at mammalian neuromuscular junctions. Recent work, however, identified emerging roles, but how LRP4 acts as a presynaptic organizer and the downstream mechanisms of LRP4 are not well understood. Here, we show that LRP4 functions presynaptically at Drosophila neuromuscular synapses, acting in motoneurons to instruct pre- and postsynaptic development. Loss of presynaptic LRP4 results in multiple defects, impairing active zone organization, synapse growth, physiological function, microtubule organization, synaptic ultrastructure and synapse maturation. We further demonstrate that LRP4 promotes most aspects of presynaptic development via a downstream SR-protein kinase, SRPK79D. These data demonstrate a function for presynaptic LRP4 as a peripheral synaptic organizer, highlight a downstream mechanism conserved with its CNS function in Drosophila, and underscore previously unappreciated but important developmental roles for LRP4 in cytoskeletal organization, synapse maturation and active zone organization. [ABSTRACT FROM AUTHOR]

Abstract Background Human patients often experience an episode of serious seizure activity, such as status epilepticus (SE), prior to the onset of temporal lobe epilepsy (TLE), suggesting that SE can trigger the development of epilepsy. Yet, the underlying mechanisms are not fully understood. The low-density lipoprotein receptor related protein (Lrp4), a receptor for proteoglycan-agrin, has been indicated to modulate seizure susceptibility. However, whether agrin-Lrp4 pathway also plays a role in the development of SE-induced TLE is not clear. Methods Lrp4 f/f mice were crossed with hGFAP-Cre and Nex-Cre mice to generate brain conditional Lrp4 knockout mice (hGFAP-Lrp4 −/− ) and pyramidal neuron specific knockout mice (Nex-Lrp4 −/− ). Lrp4 was specifically knocked down in hippocampal astrocytes by injecting AAV virus carrying hGFAP-Cre into the hippocampus. The effects of agrin-Lrp4 pathway on the development of SE-induced TLE were evaluated on the chronic seizure model generated by injecting kainic acid (KA) into the amygdala. The spontaneous recurrent seizures (SRS) in mice were video monitored. Results We found that Lrp4 deletion from the brain but not from the pyramidal neurons elevated the seizure threshold and reduced SRS numbers, with no change in the stage or duration of SRS. More importantly, knockdown of Lrp4 in the hippocampal astrocytes after SE induction decreased SRS numbers. In accord, direct injection of agrin into the lateral ventricle of control mice but not mice with Lrp4 deletion in hippocampal astrocytes also increased the SRS numbers. These results indicate a promoting effect of agrin-Lrp4 signaling in hippocampal astrocytes on the development of SE-induced TLE. Last, we observed that knockdown of Lrp4 in hippocampal astrocytes increased the extracellular adenosine levels in the hippocampus 2 weeks after SE induction. Blockade of adenosine A1 receptor in the hippocampus by DPCPX after SE induction diminished the effects of Lrp4 on the development of SE-induced TLE. Conclusion These results demonstrate a promoting role of agrin-Lrp4 signaling in hippocampal astrocytes in the development of SE-induced development of epilepsy through elevating adenosine levels. Targeting agrin-Lrp4 signaling may serve as a potential therapeutic intervention strategy to treat TLE.

Background: Human patients often experience an episode of serious seizure activity, such as status epilepticus (SE), prior to the onset of temporal lobe epilepsy (TLE), suggesting that SE can trigger the development of epilepsy. Yet, the underlying mechanisms are not fully understood. The low-density lipoprotein receptor related protein (Lrp4), a receptor for proteoglycan-agrin, has been indicated to modulate seizure susceptibility. However, whether agrin-Lrp4 pathway also plays a role in the development of SE-induced TLE is not clear. Methods: Lrp4f/f mice were crossed with hGFAP-Cre and Nex-Cre mice to generate brain conditional Lrp4 knockout mice (hGFAP-Lrp4−/−) and pyramidal neuron specific knockout mice (Nex-Lrp4−/−). Lrp4 was specifically knocked down in hippocampal astrocytes by injecting AAV virus carrying hGFAP-Cre into the hippocampus. The effects of agrin-Lrp4 pathway on the development of SE-induced TLE were evaluated on the chronic seizure model generated by injecting kainic acid (KA) into the amygdala. The spontaneous recurrent seizures (SRS) in mice were video monitored. Results: We found that Lrp4 deletion from the brain but not from the pyramidal neurons elevated the seizure threshold and reduced SRS numbers, with no change in the stage or duration of SRS. More importantly, knockdown of Lrp4 in the hippocampal astrocytes after SE induction decreased SRS numbers. In accord, direct injection of agrin into the lateral ventricle of control mice but not mice with Lrp4 deletion in hippocampal astrocytes also increased the SRS numbers. These results indicate a promoting effect of agrin-Lrp4 signaling in hippocampal astrocytes on the development of SE-induced TLE. Last, we observed that knockdown of Lrp4 in hippocampal astrocytes increased the extracellular adenosine levels in the hippocampus 2 weeks after SE induction. Blockade of adenosine A1 receptor in the hippocampus by DPCPX after SE induction diminished the effects of Lrp4 on the development of SE-induced TLE. Conclusion: These results demonstrate a promoting role of agrin-Lrp4 signaling in hippocampal astrocytes in the development of SE-induced development of epilepsy through elevating adenosine levels. Targeting agrin-Lrp4 signaling may serve as a potential therapeutic intervention strategy to treat TLE. [ABSTRACT FROM AUTHOR]

Background. The bone healing after fracture had a great impact on the patients' life quality. However, how miR-7-5p participated in fracture healing has not been investigated. Methods. For in vitro studies, the pre-osteoblast cell line MC3T3-E1 was obtained. The male C57BL/6 mice were purchased for in vivo experiments, and the fracture model was constructed. Cell proliferation was determined by CCK8 assay, and alkaline phosphatase (ALP) activity was measured by commercial kit. Histological status was evaluated using H&E and TRAP staining. The RNA and protein levels were detected via RT-qPCR and western blotting, respectively. Results. Overexpression of miR-7-5p increased cell viability and ALP activity in vitro. Moreover, in vivo studies consistently indicated that transfection of miR-7-5p improved the histological status and increased the proportion of TRAP-positive cells. Overexpression of miR-7-5p suppressed LRP4 expression while upregulated Wnt/β-catenin pathway. Conclusion. MiR-7-5p decreased LRP4 level and further activated the Wnt/β-catenin signaling, facilitating the process of fracture healing. [ABSTRACT FROM AUTHOR]

Pathogenic variants disrupting the binding between sclerostin (encoded by SOST) and its receptor LRP4 have previously been described to cause sclerosteosis, a rare high bone mass disorder. The sclerostin-LRP4 complex inhibits canonical WNT signaling, a key pathway regulating osteoblastic bone formation and a promising therapeutic target for common bone disorders, such as osteoporosis. In the current study, we crossed mice deficient for Sost (Sost−/−) with our p.Arg1170Gln Lrp4 knock-in (Lrp4KI/KI) mouse model to create double mutant Sost−/−;Lrp4KI/KI mice. We compared the phenotype of Sost−/− mice with that of Sost−/−;Lrp4KI/KI mice, to investigate a possible synergistic effect of the disease-causing p.Arg1170Trp variant in Lrp4 on Sost deficiency. Interestingly, presence of Lrp4KI alleles partially mitigated the Sost−/− phenotype. Cellular and dynamic histomorphometry did not reveal mechanistic insights into the observed phenotypic differences. We therefore determined the molecular effect of the Lrp4KI allele by performing bulk RNA sequencing on Lrp4KI/KI primary osteoblasts. Unexpectedly, mostly genes related to bone resorption or remodeling (Acp5, Rankl, Mmp9) were upregulated in Lrp4KI/KI primary osteoblasts. Verification of these markers in Lrp4KI/KI, Sost−/− and Sost−/−;Lrp4KI/KI mice revealed that sclerostin deficiency counteracts this Lrp4KI/KI effect in Sost−/−;Lrp4KI/KI mice. We therefore hypothesize that models with two inactivating Lrp4KI alleles rather activate bone remodeling, with a net gain in bone mass, whereas sclerostin deficiency has more robust anabolic effects on bone formation. Moreover, these effects of sclerostin and Lrp4 are stronger in female mice, contributing to a more severe phenotype than in males and more detectable phenotypic differences among different genotypes. [ABSTRACT FROM AUTHOR]

Background: Cenani‐Lenzsyndactyly syndrome (CLSS; OMIM 212780) is a rare autosomal recessive acral deformity, which is mainly manifested in the fusion of fingers or toes, disordered phalangeal structure, shortening or fusion of the radius and ulna, and renal hypoplasia. Case presentation: Our report described an individual with mild phenotypes from China. His parents were not consanguineous. The affected individual was non‐dysmorphic. Standard X‐ray showed that the both hands have only four metacarpal bones. The distal end of the first metacarpal bone on the right was relatively slender, and the distal phalanx was absent. Multiple phalanges and some soft tissues of both hands were fused. Exome sequencing revealed a novel biallelic c.282C⟩Avariant in low‐density lipoprotein receptor‐related protein 4 (LRP4; OMIM604270; NM_002334.4) causing p. (Asn94Lys) change in the encoded protein. This variant is predicted to be potentially pathogenic, affecting protein structure and function. Conclusion: We report a novel missense variant present in homozygosity in LRP4 to broaden the pathogenic spectrum of LRP4 in syndactyly, and exome sequencing technology is a powerful tool for genetic analysis in prenatal diagnosis and medical research, as a preferred method for the diagnosis of syndactyly and related phenotypes. [ABSTRACT FROM AUTHOR]

Congenital Myasthenic Syndromes (CMSs) are rare inherited diseases of the neuromuscular junction characterized by muscle weakness. CMSs with acetylcholinesterase deficiency are due to pathogenic variants in COLQ, a collagen that anchors the enzyme at the synapse. The two COLQ N-terminal domains have been characterized as being biochemical and functional. They are responsible for the structure of the protein in the triple helix and the association of COLQ with acetylcholinesterase. To deepen the analysis of the distal C-terminal peptide properties and understand the CMSs associated to pathogenic variants in this domain, we have analyzed the case of a 32 year old male patient bearing a homozygote splice site variant c.1281 C > T that changes the sequence of the last 28 aa in COLQ. Using COS cell and mouse muscle cell expression, we show that the COLQ variant does not impair the formation of the collagen triple helix in these cells, nor its association with acetylcholinesterase, and that the hetero-oligomers are secreted. However, the interaction of COLQ variant with LRP4, a signaling hub at the neuromuscular junction, is decreased by 44% as demonstrated by in vitro biochemical methods. In addition, an increase in all acetylcholine receptor subunit mRNA levels is observed in muscle cells derived from the patient iPSC. All these approaches point to pathophysiological mechanisms essentially characterized by a decrease in signaling and the presence of immature acetylcholine receptors. [ABSTRACT FROM AUTHOR]

A family of Pakistani origin, segregating polydactyly, and phalangeal synostosis in an autosomal dominant manner, has been investigated and presented in the present report. Whole‐exome sequencing (WES), followed by segregation analysis using Sanger sequencing, revealed a heterozygous missense variant [c.G1696A, p.(Gly566Ser)] in the LRP4 gene located on human chromosome 11p11.2. Homology protein modeling revealed the mutant Ser566 generated new interactions with at least four other amino acids and disrupted protein folding and function. Our findings demonstrated the first direct evidence of involvement of LRP4 in causing polydactyly and phalangeal synostosis in the same family. This study highlighted the importance of inclusion of LRP4 gene in screening individuals presenting polydactyly in hands and feet, and phalangeal synostosis in the same family. [ABSTRACT FROM AUTHOR]

Background: Intervertebral disc degeneration (IVDD) is a leading cause of low back pain (LBP). The pathological process of IVDD is associated with inflammatory reactions and extracellular matrix (ECM) disorders. Digoxin is widely used for treating heart failure, and it has been reported to have antiinflammatory effects. Objective: This study is to investigate the role of digoxin in the pathogenesis of intervertebral disc degeneration as well as the involved molecular mechanism, particularly the potential target protein. Methods: We exploited a rat needle model to investigate digoxin's role in intervertebral disc degeneration in vivo. Safranin O staining was used to measure cartilaginous tissue in the intervertebral disc. The morphological changes of intervertebral discs in animal models were determined by Hematoxylin-Eosin (H&E) staining and the pathological score. Primary nucleus pulposus cells (NP cells) from intervertebral discs of patients and murine were used in the present study. Western-Blotting assay, Real-time PCR assay, immunofluorescence staining, and immunochemistry were used to detect the role of digoxin in anti-TNF-α-induced inflammatory effects in vitro. Transfection of siRNA was used to regulate low-density lipoprotein receptor-related protein 4 (LRP4) expression in NP cells to investigate the potential protein target of digoxin. Results: Digoxin protected against intervertebral disc degeneration in rat needle models. Digoxin was found to exert its disc-protective effects through at least three different pathways by a) suppressing TNF-a-induced inflammation, b) attenuating ECM destruction, c) significantly promoting ECM anabolism. Additionally, LRP4 was found to be the downstream molecule of digoxin in NP cells for anti-inflammation and regulation of ECM metabolism. The knockdown of LRP4 downregulated the protective effect of digoxin in NP cells. Conclusion: These findings suggest that digoxin may be a potential therapeutic agent for intervertebral disc degeneration through anti-catabolism and proanabolism. Digoxin might also work as an alternative for other inflammationrelated diseases. [ABSTRACT FROM AUTHOR]

MuSK is a receptor tyrosine kinase (RTK) that plays essential roles in the formation and maintenance of the neuromuscular junction. Distinct from most members of RTK family, MuSK activation requires not only its cognate ligand agrin but also its coreceptors LRP4. However, how agrin and LRP4 coactivate MuSK remains unclear. Here, we report the cryo-EM structure of the extracellular ternary complex of agrin/LRP4/MuSK in a stoichiometry of 1:1:1. This structure reveals that arc-shaped LRP4 simultaneously recruits both agrin and MuSK to its central cavity, thereby promoting a direct interaction between agrin and MuSK. Our cryo-EM analyses therefore uncover the assembly mechanism of agrin/LRP4/MuSK signaling complex and reveal how MuSK receptor is activated by concurrent binding of agrin and LRP4. [ABSTRACT FROM AUTHOR]

Sclerostin is an inhibitor of the osteogenic Wnt/β‐catenin signaling pathway that also has an endocrine role in regulating adipocyte differentiation and metabolism. Additionally, subcutaneous white adipose tissue (scWAT) sclerostin content decreases following exercise training (EXT). Therefore, we hypothesized that EXT‐induced reductions in adipose tissue sclerostin may play a role in regulating adaptations in body composition and whole‐body metabolism. To test this hypothesis, 10‐week‐old male C57BL/6J mice were either sedentary (SED) or performing 1 hour of treadmill running at ~65% to 70% maximum oxygen consumption (VO2max) 5 day/week (EXT) for 4 weeks and had subcutaneous injections of either saline (C) or recombinant sclerostin (S) (0.1 mg/kg body mass) 5 day/week; thus, making four groups (SED‐C, EXT‐C, SED‐S, and EXT‐S; n = 12/group). No differences in body mass were observed between experimental groups, whereas food intake was higher in EXT (p = 0.03) and S (p = 0.08) groups. There was a higher resting energy expenditure in all groups compared to SED‐C. EXT‐C had increased lean mass and decreased fat mass percentage compared to SED‐C and SED‐S. No differences in body composition were observed in either the SED‐S or EXT‐S groups. Lower scWAT (inguinal), epididymal white adipose tissue (eWAT) (visceral epididymal) mass, and scWAT adipocyte cell size and increased percentage of multilocular cells in scWAT were observed in the EXT‐C group compared to SED‐C, whereas lower eWAT was only observed in the EXT‐S group. EXT mice had increased scWAT low‐density lipoprotein receptor‐related protein 4 (Lrp4) and mitochondrial content and sclerostin treatment only inhibited increased Lrp4 content with EXT. Together, these results provide evidence that reductions in resting sclerostin with exercise training may influence associated alterations in energy metabolism and body composition, particularly in scWAT. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR). [ABSTRACT FROM AUTHOR]

Osteoblast Wnt/β‐catenin signaling conditions skeletal development and health. Bone formation is stimulated when on the osteoblast surface a Wnt binds to low‐density lipoprotein receptor‐related protein 5 (LRP5) or 6 (LRP6), in turn coupled to a frizzled receptor. Sclerostin and dickkopf1 inhibit osteogenesis if either links selectively to the first β‐propeller of LRP5 or LRP6, thereby disassociating these cognate co‐receptors from the frizzled receptor. Sixteen heterozygous mutations identified since 2002 within LRP5 and three heterozygous mutations identified since 2019 within LRP6 prevent this binding of sclerostin or dickkopf1 and account for the exceptionally rare, but highly instructive, autosomal dominant disorders called LRP5 and LRP6 high bone mass (HBM). Herein, we characterize LRP6 HBM in the first large affected family. Their novel heterozygous LRP6 missense mutation (c.719C>T, p.Thr240Ile) was present in two middle‐aged sisters and three of their sons. They considered themselves healthy. Their broad jaw and torus palatinus developed during childhood and, contrary to the two previous reports of LRP6 HBM, the appearance of their adult dentition was unremarkable. Skeletal modeling, defined radiographically, supported classification as an endosteal hyperostosis. Areal bone mineral density (g/cm2) of the lumbar spine and total hip featured accelerated increases reaching Z‐scores of ~ +8 and +6, respectively, although biochemical markers of bone formation were normal. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research. [ABSTRACT FROM AUTHOR]

The specification of the forebrain relies on the precise regulation of WNT/ß-catenin signalling to support neuronal progenitor cell expansion, patterning, and morphogenesis. Imbalances in WNT signalling activity in the early neuroepithelium lead to congenital disorders, such as neural tube defects (NTDs). LDL receptor-related protein (LRP) family members, including the well-studied receptors LRP5 and LRP6, play critical roles in modulating WNT signalling capacity through tightly regulated interactions with their co-receptor Frizzled, WNT ligands, inhibitors and intracellular WNT pathway components. However, little is known about the function of LRP4 as a potential modulator of WNT signalling in the central nervous system. In this study, we investigated the role of LRP4 in the regulation of WNT signalling during early mouse forebrain development. Our results demonstrate that LRP4 can modulate LRP5- and LRP6-mediated WNT signalling in the developing forebrain prior to the onset of neurogenesis at embryonic stage 9.5 and is therefore essential for accurate neural tube morphogenesis. Specifically, LRP4 functions as a genetic modifier for impaired mitotic activity and forebrain hypoplasia, but not for NTDs in LRP6-deficient mutants. In vivo and in vitro data provide evidence that LRP4 is a key player in fine-tuning WNT signalling capacity and mitotic activity of mouse neuronal progenitors and of human retinal pigment epithelial (hTERT RPE-1) cells. Our data demonstrate the crucial roles of LRP4 and LRP6 in regulating WNT signalling and forebrain development and highlight the need to consider the interaction between different signalling pathways to understand the underlying mechanisms of disease. The findings have significant implications for our mechanistic understanding of how LRPs participate in controlling WNT signalling.

Abstract Background Low-density lipoprotein receptor-related protein 4 (LRP4) plays a critical role in the central nervous system (CNS), including hippocampal synaptic plasticity, maintenance of excitatory synaptic transmission, fear regulation, as well as long-term potentiation (LTP). Results In this study, we found that Lrp4 was highly expressed in layer II of the piriform cortex. Both body weight and brain weight decreased in Lrp4 ECD/ECD mice without TMD (Transmembrane domain) and ICD (intracellular domain) of LRP4. However, in the piriform cortical neurons of Lrp4 ECD/ECD mice, the spine density increased, and the frequency of both mEPSC (miniature excitatory postsynaptic current) and sEPSC (spontaneous excitatory postsynaptic current) was enhanced. Intriguingly, finding food in the buried food-seeking test was prolonged in both Lrp4 ECD/ECD mice and Lrp4 cKO (conditional knockout of Lrp4 in the piriform cortex) mice. Conclusions This study indicated that the full length of LRP4 in the piriform cortex was necessary for maintaining synaptic plasticity and the integrity of olfactory function.

Congenital Myasthenic Syndromes (CMSs) are rare inherited diseases of the neuromuscular junction characterized by muscle weakness. CMSs with acetylcholinesterase deficiency are due to pathogenic variants in COLQ, a collagen that anchors the enzyme at the synapse. The two COLQ N-terminal domains have been characterized as being biochemical and functional. They are responsible for the structure of the protein in the triple helix and the association of COLQ with acetylcholinesterase. To deepen the analysis of the distal C-terminal peptide properties and understand the CMSs associated to pathogenic variants in this domain, we have analyzed the case of a 32 year old male patient bearing a homozygote splice site variant c.1281 C > T that changes the sequence of the last 28 aa in COLQ. Using COS cell and mouse muscle cell expression, we show that the COLQ variant does not impair the formation of the collagen triple helix in these cells, nor its association with acetylcholinesterase, and that the hetero-oligomers are secreted. However, the interaction of COLQ variant with LRP4, a signaling hub at the neuromuscular junction, is decreased by 44% as demonstrated by in vitro biochemical methods. In addition, an increase in all acetylcholine receptor subunit mRNA levels is observed in muscle cells derived from the patient iPSC. All these approaches point to pathophysiological mechanisms essentially characterized by a decrease in signaling and the presence of immature acetylcholine receptors.

Isolated hand syndactyly is a common limb malformation with limited known genetic etiology. We used exome sequencing to discover two novel variants, chr11 g.46896373C>G; p.D1403H and chr11 g.46893078G>T; p.Q1564K, in LRP4 in a child with isolated bilateral syndactyly of the third and fourth fingers. Each variant was inherited from a different parent and neither parent was affected. Variants in LRP4 have been previously associated with syndactyly in Cenani-Lenz syndactyly syndrome and Sclerosteosis 2, but have not been reported in individuals with isolated syndactyly. LRP4 inhibits LRP6/LRP5-mediated activation of canonical Wnt signaling and mediates sclerostin-dependent inhibition of bone formation. p.D1403H and p.Q1564K are located within the fourth β-propeller of the extracellular protein domain that has yet to be associated with human disease. Functional analyses of p.D1403H and p.Q1564K show that they significantly decrease LRP4's inhibition of Wnt signaling. These results suggest that variants in the fourth β-propeller of the extracellular protein domain may cause a phenotype distinct from previously characterized LRP4 variants.

Neuronal agrin, a heparan sulphate proteoglycan secreted by the α-motor neurons, promotes the formation and maintenance of the neuromuscular junction by binding to Lrp4 and activating muscle-specific kinase (MuSK). Neuronal agrin also promotes myogenesis by enhancing differentiation and maturation of myotubes, but its effect on proliferating human myoblasts, which are often considered to be unresponsive to agrin, remains unclear. Using primary human myoblasts, we determined that neuronal agrin induced transient dephosphorylation of ERK1/2, while c-Abl, STAT3, and focal adhesion kinase were unresponsive. Gene silencing of Lrp4 and MuSK markedly reduced the BrdU incorporation, suggesting the functional importance of the Lrp4/MuSK complex for myoblast proliferation. Acute and chronic treatments with neuronal agrin increased the proliferation of human myoblasts in old donors, but they did not affect the proliferation of myoblasts in young donors. The C-terminal fragment of agrin which lacks the Lrp4-binding site and cannot activate MuSK had a similar age-dependent effect, indicating that the age-dependent signalling pathways activated by neuronal agrin involve the Lrp4/MuSK receptor complex as well as an Lrp4/MuSK-independent pathway which remained unknown. Collectively, our results highlight an age-dependent role for neuronal agrin in promoting the proliferation of human myoblasts. [ABSTRACT FROM AUTHOR]

Abstract Background The neuromuscular junction (NMJ) is a peripheral synapse critical to muscle contraction. Like acetylcholine receptors (AChRs), many essential proteins of NMJ are extremely concentrated at the postjunctional membrane. However, the mechanisms of synapse-specific concentration are not well understood; furthermore, it is unclear whether signaling molecules critical to NMJ formation and maintenance are also locally transcribed. Results We studied the β-gal activity encoded by a lacZ cassette driven by the promoter of the Lrp4 gene. As reported for Lrp4 mRNA, β-gal was in the central region in embryonic muscles and at the NMJ after its formation. However, β-gal was no longer in the central areas of muscle fibers in Lrp4 or MuSK mutant mice, indicating a requirement of Lrp4/MuSK signaling. This phenotype could be rescued by transgenic expression of LRP4 with a transmembrane domain but not soluble ECD in Lrp4 mutant mice. β-gal and AChR clusters were distributed in a broader region in lacZ/ECD than that of heterozygous lacZ/+ mice, indicating an important role of the transmembrane domain in Lrp4 signaling. Synaptic β-gal activity became diffused after denervation or treatment with µ-conotoxin, despite its mRNA was increased, indicating synaptic Lrp4 mRNA enrichment requires muscle activity. β-gal was also diffused in aged mice but became re-concentrated after muscle stimulation. Finally, Lrp4 mRNA was increased in C2C12 myotubes by Wnt ligands in a manner that could be inhibited by RKI-1447, an inhibitor of ROCK in Wnt non-canonical signaling. Injecting RKI-1447 into muscles of adult mice diminished Lrp4 synaptic expression. Conclusions This study demonstrates that synapse-specific enrichment of Lrp4 mRNA requires a coordinated interaction between Lrp4/MuSK signaling, muscle activity, and Wnt non-canonical signaling. Thus, the study provides a new mechanism for Lrp4 mRNA enrichment. It also provides a potential target for the treatment of NMJ aging and other NMJ-related diseases.

Background: Low-density lipoprotein receptor-related protein 4 (LRP4) plays a critical role in the central nervous system (CNS), including hippocampal synaptic plasticity, maintenance of excitatory synaptic transmission, fear regulation, as well as long-term potentiation (LTP). Results: In this study, we found that Lrp4 was highly expressed in layer II of the piriform cortex. Both body weight and brain weight decreased in Lrp4ECD/ECD mice without TMD (Transmembrane domain) and ICD (intracellular domain) of LRP4. However, in the piriform cortical neurons of Lrp4ECD/ECD mice, the spine density increased, and the frequency of both mEPSC (miniature excitatory postsynaptic current) and sEPSC (spontaneous excitatory postsynaptic current) was enhanced. Intriguingly, finding food in the buried food-seeking test was prolonged in both Lrp4ECD/ECD mice and Lrp4 cKO (conditional knockout of Lrp4 in the piriform cortex) mice. Conclusions: This study indicated that the full length of LRP4 in the piriform cortex was necessary for maintaining synaptic plasticity and the integrity of olfactory function. [ABSTRACT FROM AUTHOR]

Objetivo: LRP4 es un facilitador esencial en la inhibición específica de esclerostina de la vía canónica de Wnt. Mutaciones en LRP4 se han asociado a diversas patologías entre las cuales se incluyen la patología de crecimiento óseo, esclerosteosis y la malformación de Chiari tipo I (MCI). Material y métodos: Se ha re-secuenciado el gen LRP4 en dos pequeñas cohortes de pacientes con el fenotipo de alta masa ósea (HBM) y con MCI con el objetivo de encontrar variantes causales. Resultados: Entre las mutaciones encontradas destacamos: 1) una mutación de cambio de sentido (missense) en un paciente con MCI, que no cosegrega con el fenotipo en la familia; 2) una mutación intrónica no descrita previamente (c.3364+16A>C) en una mujer con HBM; y 3) una mutación intrónica en una mujer con HBM cuya frecuencia en población control europea es muy baja. Conclusiones: Aunque no hemos encontrado variantes en LRP4 que expliquen el fenotipo HBM o MCI en los pacientes estudiados, animamos a otros investigadores a que analicen el gen LRP4 en sus pacientes ya que es un buen candidato funcional de ambos fenotipos.

Abstract The low-density lipoprotein receptor-related protein 4 (LRP4) is essential for inducing the neuromuscular junction (NMJ) formation in muscle fibers, and LRP4 plays a critical role in dendritic development and synaptogenesis in the central nervous system (CNS). As a single transmembrane protein, LRP4 contains an enormously sizeable extracellular domain (ECD), containing multiple LDLα repeats in the N-terminal of ECD. LRP4 only with extracellular domain acts as a similar mechanism of full-length LRP4 in muscles to stimulate acetylcholine receptor clustering. In this study, we elucidated that LDLα repeats of LRP4 maintained the body weight and survival rate. Dendritic branches of the pyramidal neurons in Lrp4-null mice with LRP4 LDLα repeats residue were more than in Lrp4-null mice without residual LRP4 domain. Supplement with conditioned medium from LRP4 LDLα overexpression cells, the primary culture pyramidal neurons achieved strong dendritic arborization ability. Besides, astrocytes with LRP4 LDLα repeats residue could promote pyramidal neuronal dendrite arborization in the primary co-cultured system. These observations signify that LRP4 LDLα repeats play a prominent underlying role in dendrite arborization.

Induction of bone formation by Wnt ligands is inhibited when sclerostin (Scl), an osteocyte-produced antagonist, binds to its receptors, the low-density lipoprotein receptor-related proteins 5 or 6 (LRP5/6). Recently, it was shown that enhanced inhibition is achieved by Scl binding to the co-receptor LRP4. However, it is not clear if the binding of Scl to LRP4 facilitates Scl binding to LRP5/6 or inhibits the Wnt pathway in an LRP5/6-independent manner. Here, using the yeast display system, we demonstrate that Scl exhibits a stronger binding affinity for LRP4 than for LRP6. Moreover, we found stronger Scl binding to LRP6 in the presence of LRP4. We further show that a Scl mutant (SclN93A), which tightly binds LRP4 but not LRP6, does not inhibit the Wnt pathway on its own. We demonstrate that SclN93A competes with Scl for a common binding site on LRP4 and antagonizes Scl inhibition of the Wnt signaling pathway in osteoblasts in vitro. Finally, we demonstrate that 2 weeks of bi-weekly subcutaneous injections of SclN93A fused to the fragment crystallizable (Fc) domain of immunoglobulin (SclN93AFc), which retains the antagonistic activity of the mutant, significantly increases bone formation rate and enhances trabecular volumetric bone fraction, trabecular number, and bone length in developing mice. Our data show that LRP4 serves as an anchor that facilitates Scl–LRP6 binding and that inhibition of the Wnt pathway by Scl depends on its prior binding to LRP4. We further provide evidence that compounds that inhibit Scl–LRP4 interactions offer a potential strategy to promote anabolic bone functions. [ABSTRACT FROM AUTHOR]

Myasthenia gravis (MG) is a disease caused by pathogenic autoantibodies against the neuromuscular junction and is characterized by muscle weakness. Most MG patients produce antibodies against the acetylcholine receptor (AChR), but a subset of patients have been found to produce autoantibodies against other components of the neuromuscular junction such as muscle specific tyrosine kinase (MuSK) and low-density lipoprotein receptor-related protein 4 (LRP4). The pathogenicity of these autoantibodies has been studied using polyclonal IgG or serum from MG patients; however, pathogenic B cells and monoclonal antibodies from these patients have rarely been investigated because of the difficulty in isolating them. Recently, isolation of pathogenic B cells from MuSK-MG patients and the subsequent generation of monoclonal pathogenic antibodies from these cells, was reported. These data revealed the existence of pathogenic IgG3 and IgG4 antibodies and identified a pathogenic mechanism alternative to the inhibition of MuSK phosphorylation. This review discusses research concerning pathogenic B cells in MG patients and rituximab therapy specifically depleting B cells. Accumulating studies show rituximab therapy is more effective in MuSK-MG patients than in AChR-MG patients. Advances in molecular biology may lead to greater understanding of pathogenic B cells in MG patients and thus potentially lead to the development of novel therapies for MG.

The receptor tyrosine kinase MuSK, its co-receptor Lrp4 and the Agrin ligand constitute a signaling pathway that is crucial in axial muscle for neuromuscular synapse development, yet whether this pathway functions similarly in appendicular muscle is unclear. Here, using the larval zebrafish pectoral fin, equivalent to tetrapod forelimbs, we show that, similar to axial muscle, developing appendicular muscles form aneural acetylcholine receptor (AChR) clusters prior to innervation. As motor axons arrive, neural AChR clusters form, eventually leading to functional synapses in a MuSK-dependent manner.We find that loss of Agrin or Lrp4 function, which abolishes synaptic AChR clusters in axial muscle, results in enlarged presynaptic nerve regions and progressively expanding appendicular AChRclusters,mimicking the consequences of motoneuron ablation. Moreover, musk depletion in lrp4 mutants partially restores synaptic AChR patterning. Combined, our results provide compelling evidence that, in addition to the canonical pathway in which Agrin/Lrp4 stimulates MuSK activity, Agrin/Lrp4 signaling in appendicular muscle constrains MuSK-dependent neuromuscular synapse organization. Thus, we reveal a previously unappreciated role for Agrin/Lrp4 signaling, thereby highlighting distinct differences between axial and appendicular synapse development. [ABSTRACT FROM AUTHOR]

Neuronal agrin, a heparan sulphate proteoglycan secreted by the α-motor neurons, promotes the formation and maintenance of the neuromuscular junction by binding to Lrp4 and activating muscle-specific kinase (MuSK). Neuronal agrin also promotes myogenesis by enhancing differentiation and maturation of myotubes, but its effect on proliferating human myoblasts, which are often considered to be unresponsive to agrin, remains unclear. Using primary human myoblasts, we determined that neuronal agrin induced transient dephosphorylation of ERK1/2, while c-Abl, STAT3, and focal adhesion kinase were unresponsive. Gene silencing of Lrp4 and MuSK markedly reduced the BrdU incorporation, suggesting the functional importance of the Lrp4/MuSK complex for myoblast proliferation. Acute and chronic treatments with neuronal agrin increased the proliferation of human myoblasts in old donors, but they did not affect the proliferation of myoblasts in young donors. The C-terminal fragment of agrin which lacks the Lrp4-binding site and cannot activate MuSK had a similar age-dependent effect, indicating that the age-dependent signalling pathways activated by neuronal agrin involve the Lrp4/MuSK receptor complex as well as an Lrp4/MuSK-independent pathway which remained unknown. Collectively, our results highlight an age-dependent role for neuronal agrin in promoting the proliferation of human myoblasts.

Myasthenia gravis (MG) is an acquired autoimmune disorder caused by autoantibodies binding acetylcholine receptors (AChR), muscle‐specific kinase (MuSK), agrin or low‐density lipoprotein receptor‐related protein 4 (Lrp4). These autoantibodies inhibit neuromuscular transmission by blocking the function of these proteins and thereby cause fluctuating skeletal muscle weakness. Several reports suggest that these autoantibodies might also affect the central nervous system (CNS) in MG patients. A comprehensive overview of the timing and localization of the expression of MG‐related antigens in other organs is currently lacking. To investigate the spatio‐temporal expression of MG‐related genes outside skeletal muscle, we used in silico tools to assess public expression databases. Acetylcholine esterase, nicotinic AChR α1 subunit, agrin, collagen Q, downstream of kinase‐7, Lrp4, MuSK and rapsyn were included as MG‐related genes because of their well‐known involvement in either congenital or autoimmune MG. We investigated expression of MG‐related genes in (1) all human tissues using GTEx data, (2) specific brain regions, (3) neurodevelopmental stages, and (4) cell types using datasets from the Allen Institute for Brain Sciences. MG‐related genes show heterogenous spatio‐temporal expression patterns in the human body as well as in the CNS. For each of these genes, several (new) tissues, brain areas and cortical cell types with (relatively) high expression were identified suggesting a potential role for these genes outside skeletal muscle. The possible presence of MG‐related antigens outside skeletal muscle suggests that autoimmune MG, congenital MG or treatments targeting the same proteins may affect MG‐related protein function in other organs. [ABSTRACT FROM AUTHOR]

Background: The neuromuscular junction (NMJ) is a peripheral synapse critical to muscle contraction. Like acetylcholine receptors (AChRs), many essential proteins of NMJ are extremely concentrated at the postjunctional membrane. However, the mechanisms of synapse-specific concentration are not well understood; furthermore, it is unclear whether signaling molecules critical to NMJ formation and maintenance are also locally transcribed. Results: We studied the β-gal activity encoded by a lacZ cassette driven by the promoter of the Lrp4 gene. As reported for Lrp4 mRNA, β-gal was in the central region in embryonic muscles and at the NMJ after its formation. However, β-gal was no longer in the central areas of muscle fibers in Lrp4 or MuSK mutant mice, indicating a requirement of Lrp4/MuSK signaling. This phenotype could be rescued by transgenic expression of LRP4 with a transmembrane domain but not soluble ECD in Lrp4 mutant mice. β-gal and AChR clusters were distributed in a broader region in lacZ/ECD than that of heterozygous lacZ/+ mice, indicating an important role of the transmembrane domain in Lrp4 signaling. Synaptic β-gal activity became diffused after denervation or treatment with µ-conotoxin, despite its mRNA was increased, indicating synaptic Lrp4 mRNA enrichment requires muscle activity. β-gal was also diffused in aged mice but became re-concentrated after muscle stimulation. Finally, Lrp4 mRNA was increased in C2C12 myotubes by Wnt ligands in a manner that could be inhibited by RKI-1447, an inhibitor of ROCK in Wnt non-canonical signaling. Injecting RKI-1447 into muscles of adult mice diminished Lrp4 synaptic expression. Conclusions: This study demonstrates that synapse-specific enrichment of Lrp4 mRNA requires a coordinated interaction between Lrp4/MuSK signaling, muscle activity, and Wnt non-canonical signaling. Thus, the study provides a new mechanism for Lrp4 mRNA enrichment. It also provides a potential target for the treatment of NMJ aging and other NMJ-related diseases. [ABSTRACT FROM AUTHOR]

Myasthenia gravis (MG) is a heterogeneous condition, characterized by autoantibodies (Abs) that target functionally important structures within neuromuscular junctions (NMJ), thus affecting nerve-to-muscle transmission. MG patients are more often now subgrouped based on the profile of serum autoantibodies, which segregate with clinical presentation, immunopathology, and their response to therapies. The serological testing plays an essential role in confirming MG diagnosis and guiding disease management, although a small percentage of MG patients remain negative for antibodies. With the advancements in new highly effective pathophysiologically-specific immunotherapeutic options, it has become increasingly important to identify the specific Abs responsible for the pathogenicity in individual MG patients. There are several new assays and protocols being developed for the improved detection of Abs in MG patients. This review focuses on the divergent immunopathological mechanisms in MG, and discusses their relevance to improved diagnostic and treatment. We propose a comprehensive “reflex testing,” algorithm for the presence of MG autoantibodies, and foresee that in the near future, the convenience and specificity of novel assays will permit the clinicians to consider them into routine systematic testing, thus stimulating laboratories to make these tests available. Moreover, adopting treatment driven testing algorithms will be crucial to identify subgroups of patients potentially benefiting from novel immunotherapies for MG.

Background and Objectives: Von Willebrand factor (VWF) is a plasma glycoprotein involved in primary hemostasis but recent data suggest additional roles beyond hemostasis in angiogenesis and potentially in vascular smooth muscle cell (VSMC) proliferation. Our aim was to investigate how VWF can modulate VSMC proliferation and identified the underlying mechanisms and the in vivo pathophysiological relevance. Methods and Results: Cultured aortic VSMCs proliferation and migration were increased in the presence of VWF. VSMCs treatment with a siRNA targeting αv integrin or the RGT-peptide blocking αvβ3 signaling completely inhibited proliferation. VWF did not bind directly to αvβ3 on VSMCs. We identified that VWF A2 domain was able to bind VSMCs. Since the low-density lipoprotein-related receptor protein (LRP) family are known to act as co-receptors we hypothesized the involvement of a member in the signaling pathway. Using the universal LRP-inhibitor (RAP), we confirmed LRP-mediated VSMC proliferation. siRNA experiments and proximity ligation assay staining identified LRP4 as the VWF-counterreceptor on VSMCs and showed co-localization between αvβ3 and LRP4. Carotid ligations were applied to VWF +/+ and −/− mice and intimal hyperplasia (IH) was measured. Less VWF−/− mice developed IH compared to VWF +/+ mice. Finally, the proliferative effect of VWF was confirmed in human atherosclerotic lesions from different vessels (aortas, carotids) showing a proximity between VWF and a-SM actin positive cells. Conclusions: VWF mediates VSMC proliferation through its A2 domain binding to the LRP4 receptor and integrin αvβ3 signaling. The decreased IH following vascular injury suggests that targeting VWF-LRP4 interactions may contribute to limit remodeling.

Cenani Lenz syndrome is a rare autosomal recessive disorder associated with variable degree of limb malformations, dysmorphism, and renal agenesis. It is caused due to pathogenic variants in the LRP4 gene, which plays an important role in limb and renal development. Mutations in the APC gene have also been occasionally associated with CLS. The phenotypic spectrum ranges from mild to very severe perinatal lethal type depending on the type of variant. We report a pathogenic variant, c.2710 del T (p.Trp904GlyfsTer5) in theLRP4 gene, in a fetus with lethal Cenani Lenz syndrome with antenatal presentation of tetraphocomelia and symmetrical involvement of hands and feet. [ABSTRACT FROM AUTHOR]

Objetive: LRP4 is an essential facilitator in sclerostin-specific inhibition of the canonical Wnt pathway. Mutations in LRP4 have been associated with various conditions, including bone growth disease, sclerosteosis, and Chiari type I malformation (CMI). Material and methods: The LRP4 has been re-sequenced in two patient cohorts with high bone mass phenotype (HBM) and with CMI aimed at finding causal variants. Results: Among the mutations found, we would highlight: 1) a missense mutation in a patient with CMI, which does not co-segregate with the phenotype in the family; 2) a previously undescribed intronic mutation (c.3364+16A>C) in a woman with HBM; and 3) an intronic mutation in a woman with HBM with a very low frequency in the European control population. Conclusions: Although we have not found variants in LRP4 to explain the HBM or CMI phenotype in the patients studied, we encourage other researchers to analyze the LRP4 gene in their patients as it is a good functional candidate for both phenotypes. [ABSTRACT FROM AUTHOR]

The low-density lipoprotein receptor-related protein 4 (LRP4) is essential for inducing the neuromuscular junction (NMJ) formation in muscle fibers, and LRP4 plays a critical role in dendritic development and synaptogenesis in the central nervous system (CNS). As a single transmembrane protein, LRP4 contains an enormously sizeable extracellular domain (ECD), containing multiple LDLα repeats in the N-terminal of ECD. LRP4 only with extracellular domain acts as a similar mechanism of full-length LRP4 in muscles to stimulate acetylcholine receptor clustering. In this study, we elucidated that LDLα repeats of LRP4 maintained the body weight and survival rate. Dendritic branches of the pyramidal neurons in Lrp4-null mice with LRP4 LDLα repeats residue were more than in Lrp4-null mice without residual LRP4 domain. Supplement with conditioned medium from LRP4 LDLα overexpression cells, the primary culture pyramidal neurons achieved strong dendritic arborization ability. Besides, astrocytes with LRP4 LDLα repeats residue could promote pyramidal neuronal dendrite arborization in the primary co-cultured system. These observations signify that LRP4 LDLα repeats play a prominent underlying role in dendrite arborization. [ABSTRACT FROM AUTHOR]

Myasthenia gravis (MG) is a heterogeneous condition, characterized by autoantibodies (Abs) that target functionally important structures within neuromuscular junctions (NMJ), thus affecting nerve-to-muscle transmission. MG patients are more often now subgrouped based on the profile of serum autoantibodies, which segregate with clinical presentation, immunopathology, and their response to therapies. The serological testing plays an essential role in confirming MG diagnosis and guiding disease management, although a small percentage of MG patients remain negative for antibodies. With the advancements in new highly effective pathophysiologically-specific immunotherapeutic options, it has become increasingly important to identify the specific Abs responsible for the pathogenicity in individual MG patients. There are several new assays and protocols being developed for the improved detection of Abs in MG patients. This review focuses on the divergent immunopathological mechanisms in MG, and discusses their relevance to improved diagnostic and treatment. We propose a comprehensive "reflex testing," algorithm for the presence of MG autoantibodies, and foresee that in the near future, the convenience and specificity of novel assays will permit the clinicians to consider them into routine systematic testing, thus stimulating laboratories to make these tests available. Moreover, adopting treatment driven testing algorithms will be crucial to identify subgroups of patients potentially benefiting from novel immunotherapies for MG. [ABSTRACT FROM AUTHOR]

Synapse formation is a very elaborate process dependent upon accurate coordination of pre and post-synaptic specialization, requiring multiple steps and a variety of receptors and signaling molecules. Due to its relative structural simplicity and the ease in manipulation and observation, the neuromuscular synapse or neuromuscular junction (NMJ)—the connection between motor neurons and skeletal muscle—represents the archetype junction system for studying synapse formation and conservation. This junction is essential for survival, as it controls our ability to move and breath. NMJ formation requires coordinated interactions between motor neurons and muscle fibers, which ultimately result in the formation of a highly specialized post-synaptic architecture and a highly differentiated nerve terminal. Furthermore, to ensure a fast and reliable synaptic transmission following neurotransmitter release, ligand-gated channels (acetylcholine receptors, AChRs) are clustered on the post-synaptic muscle cell at high concentrations in sites opposite the presynaptic active zone, supporting a direct role for nerves in the organization of the post-synaptic membrane architecture. This organized clustering process, essential for NMJ formation and for life, relies on key signaling molecules and receptors and is regulated by soluble extracellular molecules localized within the synaptic cleft. Notably, several mutations as well as auto-antibodies against components of these signaling complexes have been related to neuromuscular disorders. The recent years have witnessed strong progress in the understanding of molecular identities, architectures, and functions of NMJ macromolecules. Among these, prominent roles have been proposed for neural variants of the proteoglycan agrin, its receptor at NMJs composed of the lipoprotein receptor-related protein 4 (LRP4) and the muscle-specific kinase (MuSK), as well as the regulatory soluble synapse-specific protease Neurotrypsin. In this review we summarize the current state of the art regarding molecular structures and (agrin-dependent) canonical, as well as (agrin-independent) non-canonical, MuSK signaling mechanisms that underscore the formation of neuromuscular junctions, with the aim of providing a broad perspective to further stimulate molecular, cellular and tissue biology investigations on this fundamental intercellular contact.

Myasthenia gravis (MG) is the most common autoimmune disorder affecting the neuromuscular junction, characterized by skeletal muscle weakness and fatigability. It is caused by autoantibodies targeting proteins of the neuromuscular junction; ~85% of MG patients have autoantibodies against the muscle acetylcholine receptor (AChR-MG), whereas about 5% of MG patients have autoantibodies against the muscle specific kinase (MuSK-MG). In the remaining about 10% of patients no autoantibodies can be found with the classical diagnostics for AChR and MuSK antibodies (seronegative MG, SN-MG). Since serological tests are relatively easy and non-invasive for disease diagnosis, the improvement of methods for the detection of known autoantibodies or the discovery of novel autoantibody specificities to diminish SN-MG and to facilitate differential diagnosis of similar diseases, is crucial. Radioimmunoprecipitation assays (RIPA) are the staple for MG antibody detection, but over the past years, using cell-based assays (CBAs) or improved highly sensitive RIPAs, it has been possible to detect autoantibodies in previously SN-MG patients. This led to the identification of more patients with antibodies to the classical antigens AChR and MuSK and to the third MG autoantigen, the low-density lipoprotein receptor-related protein 4 (LRP4), while antibodies against other extracellular or intracellular targets, such as agrin, Kv1.4 potassium channels, collagen Q, titin, the ryanodine receptor and cortactin have been found in some MG patients. Since the autoantigen targeted determines in part the clinical manifestations, prognosis and response to treatment, serological tests are not only indispensable for initial diagnosis, but also for monitoring treatment efficacy. Importantly, knowing the autoantibody profile of MG patients could allow for more efficient personalized therapeutic approaches. Significant progress has been made over the past years toward the development of antigen-specific therapies, targeting only the specific immune cells or autoantibodies involved in the autoimmune response. In this review, we will present the progress made toward the development of novel sensitive autoantibody detection assays, the identification of new MG autoantigens, and the implications for improved antigen-specific therapeutics. These advancements increase our understanding of MG pathology and improve patient quality of life by providing faster, more accurate diagnosis and better disease management. [ABSTRACT FROM AUTHOR]

BACKGROUND: Low-density lipoprotein receptor-related protein 4 (LRP4) has been reported to be implicated in multiple types of cancers. However, the significance of LRP4 in gastric cancer (GC) remains poorly elucidated. Therefore, it's urgent to investigate the importance and underlying mechanisms of LRP4 in GC. OBJECTIVE: To investigate the clinical roles of LRP4 in GC. METHODS: The LRP4 mRNA and miR-140-5p was measured by qRT-PCR. The protein expression was determined Western blot. Kaplan-Meier survival curves and Cox proportional hazard regression models were performed to evaluate prognosis. RESULTS: We demonstrated that LRP4 mRNA and protein was up-regulated in GC tissues for the first time. Its high expression was significantly correlated with malignant clinical features including TNM stage and lymph-node metastasis and poor prognosis for GC patients. LRP4 promotes migration, invasion and epithelial-mesenchymal transition (EMT) progress of GC cells. Mechanically, LRP4 regulated PI3K/AKT in GC cells. AKT inhibitors reversed the effects of LRP4. Finally, LRP4 was regulated by miR-140-5p in GC. CONCLUSIONS: Our findings showed that LRP4 has an important function in GC progression and promotes GC migration, invasion and EMT by regulating PI3K/AKT under regulation of miR-140-5p, providing a potential therapeutic target for GC. [ABSTRACT FROM AUTHOR]

Introduction: Our aim in this study was to identify the prevalence and clinical characteristics of LRP4/agrin-antibody-positive double-seronegative myasthenia gravis (DNMG).Methods: DNMG patients at 16 sites in the United States were tested for LRP4 and agrin antibodies, and the clinical data were collected.Results: Of 181 DNMG patients, 27 (14.9%) were positive for either low-density lipoprotein receptor-related protein 4 (LRP4) or agrin antibodies. Twenty-three DNMG patients (12.7%) were positive for both antibodies. More antibody-positive patients presented with generalized symptoms (69%) compared with antibody-negative patients (43%) (P ≤ .02). Antibody-positive patients' maximum classification on the Myasthenia Gravis Foundation of America (MGFA) scale was significantly higher than that for antibody-negative patients (P ≤ .005). Seventy percent of antibody-positive patients were classified as MGFA class III, IV, or V compared with 39% of antibody-negative patients. Most LRP4- and agrin-antibody-positive patients (24 of 27, 89%) developed generalized myathenia gravis (MG), but with standard MG treatment 81.5% (22 of 27) improved to MGFA class I or II during a mean follow-up of 11 years.Discussion: Antibody-positive patients had more severe clinical disease than antibody-negative patients. Most DNMG patients responded to standard therapy regardless of antibody status. [ABSTRACT FROM AUTHOR]

Agrin is a crucial factor that induces postsynaptic differentiation at neuromuscular junctions (NMJs), but how secreted agrin is locally deposited in the context of extracellular matrix (ECM) environment and its function in presynaptic differentiation remain largely unclear. Here, we report that the proteolytic activity of neuronal membranetype 1 matrix metalloproteinase (MT1-MMP; also known as MMP14) facilitates agrin deposition and signaling during presynaptic development at NMJs. Firstly, agrin deposition along axons exhibits a time-dependent increase in cultured neurons that requires MMPmediated focal ECM degradation. Next, local agrin stimulation induces the clustering of mitochondria and synaptic vesicles, two well-known presynaptic markers, and regulates vesicular trafficking and surface insertion of MT1-MMP. MMP inhibitor or MT1-MMP knockdown suppresses agrin-induced presynaptic differentiation, which can be rescued by treatment with the ectodomain of lowdensity lipoprotein receptor-related protein 4 (Lrp4). Finally, neuronal MT1-MMP knockdown inhibits agrin deposition and nerve-induced acetylcholine receptor clustering in nerve-muscle co-cultures and affects synaptic structures at Xenopus NMJs in vivo. Collectively, our results demonstrate a previously unappreciated role of agrin, as well as dual functions of neuronal MT1-MMP proteolytic activity in orchestrating agrin deposition and signaling, in presynaptic development. [ABSTRACT FROM AUTHOR]

Amyloid-β (Aβ) deposition occurs years before cognitive symptoms appear and is considered a cause of Alzheimer's disease (AD). The imbalance of Aβ production and clearance leads to Aβ accumulation and Aβ deposition. Increasing evidence indicates an important role of astrocytes, the most abundant cell type among glial cells in the brain, in Aβ clearance. We explored the role of low-density lipoprotein receptor-related protein 4 (LRP4), a member of the LDLR family, in AD pathology. We show that Lrp4 is specifically expressed in astrocytes and its levels in astrocytes were higher than those of Ldlr and Lrp1, both of which have been implicated in Aβ uptake. LRP4 was reduced in postmortem brain tissues of AD patients. Genetic deletion of the Lrp4 gene augmented Aβ plaques in 5xFAD male mice, an AD mouse model, and exacerbated the deficits in neurotransmission, synchrony between the hippocampus and PFC, and cognition. Mechanistically, LRP4 promotes Aβ uptake by astrocytes likely by interacting with ApoE. Together, our study demonstrates that astrocytic LRP4 plays an important role in Aβ pathology and cognitive function. [ABSTRACT FROM AUTHOR]

Myasthenia gravis (MG) is a disease of the postsynaptic neuromuscular junction (NMJ) where nicotinic acetylcholine (ACh) receptors (AChRs) are targeted by autoantibodies. Search for other pathogenic antigens has detected the antibodies against muscle-specific tyrosine kinase (MuSK) and low-density lipoprotein-related protein 4 (Lrp4), both causing pre- and post-synaptic impairments. Agrin is also suspected as a fourth pathogen. In a complex NMJ organization centering on MuSK: (1) the Wnt non-canonical pathway through the Wnt-Lrp4-MuSK cysteine-rich domain (CRD)-Dishevelled (Dvl, scaffold protein) signaling acts to form AChR prepatterning with axonal guidance; (2) the neural agrin-Lrp4-MuSK (Ig1/2 domains) signaling acts to form rapsyn-anchored AChR clusters at the innervated stage of muscle; (3) adaptor protein Dok-7 acts on MuSK activation for AChR clustering from "inside" and also on cytoskeleton to stabilize AChR clusters by the downstream effector Sorbs1/2; (4) the trans-synaptic retrograde signaling contributes to the presynaptic organization via : (i) Wnt-MuSK CRD-Dvl-β catenin-Slit 2 pathway; (ii) Lrp4; and (iii) laminins. The presynaptic Ca2+ homeostasis conditioning ACh release is modified by autoreceptors such as M1-type muscarinic AChR and A2A adenosine receptors. The post-synaptic structure is stabilized by: (i) laminin-network including the muscle-derived agrin; (ii) the extracellular matrix proteins (including collagen Q/perlecan and biglycan which link to MuSK Ig1 domain and CRD); and (iii) the dystrophin-associated glycoprotein complex. The study on MuSK ectodomains (Ig1/2 domains and CRD) recognized by antibodies suggested that the MuSK antibodies were pathologically heterogeneous due to their binding to multiple functional domains. Focussing one of the matrix proteins, biglycan which functions in the manner similar to collagen Q, our antibody assay showed the negative result in MG patients. However, the synaptic stability may be impaired by antibodies against MuSK ectodomains because of the linkage of biglycan with MuSK Ig1 domain and CRD. The pathogenic diversity of MG is discussed based on NMJ signaling molecules. [ABSTRACT FROM AUTHOR]

The LRP4 gene encodes the highly conserved low‐density lipoprotein receptor‐related protein 4 (LRP4), which acts as a co‐receptor for sclerostin. Sclerostin and LRP4 negatively regulate WNT/β‐catenin signaling pathway and lack of their inhibitory activity leads to constant osteoblastic differentiation. Consequently, increased bone formation occurs, which in the case of LRP4 mutations results in sclerosteosis type 2 (SOST2). Alterations within the LRP4 may also cause Cenani‐Lenz syndactyly syndrome (CLSS), congenital myasthenia or isolated syndactyly. Here, we have reported a patient, in whom we found a novel homozygous splice‐site variant c.1048+6T>C in LRP4 using whole exome sequencing. The patient was initially misdiagnosed with isolated CLSS‐like or Malik‐Percin‐like syndactyly. However, we have finally refined the diagnosis after comprehensive radiological examination and molecularly confirmed SOST2. Additionally, we have pointed here to the splicing variants as important causative alterations that may be overlooked in the molecular analysis due to the lack of advanced, reliable algorithms, built‐into the standard diagnostic pipelines. Using advanced in silico prediction tools of splice‐site alterations, including Alamut Visual software, we have demonstrated that the c.1048+6T>C LRP4 variant affects the native donor site and impairs an SC35 enhancer activity. Based on our experience, we recommend comprehensive radiological imaging, including X‐ray of the skull in each case of isolated syndactyly resulting from pathogenic variants of LRP4. We suggest that all previously reported patients carrying biallelic LRP4 mutations, who were diagnosed with isolated syndactyly, could actually present with SOST2 that had been unrecognized due to the incomplete clinical and radiological assessment. [ABSTRACT FROM AUTHOR]

[Display omitted] • In peripheral nervous system, LRP4 related signaling regulates the formation of neuromuscular junction. • In the central nervous system, LRP4 may modulate the release of ATP and regulate a range of energy metabolic processes, such as dendrite and synapse formation. • LRP4 related signaling plays critical roles in many neurological diseases. The mechanism of action of low-density lipoprotein receptor related protein 4 (LRP4) is mediated largely via the Agrin-LRP4-MuSK signalling pathway in the nervous system. LRP4 contributes to the development of synapses in the peripheral nervous system (PNS). It interacts with signalling molecules such as the amyloid beta-protein precursor (APP) and the wingless type protein (Wnt). Its mechanisms of action are complex and mediated via interaction between the pre-synaptic motor neuron and post-synaptic muscle cell in the PNS, which enhances the development of the neuromuscular junction (NMJ). LRP4 may function differently in the central nervous system (CNS) than in the PNS, where it regulates ATP and glutamate release via astrocytes. It may affect the growth and development of the CNS by controlling the energy metabolism. LRP4 interacts with Agrin to maintain dendrite growth and density in the CNS. The goal of this article is to review the current studies involving relevant LRP4 signaling pathways in the nervous system. The review also discusses the clinical and etiological roles of LRP4 in neurological illnesses, such as myasthenia gravis, Alzheimer's disease and epilepsy. In this review, we provide a theoretical foundation for the pathogenesis and therapeutic application of LRP4 in neurologic diseases. [ABSTRACT FROM AUTHOR]

LRP4 is expressed in many organs. It mediates SOST-dependent inhibition of bone formation and acts as an inhibitor of WNT signaling. It is also a postsynaptic end plate cell surface receptor at the neuromuscular junction and is central to its development, maintenance, and function. Pathogenic variants of LRP4 that specifically affect the canonical WNT signaling pathway are known to be associated with Cenani-Lenz syndactyly syndrome or the overlapping condition sclerosteosis. However, site-specific pathogenic variants of LRP4 have been associated with the congenital myasthenic syndrome (CMS) type 17 with no abnormal bone phenotype. Only two studies reported biallelic variants of LRP4 associated with CMS17 that presented during childhood. All three reported variants (NM_002334.4: p.Glu1233Ala, p.Glu1233Lys, or p.Arg1277His) are located within the 3′-edge of the third β-propeller domain of LRP4. We report on a patient with a biallelic variant of the LRP4 gene presenting with a severe and neonatal lethal phenotype; we also provide a literature review of the previously reported patients. A female neonate, born to healthy consanguineous parents, presented with severe hypotonia, congenital diaphragmatic hernia, pulmonary hypertension, and progressive hypoxemia. Two of her siblings presented with a similar condition in the past, and all three died shortly after birth. Clinical exome sequencing revealed homozygosity for the pathogenic variant NM_002334.4:c.3698A > C (p.[Glu1233Ala]). [ABSTRACT FROM AUTHOR]