SummarySynapses, as fundamental units of the neural circuitry, enable complex behaviors. The neuromuscular junction (NMJ) is a synapse type that forms between motoneurons and skeletal muscle fibers and that exhibits a high degree of subcellular specialization. Aided by genetic techniques and suitable animal models, studies in the past decade have brought significant progress in identifying NMJ components and assembly mechanisms. This review highlights recent advances in the study of NMJ development, focusing on signaling pathways that are activated by diffusible cues, which shed light on synaptogenesis in the brain and contribute to a better understanding of muscular dystrophy. Key words: Neural development, Neuromuscular junction, Retrograde signaling, Synapse formation IntroductionThe brain contains billions of nerve cells, or neurons, which receive and integrate signals from the environment, and which govern the body's responses. Nervous system activity is made possible by synapses, contacts formed either between neurons or between a neuron and a target cell. Synapses are asymmetric structures in which neurotransmitter molecules are released from the presynaptic membrane and activate receptors on the postsynaptic membrane, thus establishing neuronal communication. As such, synapses are fundamental units of neural circuitry and enable complex behaviors. The neuromuscular junction (NMJ) is a type of synapse formed between motoneurons and skeletal muscle fibers. Large and easily accessed experimentally, this peripheral synapse has contributed greatly to the understanding of the general principles of synaptogenesis and to the development of potential therapeutic strategies for muscular disorders. The NMJ uses different neurotransmitters in different species; for example, acetylcholine (ACh) in vertebrates and glutamate in Drosophila, both of which are excitatory and cause muscle contraction. In Caenorhabditis elegans, there are two types of NMJs: at excitatory NMJs, ACh causes muscle contraction, whereas inhibitory NMJs release g-aminobutyric acid (GABA) to cause muscle relaxation. Motor nerve terminals differentiate to form presynaptic active zones, where synaptic vesicles dock and release neurotransmitters. On the apposed postsynaptic membranes, neurotransmitter receptors are packed at high densities. Aided by genetic techniques and by the use of suitable animal models, including rodents, zebrafish, Drosophila and C. elegans, studies in the past decade have brought significant progress, not only in identifying components present in pre-and postsynaptic membranes, but also in understanding the mechanisms that underpin NMJ assembly. This review highlights recent advances in the study of NMJ development, focusing on signaling pathways that are activated by diffusible cues from motor nerves and muscle fibers. Readers are referred to other outstanding reviews for a broad view of NMJ development (see Froehner, 1993;Hall and Sanes, 1993;Kummer et al., 2006;Salpeter and Loring, 1985;Schaeffer et al., 2001). NMJ formati...

Developments in miniaturized microscopes have enabled visualization of brain activities and structural dynamics in animals engaging in self-determined behaviors. However, it remains a challenge to resolve activity at single dendritic spines in freely behaving animals. Here, we report the design and application of a fast high-resolution, miniaturized two-photon microscope (FHIRM-TPM) that accomplishes this goal. With a headpiece weighing 2.15 g and a hollow-core photonic crystal fiber delivering 920-nm femtosecond laser pulses, the FHIRM-TPM is capable of imaging commonly used biosensors (GFP and GCaMP6) at high spatiotemporal resolution (0.64 μm laterally and 3.35 μm axially, 40 Hz at 256 × 256 pixels for raster scanning and 10,000 Hz for free-line scanning). We demonstrate the microscope's robustness with hour-long recordings of neuronal activities at the level of spines in mice experiencing vigorous body movements.

SUMMARY Neuromuscular junction (NMJ) formation requires precise interaction between motoneurons and muscle fibers. LRP4 is a receptor of agrin that is thought to act incis to stimulate MuSK in muscle fibers for postsynaptic differentiation. Here we dissected the roles of LRP4 in muscle fibers and motoneurons in NMJ formation by cell-specific mutation. Studies of muscle-specific mutants suggest that LRP4 is involved in deciding where to form AChR clusters in muscle fibers, postsynaptic differentiation, and axon terminal development. LRP4 in HEK293 cells increased synapsin or SV2 puncta in contacting axons of co-cultured neurons, suggesting a synaptogenic function. Analysis of LRP4 muscle and motoneuron double mutants and mechanistic studies suggest that NMJ formation may also be regulated by LRP4 in motoneurons, which could serve as agrin’s receptor in trans to induce AChR clusters. These observations uncovered distinct roles of LRP4 in motoneurons and muscles in NMJ development.

Myasthenia gravis (MG) is the most common disorder affecting the neuromuscular junction (NMJ). MG is frequently caused by autoantibodies against acetylcholine receptor (AChR) and a kinase critical for NMJ formation, MuSK; however, a proportion of MG patients are double-negative for anti-AChR and anti-MuSK antibodies. Recent studies in these subjects have identified autoantibodies against low-density lipoprotein receptor-related protein 4 (LRP4), an agrin receptor also critical for NMJ formation. LRP4 autoantibodies have not previously been implicated in MG pathogenesis. Here we demonstrate that mice immunized with the extracellular domain of LRP4 generated anti-LRP4 antibodies and exhibited MG-associated symptoms, including muscle weakness, reduced compound muscle action potentials (CMAPs), and compromised neuromuscular transmission. Additionally, fragmented and distorted NMJs were evident at both the light microscopic and electron microscopic levels. We found that anti-LRP4 sera decreased cell surface LRP4 levels, inhibited agrin-induced MuSK activation and AChR clustering, and activated complements, revealing potential pathophysiological mechanisms. To further confirm the pathogenicity of LRP4 antibodies, we transferred IgGs purified from LRP4-immunized rabbits into naive mice and found that they exhibited MG-like symptoms, including reduced CMAP and impaired neuromuscular transmission. Together, these data demonstrate that LRP4 autoantibodies induce MG and that LRP4 contributes to NMJ maintenance in adulthood.

Neurotransmission requires precise control of neurotransmitter release from axon terminals. This process is regulated by glial cells; however, underlying mechanisms are not fully understood. Here we report that glutamate release in the brain is impaired in mice lacking low density lipoprotein receptor-related protein 4 (Lrp4), a protein critical for neuromuscular junction formation. Electrophysiological studies indicate compromised release probability in astrocyte-specific Lrp4 knockout mice. Lrp4 mutant astrocytes suppress glutamate transmission by enhancing the release of ATP, whose levels are elevated in the hippocampus of Lrp4 mutant mice. Consequently, the mutant mice are impaired in locomotor activity and spatial memory and are resistant to seizure induction. These impairments could be ameliorated by adenosine A1 receptor antagonist. The results reveal a critical role of Lrp4, in response to agrin, in modulating astrocytic ATP release and synaptic transmission. Our study provides insight into the interaction between neurons and astrocytes for synaptic homeostasis and/or plasticity.

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Highlights d Lipophilic statins and lipophilic bisphosphonates are potent vaccine adjuvants d Modulation of post-translational protein prenylation confers adjuvanticity d Decreased protein prenylation augments antigen preservation and presentation d Statin-or bisphosphonate-mediated vaccination synergizes with anti-PD1 against cancer

Bone mass is maintained by balanced activity of osteoblasts and osteoclasts. Lrp4 (low-density lipoprotein receptor related protein 4) is a member of the LDL receptor family, whose mutations have been identified in patients with high-bone-mass disorders, such as sclerosteosis and van Buchem diseases. However, it remains unknown whether and how Lrp4 regulates bone-mass homeostasis in vivo. Here we provide evidence that Lrp4-null mutation or specific mutation in osteoblast-lineage cells increased cortical and trabecular bone mass, which was associated with elevated bone formation and impaired bone resorption. This phenotype was not observed in osteoclast-selective Lrp4 knockout mice. Mechanistic studies indicate that loss of Lrp4 function in osteoblast-lineage cells increased serum levels of sclerostin, a key factor for bone-mass homeostasis that interacts with Lrp4, but abolished the inhibition of Wnt/β-catenin signaling and osteoblastic differentiation by sclerostin. Concomitantly, sclerostin induction of RANKL (receptor activator of nuclear kappa B ligand) was impaired, leading to a lower ratio of RANKL over OPG (osteoprotegerin) (a key factor for osteoclastogenesis). Taken together, these results support the view for Lrp4 as a receptor of sclerostin to inhibit Wnt/β-catenin signaling and bone formation and identify Lrp4 as a critical player in bonemass homeostasis.one remodeling is a dynamic process essential for maintenance of skeletal integrity and bone homeostasis (1). Bone mass is tightly regulated by bone-forming osteoblasts (OBs) and bone-resorbing osteoclasts (OCs). OBs are differentiated from bone marrow stromal cells (BMSCs) or mesenchymal progenitor cells, whereas OCs are derived from hematopoietic bone marrow macrophages or myeloid monocytes (BMMs). The balance of bone formation and resorption is critical for maintenance of healthy bone mass. The imbalance of bone formation and resorption could result in high-bone-mass disorders such as sclerosteosis and van Buchem disease or bone loss such as osteoporosis.The canonical Wnt/β-catenin signaling is critical to regulate bone-mass homeostasis (1, 2). Binding of Wnt ligands to a dualreceptor complex of frizzled and Lrp5/6 leads to accumulation of cytoplasmic β-catenin and translocation of β-catenin into the nucleus to regulate gene expression. This pathway is required for commitment of mesenchymal stem cells to the OB lineage, OB precursor cell proliferation and differentiation, and OC genesis and activation (1-3). Clinically, Lrp5 mutations are associated with the osteoporosis-pseudoglioma syndrome, a low-bone-mass disorder (4), as well as with high-bone-mass disorders (5, 6).Lrp4 is a member of LDL family protein, containing a large extracellular region with multiple LDLa, EGF-like, and β-propeller repeats, a transmembrane domain, and a short C-terminal region. Lrp4 is a receptor of agrin (7,8), critical for neuromuscular junction formation. Mice lacking Lrp4 (null allele) die at birth because of inability to breathe (9). Lrp4 is also highly related to...