Arnab Barik

Antibodies against low-density lipoprotein receptor–related protein 4 induce myasthenia gravis

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.

LRP4 Is Critical for Neuromuscular Junction Maintenance

The neuromuscular junction (NMJ) is a synapse between motor neurons and skeletal muscle fibers, and is critical for control of muscle contraction. Its formation requires neuronal agrin that acts by binding to LRP4 to stimulate MuSK. Mutations have been identified in agrin, MuSK, and LRP4 in patients with congenital myasthenic syndrome, and patients with myasthenia gravis develop antibodies against agrin, LRP4, and MuSK. However, it remains unclear whether the agrin signaling pathway is critical for NMJ maintenance because null mutation of any of the three genes is perinatal lethal. In this study, we generated imKO mice, a mutant strain whose LRP4 gene can be deleted in muscles by doxycycline (Dox) treatment. Ablation of the LRP4 gene in adult muscle enabled studies of its role in NMJ maintenance. We demonstrate that Dox treatment of P30 mice reduced muscle strength and compound muscle action potentials. AChR clusters became fragmented with diminished junctional folds and synaptic vesicles. The amplitude and frequency of miniature endplate potentials were reduced, indicating impaired neuromuscular transmission and providing cellular mechanisms of adult LRP4 deficiency. We showed that LRP4 ablation led to the loss of synaptic agrin and the 90 kDa fragments, which occurred ahead of other prejunctional and postjunctional components, suggesting that LRP4 may regulate the stability of synaptic agrin. These observations demonstrate that LRP4 is essential for maintaining the structural and functional integrity of the NMJ and that loss of muscle LRP4 in adulthood alone is sufficient to cause myasthenic symptoms.

Crosstalk between Agrin and Wnt signaling pathways in development of vertebrate neuromuscular junction

Neuromuscular junction (NMJ) is a cholinergic synapse where motor neurons elicit muscle contraction. Agrin and its coreceptors LRP4 and MuSK are critical for vertebrate NMJ formation. This paper reviews recent evidence for Wnts and Wnt signaling molecules in NMJ formation including a possible retrograde mechanism by muscle β‐catenin. We also present data that Wnt3a, 7a, 8a and 10b could inhibit agrin‐mediated AChR clustering. Together with the stimulating effect of Wnt9a, 9b, 10b, 11 and 16 on AChR clustering in the absence of agrin, these results suggest diverse roles for Wnt ligands in NMJ development.

Lrp4 in astrocytes modulates glutamatergic transmission

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

Slit2 as a β-catenin/Ctnnb1-dependent retrograde signal for presynaptic differentiation

Neuromuscular junction formation requires proper interaction between motoneurons and muscle cells. β-Catenin (Ctnnb1) in muscle is critical for motoneuron differentiation; however, little is known about the relevant retrograde signal. In this paper, we dissected which functions of muscle Ctnnb1 are critical by an in vivo transgenic approach. We show that Ctnnb1 mutant without the transactivation domain was unable to rescue presynaptic deficits of Ctnnb1 mutation, indicating the involvement of transcription regulation. On the other hand, the cell-adhesion function of Ctnnb1 is dispensable. We screened for proteins that may serve as a Ctnnb1-directed retrograde factor and identified Slit2. Transgenic expression of Slit2 specifically in the muscle was able to diminish presynaptic deficits by Ctnnb1 mutation in mice. Slit2 immobilized on beads was able to induce synaptophysin puncta in axons of spinal cord explants. Together, these observations suggest that Slit2 serves as a factor utilized by muscle Ctnnb1 to direct presynaptic differentiation. DOI: http://dx.doi.org/10.7554/eLife.07266.001

Third-Party Bioluminescence Resonance Energy Transfer Indicates Constitutive Association of Membrane Proteins: Application to Class A G-Protein-Coupled Receptors and G-Proteins

Many of the molecules that mediate G-protein signaling are thought to constitutively associate with each other in variably stable signaling complexes. Much of the evidence for signaling complexes has come from Förster resonance energy transfer and bioluminescence resonance energy transfer (BRET) studies. However, detection of constitutive protein association with these methods is hampered by nonspecific energy transfer that occurs when donor and acceptor molecules are in close proximity by chance. We show that chemically-induced recruitment of local third-party BRET donors or acceptors reliably separates nonspecific and specific BRET. We use this method to reexamine the constitutive association of class A G-protein-coupled receptors (GPCRs) with other GPCRs and with heterotrimeric G-proteins. We find that beta2 adrenoreceptors constitutively associate with each other and with several other class A GPCRs. In contrast, GPCRs and G-proteins are unlikely to exist in stable constitutive preassembled complexes.

Schwann Cells in Neuromuscular Junction Formation and Maintenance

The neuromuscular junction (NMJ) is a tripartite synapse that is formed by motor nerve terminals, postjunctional muscle membranes, and terminal Schwann cells (TSCs) that cover the nerve-muscle contact. NMJ formation requires intimate communications among the three different components. Unlike nerve-muscle interaction, which has been well characterized, less is known about the role of SCs in NMJ formation and maintenance. We show that SCs in mice lead nerve terminals to prepatterned AChRs. Ablating SCs at E8.5 (i.e., prior nerve arrival at the clusters) had little effect on aneural AChR clusters at E13.5, suggesting that SCs may not be necessary for aneural clusters. SC ablation at E12.5, a time when phrenic nerves approach muscle fibers, resulted in smaller and fewer nerve-induced AChR clusters; however, SC ablation at E15.5 reduced AChR cluster size but had no effect on cluster density, suggesting that SCs are involved in AChR cluster maturation. Miniature endplate potential amplitude, but not frequency, was reduced when SCs were ablated at E15.5, suggesting that postsynaptic alterations may occur ahead of presynaptic deficits. Finally, ablation of SCs at P30, after NMJ maturation, led to NMJ fragmentation and neuromuscular transmission deficits. Miniature endplate potential amplitude was reduced 3 d after SC ablation, but both amplitude and frequency were reduced 6 d after. Together, these results indicate that SCs are not only required for NMJ formation, but also necessary for its maintenance; and postsynaptic function and structure appeared to be more sensitive to SC ablation. SIGNIFICANCE STATEMENT Neuromuscular junctions (NMJs) are critical for survival and daily functioning. Defects in NMJ formation during development or maintenance in adulthood result in debilitating neuromuscular disorders. The role of Schwann cells (SCs) in NMJ formation and maintenance was not well understood. We genetically ablated SCs during development and after NMJ formation to investigate the consequences of the ablation. This study reveals a critical role of SCs in NMJ formation as well as maintenance.

Enzymatic Activity of the Scaffold Protein Rapsyn for Synapse Formation

Neurotransmission is ensured by a high concentration of neurotransmitter receptors at the postsynaptic membrane. This is mediated by scaffold proteins that bridge the receptors with cytoskeleton. One such protein is rapsyn (receptor-associated protein at synapse), which is essential for acetylcholine receptor (AChR) clustering and NMJ (neuromuscular junction) formation. We show that the RING domain of rapsyn contains E3 ligase activity. Mutation of the RING domain that abolishes the enzyme activity inhibits rapsyn- as well as agrin-induced AChR clustering in heterologous and muscle cells. Further biological and genetic studies support a working model where rapsyn, a classic scaffold protein, serves as an E3 ligase to induce AChR clustering and NMJ formation, possibly by regulation of AChR neddylation. This study identifies a previously unappreciated enzymatic function of rapsyn and a role of neddylation in synapse formation, and reveals a potential target of therapeutic intervention for relevant neurological disorders.

ERBB3-mediated regulation of Bergmann glia proliferation in cerebellar lamination

Cortical lamination is crucial for the assembly of cerebellar circuitry. In this process, granule neurons (GNs) migrate along Bergmann glia (BG), which are specialized astroglial cells, from the external granule layer to the internal granule layer. However, the molecular mechanisms underlying BG development are not well understood. Here, we show that GFAP::Cre;Erbb3F/F mice, which lack Erbb3 in both radial glia and neurons, exhibit impairments in balance and motor coordination. Cerebellar lamination is aberrant, with misplaced Purkinje neurons and GN clusters. These phenotypes were not observed in Math1::CreERT2;Erbb3F/F mice, where the Erbb3 gene was deleted in GNs, suggesting involvement of non-neuronal Erbb3 in cerebellar lamination. Mechanistic studies indicate that ERBB3 is crucial for the proliferation of BG, which are required for GN migration. These observations identify a crucial role for ERBB3 in cerebellar lamination and reveal a novel mechanism that regulates BG development. Summary: Erbb3 knockout mice show cerebellar lamination and subsequent behavioural defects due to impaired proliferation of Bergmann glia - the scaffolds for granule neuron migration.

Regulation of synapse development by Vgat deletion from ErbB4-positive interneurons

GABA signaling has been implicated in neural development; however, in vivo genetic evidence is missing because mutant mice lacking GABA activity die prematurely. Here, we studied synapse development by ablating vesicular GABA transporter (Vgat) in ErbB4+ interneurons. We show that inhibitory axo–somatic synapses onto pyramidal neurons vary from one cortical layer to another; however, inhibitory synapses on axon initial segments (AISs) were similar across layers. Conversely, parvalbumin-positive (PV+)/ErbB4+ interneurons and PV-only interneurons receive a higher number of inhibitory synapses from PV+ErbB4+ interneurons compared with ErbB4-only interneurons. Vgat deletion from ErbB4+ interneurons reduced axo–somatic or axo–axonic synapses from PV+ErbB4+ interneurons onto excitatory neurons. This effect was associated with corresponding changes in neurotransmission. However, the Vgat mutation seemed to have little effect on inhibitory synapses onto PV+ and/or ErbB4+ interneurons. Interestingly, perineuronal nets, extracellular matrix structures implicated in maturation, survival, protection, and plasticity of PV+ interneurons, were increased in the cortex of ErbB4-Vgat−/− mice. No apparent difference was observed between males and females. These results demonstrate that Vgat of ErbB4+ interneurons is essential for the development of inhibitory synapses onto excitatory neurons and suggest a role of GABA in circuit assembly. SIGNIFICANCE STATEMENT GABA has been implicated in neural development, but in vivo genetic evidence is missing because mutant mice lacking GABA die prematurely. Here, we ablated Vgat in ErbB4+ interneurons in an inducible manner. We provide evidence that the formation of inhibitory and excitatory synapses onto excitatory neurons requires Vgat in interneurons. In particular, inhibitory axo–somatic and axo–axonic synapses are more vulnerable. Our results suggest a role of GABA in circuit assembly.