Diverging roles for Lrp4 and Wnt signaling in neuromuscular synapse development during evolution.

Motor axons approach muscles that are prepatterned in the prospective synaptic region. In mice, prepatterning of acetylcholine receptors requires Lrp4, a LDLR family member, and MuSK, a receptor tyrosine kinase. Lrp4 can bind and stimulate MuSK, strongly suggesting that association between Lrp4 and MuSK, independent of additional ligands, initiates prepatterning in mice. In zebrafish, Wnts, which bind the Frizzled (Fz)-like domain in MuSK, are required for prepatterning, suggesting that Wnts may contribute to prepatterning and neuromuscular development in mammals. We show that prepatterning in mice requires Lrp4 but not the MuSK Fz-like domain. In contrast, prepatterning in zebrafish requires the MuSK Fz-like domain but not Lrp4. Despite these differences, neuromuscular synapse formation in zebrafish and mice share similar mechanisms, requiring Lrp4, MuSK, and neuronal Agrin but not the MuSK Fz-like domain or Wnt production from muscle. Our findings demonstrate that evolutionary divergent mechanisms establish muscle prepatterning in zebrafish and mice.

Z-disc-associated, alternatively spliced, PDZ motif-containing protein (ZASP) mutations in the actin-binding domain cause disruption of skeletal muscle actin filaments in myofibrillar myopathy.

The core of skeletal muscle Z-discs consists of actin filaments from adjacent sarcomeres that are cross-linked by α-actinin homodimers. Z-disc-associated, alternatively spliced, PDZ motif-containing protein (ZASP)/Cypher interacts with α-actinin, myotilin, and other Z-disc proteins via the PDZ domain. However, these interactions are not sufficient to maintain the Z-disc structure. We show that ZASP directly interacts with skeletal actin filaments. The actin-binding domain is between the modular PDZ and LIM domains. This ZASP region is alternatively spliced so that each isoform has unique actin-binding domains. All ZASP isoforms contain the exon 6-encoded ZASP-like motif that is mutated in zaspopathy, a myofibrillar myopathy (MFM), whereas the exon 8-11 junction-encoded peptide is exclusive to the postnatal long ZASP isoform (ZASP-LΔex10). MFM is characterized by disruption of skeletal muscle Z-discs and accumulation of myofibrillar degradation products. Wild-type and mutant ZASP interact with α-actin, α-actinin, and myotilin. Expression of mutant, but not wild-type, ZASP leads to Z-disc disruption and F-actin accumulation in mouse skeletal muscle, as in MFM. Mutations in the actin-binding domain of ZASP-LΔex10, but not other isoforms, cause disruption of the actin cytoskeleton in muscle cells. These isoform-specific mutation effects highlight the essential role of the ZASP-LΔex10 isoform in F-actin organization. Our results show that MFM-associated ZASP mutations in the actin-binding domain have deleterious effects on the core structure of the Z-discs in skeletal muscle.

Initiation of synapse formation by Wnt-induced MuSK endocytosis.

In zebrafish, the MuSK receptor initiates neuromuscular synapse formation by restricting presynaptic growth cones and postsynaptic acetylcholine receptors (AChRs) to the center of skeletal muscle cells. Increasing evidence suggests a role for Wnts in this process, yet how muscle cells respond to Wnt signals is unclear. Here, we show that in vivo, wnt11r and wnt4a initiate MuSK translocation from muscle membranes to recycling endosomes and that this transition is crucial for AChR accumulation at future synaptic sites. Moreover, we demonstrate that components of the planar cell polarity pathway colocalize to recycling endosomes and that this localization is MuSK dependent. Knockdown of several core components disrupts MuSK translocation to endosomes, AChR localization and axonal guidance. We propose that Wnt-induced trafficking of the MuSK receptor to endosomes initiates a signaling cascade to align pre- with postsynaptic elements. Collectively, these findings suggest a general mechanism by which Wnt signals shape synaptic connectivity through localized receptor endocytosis.

Characterization of non-alpha retinal ganglion cell injury responses reveals a possible block to restoring ipRGC function.

Visual impairment caused by retinal ganglion cell (RGC) axon damage or degeneration affects millions of individuals throughout the world. While some progress has been made in promoting long-distance RGC axon regrowth following injury, it remains unclear whether RGC axons can properly reconnect with their central targets to restore visual function. Additionally, the regenerative capacity of many RGC subtypes remains unknown in part due to a lack of available genetic tools. Here, we use a new mouse line, Sema6ACreERT2, that labels On direction-selective RGCs (oDSGCs) and characterize the survival and regenerative potential of these cells following optic nerve crush (ONC). In parallel, we use a previously characterized mouse line, Opn4CreERT2, to answer these same questions for M1 intrinsically photosensitive RGCs (ipRGCs). We find that both M1 ipRGCs and oDSGCs are resilient to injury but do not display long-distance axon regrowth following Lin28a overexpression. Unexpectedly, we found that M1 ipRGC, but not oDSGC, intraretinal axons exhibit ectopic branching and are misaligned near the optic disc between one- and three-weeks following injury. Additionally, we observe that numerous ectopic presynaptic specializations associate with misguided ipRGC intraretinal axons. Taken together, these results reveal insights into the injury response of M1 ipRGCs and oDSGCs, providing a foundation for future efforts seeking to restore visual system function following injury.

Genetic access to neurons in the accessory optic system reveals a role for Sema6A in midbrain circuitry mediating motion perception.

The accessory optic system (AOS) detects retinal image slip and reports it to the oculomotor system for reflexive image stabilization. Here, we characterize two Cre lines that permit genetic access to AOS circuits responding to vertical motion. The first (Pcdh9-Cre) labels only one of the four subtypes of ON direction-selective retinal ganglion cells (ON-DS RGCs), those preferring ventral retinal motion. Their axons diverge from the optic tract just behind the chiasm and selectively innervate the medial terminal nucleus (MTN) of the AOS. Unlike most RGC subtypes examined, they survive after optic nerve crush. The second Cre-driver line (Pdzk1ip1-Cre) labels postsynaptic neurons in the MTN. These project predominantly to the other major terminal nucleus of the AOS, the nucleus of the optic tract (NOT). We find that the transmembrane protein semaphorin 6A (Sema6A) is required for the formation of axonal projections from the MTN to the NOT, just as it is for the retinal innervation of the MTN. These new tools permit manipulation of specific circuits in the AOS and show that Sema6A is required for establishing AOS connections in multiple locations.

The synaptic receptor Lrp4 promotes peripheral nerve regeneration.

Early during PNS regeneration, regenerating axons emerge from the proximal nerve stump, yet whether they extend simultaneously or whether pioneering axons establish a path for follower axons remains unknown. Moreover, the molecular mechanisms underlying robust regeneration are incompletely understood. Using live imaging, we demonstrate that in zebrafish pioneering axons establish a regenerative path for follower axons. We find this process requires the synaptic receptor lrp4, and in lrp4 mutants pioneers are unaffected while follower axons frequently stall at the injury gap, providing evidence for molecular diversity between pioneering and follower axons in regeneration. We demonstrate that Lrp4 promotes regeneration through an axon extrinsic mechanism and independent of membrane anchoring and MuSK co-receptor signaling essential for synaptic development. Finally, we show that Lrp4 coordinates the realignment of denervated Schwann cells with regenerating axons, consistent with a model by which Lrp4 is repurposed to promote sustained peripheral nerve regeneration via axon-glia interactions.