InteractiveFly: GeneBrief

LDL receptor related protein: Biological Overview | References

Precise coordination of synaptic connections ensures proper information flow within circuits. The activity of presynaptic organizing molecules signaling to downstream pathways is essential for such coordination, though such entities remain incompletely known. This study shows that LRP4 (CG8909), a conserved transmembrane protein known for its postsynaptic roles, functions presynaptically as an organizing molecule. In the Drosophila brain, LRP4 localizes to the nerve terminals at or near active zones. Loss of presynaptic LRP4 reduces excitatory (not inhibitory) synapse number, impairs active zone architecture, and abolishes olfactory attraction. The latter of which can be suppressed by reducing presynaptic GABAB receptors. LRP4 overexpression increases synapse number in excitatory and inhibitory neurons, suggesting an instructive role and a common downstream synapse addition pathway. Mechanistically, LRP4 functions via the conserved kinase SRPK79D to ensure normal synapse number and behavior. This highlights a presynaptic function for LRP4, enabling deeper understanding of how synapse organization is coordinated (Mosca, 2017).

Multiple levels of synaptic organization ensure accurate, controlled information flow through neuronal circuits. Neurons must first make an appropriate number of synaptic connections with their postsynaptic partners. Each of these synaptic connections must have appropriate strength that can be modified by plasticity and homeostasis as a result of experience and activity changes. Further, there must be an appropriate balance between excitatory and inhibitory synapses. Finally, recent work has shown that these connections also occupy precise locations with regards to the three-dimensional structure of the synaptic neuropil. Indeed, circuit models for diverse neuronal ensembles fail to recapitulate functional patterns unless these aspects are accounted for. The misregulation of any one of these organizational parameters can result in neurodevelopmental disorders and intellectual disabilities like autism, epilepsy, and other synaptopathies. Revealing the molecular mechanisms that ensure all of these facets are achieved is a critical step in understanding circuit assembly and function (Mosca, 2017).

Synaptic organizers like Neurexins / Neuroligins, Teneurins, protein tyrosine phosphatases (PTPs), leucine rich repeat transmembrane proteins (LRRTMs), and Ephrin / Eph receptors, among others, ensure the proper number, distribution, and function of synaptic connections. Loss-of-function mutations in these key synaptogenic molecules have deleterious structural, functional, and organizational consequences for synapses and circuits. At the vertebrate neuromuscular junction, one of these critical organizers is LRP4. There, it forms a receptor complex with MuSK in muscle fibers to promote clustering of acetylcholine receptors in response to motoneuron-derived agrin (Zhang, 2008; Kim, 2008; Weatherbee, 2006). Muscle LRP4 can also function as a retrograde signal with an unknown motoneuron receptor to regulate presynaptic differentiation (Yumoto, 2012). In these roles, the known functions from LRP4 are overwhelmingly postsynaptic. However, a number of lines of evidence suggest a broader role, beyond postsynaptic, for LRP4. First, motoneuron-derived LRP4 can regulate presynaptic differentiation, demonstrating a role for neuronal LRP4 (Wu, 2012). Second, in the vertebrate central nervous system (CNS), agrin is not essential for synapse formation (Daniels, 2012) though LRP4 can regulate synaptic plasticity, development, and cognitive function (Gomez, 2014; Pohlkamp, 2015), through functioning in astrocytes in some cases. In this vein, the Drosophila genome contains an LRP4 homologue, but no clear agrin or MuSK homologues, so any role for LRP4 there must be agrin-independent (Mosca, 2017).

This study shows in the Drosophila CNS that LRP4 is a presynaptic protein that regulates the number, architecture, and function of synapses. LRP4 functions largely through the conserved, presynaptic SR-protein kinase, SRPK79D. LRP4 and SRPK79D interact genetically and epistatically, as SRPK79D overexpression can suppress lrp4-related phenotypes. Unexpectedly, this role for LRP4 occurs preferentially in excitatory neurons, as impairing lrp4 in inhibitory neurons has no effect. As little is known about the presynaptic determinants (save neurotransmitter-related enzymes and transporters) of excitatory versus inhibitory synapses, this may suggest a new mode for distinguishing such synapses from the presynaptic side. Thus, LRP4 may represent a conserved synaptic organizer that functions presynaptically, cell autonomously, and independently of agrin to coordinate synapse number and function (Mosca, 2017).

Understanding how synaptic organizers regulate the number and function of synapses in the CNS is a central goal of molecular neurobiology. This study identifies LRP4 as a synaptic protein whose expression may be preferential for excitatory neurons in the Drosophila CNS. Though well-known as the postsynaptic agrin receptor at the mouse NMJ, this study describes an agrin-independent, presynaptic role for LRP4. In the Drosophila CNS, LRP4 functions presynaptically to regulate the number of active zones in presynaptic ORNs and acetylcholine receptor clusters in the PNs postsynaptic to those ORNs. Moreover, LRP4 also controls the morphology of individual active zones: lrp4 mutant T-bars exhibit striking defects in patterning and biogenesis. These defects are specific for excitatory neurons, as inhibitory neuron synapses in the antennal lobe remain unaffected. Overexpression of LRP4, however, can increase synapse number cell autonomously in both excitatory and inhibitory neurons, suggesting that both share common mechanisms for synapse addition. The role for LRP4 further extends to higher order olfactory neuropil in the lateral horn, suggesting that it may serve a general role in synaptic organization. Underscoring the functional importance of LRP4, its perturbation in excitatory ORNs abrogated olfactory attraction behavior. The suppression of the behavioral phenotype by reducing presynaptic inhibition onto ORNs further suggests that a proper level of excitatory drive is important for functional circuit output. To mediate both morphological and behavioral effects, LRP4 likely functions through SRPK79D, a conserved SR-protein kinase whose loss-of-function phenotypes resemble those of lrp4, whose synaptic localization depends on LRP4, who interacts genetically with and is physically in proximity to LRP4, and whose overexpression suppresses the phenotypes associated with loss of lrp4 (Mosca, 2017).

Imbalances in excitation and inhibition lead to epileptic states and social dysfunction, and may also underlie many autism spectrum disorders. The mechanisms that maintain this balance are incompletely understood, though likely involve multiple aspects including the number of each type of neuron, their firing rates, release probabilities, synaptic strength, and neurotransmitter receptor sensitivities. Such regulation likely requires distinguishing excitatory from inhibitory neurons at both pre- and postsynaptic levels. Excitatory and inhibitory synapses are identified postsynaptically by distinct neurotransmitter receptor, scaffolding protein, and adhesion molecule repertoires. Postsynaptic factors like Neuroligin 2, Gephyrin, and Slitrk3 organize inhibitory GABAergic synapses while LRRTMs organize excitatory synapses. Thus, postsynaptic regulation can occur by differential modulation of these factors. Little is known, however, about the presynaptic identifiers of excitatory versus inhibitory neurons. Recent work identified Punctin / MADD-4 as a determinant of excitatory versus inhibitory neuromuscular synapses in C. elegans, though as a secreted factor that functions via postsynaptic interaction. Further, Glypican4 can localize to excitatory presynaptic terminals and interact with LRRTM4 but its synaptogenic activity is also provided by astrocytes and thus is not neuronal specific. Proteomic comparisons suggest few differences beyond those pertaining to neurotransmitter synthesis enzymes and transporters. But these components may not be sufficient to distinguish presynaptic excitatory from inhibitory neurons. In the Drosophila olfactory system, for example, glutamate can be inhibitory when its postsynaptic partners express glutamate-gated chloride channels. This suggests that pre- and postsynaptic regulators may exist to distinguish excitatory and inhibitory synapses, though it is unclear what those presynaptic regulators might be (Mosca, 2017).

These data suggests that LRP4 may be a candidate presynaptic organizer specific for excitatory connections. LRP4 is expressed in a subset of excitatory cholinergic neurons, excluded from inhibitory GABAergic neurons, and expressed in a subset of glutamatergic neurons that may be excitatory or inhibitory. Though it cannot be ruled out that inhibitory neuron expression in the case of the glutamatergic subset, the phenotypes associated with LRP4 perturbation are consistent with an excitatory neuron-specific role. Thus, LRP4 may not only serve an identifying role at excitatory synapses, but also a functional one. Loss of lrp4 results in fewer excitatory synapses but has no effect on inhibitory synapses. However, both excitatory and inhibitory neurons show increased synapse number with lrp4 overexpression. This shared competency suggests that both neurons contain machinery that can be engaged downstream of LRP4 (or the cell surface) to add synapses. Thus, proteins like LRP4 may represent identifiers of excitatory or inhibitory terminals that function by engaging common mechanisms to add synapses (Mosca, 2017).

At the mouse NMJ, LRP4 is the well-established postsynaptic receptor for motoneuron-derived Agrin and regulates synapse formation and maintenance. However, additional roles for LRP4 exist at the level of the presynaptic motoneuron. A retrograde signal composed of LRP4 from the postsynaptic muscle interacts with an unknown receptor in the motoneuron to regulate presynaptic differentiation. Thus, at the mouse NMJ, postsynaptic LRP4 has both cell-autonomous and non-cell autonomous roles. In addition, presynaptic LRP4 has been implicated to regulate acetylcholine receptor clustering via MMP-mediated proteolytic cleavage (Mosca, 2017).

In the mouse CNS, LRP4 regulates synaptic physiology (Gomez, 2014; Pohlkamp, 2015), learning and memory, fear conditioning, and CA1 spine density (Gomez, 2014). Though CNS LRP4 most commonly associates with postsynaptic densities (Tian, 2006), it also fractionates with synaptophysin-positive membranes (Gomez, 2014). Indeed, the observed CNS phenotypes have not been localized to a particular pool of LRP4. Identification of Drosophila LRP4 as a key player in CNS synaptogenesis, however, posits a cell-autonomous presynaptic role. While an additional, perhaps concurrent, postsynaptic role cannot be ruled out, this work is the first to demonstrate clear cell-autonomous presynaptic functions for LRP4. Indeed, LRP4 is expressed in PNs and may localize to PN dendrites within the antennal lobe. In such a case, it could function either presynaptically, at dendrodendritic presynapses or as a postsynaptic factor. Moreover, as the Drosophila genome lacks clear Agrin and MuSK homologs, this suggests a synaptic function of LRP4 that evolutionarily precedes Agrin and MuSK recruitment to vertebrate NMJ synaptogenesis (Mosca, 2017).

It remains open whether this presynaptic function is conserved in the mammalian CNS and, if so, what signal LRP4 receives. In Drosophila, the signal cannot be Agrin and in the mammalian CNS, Agrin is not essential for CNS synapse formation. Thus, the Agrin-independence of CNS LRP4 may be conserved across systems. Moreover, the finding that LRP4 promotes excitatory, but not inhibitory, synapse formation and function is consistent with reduced excitatory but normal inhibitory input in hippocampal CA1 neurons of lrp4 mutant mice (Gomez, 2014). Moreover, this study found that LRP4 in the Drosophila CNS functions through the SR-protein kinase SRPK79D. Impaired srpk79D function reduces synapse number and overexpression can suppress the functional and morphological defects associated with lrp4 loss. This kinase is evolutionarily conserved and the three mammalian homologues are widely expressed in the mouse brain, including in the hippocampus. From yeast to human, SRPKs regulate spliceosome assembly and gene expression but have not been studied in mammalian synapse formation. It will be interesting to test if these kinases also function in the mammalian CNS. Combined, however, these commonalities suggest a basic conservation between invertebrate and vertebrate systems for future study (Mosca, 2017).

Recent work implicated LRP4 in both amyotrophic lateral sclerosis (ALS) and myasthenia gravis (MG), two debilitating motor disorders with a worldwide prevalence of ~1/5000. Distinct ALS and MG populations are seropositive for LRP4 autoantibodies and double seronegative for Agrin or MuSK, suggesting that seropositivity is not a byproduct of generalized NMJ breakdown. Further, injection of LRP4 function-blocking antibodies into mice recapitulates MG. Beyond peripheral symptoms, cognitive impairment (besides that as frontotemporal dementia) also occurs in a subset of ALS patients. Thus, understanding the roles of LRP4 in the peripheral and central nervous systems has marked clinical significance. The identification of an evolutionarily conserved kinase, SRPK79D, as a downstream target of LRP4 signaling may offer a window into those roles. As SRPK79D overexpression suppresses the behavioral and the synaptic phenotypes of lrp4 loss, if it functions similarly in the mammalian CNS, SRPKs could be a target for therapeutics. Further investigation of how LRP4 functions in the CNS will provide new insight not only into the cognitive aspects of these debilitating motor disorders, but also into the fundamental aspects of excitatory synapse formation (Mosca, 2017).

Neuronal LRP4 regulates synapse formation in the developing CNS

The low-density lipoprotein receptor-related protein 4 (LRP4) is essential in muscle fibers for the establishment of the neuromuscular junction. This study shows that LRP4 is also expressed by embryonic cortical and hippocampal neurons, and that downregulation of LRP4 in these neurons causes a reduction in density of synapses and number of primary dendrites. Accordingly, overexpression of LRP4 in cultured neurons had the opposite effect inducing more but shorter primary dendrites with an increased number of spines. Transsynaptic tracing mediated by rabies virus revealed a reduced number of neurons presynaptic to the cortical neurons in which LRP4 was knocked down. Moreover, neuron-specific knockdown of LRP4 by in utero electroporation of LRP4 miRNA in vivo also resulted in neurons with fewer primary dendrites and a lower density of spines in the developing cortex and hippocampus. Collectively, these results demonstrate an essential and novel role of neuronal LRP4 in dendritic development and synaptogenesis in the CNS (Karakatsani, 2017).

LRP4 has a fundamental role during formation, maintenance and regeneration of the NMJ. Moreover, cumulative evidence indicates that LRP4 plays crucial roles in synaptic function in the CNS. However, the molecular mechanisms regulating these functions are still mostly unknown. A recent study indicated that, in Drosophila, presynaptic expression of LRP4 regulates synapse number. Studies in LRP4-deficient mice rescued from perinatal death by re-expression of LRP4 in the neuromuscular system, as well as mice with a selective loss of the intracellular and the transmembrane domains showed a significantly decreased density of dendritic spines in the CA3-hippocampal region, but had no gross anatomical abnormalities in the prenatal and adult hippocampus, cortex and cerebellum. In addition, Lrp4 mRNA is present in the neocortex, hippocampus, cerebellum and olfactory bulb. A recent study reported that in adult mice β-galactosidase expression driven by the endogenous Lrp4 promoter is mostly detectable in astrocytes, which is in concordance with current data showing that LRP4 levels, detected by western blotting, are higher in astrocytes than in neurons. Moreover, it was demonstrated that the activation of astroglial LRP4 by neuronal agrin induced the release of ATP, which modulates the synaptic activity of hippocampal pyramidal neurons. Consistently, mice with a conditional knockout for astroglial Lrp4 mRNA exhibited reduced glutamatergic synaptic transmission in neurons from the CA3 region. However, depletion of LRP4 expression in astrocytes did not lead to a decreased density of dendritic spines in the hippocampus, suggesting that other cell sources of LRP4 (i.e. neurons, oligodendrocytes or microglia) play additional roles in the CNS. The current study revealed that reduced LRP4 expression specifically in cortical and hippocampal neurons in vitro and in vivo led to a reduced density of spines and dendrite-associated synapse-like specializations, including bassoon- and synaptobrevin2-positive puncta. Accordingly, increased LRP4 expression in cultured CNS neurons resulted in an increase in the number of spines. As the experiments reported in this study affected LRP4 expression only in neurons (due to the neuron-specific synapsin promoter), the altered spine density that was observed must be caused by changes in neuronally expressed LRP4. Moreover, using a rabies virus-mediated monosynaptic tracing technique, it was demonstrated that the decreased number of synapse-like specializations and dendritic spines mediated by the expression of LRP4 miRNAs is paralleled by a decrease in the number of synaptic inputs as well, and therefore functional synapses (Karakatsani, 2017).

Hippocampal and cortical neurons in culture also responded to reduced LRP4 expression by changes in dendritic morphology. Fewer but longer dendritic processes were generated when the density of synapse-like specializations was decreased. Overexpression of LRP4 in these neurons had the opposite effect, i.e., more but shorter dendrites extended from the soma with a concomitant increase in synaptic density. Moreover, knockdown of neuronal LRP4 also led to a decrease in the number of dendritic branches in the CA1 area of the hippocampus in vivo. Interestingly, the effect of LRP4 knockdown and overexpression on dendrite numbers disappears in cultured neurons over time. This could explain why changes in dendritic morphology were not previously detected in adult LRP4-deficient brains. However, changes in dendrite length remain evident after 21 days of maturation in vitro, suggesting that LRP4 levels would still have a long-term impact on neuronal arbor (Karakatsani, 2017).

Previous studies have shown that dendritogenesis is a highly dynamic process that is influenced by synaptic activity as well as by signals from the immediate environment. The opposite effects of LRP4 overexpression and knockdown on the number of primary dendrites, their length and on spine/synapse density suggest that all these effects might be mechanistically linked. One possibility is that the increase in the dendritic length observed in neurons with reduced levels of LRP4 is a consequence of the decrease in synaptic density. However, this is unlikely to be the case as it was observed that the effect of LRP4 on dendritic arborization takes place before the development of synaptic specializations. Another possibility is that LRP4 regulates the dynamics of cytoskeleton assembly, as rearrangements of cytoskeletal architecture is crucial for both dendrite morphogenesis and development of presynaptic terminals. Supporting this hypothesis, it was observed that LRP4 overexpression results in a decreased mobility of dendrite terminals, blocking both extension and retraction of growing dendrites. Accordingly, immunoreactivity for LRP4 was detected in dendritic growth cones. However, further studies are needed to determine the molecular signals regulating these processes (Karakatsani, 2017).

At the NMJ, the interaction of LRP4 with agrin is crucial for synaptogenesis (Tintignac, 2015). In CNS neurons, anti-agrin antibodies or addition of a soluble C-terminal agrin fragment reversed the effects of LRP4 overexpression. As both treatments would interfere with the TM-agrin-LRP4 interaction either by blocking agrin binding to LRP4 (antibodies) or by competing with the ligand-binding site of LRP4 (soluble agrin fragment), these results implicate TM-agrin as one potential binding partner for LRP4 in the CNS. Thus, TM-agrin-LRP4 interactions might shape dendritic morphology and induce the formation of synapses in cultured neurons of the CNS. This hypothesis is further strengthened by the observation that agrin accumulated at contact sites between dendrites of neurons with increased LRP4 expression and axons overexpressing agrin, whereas agrin did not aggregate at contact sites with neurons in which LRP4 expression was reduced. Interestingly, neurons in the brain of agrin-/- mice, in which the perinatal lethality has been rescued by motoneuron-specific expression of agrin, also develop fewer synapses and shorter dendrites (Karakatsani, 2017).

Although this study focused on cortical and hippocampal neurons, LRP4 has a widespread expression pattern in the CNS, including astrocytes and neuronal populations outside of the forebrain, such as the cerebellum and the olfactory bulb. It is therefore conceivable that LRP4 generally regulates dendritogenesis and synapse formation in many regions of the brain beyond the cerebral cortex and hippocampus. This study provides the first indications of a function for LRP4 in dendrite and synapse formation in developing forebrain neurons, opening up avenues for elucidating the underlying molecular mechanisms in these and other regions of the CNS. These results also suggest that at the molecular level, synaptogenesis at the NMJ and in the CNS are more similar than previously anticipated (Karakatsani, 2017).

Agrin as a mechanotransduction signal regulating YAP through the Hippo pathway

The Hippo pathway effectors YAP and TAZ (see Drosophila Yorkie) act as nuclear sensors of mechanical signals in response to extracellular matrix (ECM) cues. However, the identity and nature of regulators in the ECM and the precise pathways relaying mechanoresponsive signals into intracellular sensors remain unclear. This study uncovered a functional link between the ECM proteoglycan Agrin and the transcriptional co-activator YAP. Importantly, Agrin transduces matrix and cellular rigidity signals that enhance stability and mechanoactivity of YAP through the integrin-focal adhesion- and Lrp4/MuSK receptor-mediated signaling pathways. Agrin antagonizes focal adhesion assembly of the core Hippo components by facilitating ILK-PAK1 (see Drosophila Pak) signaling and negating the functions of Merlin and LATS1/2 (see Drosophila Merlin and Warts). It was further shown that Agrin promotes oncogenesis through YAP-dependent transcription and is clinically relevant in human liver cancer. It is proposed that Agrin acts as a mechanotransduction signal in the ECM (Chakraborty, 2017).

Multiple modes of Lrp4 function in modulation of Wnt/beta-catenin signaling during tooth development

During development and homeostasis, precise control of Wnt/beta-catenin signaling is in part achieved by secreted and membrane proteins that negatively control activity of the Wnt co-receptors Lrp5 and Lrp6. Lrp4 is related to Lrp5/6 and is implicated in modulation of Wnt/beta-catenin signaling, presumably through its ability to bind to the Wise (Sostdc1)/sclerostin (Sost) family of Wnt antagonists. To gain insights into the molecular mechanisms of Lrp4 function in modulating Wnt signaling, this study performed an array of genetic analyses in murine tooth development, where Lrp4 and Wise play important roles. Genetic evidence is provided that Lrp4 mediates the Wnt inhibitory function of Wise and also modulates Wnt/beta-catenin signaling independently of Wise. Chimeric receptor analyses raise the possibility that the Lrp4 extracellular domain interacts with Wnt ligands, as well as the Wnt antagonists. Diverse modes of Lrp4 function are supported by severe tooth phenotypes of mice carrying a human mutation known to abolish Lrp4 binding to Sost. These data suggest a model whereby Lrp4 modulates Wnt/beta-catenin signaling via interaction with Wnt ligands and antagonists in a context-dependent manner (Ahn, 2017).

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. This study 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. These findings provide insight into the interaction between neurons and astrocytes for synaptic homeostasis and/or plasticity (Sun, 2016).

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. This study shows 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. These findings demonstrate that evolutionary divergent mechanisms establish muscle prepatterning in zebrafish and mice (Remedio, 2016).

Lrp4 domains differentially regulate limb/brain development and synaptic plasticity

Apolipoprotein E (ApoE) genotype is the strongest predictor of Alzheimer's Disease (AD) risk. ApoE is a cholesterol transport protein that binds to members of the Low-Density Lipoprotein (LDL) Receptor family, which includes LDL Receptor Related Protein 4 (Lrp4). Lrp4, together with one of its ligands Agrin and its co-receptors Muscle Specific Kinase (MuSK) and Amyloid Precursor Protein (APP), regulates neuromuscular junction (NMJ) formation. All four proteins are also expressed in the adult brain, and APP, MuSK, and Agrin are required for normal synapse function in the CNS. This study shows that Lrp4 is also required for normal hippocampal plasticity. In contrast to the closely related Lrp8/Apoer2, the intracellular domain of Lrp4 does not appear to be necessary for normal expression and maintenance of long-term potentiation at central synapses or for the formation and maintenance of peripheral NMJs. However, it does play a role in limb development (Pohlkamp, 2015).

The MuSK activator agrin has a separate role essential for postnatal maintenance of neuromuscular synapses

The motoneural control of skeletal muscle contraction requires the neuromuscular junction (NMJ), a midmuscle synapse between the motor nerve and myotube. The formation and maintenance of NMJs are orchestrated by the muscle-specific receptor tyrosine kinase (MuSK). Motor neuron-derived agrin activates MuSK via binding to MuSK's coreceptor Lrp4, and genetic defects in agrin underlie a congenital myasthenic syndrome (an NMJ disorder). However, MuSK-dependent postsynaptic differentiation of NMJs occurs in the absence of a motor neuron, indicating a need for nerve/agrin-independent MuSK activation. Previous work identified the muscle protein Dok-7 as an essential activator of MuSK. Although NMJ formation requires agrin under physiological conditions, it is dispensable for NMJ formation experimentally in the absence of the neurotransmitter acetylcholine, which inhibits postsynaptic specialization. Thus, it was hypothesized that MuSK needs agrin together with Lrp4 and Dok-7 to achieve sufficient activation to surmount inhibition by acetylcholine. Forced expression of Dok-7 in muscle enhanced MuSK activation in mice lacking agrin or Lrp4 and restored midmuscle NMJ formation in agrin-deficient mice, but not in Lrp4-deficient mice, probably due to the loss of Lrp4-dependent presynaptic differentiation. However, these NMJs in agrin-deficient mice rapidly disappeared after birth, and postsynaptic specializations emerged ectopically throughout myotubes whereas exogenous Dok-7-mediated MuSK activation was maintained. These findings demonstrate that the MuSK activator agrin plays another role essential for the postnatal maintenance, but not for embryonic formation, of NMJs and also for the postnatal, but not prenatal, midmuscle localization of postsynaptic specializations, providing physiological and pathophysiological insight into NMJ homeostasis (Tezuka, 2014).

Synaptic plasticity and cognitive function are disrupted in the absence of Lrp4

Lrp4, the muscle receptor for neuronal Agrin, is expressed in the hippocampus and areas involved in cognition. The function of Lrp4 in the brain, however, is unknown, as Lrp4-/- mice fail to form neuromuscular synapses and die at birth. Lrp4-/- mice, rescued for Lrp4 expression selectively in muscle, survive into adulthood and showed profound deficits in cognitive tasks that assess learning and memory. To learn whether synapses form and function aberrantly, electrophysiological and anatomical methods were used to study hippocampal CA3-CA1 synapses. In the absence of Lrp4, the organization of the hippocampus appeared normal, but the frequency of spontaneous release events and spine density on primary apical dendrites were reduced. CA3 input was unable to adequately depolarize CA1 neurons to induce long-term potentiation. These studies demonstrate a role for Lrp4 in hippocampal function and suggest that patients with mutations in Lrp4 or auto-antibodies to Lrp4 should be evaluated for neurological deficits (Gomez, 2014).

Wnt proteins regulate acetylcholine receptor clustering in muscle cells

The neuromuscular junction (NMJ) is a cholinergic synapse that rapidly conveys signals from motoneurons to muscle cells and exhibits a high degree of subcellular specialization characteristic of chemical synapses. NMJ formation requires agrin and its coreceptors LRP4 and MuSK. Increasing evidence indicates that Wnt signaling regulates NMJ formation in Drosophila, C. elegans and zebrafish. This study systematically studied the effect of all 19 different Wnts in mammals on acetylcholine receptor (AChR) cluster formation. Five Wnts (Wnt9a, Wnt9b, Wnt10b, Wnt11, and Wnt16) were identified that are able to stimulate AChR clustering, of which Wnt9a and Wnt11 are expressed abundantly in developing muscles. Using Wnt9a and Wnt11 as example, Wnt induction of AChR clusters was demonstrated to be dose-dependent and non-additive to that of agrin, suggesting that Wnts may act via similar pathways to induce AChR clusters. Evidence that Wnt9a and Wnt11 bind directly to the extracellular domain of MuSK, to induce MuSK dimerization and subsequent tyrosine phosphorylation of the kinase. In addition, Wnt-induced AChR clustering requires LRP4. These results identify Wnts as new players in AChR cluster formation, which act in a manner that requires both MuSK and LRP4, revealing a novel function of LRP4 (Zhang, 2012).

Lrp4 is a retrograde signal for presynaptic differentiation at neuromuscular synapses

Motor axons receive retrograde signals from skeletal muscle that are essential for the differentiation and stabilization of motor nerve terminals. Identification of these retrograde signals has proved elusive, but their production by muscle depends on the receptor tyrosine kinase, MuSK (muscle, skeletal receptor tyrosine-protein kinase), and Lrp4 (low-density lipoprotein receptor (LDLR)-related protein 4), an LDLR family member that forms a complex with MuSK, binds neural agrin and stimulates MuSK kinase activity. This study shows that Lrp4 also functions as a direct muscle-derived retrograde signal for early steps in presynaptic differentiation. Lrp4 is shown to be necessary, independent of MuSK activation, for presynaptic differentiation in vivo, and Lrp4 is shown to bind to motor axons and induces clustering of synaptic-vesicle and active-zone proteins. Thus, Lrp4 acts bidirectionally and coordinates synapse formation by binding agrin, activating MuSK and stimulating postsynaptic differentiation, and functioning in turn as a muscle-derived retrograde signal that is necessary and sufficient for presynaptic differentiation (Yumoto, 2012).

Distinct roles of muscle and motoneuron LRP4 in neuromuscular junction formation

Neuromuscular junction (NMJ) formation requires precise interaction between motoneurons and muscle fibers. LRP4 is a receptor of agrin that is thought to act in cis to stimulate MuSK in muscle fibers for postsynaptic differentiation. This study 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 cocultured 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 (Wu, 2012).

Search PubMed for articles about Drosophila Lrp4

Ahn, Y., Sims, C., Murray, M. J., Kuhlmann, P. K., Fuentes-Antras, J., Weatherbee, S. D. and Krumlauf, R. (2017). Multiple modes of Lrp4 function in modulation of Wnt/beta-catenin signaling during tooth development. Development 144(15): 2824-2836. PubMed ID: 28694256

Chakraborty, S., Njah, K., Pobbati, A. V., Lim, Y. B., Raju, A., Lakshmanan, M., Tergaonkar, V., Lim, C. T. and Hong, W. (2017). Agrin as a Mechanotransduction Signal Regulating YAP through the Hippo Pathway. Cell Rep 18(10): 2464-2479. PubMed ID: 28273460

Daniels, M. P. (2012). The role of agrin in synaptic development, plasticity and signaling in the central nervous system. Neurochem Int 61(6): 848-853. PubMed ID: 22414531

Gomez, A. M., Froemke, R. C. and Burden, S. J. (2014). Synaptic plasticity and cognitive function are disrupted in the absence of Lrp4. Elife 3: e04287. PubMed ID: 25407677

Karakatsani, A., Marichal, N., Urban, S., Kalamakis, G., Ghanem, A., Schick, A., Zhang, Y., Conzelmann, K. K., Ruegg, M. A., Berninger, B., Ruiz de Almodovar, C., Gascon, S. and Kroger, S. (2017). Neuronal LRP4 regulates synapse formation in the developing CNS. Development 144(24): 4604-4615. PubMed ID: 29061639

Kim, N., Stiegler, A. L., Cameron, T. O., Hallock, P. T., Gomez, A. M., Huang, J. H., Hubbard, S. R., Dustin, M. L. and Burden, S. J. (2008). Lrp4 is a receptor for Agrin and forms a complex with MuSK. Cell 135(2): 334-342. PubMed ID: 18848351

Mosca, T. J., Luginbuhl, D. J., Wang, I. E. and Luo, L. (2017). Presynaptic LRP4 promotes synapse number and function of excitatory CNS neurons. Elife 6. PubMed ID: 28606304

Pohlkamp, T., Durakoglugil, M., Lane-Donovan, C., Xian, X., Johnson, E. B., Hammer, R. E. and Herz, J. (2015). Lrp4 domains differentially regulate limb/brain development and synaptic plasticity. PLoS One 10(2): e0116701. PubMed ID: 25688974

Remedio, L., Gribble, K. D., Lee, J. K., Kim, N., Hallock, P. T., Delestree, N., Mentis, G. Z., Froemke, R. C., Granato, M. and Burden, S. J. (2016). Diverging roles for Lrp4 and Wnt signaling in neuromuscular synapse development during evolution. Genes Dev 30(9): 1058-1069. PubMed ID: 27151977

Sun, X. D., Li, L., Liu, F., Huang, Z. H., Bean, J. C., Jiao, H. F., Barik, A., Kim, S. M., Wu, H., Shen, C., Tian, Y., Lin, T. W., Bates, R., Sathyamurthy, A., Chen, Y. J., Yin, D. M., Xiong, L., Lin, H. P., Hu, J. X., Li, B. M., Gao, T. M., Xiong, W. C. and Mei, L. (2016). Lrp4 in astrocytes modulates glutamatergic transmission. Nat Neurosci 19(8): 1010-1018. PubMed ID: 27294513

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date revised: 25 September 2017

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