InteractiveFly: GeneBrief

Multiplexin: Biological Overview | References

BIOLOGICAL OVERVIEW

At synapses in organisms ranging from fly to human, a decrease in postsynaptic neurotransmitter receptor function elicits a homeostatic increase in presynaptic release that restores baseline synaptic efficacy. This process, termed presynaptic homeostasis, requires a retrograde, trans-synaptic signal of unknown identity. Multiplexin was identified in a forward genetic screen for homeostatic plasticity genes. Multiplexin is the Drosophila homolog of Collagen XV/XVIII, a matrix protein that can be proteolytically cleaved to release Endostatin, an antiangiogenesis signaling factor. This study demonstrates that Multiplexin is required for normal calcium channel abundance, presynaptic calcium influx, and neurotransmitter release. Remarkably, Endostatin has a specific activity, independent of baseline synapse development, that is required for the homeostatic modulation of presynaptic calcium influx and neurotransmitter release. These data support a model in which proteolytic release of Endostatin signals trans-synaptically, acting in concert with the presynaptic CaV2.1 calcium channel, to promote presynaptic homeostasis (Wang, 2014).

The nervous system is continually modified by experience. Given the tremendous complexity of the nervous system, it is astounding that robust and reproducible neural function can be sustained throughout life. It is now apparent that homeostatic signaling systems stabilize the excitable properties of nerve and muscle and, thereby, constrain how the nervous system can be altered by experience or crippled by disease. The Drosophila neuromuscular junction (NMJ) has emerged as a powerful model system to dissect the underlying mechanisms that achieve the homeostatic modulation of presynaptic neurotransmitter release. At the Drosophila NMJ, inhibition of postsynaptic glutamate receptor function causes a homeostatic increase in presynaptic neurotransmitter release that precisely restores muscle excitation to baseline levels. This phenomenon is conserved from fly to human. Importantly, presynaptic homeostasis has also been observed at mammalian central synapses in vitro in response to differences in target innervation and altered postsynaptic excitability and following chronic inhibition of neural activity (Wang, 2014).

Despite progress in identifying presynaptic effector proteins that are required for the expression of presynaptic homeostasis (Davis, 2013), the identity of the retrograde signaling system remains unknown. Numerous neurotrophic factors, such as nerve growth factor; brain-derived neurotrophic factor (BDNF); and glia-derived neurotrophic factor, as well as nitric oxide, endocannabinoids, and adhesion molecules, are identified as retrograde signals that regulate presynaptic cell survival, differentiation, and biophysical properties in an activity-dependent manner. Among these molecules, BDNF has been implicated in the trans-synaptic control of presynaptic release in cultured hippocampal neurons. Previous work demonstrated that a bone morphogenetic protein (BMP) ligand (Glass bottom boat) is released from muscle, activates a type II BMP receptor at the presynaptic terminal, and is required for the growth of the presynaptic nerve terminal. This BMP signaling system is also necessary for presynaptic homeostasis. However, the BMP signaling system is a permissive signal that acts at the motoneuron cell body (Wang, 2014).

A large-scale, electrophysiology-based forward genetic screen for mutations that block presynaptic homeostasis (Dickman, 2009 and Müller, 2011) identified multiplexin as a candidate homeostatic plasticity gene. Drosophila Multiplexin is the homolog of human Collagen XV and XVIII, matrix molecules that are expressed ubiquitously in various vascular and epithelial basement membranes throughout the body (Seppinen, 2011). Mutations in the human COL18A1 gene cause Knobloch syndrome, characterized by retinal detachment, macular abnormalities, and occipital encephalocele. Patients with Knobloch syndrome are also predisposed to epilepsy, highlighting the critical function of Collagen XVIII in the central nervous system. Moreover, the C-terminal of Collagen XVIII, encoding an Endostatin domain, can be cleaved proteolytically (Chang, 2005, Felbor, 2000; Heljasvaara, 2005) and functions as an antiangiogenesis factor to inhibit tumor progression (Dhanabal, 1999, O'Reilly, 1997; Yamaguchi, 1999). Endostatin inhibits angiogenesis by interacting with various downstream signaling factors, including vascular endothelial growth factor receptors (Kim, 2002), integrins (Wickström, 2002), and Wnt signaling molecules (Hanai, 2002). Little is known regarding the function of multiplexin in the nervous system. This study provides evidence that Endostatin, a proteolytic cleavage product of Drosophila Multiplexin, functions as a trans-synaptic signaling molecule that is essential for the homeostatic modulation of presynaptic neurotransmitter release at the Drosophila NMJ (Wang, 2014).

Loss of Endostatin blocks the homeostatic modulation of presynaptic calcium influx and presynaptic neurotransmitter release. This activity is remarkably specific to presynaptic homeostasis, since loss of Endostatin has no effect on baseline neurotransmission or synapse morphology. Endostatin also interacts genetically with the pore-forming subunit of the CaV2.1 calcium channel and is required for the homeostatic increase of presynaptic calcium influx during synaptic homeostatic plasticity. Finally, transgenic overexpression of Endostatin is sufficient to rescue synaptic homeostasis and baseline neurotransmitter release when it is supplied to either the presynaptic or postsynaptic side of the synapse. Although deletion of Endostatin does not impair baseline transmission, overexpression of Endostatin in the dmpf07253 mutant is sufficient to restore baseline transmission release even in the absence of the Thrombospondin-like domain. As a working model, it is proposed that inhibition of postsynaptic glutamate receptors initiates the proteolytic cleavage of Multiplexin, which resides in the synaptic cleft. It is further proposed that release of Endostatin acts upon presynaptic calcium channels, directly or indirectly, to potentiate calcium influx and presynaptic neurotransmitter release. This model is consistent with data from other systems demonstrating that activation of Endostatin requires proteolytic cleavage of Collagen XVIII by matrix metalloproteases (MMPs) and cysteine cathepsins. Moreover, only free Endostatin released by cleavage functions as an antiangiogenesis factor (Heljasvaara, 2005; Wang, 2014).

The means by which presynaptic calcium channel function is modulated by Endostatin remains to be elucidated. Recently, it has been shown that a presynaptic Deg/ENaC channel is also necessary for the homeostatic modulation of presynaptic release (Younger, 2013). In this previous study, a model is presented in which ENaC channel insertion causes a sodium leak and modest depolarization of the presynaptic resting membrane potential that, in turn, potentiates presynaptic calcium influx. One possibility is that the interaction of Endostatin with the presynaptic CaV2.1 channels enables the channels to respond to low-voltage modulation. This would be consistent with both Endostatin and the ENaC channel being strictly necessary for presynaptic homeostasis. It remains formally possible that Endostatin stabilizes presynaptic ENaC channels and, thereby, influences presynaptic calcium influx. For example, it was demonstrated that the interaction between ENaC channels and extracellular collagens mediates the mechanosensory transduction in the touch reception systems (Chalfie, 2009; Liu, 1996; Wang, 2014).

Activation of Endostatinin in other systems requires proteolytical cleavage of Collagen XVIII by MMPs and cysteine cathepsins. This raises an intriguing possibility that, during synaptic homeostasis, Multiplexin could be cleaved by synaptic MMPs, releasing Endostatin to trigger a homeostatic change in presynaptic release. In this model, inhibition of postsynaptic glutamate receptors would lead to the activation of MMPs within the synaptic cleft. Thus, the retrograde signal would be a multistage system, providing opportunity for both amplification and multilevel control of the signaling event. At glutamatergic synapses in hippocampal neurons, proteolytic cleavage of neuroligin-1, a synaptic adhesion molecule residing in postsynaptic terminals, is triggered by postsynaptic NMDA receptor activation. Cleavage of neuroligin-1 depresses presynaptic transmission by reducing presynaptic release probability in a trans-synaptic manner (Peixoto, 2012). Thus, the activity-dependent cleavage of cell adhesion and extracellular matrix proteins could provide a robust and evolutionarily conserved feedback paradigm for trans-synaptic signaling to regulate synaptic efficacy in diverse neuronal circuits (Wang, 2014).

Discoidin domain receptor regulates ensheathment, survival, and caliber of peripheral axons

Most invertebrate axons and small caliber axons in mammalian peripheral nerves are unmyelinated but still ensheathed by glia. This study used Drosophila wrapping glia to study the development and function of non-myelinating axon ensheathment, which is poorly understood. Selective ablation of these glia from peripheral nerves severely impaired larval locomotor behavior. In an in vivo RNAi screen to identify glial genes required for axon ensheathment, the conserved receptor tyrosine kinase Discoidin domain receptor (Ddr) was identified. In larval peripheral nerves, loss of Ddr resulted in severely reduced ensheathment of axons and reduced axon caliber, and a strong dominant genetic interaction was found between Ddr and the type XV/XVIII collagen Multiplexin (Mp), suggesting Ddr functions as a collagen receptor to drive axon wrapping. In adult nerves, loss of Ddr decreased long-term survival of sensory neurons and significantly reduced axon caliber without overtly affecting ensheathment. These data establish essential roles for non-myelinating glia in nerve development, maintenance, and function, and identify Ddr as a key regulator of axon-glia interactions during ensheathment and establishment of axon caliber (Corty, 2022).

Non-myelinating ensheathment of axons is a conserved but understudied feature of the PNS. Although this type of multi-axonal ensheathment has been less studied compared with myelination, a growing body of evidence indicates it is important for the health and function of neurons and axons in the periphery. For example, Schwann cell-specific loss of the transmembrane receptor LDL receptor related protein-1 (LRP1) causes both thin myelin and abnormal Remak bundle structure. These conditional knockout animals also showed a lowered pain threshold, suggesting that the physiology of nociceptor neurons is impaired when Remak ensheathment is disrupted. Disrupting metabolism in Schwann cells causes progressive axon loss, with small unmyelinated fibers dying first, before myelinated fibers begin to show signs of degeneration. In the fly, disruption of axonal wrapping leads to uncoordinated behavioral responses that hint at aberrant ephaptic coupling between neighboring axons in nerves when not properly separated (Kottmeier, 2020). Such coupling could cause the inappropriate activation of sensory or nociceptive neurons underlying peripheral neuropathies. Previous studies lab have shown that wrapping glia are required to clear neuronal debris after nerve injury and mediate injury signaling between injured and intact 'bystander' neurons, which might be important for functional recovery after nerve trauma. These and other findings suggest that Remak-type ensheathment and axon-glia signaling of unmyelinated fibers play a variety of underappreciated roles in peripheral nerve physiology that contribute to the pathophysiology of a number of PNS disorders, including debilitating peripheral neuropathies and responses to nerve injury (Corty, 2022).

To gain insight into non-myelinating ensheathment, the Drosophila peripheral nerves were used to identify a molecular pathway important for the development and function multi-axonal ensheathment. A new Split-Gal4 intersectional driver was generated to target wrapping glia more specifically for functional and behavioral studies in order to improve understanding of whether and how wrapping glia support axon health, physiology and, ultimately, circuit function. Finally, this study uncovered roles for glia in mediating long-term neuronal survival and driving increased axon caliber that are separable from overt effects on wrapping, demonstrating that non-myelinating ensheathing glia perform crucial, previously unappreciated, roles in nervous system development, maintenance and function (Corty, 2022).

A main advantage of Drosophila is the ability to conduct large-scale in vivo screens. Use was made of available UAS-RNAi libraries to carry out a broad screen for regulators of axonal ensheathment in intact nerves. This morphological screen was sensitive enough to identify genes previously implicated in wrapping glia development, including vn, LanB1 and mys, validating the approach. Moreover, in the case of Ddr, it was possible to identify an important regulator of ensheathment that a simple behavioral or lethality screen would have missed in light of follow-up behavioral testing. Knockdown of Ddr in wrapping glia resulted in reduced glial membrane coverage in nerve cross-sections by fluorescence microscopy. Similar phenotypes were observed in Ddr loss-of-function animals and could be rescued by resupplying Ddr specifically in wrapping glia, confirming the specificity of the RNAi results. TEM clearly showed that reduced glial membrane coverage at the light level corresponds to decreased axon wrapping (Corty, 2022).

Although neither of the vertebrate homologs, Ddr1 and Ddr2, has been explicitly implicated in glial development, several lines of evidence suggests that Ddr1 may have a conserved role in vertebrate glial development or function. Ddr1 is highly expressed in the mouse oligodendrocyte lineage starting from when the cells begin to associate with axons, is upregulated in newly formed oligodendrocytes after cuprizone treatment, and is expressed in both myelinating and Remak Schwann cells. Moreover, DDR1 is expressed in human oligodendrocytes and myelin, and variants in the human gene have been correlated with abnormal white matter and schizophrenia (Corty, 2022).

Vertebrate Ddr1 and Ddr2 are potently activated by collagens in vitro, prompting an investigation of whether collagens were involved with Ddr function in fly nerves. Knockdown of the Drosophila collagen Mp specifically in wrapping glia but not in neurons was found to disrupted ensheathment. Together with the established roles for vertebrate Ddr1 and Ddr2 as collagen receptors, the strong genetic interaction observed between Ddr and Mp is consistent with a model in which Mp acts as a collagen ligand for Ddr during axonal ensheathment. Although the Mp-GFP protein trap shows diffuse Mp expression throughout the nerve, it remains unclear precisely which cell type(s) within the nerve are producing it. Previous reports indicate that Mp can be expressed in the outer peripheral glia layers, so they may provide some Mp to the wrapping glia. However, the strong ensheathment defect seen when Mp is knocked down exclusively in wrapping glia indicates that wrapping glia themselves are likely to be the primary, relevant source of the Mp required for their own morphogenesis. Schwann cells similarly rely on components of their own basal lamina to regulate their development. For example, laminin-211 serves as a ligand for GPR126 to promote myelination. Mp is the sole Drosophila homolog of collagen types XV/XVIII, containing a central helical collagen region with a cleavable N-terminal thrombospondin-like domain and C-terminal endostatin-like domain. Collagen 15a1 and 18a1 are expressed in mouse peripheral nerves and Col15a1 mutants have radial-sorting defects, suggesting that the role of Mp in promoting axon wrapping is likely conserved. In fact, Mp appears to play multiple roles in nerve biology. For example, Mp secreted by the outer glia layers acts via its cleaved endostatin domain to modulate homeostatic plasticity at motor neuron synapses. How Ddr activation within wrapping glia ultimately drives axon wrapping still remains to be determined, but Ddr joins two other receptor tyrosine kinases - EGFR and FGFR - as important and conserved regulators of axon ensheathment. As a non-canonical collagen receptor, Ddr may also interact with other collagen receptors, such as integrins (known to play roles in wrapping glia development), to sense and remodel the extracellular matrix and permit extension of glia processes between axons, similar to its roles in promoting tumor metastasis (Corty, 2022).

The nrv2-Gal4 driver has been the standard method to genetically target wrapping glia for morphological studies, but it is imperfect for manipulation of wrapping glia in ablation or behavioral assays owing to its expression in several subtypes of CNS glia. This study generated a new Split-Gal4 intersectional driver that drives exclusively in wrapping glia. This allowed performing of precise ablation of wrapping glia that led to severely impaired larval locomotion, indicating that the wrapping glia are essential for basic crawling circuit function. This phenotype was particularly striking in light of that fact that no clear crawling defect was observed in Ddr mutant larvae, even though wrapping was severely impaired. It may be possible that non-contact-mediated mechanisms, such as one or more secreted factors, constitute the essential contribution of wrapping glia to axon health and physiology. Alternatively, perhaps even a small amount of direct glia-axon contact may be sufficient to support neuron health and axon function. This would be consistent with the lack of overt behavioral defects in newly hatched first instar larvae, which have poor wrapping compared with later stages, and even in wild-type third instar larvae, in which not every axon is individually wrapped. It is also consistent with our findings that many nerves in WG-ablated larvae seem to be missing axons, whereas this was not observed in Ddr mutant nerves. These results are also similar to what has been recently reported using a different approach to ablate wrapping glia, where only minor behavioral defects were observed upon FGFR signaling disruption but profound crawling defects were seen upon ablation. As with all ablation studies, it is not possible to strictly rule out unexpected negative side effects of the ablation itself; however, using a genetic approach should limit collateral damage (compared with laser or toxin approaches). Together, these data support the conclusion that even limited wrapping or simply some degree of glia-axon contact is sufficient to support axon survival and nerve function compared with no glia at all at least for the first ~5 days of larval life (Corty, 2022).

Previous studies of oligodendrocytes and Schwann cells have found that impairing glial function can result in seemingly normal wrapping and circuit function in young animals, with deficits only appearing when the system is stressed or aged. Studying wrapping in adult Drosophila allows for aging and maintenance studies that the short larval period precludes. Adult peripheral nerves are encased in a transparent but hard cuticle that allows for live imaging but makes fixation challenging. Because of the resolution limits of light microscopy, a reliable method was developed to study their ultrastructure using TEM. Ensheathment in the adult wing nerve was found to differ from that of the larva, as all axons appear to be separated by glial membranes. Surprisingly, wrapping was not obviously impaired in adult nerves of Ddr knockdown or mutant animals. One difference between larval and adult wrapping glia is the territory size of each cell. In larvae, one wrapping glia cell covers the majority of the nerve from the VNC to the muscle field. This wrapping glial cell must therefore undergo tremendous growth to keep up with nerve elongation as the animal grows, as well as radial growth to ensheathe axons. A single cell can end up covering from ~750 μm to 2.5 mm of nerve length, depending on the segment, whereas in the wing there are ~13 wrapping glia along the region of the L1 nerve that was analyzed; This is is ~400 μm long. In larval wrapping glia, there are three receptor tyrosine kinases (EGFR, the FGFR Heartless, and now Ddr) that are each required for normal ensheathment, and thus cannot fully compensate for one another. It is hypothesized that in the larva the cell is pushed to its growth limits and any perturbation in pro-wrapping signaling has a strong effect on morphology, whereas in the adult nerve the system is robust and redundant enough to withstand perturbations of single genes. Future studies of double and triple mutants may be able to test this hypothesis (Corty, 2022).

Loss of Ddr led to an increase in spontaneous neurodegeneration in the nerve as animals naturally aged. Such an uncoupling of neuron health from overt effects on myelination has been demonstrated previously. For example, Cnp1 (Cnp) mutant mice show severe age-dependent neurodegeneration, although they have grossly normal myelin with only subtle changes in myelin ultrastructure. Loss of the proteolipid PLP results in axon degeneration despite having largely normal myelin. It was found that the number of VGlut+ neurons was reduced in aged wings of Ddr knockdown animals, indicating that wrapping glial Ddr is important for long-term neuronal survival. When Ddr whole animal mutants were analyzed by TEM a small but significant reduction was found in axon profile number, which should correspond to the number of surviving neurons. Together with the increased variability observed, this suggests that absence of Ddr signaling increases the susceptibility of subpopulations of neurons to insult or injury that may underlie age-related degeneration (Corty, 2022).

Myelination can directly affect the structure and function of the axons they wrap, including controlling caliber. In general, myelination increases caliber. For example, dysmyelinated Trembler mice have reduced axon calibers compared with controls, and in the PNS caliber along a single axon can vary with reduced caliber at points without direct myelin contact, such as nodes of Ranvier. Axon caliber is an important determinant of conduction velocity but varies widely between neuronal subtypes, so achieving and maintaining appropriate caliber is crucial for proper circuit function. How non-myelinating ensheathment impacts axon caliber is not understood. This study found glial Ddr promotes increased axon caliber. This study focused on the distal twin sensilla of the margin (dTSM) neuron, so it was possible to directly compare the caliber of an identifiable axon between conditions. The reductions in caliber were similar between Ddr mutants and glial-specific DdrRNAi, supporting a non-cell-autonomous role for glial Ddr in regulating axon caliber. The effect is considerable: nearly a 50% reduction in axon caliber at 5 dpe. We hypothesize that by this time point, wild-type dTSM axons have reached their mature caliber, as it is comparable between 5 dpe and 28 dpe in comparable genetic backgrounds. In Ddr mutants, however, we observe that the relative size compared with controls changes over time, suggesting that in Ddr mutants (or knockdowns) the axon continues to increase its caliber, perhaps in an effort to achieve the optimal size, although the axons still remain ~25% smaller than wild-type axons at 28 dpe (Corty, 2022).

Two proteins, MAG, which acts to increase the caliber of myelinated axons, and CMTM6, which restricts the caliber of myelinated and unmyelinated axons, are the only proteins reported to non-cell-autonomously affect the caliber of vertebrate axons, and both do so without overtly affecting myelin. In the fly, it has been shown that a shift in the average size of axons in larval nerves when wrapping glia are absent or severely disrupted, supporting a general role for wrapping glia in promoting axon size. In the adult, this study showed that Ddr is still required for increased axon caliber even when wrapping appears intact. The exact molecular mechanism by which Ddr may promote increased caliber size remains unclear as the control of axon caliber, generally, is not well understood. Genes involved in the general regulation of cell size have been implicated as cell-autonomous determinants. For example, in the fly, S6 kinase signaling is a positive regulator of motor neuron size, including axon caliber. In mammalian axons, the phosphorylation state of neurofilaments and microtubules determines their spacing to determine caliber. Determining how glial Ddr activity ultimately influences the axonal cytoskeleton is an important next step. A 25-50% reduction in caliber would be predicted to impact conduction velocity along the dTSM axon. Given that campaniform sensilla provide essential rapid sensory feedback to fine-tune movement, it will be of interest to test conduction velocity and flight behavior in Ddr mutant animals to see how the proprioceptive circuit might be affected (Corty, 2022).

Taken together, these studies identify Ddr as an important regulator of wrapping glia development and function in the fly, with distinct roles in larval and adult wrapping glia. Ddr is essential for the normal morphological development of axon wrapping in the larvae, and also mediates important axon-glia communication that controls axon caliber growth and affects neuronal health and survival. Given its expression pattern in vertebrate oligodendrocytes and Schwann cells, it seems likely that these essential functions are conserved in vertebrates. Further study into how Ddr functions in both fly and vertebrate glia promises to increase understanding of axon ensheathment in health and disease (Corty, 2022).

Drosophila multiplexin (Dmp) modulates motor axon pathfinding accuracy

Multiplexins are multidomain collagens typically composed of an N-terminal thrombospondin-related domain, an interrupted triple helix and a C-terminal endostatin domain. They feature a clear regulatory function in the development of different tissues, which is chiefly conveyed by the endostatin domain. This domain can be found in proteolytically released monomeric and trimeric versions, and their diverse and opposed effects on the migratory behavior of epithelial and endothelial cell types have been demonstrated in cell culture experiments. The only Drosophila multiplexin displays specific features of both vertebrate multiplexins, collagens XV and XVIII. This study characterized the Drosophila multiplexin (dmp) gene and found that three main isoforms are expressed from it, one of which is the monomeric endostatin version. Generation of dmp deletion alleles revealed that Dmp plays a role in motor axon pathfinding, as the mutants exhibit ventral bypass defects of the intersegmental nerve b (ISNb) similar to other motor axon guidance mutants. Transgenic overexpression of monomeric endostatin as well as of full-length Dmp, but not trimeric endostatin, were able to rescue these defects. In contrast, trimeric endostatin increased axon pathfinding accuracy in wild type background. It is concluded that Dmp plays a modulating role in motor axon pathfinding and may be part of a buffering system that functions to avoid innervation errors (Meyer, 2009).

Axonal pathfinding is a complex process depending on the balance of various attractive and repulsive guidance cues that act on an individual growth cone. The C-terminal domains of vertebrate multiplexins have received ample attention as activators and inhibitors of endothelial and epithelial cell migration, and some evidence also exists for a role of multiplexins as modulators of neuronal migratory behavior. Multiplexins are multidomain proteins, containing an N-terminal domain with homology to the N-terminal domain of thrombospondin (TSPN), an interrupted collagen triple helix domain and a C-terminal non-collagenous domain (NC1), that subdivides into a trimerization region, a protease-sensitive hinge region and a globular endostatin (ES) domain. One of the two vertebrate multiplexins, collagen XVIII, and C. elegans collagen XVIII occur in three different isoforms that can contain further N-terminal domains. This study found that the Drosophila multiplexin (Dmp) is encoded by a complex locus, comprising CG8647 and CG33171, that encodes a TSPN domain, but no further N-terminal moieties (Meyer, 2009).

Two deletions were generated within the dmp gene, and both deletion strains show pathfinding deficiencies of the same quality. ISNb growth cones fail to detach from the main nerve tract and steer into the ventral muscle field but continue to grow on dorsally. Additionally, ISNb as well as ISN nerves sometimes disrespect segment boundaries and fuse to nerves of adjacent segments. The two deletion strains differ by the incidence of pathfinding errors, as dmp^ΔC12–25, lacking the larger part of the gene, including part of the triple helix repeat region and the NC1-encoding region, shows greater penetrance than dmp^ΔN4–11, where the second alternative N-terminus and the following triple helical repeat regions are missing. dmp^ΔN4–11 is not likely to be a null mutation since there is wild type-like transcript present from the NC1-encoding region of the gene. This situation suggests that a low dosage of a truncated Dmp version leads to an improved phenotype compared with dmp^ΔC12–25. However, an effect caused by the absence of the protein's N-terminal portion cannot be completely ruled out. For example, one can speculate about a function of a putative integrin-interacting RGD motif present therein (Meyer, 2009).

The spatially and temporally very restricted expression of dmp contrasts with the landmarks of the two collagen IV molecules, that are the only other conserved collagens in Drosophila and general important ECM constituents. In Drosophila, their expression begins in embryonic stage 12 or even earlier and hemocytes produce and disperse them all over the embryo while circulating in the hemolymph throughout the rest of embryonic development. During late embryogenesis, specific hemocytes also migrate along the ventral midline between the CNS and the epidermis, but these midline hemocytes are dispersed very asymmetrically, and it is not believed that any of the dmp expression observed originates from these cells. Hence, Dmp production is largely different from collagen IV expression, since it is restricted to limited domains of immobile cells and occurs markedly later in development (Meyer, 2009).

Both dmp deletion strains were homozygous viable. This is true for collagen XV and XVIII single knockouts in mice and even with the double knockout (Ylikarppa, 2003), as well as the C. elegans cle-1 mutation (Ackley, 2001), none of which are lethal. However, the dmp gene and two type IV collagen genes are the only collagen genes in the Drosophila genome, whereas vertebrates and nematodes possess a far greater number of collagen types and genes. This renders the dispensability of Dmp in flies even more surprising than in other organisms, and underlines that its role is very different from that of collagen IV (Meyer, 2009).

It was possible to rescue the dmp^ΔC12–25 ISNb phenotype by overexpressing the Dmp3hNC1 isoform in dmp^ΔC12–25 mutant background, while its overexpression in wild type had no effect on axon pathfinding. This indicates that the protein product from the transgene functions in a fashion similar to the endogenous protein. Monomeric Endostatin (ES) had a rescue effect on the dmp^ΔC12–25 ISNb phenotype that is indistinguishable from the effect of UAS-3hNC1, whereas UAS-NC1, which is expected to lead to the formation of trimeric ES, did not improve the phenotype. It is concluded from this that a beneficial activity resides specifically within the monomeric ES version, which is necessary and sufficient for ensuring ISNb pathfinding accuracy. Biochemical work on vertebrate collagen XVIII has identified a number of proteases capable of releasing monomeric ES. In the dmp mutants, a proteolytic release of monomeric ES might account for the rescue effect of UAS-3hNC1. This implies that ES release is not possible from UAS-NC1, since this construct does not improve the mutant phenotype. A possible explanation for this is that the triple helix region of the molecule is needed for specific enzyme-substrate recognition (Meyer, 2009).

The overexpression effect of ES in wild type background is qualitatively different from its impact on the dmp mutant phenotype. While ES overexpression was largely beneficial in the mutant, it caused a variety of phenotypes in wild type background, ranging from a defective body morphology giving the impression that morphogenesis was hampered on a general level, to wild type looking individuals and such that appeared to exhibit a specific motor axon guidance phenotype. It is suggested that the phenotypic differences between the two genotypes are due to an ES dosage effect. ES overexpression in dmp mutant background produces an ES quantity that can be fully absorbed by the standard interaction partners, whereas the ES excess in the wild type overexpression situation leads to an ES overcharge of standard or non-standard interaction partners that inhibits vital interactions with other molecules. In an alternative scenario, the detrimental effect of ES in wild type background would arise from an interaction between ectopic ES and the trunk of the Dmp molecule (Meyer, 2009).

When overexpressing Dmp3hNC1, no detrimental effect is observed, so the full-length protein dosage increase over wild type level apparently does not pose a problem. This can be due to the endogenous mechanisms that balance proteolytic generation of monomeric ES, such as the characteristics of the proteases involved, which can include their spatial and temporal distribution, quantity, substrate affinity and the resulting turnover numbers (Meyer, 2009).

Transgenic expression of trimeric ES, as formed by the NC1 transgene product, and monomeric ES had exactly converse effects on the ISNb phenotypes of wild type and dmp^ΔC12–25 mutants. Free ES was able to rescue the pathfinding defects observed in dmp^ΔC12–25 and had a detrimental effect in wild type background. In contrast, the NC1 transgene, while having no impact in the mutant background, significantly reduced the wild type ISNb error rate of 7.2% to a virtually flawless ISNb guidance with 0.75% errors. These observations indicate that the function of NC1 depends on the presence of endogenous Dmp. In one scenario, endogenous Dmp plays a role in creating a microenvironment or architecture in the basement membrane that is permissive for NC1 activity. Conversely, a basement membrane without Dmp deteriorates in quality resulting in an inactive form of NC1. Alternatively, a direct interaction between Dmp or parts of it and NC1 may be required for NC1 function. Each feature of Dmp may potentially convey the relevant interactions, including the collagen triple helix region, the TSPN-1 domain, the NC1 portion, or several of these domains cooperate. Since both the Dmp triple helix region and the ES domain contain putative glycosaminoglycan attachment attachment sites, options for functional moieties include heparan sulfate side chains (Meyer, 2009).

Another question concerns the mode of action of NC1. One possibility is that it acts, if supported by the presence of functional Dmp, by releasing ES. In this scenario, NC1 simply is an inhibited version of ES. This option, however, is contradicted by the observation that neither overexpression of ES nor of 3hNC1 improved pathfinding in wild type background. An alternative idea is that ES monomers and NC1 trimers play distinct roles. In this setting, ES cannot be released from NC1, but NC1 exerts its own action as a trimer when Dmp is present. One option for NC1 activity is that its trivalence is used to crosslink different ECM components. This might stabilize structures that contain guidance information and eventually enhance the stringency of guidance interactions (Meyer, 2009).

The results show that the wild type ISNb error rate of 7.2% can be reduced by 10-fold by the expression of additional trimeric endostatin from the UAS-NC1 transgene. This illustrates that Dmp possesses a capacity to influence pathfinding accuracy that is not fully taken advantage of in the normal wild type situation. This could be explained in that a low rate of statistically no more than one error per individual does not decrease the affected individual's fitness and hence does not exert any selective pressure (Meyer, 2009).

Another possible reason is that increasing the fidelity of guidance interactions would interfere with the system's error prevention and/or correction mechanisms. As already described, correct pathfinding depends on the appropriate balance of attractive and repulsive forces, and this system is susceptible to perturbations. The observation that nerves are occasionally misrouted in wild type is a reflection of this fact. An ability to correct errors would therefore convey a certain degree of robustness in dealing with such perturbations. Indeed, error correction has been described in the Drosophila motor axon guidance system for SNa misrouting phenotypes that occur due to axonal overexpression of FasII. In third instar larvae, incidence of this phenotype is much lower than in embryos, suggesting that errors can get corrected during larval stages. For zebrafish retinal axons, it has also been stated that errors occur in wild type, but get corrected. In the example of ISNb pathfinding, excess trimeric endostatin might impair the system's plasticity by overly stabilizing misleading guidance interactions, and thus impair correction mechanisms, and by consequence reduces plasticity of the system (Meyer, 2009).

The observation that dmp mutations lead to pathfinding defects shows that certain features of Dmp do normally act to improve guidance accuracy. Hence, Dmp seems to play a dual modulating role, and as detailed above, these roles are likely exerted by different mechanisms. On the one hand, Dmp helps leading nerves where they belong, but on the other hand, does normally not make full use of its capacity to do so. This opens up the possibility that Dmp is an agent that has multiple means to modulate axon guidance accuracy by buffering axonal navigation against perturbations. It will be interesting to see if excess NC1 is able to improve guidance phenotypes of mutants other than Dmp (Meyer, 2009).

Different effects of monomeric versus oligomeric endostatin have also been observed in C. elegans (Ackley, 2001), but in contrast the the current results, the C. elegans cle-1 neuronal migratory phenotype gets rescued by the putatively trimeric NC1 domain, whereas the ES domain does not provide rescue activity (Meyer, 2009).

Mono- and oligomeric ES have also been tested in vertebrate tissue culture systems for their effects on tubule formation and angiogenesis. Oligomeric ES is an inhibitor of tube morphogenesis in HUVEC (human umbilical vein endothelial cell) cultures, since an addition of oligomeric ES to the culture before tube formation inhibited the process, which is motility-dependent, whereas after completion of tube formation, oligomeric ES had a motogenic activity leading to dispersal of the tubular structures. Monomeric ES alone did not have any effect on cell behavior, but pre-incubation with ES inhibited the motogenic effect of oligomeric ES. In a CAM (chorioallantoic membrane) angiogenesis assay system, mono- and oligomeric ES of collagen XV and XVIII are converse in their inhibitory activity, that also depends on the cytokine used for stimulating angiogenesis. In another context, using IBE cells (intrahepatic biliary epithelial), monomeric ES even enhanced tubule formation. Hence, the action of mono- and trimeric ES seems to greatly depend on the biological context, on the cell types involved and additional signals they receive from their environment. The only common theme seems to be an antagonistic action of monomeric versus oligomeric ES. In this light, it is not surprising that the rescue activity that was observed on the Drosophila motor axon guidance phenotype is exactly opposite to the results for mechanosensory neuron (MSN) migration in C. elegans (Ackley, 2001). Notably, the biological process of axonal growth cone steering depends on directional cues and their correct interpretation, which is a fundamental difference to all other experimental systems that compared the action of mono- and oligomeric ES so far, as these systems all focused on the presence or absence of migratory activity per se. This includes the observations made for C. elegans MSN migration, as the phenotypes described by Ackley (2001) mainly consist of errors in migration distance of whole neurons and not direction. So the observations of ES effects in the Drosophila system are found in a novel biological context, and differences compared with other contexts are not surprising (Meyer, 2009).

For murine multiplexins biochemical interactions with several other ECM components have been observed. Together with the variety of biological effects described for multiplexin fragments, it seems likely that its exact mechanisms of action depends very much on the biological context. This study has provided the starting point for functionally integrating Drosophila Multiplexin into the biological process of motor axon pathfinding (Meyer, 2009).

Search PubMed for articles about Drosophila Multiplexin

Ackley, B. D., Crew, J. R., Elamaa, H., Pihlajaniemi, T., Kuo, C. J. and Kramer, J. M. (2001). The NC1/endostatin domain of Caenorhabditis elegans type XVIII collagen affects cell migration and axon guidance. J Cell Biol 152: 1219-1232. PubMed ID: 11257122

Chalfie, M. (2009). Neurosensory mechanotransduction. Nat Rev Mol Cell Biol 10: 44-52. PubMed ID: 19197331

Chang, J. H., Javier, J. A., Chang, G. Y., Oliveira, H. B. and Azar, D. T. (2005). Functional characterization of neostatins, the MMP-derived, enzymatic cleavage products of type XVIII collagen. FEBS Lett 579: 3601-3606. PubMed ID: 15978592

Corty, M. M., Hulegaard, A. L., Hill, J. Q., Sheehan, A. E., Aicher, S. A. and Freeman, M. R. (2022). Discoidin domain receptor regulates ensheathment, survival, and caliber of peripheral axons. Development. PubMed ID: 36355066

Davis, G. W. (2013). Homeostatic signaling and the stabilization of neural function. Neuron 80: 718-728. PubMed ID: 24183022abilization of neural function

Dhanabal, M., Ramchandran, R., Waterman, M. J., Lu, H., Knebelmann, B., Segal, M. and Sukhatme, V. P. (1999). Endostatin induces endothelial cell apoptosis. J Biol Chem 274: 11721-11726. PubMed ID: 10206987

Dickman, D. K. and Davis, G. W. (2009). The schizophrenia susceptibility gene dysbindin controls synaptic homeostasis. Science 326: 1127-1130. PubMed ID: 19965435

Felbor, U., Dreier, L., Bryant, R. A., Ploegh, H. L., Olsen, B. R. and Mothes, W. (2000). Secreted cathepsin L generates endostatin from collagen XVIII. EMBO J 19: 1187-1194. PubMed ID: 10716919

Hanai, J., Gloy, J., Karumanchi, S. A., Kale, S., Tang, J., Hu, G., Chan, B., Ramchandran, R., Jha, V., Sukhatme, V. P. and Sokol, S. (2002). Endostatin is a potential inhibitor of Wnt signaling. J Cell Biol 158: 529-539. PubMed ID: 12147676

Heljasvaara, R., Nyberg, P., Luostarinen, J., Parikka, M., Heikkila, P., Rehn, M., Sorsa, T., Salo, T. and Pihlajaniemi, T. (2005). Generation of biologically active endostatin fragments from human collagen XVIII by distinct matrix metalloproteases. Exp Cell Res 307: 292-304. PubMed ID: 15950618

Kim, Y. M., Hwang, S., Kim, Y. M., Pyun, B. J., Kim, T. Y., Lee, S. T., Gho, Y. S. and Kwon, Y. G. (2002). Endostatin blocks vascular endothelial growth factor-mediated signaling via direct interaction with KDR/Flk-1. J Biol Chem 277: 27872-27879. PubMed ID: 12029087

Liu, J., Schrank, B. and Waterston, R. H. (1996). Interaction between a putative mechanosensory membrane channel and a collagen. Science 273: 361-364. PubMed ID: 8662524

Meyer, F. and Moussian, B. (2009). Drosophila multiplexin (Dmp) modulates motor axon pathfinding accuracy. Dev Growth Differ 51: 483-498. PubMed ID: 19469789

Muller, M. and Davis, G. W. (2012). Transsynaptic control of presynaptic Ca(2)(+) influx achieves homeostatic potentiation of neurotransmitter release. Curr Biol 22: 1102-1108. PubMed ID: 22633807

O'Reilly, M. S., Boehm, T., Shing, Y., Fukai, N., Vasios, G., Lane, W. S., Flynn, E., Birkhead, J. R., Olsen, B. R. and Folkman, J. (1997). Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 88: 277-285. PubMed ID: 9008168

Peixoto, R. T., Kunz, P. A., Kwon, H., Mabb, A. M., Sabatini, B. L., Philpot, B. D. and Ehlers, M. D. (2012). Transsynaptic signaling by activity-dependent cleavage of neuroligin-1. Neuron 76: 396-409. PubMed ID: 23083741

Seppinen, L. and Pihlajaniemi, T. (2011). The multiple functions of collagen XVIII in development and disease. Matrix Biol 30: 83-92. PubMed ID: 21163348

Wang, T., Hauswirth, A. G., Tong, A., Dickman, D. K. and Davis, G. W. (2014). Endostatin is a trans-synaptic signal for homeostatic synaptic plasticity. Neuron 83: 616-629. PubMed ID: 25066085

Wickstrom, S. A., Alitalo, K. and Keski-Oja, J. (2002). Endostatin associates with integrin alpha5beta1 and caveolin-1, and activates Src via a tyrosyl phosphatase-dependent pathway in human endothelial cells. Cancer Res 62: 5580-5589. PubMed ID: 12359771

Yamaguchi, N., Anand-Apte, B., Lee, M., Sasaki, T., Fukai, N., Shapiro, R., Que, I., Lowik, C., Timpl, R. and Olsen, B. R. (1999). Endostatin inhibits VEGF-induced endothelial cell migration and tumor growth independently of zinc binding. EMBO J 18: 4414-4423. PubMed ID: 10449407

Ylikarppa, R., Eklund, L., Sormunen, R., Muona, A., Fukai, N., Olsen, B. R. and Pihlajaniemi, T. (2003). Double knockout mice reveal a lack of major functional compensation between collagens XV and XVIII. Matrix Biol 22: 443-448. PubMed ID: 14614990

Younger, M. A., Muller, M., Tong, A., Pym, E. C. and Davis, G. W. (2013). A presynaptic ENaC channel drives homeostatic plasticity. Neuron 79: 1183-1196. PubMed ID: 23973209

date revised: 24 Decembe 2023

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