InteractiveFly: GeneBrief

Tissue inhibitor of metalloproteases: Biological Overview | References

BIOLOGICAL OVERVIEW

Synaptogenesis is coordinated by trans-synaptic signals that traverse the specialized synaptomatrix between pre- and postsynaptic cells. Matrix metalloproteinase (Mmp) activity sculpts this environment, balanced by secreted Tissue inhibitors of Mmp (Timp). This study used the reductionist Drosophila matrix metalloproteome to test consequences of eliminating all Timp regulatory control of Mmp activity at the neuromuscular junction (NMJ). Using in situ zymography, Timp was found to limit Mmp activity at the NMJ terminal and shape extracellular proteolytic dynamics surrounding individual synaptic boutons. In newly-generated timp null mutants, NMJs exhibit architectural overelaboration with supernumerary synaptic boutons. With cell-targeted RNAi and rescue studies, postsynaptic Timp was found to limit presynaptic architecture. Functionally, timp nulls exhibit compromised synaptic vesicle cycling, with reduced, lower fidelity activity. NMJ defects manifest in impaired locomotor function. Mechanistically, Timp was found to limit BMP trans-synaptic signaling and the downstream synapse-to-nucleus signal transduction. Pharmacologically restoring Mmp inhibition in timp nulls corrects BMP signaling and synaptic properties. Genetically restoring BMP signaling in timp nulls corrects NMJ structure and motor function. Thus, Timp inhibition of Mmp proteolytic activity restricts BMP trans-synaptic signaling to coordinate synaptogenesis (Shilts, 2017).

The synaptic cleft is populated with a complex extracellular network of secreted and transmembrane proteins, yet little is known about the extracellular mechanisms that act to shape this critical cellular interface. This synaptomatrix contains integrin, heparan sulfate proteoglycan and cognate receptors for a host of known secreted and transmembrane ligands. Extracellular proteins in the cleft are extensively remodeled in parallel with intercellular changes that accompany synaptic maturation and activity-dependent plasticity. Extracellular matrix metalloproteinase (Mmp) enzymes catalyze synaptic remodeling by proteolytically cleaving the secreted and transmembrane substrates regulating synapse structural integrity, and modulating intercellular signaling between presynaptic and postsynaptic partners. Given the powerful organizing effects of these proteases, their activity must be tightly regulated. One key mechanism is secretion of tissue inhibitors of Mmps (Timps), which restrict Mmp activity to proper spatial and temporal windows. Whenever Mmp regulation is disrupted, developmental abnormalities and disease often result. Improper Mmp expression and activity is implicated in a range of neurological disorders, including schizophrenia, addiction, epilepsy and autism spectrum disorder (ASD). As the most common heritable ASD and intellectual disability, Fragile X syndrome (FXS) underscores the importance of preserving Mmp balance to control proper synaptic structure and function. Importantly, the Mmp inhibitor minocycline alleviates synaptic and behavioral phenotypes in FXS disease models, and has shown promise in clinical trials for human patients, showing that elevated Mmp activity is causally linked to FXS neuropathology (Shilts, 2017).

Since Mmp dysregulation produces pronounced synaptic defects in disease states, it was hypothesized that loss of the endogenous Mmp control mediated by Timp would disrupt synapse architecture and function. This study took advantage of the simplified Drosophila melanogaster matrix metalloproteome to test consequences of genetically ablating all Timp regulatory control over Mmp activity (Dear, 2016). In contrast to mammals, which have at least 24 Mmps and four partially redundant Timps, Drosophila has a single secreted Mmp (Mmp1), a single membrane-anchored Mmp (Mmp2) and a single Timp, all of which are highly conserved and can interact with their respective human homologs. In the Drosophila nervous system, Mmps direct both axonal targeting and dendritic remodeling. Recently, Mmp1 and Mmp2 were found to regulate trans-synaptic signaling at the neuromuscular junction (NMJ) to modulate synaptic structure and function (Dear, 2016). Moreover, trans-synaptic signaling dysregulation has been causally linked to synaptic defects in the Drosophila FXS model (Friedman, 2013). One trans-synaptic pathway important for both synaptic structure and function involves bone morphogenetic protein (BMP) signaling via the Glass Bottom Boat (Gbb) ligand. Gbb secreted from the muscle regulates NMJ structure, whereas Gbb secreted from the motor neuron regulates neurotransmission. Gbb ligand in the extracellular space surrounding synaptic boutons activates downstream phosphorylated Mothers Against Decapentaplegic (pMAD) signal transduction locally at the synapse and distantly within motor neuron nuclei of the central nervous system. Synaptic pMAD is associated with assembly of functional neurotransmission machinery at the NMJ, whereas the accumulation of nuclear pMAD promotes NMJ growth. It is hypothesized that the balance of Mmp proteolytic activity controlled by Timp guides trans-synaptic signaling pathways to modulate both NMJ synaptic structure and function (Shilts, 2017).

To test this hypothesis, the first ever timp-specific loss-of-function null alleles were generated using CRISPR/Cas9 genome editing. Generation of specific mutations was previously intractable due to a conserved nested relationship that places the timp gene within an intron of the important synaptic synapsin gene. Previously work has shown that Timp localizes in the NMJ perisynaptic space (Dear, 2016), where it shows a co-dependent relationship with both secreted Mmp1 and membrane-anchored Mmp2. The current study employed in situ zymography in living NMJs to show that Timp inhibits synaptic Mmp function and regulates the dynamics of Mmp proteolytic activity in the extracellular space surrounding synaptic boutons. Loss of Timp regulation removes a restraint on synaptic architecture, resulting in the overelaboration of boutons. Using transgenic RNA interference (RNAi) and rescue, Timp secretion from the postsynaptic muscle was shown to be required to regulate the presynaptic motor neuron architecture. In parallel, Timp was also found to control synaptic function. By employing FM1-43 dye imaging, this study found Timp modulates the speed and fidelity of the synaptic vesicle (SV) cycle driving synaptic neurotransmission, and impairs the coordinated muscle peristalsis output of neuromuscular activity. In testing trans-synaptic signaling pathways, Timp function was found to act to restrict BMP signaling, with timp null mutants showing elevated Gbb ligand levels in the extracellular space surrounding synaptic boutons and increased downstream pMAD signal transduction at both the synapse and within motor neuron nuclei. Inhibiting proteolytic activity with minocycline treatment in timp null mutants restores normal BMP signaling and significantly corrects NMJ properties and output motor function. Genetically restoring normal BMP signaling in timp null mutants corrects NMJ architecture and functional motor output, indicating that aberrant trans-synaptic signaling is the causal mechanism. Taken together, these results show that neuromuscular synapses require a responsive balance of Mmp activity controlled by Timp inhibition to restrict the BMP trans-synaptic signaling that modulates NMJ structure and function (Shilts, 2017).

Remodeling of the synaptic extracellular environment is a highly dynamic process, demanding precise spatiotemporal control in response to specific developmental and activity-dependent signals. Mmp proteolytic activity is an ideal node of regulation for the necessary responsive kinetics and specificity, with Timps controlling the timing, duration and spatial specificity of enzyme function (Yamamoto, 2015). Taking advantage of the simplified matrix metalloproteome of Drosophila, with only a single functionally conserved Timp (Wei, 2003), it was possible to eliminate all Timp function with one mutation. Using site-directed CRISPR/Cas9, the first timp null allele was generated, with targeted mutation of the timp gene, without disrupting the synapsin gene in which it is nested. Although nested genetic placement does not imply a functional relationship, the highly conserved nesting of timp in synapsin occurs across vertebrates and invertebrates, which are separated by hundreds of millions of years of evolution. The evolutionary conservation of timp nesting in synapsin is interesting, since synapsin encodes a key synaptic regulator and there is evidence of co-regulation of genes nested with Timps. In addition to timp loss-of-function mutants, no viable Mmp gain-of-function has yet been reported in Drosophila. Thus, the new CRISPR-induced timp null is a tool to characterize total Timp function as well as generally elevated Mmp activity as seen in the nervous system. Currently, there are relatively few reports concerning Timp loss in the nervous system. In mice, TIMP-2 knockout causes motor deficits (Jaworski, 2006) and expanded NMJ branching (Lluri, 2006), and TIMP-1 overexpression reduces outgrowth in cortical cells (Ould-yahoui, 2009), supporting findings of this tudy. In Drosophila, Timp overexpression inhibits NMJ growth (Dear, 2016), which again complements the current findings (Shilts, 2017).

This study uncovered key roles for Timp in controlling synaptic Mmp activity, thereby regulating NMJ structure, function and output. Muscle-secreted Timp limits synaptic Mmp proteolytic activity and shapes the distribution of Mmp activity within the synaptomatrix. This local regulation of Mmp functional dynamics has not been reported in neuronal synapses, but is consistent with known roles of Timp in non-neuronal contexts (Kessenbrock, 2010; Mittal, 2016). This study found that postsynaptic Timp limits presynaptic NMJ architecture and bouton formation. This is surprising given that individual Mmp knockdown similarly limits synaptic structure in flies and mice, but may suggest that both loss and gain of Mmp function converge phenotypically or that, collectively, Timp repression of multiple Mmp activities acts as a brake on synaptic growth. Timp also was found to regulate synaptic function, by facilitating SV endocytosis and maintaining SV cycle fidelity. In comparison, mmp mutants elevate transmission strength, also by altered SV cycling dynamics, consistent with Timp repression of Mmp function. Timp enables faster and higher fidelity muscle contraction peristalsis, driving coordinated locomotion. Motor defects have consistently been found across a range of Mmp manipulations (Brkic, 2015; Jaworski, 2006; Sidhu, 2014), although molecular mechanisms had not been identified. Taken together, these results complete a characterization of the entire Drosophila matrix metalloproteome in controlling neuromuscular synapse structure and function (Dear, 2016). The timp null synaptic phenotypes prompt a re-assessment: Mmps are not simply negative regulators of synaptic differentiation, but can promote structural development within a context-dependent mechanism (Dziembowska, 2012). This work shows Timp and Mmps interact to sculpt synapse form and function (Shilts, 2017).

Timp limits BMP trans-synaptic signals mediating communication between the muscle and motor neuron, with Timp loss elevating Gbb ligand levels. BMP ligands are well known to be sequestered by extracellular molecules, and proteolytic cleavage of these extracellular antagonists controls the distributions of signaling activity in multiple cellular contexts. In Drosophila neurons, Mmp2 regulates motor axon pathfinding and fasciculation via Mmp2-mediated proteolytic cleavage of the ECM Fibrillin/Fibulin-related Faulty Attraction (Frac) protein to enable BMP signaling. Similarly, this study found elevated BMP trans-synaptic signaling in timp mutants with Mmp proteolytic hyperactivity. Gbb secretion from the postsynaptic muscle regulates NMJ architecture, whereas Gbb released from the presynaptic motor neuron regulates neurotransmission function. These roles are consistent with the misregulation of synaptic structure and SV cycle function, respectively, seen in timp mutants with elevated Gbb signaling. The accumulation of Gbb in the perisynaptic synaptomatrix of timp null mutants drives downstream activation of pMAD signal transduction in both motor neuron synaptic terminals and motor neuron nuclei. This is consistent with pMAD activation of transcriptional programs for coordinating synapse structural and functional differentiation. Gbb secreted from the postsynaptic muscle is regulated by Timp that is also secreted from the muscle, which provides control for motor neuron terminals to expand in response to muscle growth and activity-dependent plasticity. In contrast, Mmps come from both presynaptic and postsynaptic cells (Dear, 2016). Thus, directional Timp control acts as a specific muscle-derived mechanism to regulate Gbb trans-synaptic signaling (Shilts, 2017).

Elevated BMP Gbb signaling in a Drosophila model of Troyer syndrome, a hereditary spastic paraplegia (HSP) disease, causes strikingly similar NMJ synaptic structural and functional defects to those seen upon loss of Timp (Nahm, 2013). Like the timp null mutants, spartin mutants that are causatively associated with Troyer syndrome exhibit expanded synaptic arbors and decreased FM1-43 dye SV endocytic loading with impaired motor function. Importantly, Fragile X Mental Retardation Protein (FMRP) is a downstream effector of Spartin function, limiting BMP Gbb signaling (Nahm, 2013). Loss of FMRP causes Fragile X syndrome (FXS), and reducing non-canonical BMP signaling alleviates the synaptic defects in Drosophila and mouse FXS disease models. Likewise, targeted mutation of the FXS-related S6 kinase (S6K) similarly results in both expanded synaptic architecture and decreased SV endocytosis at the Drosophila NMJ, once again resembling timp null phenotypes. As in timp mutants, there are also clear precedents for mutations of other key regulatory proteins increasing NMJ functional variability to compromise motor output function. These findings with timp demonstrate the utility of variability as a metric to uncover regulatory nodes that preserve the functional resiliency of the nervous system (Shilts, 2017).

By pharmacologically correcting timp null phenotypes with the characterized Mmp inhibitor minocycline, this study has shown that mutant defects are causally linked to Mmp hyperactivity. Alleviation of timp null phenotypes is robust, albeit partial, which may reflect experimental limitations of the drug administration, or possibly reveal other Mmp-independent Timp functions. In particular, behavioral assays of motor function show conspicuous, albeit partial, rescue, which may be evidence of an Mmp-independent contribution to motor function or, more likely, that the precise spatiotemporal dynamics of Timp at the NMJ are necessary for proper motor function. In rats, transient proteolytic activity in the synaptomatrix accompanies long-term potentiation and dendrite maturation, which corroborates the current model that Timp dynamically restricts synaptic modulation through localized ECM proteolysis. Crucially, pharmacologically balancing Mmp activity in timp null mutants with minocycline treatment restores BMP trans-synaptic signaling, and genetically correcting BMP signaling prevent synaptic and movement defects. These findings support the model that Mmp activity in the synaptomatrix, under regulation by Timp, limits BMP trans-synaptic signals, thereby controlling NMJ synaptogenesis and functional motor output (Shilts, 2017).

These studies provide an avenue for possible therapeutic treatments in a range of neurological disease states with elevated Mmp activity. In particular, Mmp hyperactivity has been causally implicated in FXS and related ASD conditions. The synaptic cytoarchitectural phenotypes of timp mutants phenocopy the Drosophila FXS model, and trans-synaptic signaling defects are causative in synaptic structural and functional defects in this disease model, including BMP signaling. By re-creating the elevated Mmp activity characterizing neurological disease conditions such as FXS, the timp genetic tools developed in this study provide insights into fundamental synaptic mechanisms with direct clinical relevance. In future studies, timp manipulations will be combined with established Drosophila disease models in order to more fully dissect contributions of Mmp-dependent trans-synaptic signaling impairments in different neurological disease states (Shilts, 2017).

Control of tissue morphogenesis by the HOX gene Ultrabithorax

Mutations in the Ultrabithorax (Ubx) gene cause homeotic transformation of the normally two-winged Drosophila into a four-winged mutant fly. Ubx encodes a HOX family transcription factor that specifies segment identity, including transformation of the second set of wings into rudimentary halteres. Ubx is known to control the expression of many genes that regulate tissue growth and patterning, but how it regulates tissue morphogenesis to reshape the wing into a haltere is still unclear. This study shows that Ubx acts by repressing the expression of two genes in the haltere, Stubble and Notopleural, both of which encode transmembrane proteases that remodel the apical extracellular matrix to promote wing morphogenesis. In addition, Ubx induces expression of the Tissue inhibitor of metalloproteases in the haltere, which prevents the basal extracellular matrix remodelling necessary for wing morphogenesis. These results provide a long-awaited explanation for how Ubx controls morphogenetic transformation (Diaz-de-la-Loza, 2020).

The results reveal how Ubx – a homeotic gene that encodes the founding member of the HOX-family of transcription factors – regulates apical and basal matrix remodelling to control epithelial morphogenesis (see Ubx controls apical and basal ECM degradation to regulate morphogenesis). Ubx strongly represses two genes encoding apical matrix proteases (Np and Sb), as well as partially repressing two genes encoding basal matrix metalloproteases (Mmp1 and Mmp2), while inducing an inhibitor of Mmp1/2 (Timp) in the haltere. In this way, Ubx prevents both apical and basal matrix remodelling in the haltere, a key event in the homeotic wing-to-haltere transformation. In addition to regulating morphogenesis, Ubx controls many other genes affecting wing growth and pattern. Together, the combined repression of morphogenesis, growth and patterning by Ubx is responsible for the full transformation of wing to haltere (Diaz-de-la-Loza, 2020).

Ubx controls apical and basal ECM degradation to regulate morphogenesis. Schematic of Ubx expression and function in Drosophila and a hypothetical four-winged ancestor. Ubx controls organ shape via regulation of aECM and bECM proteases, in addition to its known functions in regulating organ growth and patterning. These target genes have presumably evolved to be specifically regulated in the Drosophila wing and/or haltere, and must be insensitive to Ubx in four-winged ancestors (Diaz-de-la-Loza, 2020).

These findings also support the general view that transcriptional control of matrix synthesis and degradation is a conserved mechanism by which information encoded in the genome is deployed to govern the shape of tissues and organs in animals. Although this concept is broadly appreciated for the regulation of the bECM, the notion that the aECM is also developmentally regulated during tissue morphogenesis needs further investigation, particularly in mammals. Beyond animals, morphogenesis of plants, fungi and bacteria is also known to be fundamentally dependent on patterned synthesis and degradation of the cell wall, a type of ECM. Thus, genetic control of the matrix appears to be a general principle that shapes all life forms (Diaz-de-la-Loza, 2020).

Juvenile hormone and 20-hydroxyecdysone coordinately control the developmental timing of matrix metalloproteinase-induced fat body cell dissociation

Tissue remodeling is a crucial process in animal development and disease progression. Coordinately controlled by the two main insect hormones, juvenile hormone (JH) and 20-hydroxyecdysone (20E), tissues are remodeled context-specifically during insect metamorphosis. Previous work has discovered that two matrix metalloproteinases (Mmps) cooperatively induce fat body cell dissociation in Drosophila. However, the molecular events involved in this Mmps-mediated dissociation are unclear. This study reports that JH and 20E coordinately and precisely control the developmental timing of Mmps-induced fat body cell dissociation. During the larval-prepupal transition, the anti-metamorphic factor Kr-h1 was found to transduce JH signaling, which directly inhibited Mmps expression and activated expression of tissue inhibitor of metalloproteinases (timp), and thereby suppressed Mmps-induced fat body cell dissociation. It is also noted that upon a decline in the JH titer, a prepupal peak of 20E suppresses Mmps-induced fat body cell dissociation through the 20E primary-response genes, E75 and Blimp-1, which inhibited expression of the nuclear receptor and competence factor βftz-F1. Moreover, upon a decline in the 20E titer, βftz-F1 expression was induced by the 20E early-late response gene DHR3, and then βftz-F1 directly activated Mmps expression and inhibited timp expression, causing Mmps-induced fat body cell dissociation during 6-12 hrs after puparium formation. In conclusion, coordinated signaling via JH and 20E finely tunes the developmental timing of Mmps-induced fat body cell dissociation. These findings shed critical light on hormonal regulation of insect metamorphosis (Jia, 2017).

MMPs and tissue inhibitor of metalloproteinases (TIMPs) play crucial roles in regulating tissue remodeling in both vertebrates and Drosophila. Previous work has demonstrated the collaborative functions of Mmp1 and Mmp2 in inducing fat body cell dissociation in Drosophila. timp mutant adults show autolyzed tissue in the abdominal cavity and inflated wings, a phenotype consistent with the role of timp in BM integrity and remodeling. The current study clarified the role of timp in inhibiting the enzymatic activity of Mmps and thus, Mmp-induced fat body cell dissociation. In mammals, Mmps activity in vivo is controlled at different levels, including the regulation by gene expression, the zymogens activation, and the inhibition of active enzymes by TIMPs. These studies unify the important inhibitory roles of timp/TIMP in regulating tissue remodeling in both Drosophila and mammals. In addition to regulating Mmp expression, JH and 20E signals differentially regulate timp expression, with the stimulatory role of Kr-h1 and the inhibitory role of βftz-F1. Because timp inhibits the enzymatic activity of Mmps in the Drosophila fat body, it is concluded that JH and 20E coordinately control Mmps activity at both the mRNA and enzymatic levels (Jia, 2017).

Previously work has show the requirement of both JH and its receptors to inhibit fat body cell dissociation in Drosophila. This study demonstrated the ability of Kr-h1 to transduce JH signaling to decrease Mmp expression and to induce timp expression during larval-prepupal transition. Moreover, a Kr-h1-binding sites (KBS) was identified in the Mmp1 promoter, indicating that Kr-h1 directly represses Mmp1 expression. Interestingly, Kr-h1 expression gradually increases from initiation of wandering (IW) to 3 h APF when induced by JH and 20E in an overlapping manner, thus inhibiting the enzymatic activity of Mmps and Mmp-induced fat body cell dissociation during the larval-prepupal transition. Moreover, Kr-h1 acts as an anti-metamorphic factor by inhibiting 20E signaling. It is proposed, in addition to directly affecting the expression of Mmps and timp, that Kr-h1 might also indirectly regulate their expression by inhibiting 20E signaling (Jia, 2017).

Two consecutive 20E pulses control timely metamorphosis in Drosophila. Together with previous findings, the current results show that the conserved 20E transcriptional cascade precisely controls the timing of Mmp-induced fat body cell dissociation. In general, the first 20E signal pulse plays an inhibitory role during the larval-prepupal transition; however, it is a prerequisite for the expression of βftz-F1, which induces the second 20E signal pulse during the prepupal-pupal transition and the expression of Mmps. Because of the requirement for the first 20E signal pulse, blockade of the 20E receptor prevents fat body cell dissociation. When JH titer declines, the prepupal peak of 20E activates expression of two 20E primary-response genes, E75 and Blimp-1, to inhibit fat body cell dissociation: E75 represses DHR3 transactivation of βftz-F1 expression, and Blimp-1 directly represses βftz-F1 expression. During the prepupal-pupal transition, DHR3 directly induces βftz-F1 expression from 6 h APF to 12 APF. Before pupation, βftz-F1 induces Mmp expression and represses timp expression. Moreover, an FBS was identified in the Mmp2 promoter, demonstrating that βftz-F1 directly induces Mmp2 expression. Finally, within 6 h before pupation, Mmp1 and Mmp2 cooperatively induce fat body cell dissociation, with each assuming a distinct role (Jia, 2017).

Insect metamorphosis is coordinately controlled by JH and 20E, whereas the hormonal control of tissue remodeling is strictly context-specific. Different larval tissues and adult organs might have distinct, yet precise, developmental fates and timing. Knowledge regarding this question is poor. Based on previous preliminary information, this study clarified the detailed molecular mechanisms by which JH and 20E precisely control the developmental timing of Mmp-induced fat body cell dissociation at both mRNA and enzymatic levels in Drosophila, and a working model is provided of hormonal control of tissue remodeling in animals (see Model showing developmental timing of Mmp-induced fat body cell dissociation is coordinately and precisely controlled by JH and 20E in Drosophila). In summary, at first, Kr-h1 transduces JH signaling to inhibit Mmp-induced fat body cell dissociation during larval-prepupal transition. Then when JH titer declines, the prepupal peak of 20E suppresses Mmp-induced fat body cell dissociation through E75 and Blimp-1, which inhibit βftz-F1 expression. Finally, until 20E titer declines, DHR3 induces βftz-F1 expression, and βftz-F1 covers the 20E-triggered transcriptional cascade to activate Mmp-induced fat body cell dissociation within 6 h before pupation. This study provides an excellent sample for better understanding the hormonal regulation of insect metamorphosis (Jia, 2017).

Two matrix metalloproteinase classes reciprocally regulate synaptogenesis

Synaptogenesis requires orchestrated intercellular communication between synaptic partners, with trans-synaptic signals necessarily traversing the extracellular synaptomatrix separating presynaptic and postsynaptic cells. Extracellular matrix metalloproteinases (Mmps) regulated by secreted tissue inhibitors of metalloproteinases (Timps), cleave secreted and membrane-associated targets to sculpt the extracellular environment and modulate intercellular signaling. This study tested Mmp roles at the neuromuscular junction (NMJ) model synapse in the reductionist Drosophila system, which contains just two Mmps (secreted Mmp1 and GPI-anchored Mmp2) and one secreted Timp. All three matrix metalloproteome components co-dependently localize in the synaptomatrix. Both Mmp1 and Mmp2 independently restrict synapse morphogenesis and functional differentiation. Surprisingly, either dual knockdown or simultaneous inhibition of the two Mmp classes together restores normal synapse development, identifying a novel reciprocal suppression mechanism. The two Mmp classes co-regulate a Wnt trans-synaptic signaling pathway modulating structural and functional synaptogenesis, including the GPI-anchored heparan sulfate proteoglycan (HSPG) Wnt co-receptor Dally-like Protein (Dlp), cognate receptor Frizzled-2 and Wingless ligand. Loss of either Mmp1 or Mmp2 reciprocally misregulates Dlp at the synapse, with normal signaling restored by co-removal of both Mmp classes. Correcting Wnt co-receptor Dlp levels in both mmp mutants prevents structural and functional synaptogenic defects. Taken together, these results identify a novel Mmp mechanism that fine-tunes HSPG co-receptor function to modulate Wnt signaling to coordinate synapse structural and functional development (Dear, 2015).

A large number of Mmps are expressed in the mammalian nervous system, with roles in neurodevelopment, plasticity and neurological disease. Understanding how each Mmp individually and combinatorially functions is hindered by genetic redundancy and compensatory mechanisms. This study exploited the Drosophila system to analyze a matrix metalloproteome containing just one member of each conserved component: one secreted Mmp, one membrane-tethered Mmp and one Timp. Both Mmp classes were found to attenuate structural and functional synaptic development, with electrophysiological, ultrastructural and molecular roles in both presynaptic and postsynaptic cells. A surprising discovery is that the Mmp classes suppress each other's requirements at the synapse. From discrete activities to redundancy, cooperation and now reciprocal suppression, studies continue to reveal how Mmps interact to regulate developmental processes. This study shows that the two Mmp classes play separable yet interactive roles in sculpting NMJ development. During the writing of this manuscript, a genomic Mmp2 rescue line was produced (Wang, 2014), which will be critical in further testing this interactive mechanism. It will be interesting to determine whether the Mmp suppressive mechanism is used in other developmental contexts, other intercellular signaling pathways and in mammalian models. Mammalian Mmp9 regulates synapse architecture and also postsynaptic glutamate receptor expression and/or localization. Likewise, mammalian Mmp7 regulates both presynaptic properties and postsynaptic glutamate receptor subunits. Thus, the dual roles of Mmps in pre- and postsynaptic compartments appear to be evolutionarily conserved (Dear, 2015).

Previous work demonstrated that Mmp1 and Mmp2 both regulate motor axon pathfinding in Drosophila embryos, albeit to different degrees and in this study, double Mmp mutants still exhibited defasciculated nerve bundles that separate prematurely. Consistently, both Mmp single mutants display excessive terminal axon branching at the postembryonic NMJ, but here the defect is fully alleviated by the removal of both Mmps. Other studies have either not identified, or not tested, a similar Mmp interaction, suggesting that reciprocal suppression might be specific to synaptogenesis. However, there are numerous reports that highlight the importance of Mmp and Timp balance. Mmp:Timp ratios can influence protease activation, localization, substrate specificity and Timp signaling and are commonly used as predictive clinical correlates in disease pathology. At the Drosophila NMJ, a similar reciprocal suppression interaction between pgant glycosyltransferases involved in O-linked glycosylation regulates synaptogenesis via integrin-tenascin trans-synaptic signaling. A recent study reported that pgant activity protects substrates from Furin-mediated proteolysis, which is a protease responsible for processing or activating Drosophila Mmp1 and Mmp2. Thus, Mmp proteolytic and glycan mechanisms could converge within the NMJ synaptomatrix to regulate trans-synaptic signaling (Dear, 2015).

New antibody tools produced in this study provide the means to interrogate an entire matrix metalloproteome, and will be important for testing Mmp and Timp functions throughout Drosophila. Many Mmps are both developmentally and activity regulated, with highly context-dependent functions. Future work will temporally dissect this mechanism at the developing NMJ and investigate how activity might regulate Mmp localization and function. It will be informative to correlate synaptogenic Mmp requirements with Mmp enzymatic activity by using in situ zymography assays, although non-enzymatic roles are certainly also possible. Lack of ultrastructure defects in Mmp mutant NMJs suggests that Drosophila Mmps have primarily instructive functions at the synapse, rather than broad proteolytic roles in ECM degradation. Consistently, Drosophila Mmp2 instructs motor axon pathfinding via a BMP intercellular signaling mechanism. Conversely, Mmp2 functions permissively in basement membrane degradation while shaping dendritic arbors. Because synaptic bouton size is reduced in mmp1 mutants, Mmp1 activity might degrade a prohibitive physical barrier at the NMJ. However, the results indicate a primary Mmp role in regulating intercellular signaling during synaptic development (Dear, 2015).

HSPG co-receptors of trans-synaptic ligands are key modulators of NMJ synaptogenesis and HSPGs are also established substrates of both mammalian and Drosophila Mmps. Mmp1 and Mmp2 differentially regulate the HSPG Dlp co-receptor to restrict the Wnt Wg trans-synaptic signaling driving structural and functional NMJ development. How might both increased and decreased levels of the Dlp co-receptor yield increased FNI pathway signal transduction? Regulation of Wnt signaling interactions ligands, co-receptors and receptors is managed at many levels. The 'Wg exchange factor model' provides a mechanistic framework for understanding the suppressive interactions of Mmp. In this mechanism, a low Dlp:Frz2 ratio helps the Frz2 receptor obtain more Wg, whereas a high Dlp:Frz2 ratio prevents Frz2 from capturing Wg as Dlp competes and sequesters Wg away from Frz2. Importantly, however, Dlp exhibits a context-dependent, bimodal role as both activator and repressor. Indeed, previous studies show these mechanisms are a key driving force in Wg signal transduction at the Drosophila NMJ (Dani, 2012; Friedman, 2013). In mmp1 mutants, Wg and Dlp are both reduced, resulting in a low Dlp:Frz2 ratio and elevated FNI. In mmp2 mutants, Dlp is spatially diffuse and Frz2 is increased, similarly resulting in a low Dlp:Frz2 ratio and elevated FNI. Balance is reset with Mmp co-removal because neither form of Mmp-induced HSPG tuning occurs. In this regard, it might be predicted that Dlp reduction in mmp2 mutants would only further increase FNI and therefore structural and functional defects. It is likely that absolute Dlp levels are the important driving factor in synaptogenesis and/or that Dlp exhibits bimodal functions in synaptic development (Dear, 2015).

Interestingly, a recent mouse study showed the Mmp3 hemopexin domain promotes Wnt signaling by inhibiting a negative Wnt regulator, raising the possibility that Mmps can act as molecular switches (or in feedback loops) dictating Wnt transduction. Another study suggests that Wnt signaling can directly mediate co-regulation of heparanase and Mmps. Indeed, both neural activity and intercellular signaling can stimulate Mmp-dependent ectodomain shedding of plasma membrane target proteins, thereby directly regulating the surface abundance of HSPGs and receptors, as well as other Mmps, which thus reciprocally modulate intra- and extracellular organization. From this model, the spatial arrangement of Dlp could be affected by co-regulated sheddase activity that is differentially altered in mmp1 and mmp2 mutants. Specifically, Mmp2 could shed Dlp, resulting in an increased area of Dlp expression in mmp2 mutants and loss of Mmp2 regulation by Mmp1 could result in aberrant Dlp restriction in mmp1 mutants, with Mmp co-removal remediating the Dlp domain thereby restoring normal Wnt trans-synaptic signaling. Future work will test the reciprocal impacts of Wnt signaling on Mmp expression and/or function in the context of synaptic development (Dear, 2015).

Emerging evidence suggests HSPG glycosaminoglycan (GAG) chains function as allosteric regulators of Mmps, with GAG content or composition influencing the localization and substrate specificity of Mmp. Indeed, Wg signaling is sensitive to perturbations in HSPG chain biosynthesis and HS modifying enzymes, which modulate both NMJ structure and function. It is easy to envision how tissue- and development-stage-specific HS modifications could coordinate HSPG/Mmp-dependent functions, thereby differentially regulating diverse signaling events, which enable context-specific responses instructed by the extracellular environment. Future work will examine how dual inputs of the HSPG co-receptor function and how Mmp proteolytic cleavage coordinates Wnt trans-synaptic signaling during synaptogenesis, particularly in the context of the Fragile X syndrome (FXS) disease model. Given that both loss or inhibition Mmp and correction of HSPG elevation independently alleviate synaptic defects in the FXS disease state, the overlapping mechanism provides an exciting avenue to therapeutic interventions for FXS and, potentially, related intellectual disability and autism spectrum disorders (Dear, 2015).

Drosophila TIMP is a potent inhibitor of MMPs and TACE: similarities in structure and function to TIMP-3

The four tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors that regulate the activity of matrix metalloproteinases (MMPs) and certain disintegrin and metalloproteinase (ADAM) family proteases in mammals. The protease inhibitory activity is present in the N-terminal domains of TIMPs (N-TIMPs). In this work, the N-terminal inhibitory domain of the only TIMP produced by Drosophila (dN-TIMP) was expressed in Escherichia coli and folded in vitro. The purified recombinant protein is a potent inhibitor of human MMPs, including membrane-type 1-MMP, although it lacks a disulfide bond that is conserved in all other known N-TIMPs. Titration with the catalytic domain of human MMP-3 [MMP-3(DeltaC)] showed that dN-TIMP prepared by this method is correctly folded and fully active. dN-TIMP also inhibits, in vitro, the activity of the only two MMPs of Drosophila, dm1- and dm2-MMPs, indicating that the Drosophila TIMP is an endogenous inhibitor of the Drosophila MMPs. dN-TIMP resembles mammalian N-TIMP-3 in strongly inhibiting human tumor necrosis factor-alpha-converting enzyme (TACE/ADAM17) but is a weak inhibitor of human ADAM10. Models of the structures of dN-TIMP and N-TIMP-3 are strikingly similar in surface charge distribution, which may explain their functional similarity. Although the gene duplication events that led to the evolutionary development of the four mammalian TIMPs might be expected to be associated with functional specialization, Timp-3 appears to have conserved most of the functions of the ancestral TIMP gene (Wei, 2003).

Search PubMed for articles about Drosophila Timp

Brkic, M., Balusu, S., Libert, C. and Vandenbroucke, R. E. (2015). Friends or Foes: Matrix Metalloproteinases and Their Multifaceted Roles in Neurodegenerative Diseases. Mediators Inflamm 2015: 620581. PubMed ID: 26538832

Dani, N. and Broadie, K. (2012). Glycosylated synaptomatrix regulation of trans-synaptic signaling. Dev Neurobiol 72(1): 2-21. PubMed ID: 21509945

Dear, M. L., Dani, N., Parkinson, W., Zhou, S. and Broadie, K. (2015). Two matrix metalloproteinase classes reciprocally regulate synaptogenesis. Development 143(1):75-87. PubMed ID: 26603384

Diaz-de-la-Loza, M. D., Loker, R., Mann, R. S., Thompson, B. J. (2020). Control of tissue morphogenesis by the HOX gene Ultrabithorax. Development, 147(5) PubMed ID: 32122911

Dziembowska, M. and Wlodarczyk, J. (2012). MMP9: a novel function in synaptic plasticity. Int J Biochem Cell Biol 44(5): 709-713. PubMed ID: 22326910

Friedman, S. H., Dani, N., Rushton, E. and Broadie, K. (2013). Fragile X mental retardation protein regulates trans-synaptic signaling in Drosophila. Dis Model Mech 6(6): 1400-1413. PubMed ID: 24046358

Jaworski, D. M., Soloway, P., Caterina, J. and Falls, W. A. (2006). Tissue inhibitor of metalloproteinase-2(TIMP-2)-deficient mice display motor deficits. J Neurobiol 66(1): 82-94. PubMed ID: 16216006

Jia, Q., Liu, S., Wen, D., Cheng, Y., Bendena, W. G., Wang, J. and Li, S. (2017). Juvenile hormone and 20-hydroxyecdysone coordinately control the developmental timing of matrix metalloproteinase-induced fat body cell dissociation. J Biol Chem 292(52): 21504-21516. PubMed ID: 29118190

Kessenbrock, K., Plaks, V. and Werb, Z. (2010). Matrix metalloproteinases: regulators of the tumor microenvironment. Cell 141(1): 52-67. PubMed ID: 20371345

Lluri, G., Langlois, G. D., McClellan, B., Soloway, P. D. and Jaworski, D. M. (2006). Tissue inhibitor of metalloproteinase-2 (TIMP-2) regulates neuromuscular junction development via a beta1 integrin-mediated mechanism. J Neurobiol 66(12): 1365-1377. PubMed ID: 16967503

Mittal, R., Patel, A. P., Debs, L. H., Nguyen, D., Patel, K., Grati, M., Mittal, J., Yan, D., Chapagain, P. and Liu, X. Z. (2016). Intricate Functions of Matrix Metalloproteinases in Physiological and Pathological Conditions. J Cell Physiol 231(12): 2599-2621. PubMed ID: 27187048

Nahm, M., Lee, M. J., Parkinson, W., Lee, M., Kim, H., Kim, Y. J., Kim, S., Cho, Y. S., Min, B. M., Bae, Y. C., Broadie, K. and Lee, S. (2013). Spartin regulates synaptic growth and neuronal survival by inhibiting BMP-mediated microtubule stabilization. Neuron 77(4): 680-695. PubMed ID: 23439121

Ould-yahoui, A., Tremblay, E., Sbai, O., Ferhat, L., Bernard, A., Charrat, E., Gueye, Y., Lim, N. H., Brew, K., Risso, J. J., Dive, V., Khrestchatisky, M. and Rivera, S. (2009). A new role for TIMP-1 in modulating neurite outgrowth and morphology of cortical neurons. PLoS One 4(12): e8289. PubMed ID: 20011518

Shilts, J. and Broadie, K. (2017). Secreted tissue inhibitor of matrix metalloproteinase restricts trans-synaptic signaling to coordinate synaptogenesis. J Cell Sci 130(14):2344-2358. PubMed ID: 28576972

Sidhu, H., Dansie, L. E., Hickmott, P. W., Ethell, D. W. and Ethell, I. M. (2014). Genetic removal of matrix metalloproteinase 9 rescues the symptoms of fragile X syndrome in a mouse model. J Neurosci 34(30): 9867-9879. PubMed ID: 25057190

Wang, X. and Page-McCaw, A. (2014). A matrix metalloproteinase mediates long-distance attenuation of stem cell proliferation. J Cell Biol 206(7): 923-936. PubMed ID: 25267296

Wei, S., Xie, Z., Filenova, E. and Brew, K. (2003). Drosophila TIMP is a potent inhibitor of MMPs and TACE: similarities in structure and function to TIMP-3. Biochemistry 42(42): 12200-12207. PubMed ID: 14567681

Yamamoto, K., Murphy, G. and Troeberg, L. (2015). Extracellular regulation of metalloproteinases. Matrix Biol 44-46: 255-263. PubMed ID: 25701651

date revised: 22 September 2017

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