Neprilysin 4: Biological Overview | References
Gene name - Neprilysin 4
Cytological map position -
Function - enzyme
Keywords - endopeptidase involved in the extra-cellular metabolism of signalling peptides, metallopeptidase, regulates insulin-like peptide (ILP) expression and food intake, required in the mushroom body for middle-term and long-term memory
Symbol - Nep4
FlyBase ID: FBgn0038818
Genetic map position - chr3R:20,652,567-20,659,606
NCBI classification - Peptidase family M13
FlyBase gene group - Nephrilysin
Cellular location - transmembrane
Insulin and IGF signaling are critical to numerous developmental and physiological processes, with perturbations being pathognomonic of various diseases, including diabetes. Although the functional roles of the respective signaling pathways have been extensively studied, the control of insulin production and release is only partially understood. This study shows that in Drosophila expression of insulin-like peptides is regulated by neprilysin activity. Concomitant phenotypes of altered expression of the metallopeptidase neprilysin, included impaired food intake, reduced body size, and characteristic changes in the metabolite composition. Ectopic expression of a catalytically inactive mutant did not elicit any of the phenotypes, which confirms abnormal peptide hydrolysis as a causative factor. A screen for corresponding substrates of the neprilysin identified distinct peptides that regulate insulin-like peptide expression, feeding behavior, or both. The high functional conservation of neprilysins and their substrates renders the characterized principles applicable to numerous species, including higher eukaryotes and humans (Hallier, 2016).
Neprilysins are highly conserved ectoenzymes that cleave and thereby inactivate many physiologically relevant peptides in the extracellular space, thus contributing considerably to the maintenance of peptide homeostasis in this compartment. Members of the neprilysin family generally consist of a short N-terminal cytoplasmic domain, a membrane spanning region, and a large extracellular domain with two highly conserved sequence motifs (HExxH; ExxA/GD) critical for zinc coordination, catalysis, and substrate or inhibitor binding (Oefner, 2000). Because of these characteristics, neprilysins are classified as M13 zinc metallopeptidases. For human Neprilysin (NEP), the most well-characterized family member, identified substrates include endothelins, angiotensins I and II, enkephalins, bradykinin, atrial natriuretic peptide, substance P, and the amyloid-beta peptide (Turner, 2001). Because of this high substrate variability, NEP activity has been implicated in the pathogenesis of hypertension, analgesia, cancer, and Alzheimer's disease. Recent clinical trials have demonstrated significant efficacy of Neprilysin inhibitors in the treatment of certain indications. However, despite the clinical relevance of the neprilysins, the physiological function and in vivo substrates of most family members are unknown (Hallier, 2016).
In Drosophila melanogaster, at least five neprilysin genes are expressed (Meyer, 2011; Sitnik, 2014), two of the corresponding protein products, Nep2 and Nep4, were reported to be enzymatically active (Bland, 2007; Meyer, 2009; Thomas, 2005). With respect to Nep4, a critical function of the enzyme's non-catalytic intracellular N-terminus has been demonstrated: when present in excess, the domain induces severe muscle degeneration concomitant with lethality during late larval development. Because the intracellular domain interacts with a carbohydrate kinase, impaired energy metabolism has been proposed as the underlying cause of the phenotype (Panz, 2012). In addition, Nep2 has been implicated in the regulation of locomotion and geotactic behavior (Bland, 2009), and neprilysin activity in general appears to be critical to the formation of middle- and long-term memory (Turrel, 2016), as well as to the regulation of pigment dispersing factor (PDF) signaling within circadian neural circuits (Isaac, 2007). However, despite these experiments and recent findings that suggest a critical role of neprilysins in reproduction (Sitnik, 2014), the physiological functionality of these enzymes is still far from being understood. In this respect, the lack of identified substrates with in vivo relevance is a major hindrance (Hallier, 2016).
This study describes the identification of numerous novel substrates of Drosophila Neprilysin 4 (Nep4) and provide evidence that Nep4-mediated peptide hydrolysis regulates insulin-like peptide (ILP) expression and food intake. These results establish a correlation between neprilysin activity and ILP expression and thus clarify understanding of the complex mechanisms that control the production and release of these essential peptides (Hallier, 2016).
While the functional roles of insulin-like peptides (ILPs) and the corresponding insulin- and IGF-signaling have been intensively studied, the control of ILP production and release is not well understood. This study demonstrates that modulating the expression of a Drosophila neprilysin interferes with the expression of insulin-like peptides, thus establishing a correlation between neprilysin activity and the regulation of insulin signaling. A high physiological relevance is confirmed by the fact that altering nep4 expression phenocopies characteristic effects of IPC ablation, including reduced size and weight of corresponding animals, as well as increased levels of carbohydrates such as glucose and fructose. The result that the levels of these sugars are increased, although food intake rates are reduced presumably reflects the physiological impact of the diminished ilp expression that is also obvious in corresponding animals. In this respect, the impaired insulin signaling likely results in inefficient metabolization and thus accumulation of the sugars, which overcompensates the diametrical effects of reduced food intake. By identifying 16 novel peptide substrates of Nep4, the majority of which are involved in regulating dilp expression or feeding behavior, and by localizing the peptidase to the surface of body wall muscles and IPCs within the larval CNS, this study provides initial evidence that neprilysin-mediated hydrolysis of hemolymph circulating as well as CNS intrinsic peptides is the physiological basis of the described phenotypes. The finding that only the catalytically active enzyme affected dilp expression whereas the inactive construct did not, substantiates this evidence because it confirms aberrant enzymatic activity and thus abnormal peptide hydrolysis as a causative parameter. Interestingly, the strongest effects on size and dilp expression were observed with muscle-specific overexpression of Nep4; overexpression of the peptidase in the CNS was less detrimental. These results indicate that hemolymph circulating peptides accessible to muscle-bound Nep4 are mainly responsible for the observed effects, while CNS intrinsic peptide signaling is less relevant. The fact that all peptides cleaved by Nep4 could be released into the hemolymph, either from enteroendocrine cells or from neurohormonal release sites (Nassel, 2010), substantiates this indication. Since the Drosophila midgut is the source of several neuropeptides, it is conceivable that a main reason for the observed phenotypes is aberrant cleavage of certain gut-derived peptides that are required for proper midgut-IPC communication. Allatostatin A, neuropeptide F, diuretic hormone 31, and some tachykinins are produced by endocrine cells of the gut. Interestingly, all have been implicated in regulating dilp expression and/or feeding behavior, and most of them, namely allatostatin A1-4, diuretic hormone 31, and tachykinin 1, 2, 4, and 5, were cleaved by Nep4, indicating enzyme-substrate relationships. Thus, these results suggest that Nep4 activity at the surface of muscle cells is necessary to maintain homeostasis of distinct hemolymph circulating signaling peptides, probably gut-derived, thereby ensuring proper midgut-IPC communication. On the other hand, fat body-IPC feedback may be affected as well. However, the only factors known to mediate this process, Unpaired 2, DILP6, and Stunted have molecular masses of more than 5 kDa, and thus exceed the maximum mass of a putative neprilysin substrate. Consequently, a direct regulatory influence of Nep4 on Unpaired 2, DILP6, or Stunted activity appears unlikely (Hallier, 2016).
In addition to body wall muscles, nep4 is expressed in numerous cells of the central nervous system, predominantly in glial cells. Interestingly, compared to the muscle-specific effects, modulating nep4 expression in this tissue has distinct and less severe effects on dilp expression. This result suggests that CNS intrinsic Nep4 activity affects different neuropeptide regulatory systems than the corresponding muscle-bound activity. Considering the rather broad expression in glial cells, it is furthermore likely that the CNS regulation affects more than one system. However, localization at the IPC surface clearly supports a direct function in the regulation of dilp expression. In this context, spatial proximity of the peptidase may be necessary to ensure low ligand concentrations and thus tight regulation of specific neuropeptide receptors present at the surface of IPCs. Such receptors include an allatostatin A receptor (Dar-2), a tachykinin receptor (DTKR), and the short neuropeptide F receptor (sNPFR). All are essential to proper dilp expression . Interestingly, with respect to sNPFR, corresponding ligands (sNPF11-11, sNPF14-11, and sNPF212-19) exhibit very high-binding affinities, with IC50 values in the low nanomolar range, a finding that further emphasizes the need for effective ligand clearance mechanisms in order to prevent inadvertent receptor activation. Localization of Nep4 to the surface of IPCs and confirmation of Dar-2, DTKR, and sNPFR ligands as substrates of the peptidase strongly indicate that Nep4 participates in such clearance mechanisms (Hallier, 2016).
Of note, sNPF species were detected in both, CNS and hemolymph preparations, with neuroendocrine functions of the respective peptides being suggested. The dual localization is interesting because both compartments are accessible to Nep4, either to the CNS resident or to the muscle-bound enzyme. Significantly, sNPF is a potent regulator of dilp expression. Increased sNPF levels result in upregulation of dilp expression, and decreased sNPF levels have the opposite effect. The fact that these results inversely correlate with the effects of modulating nep4 expression suggests a functional relationship between sNPF and the neprilysin. Nep4-mediated cleavage of distinct sNPF species represents further evidence for this relationship (Hallier, 2016).
Besides sNPF, Nep4 also cleaves corazonin, drosulfakinins, and allatostatin A. Interestingly, corazonin promotes food intake, while allatostatin A and drosulfakinins inhibit it. This regulatory activity on peptides with opposing physiological functions indicates that Nep4 affects multiple aspects of feeding control, rather than promoting or inhibiting food intake in a mutually exclusive manner. The finding that both, nep4 knockdown and overexpression larvae exhibit reduced food intake supports this indication since it suggests that regular Nep4 activity adjusts the general peptide homeostasis in a manner that promotes optimal food intake, with deviations in either direction being deteriorative. The result that nep4 knockdown animals exhibit reduced food intake for only up to 20 min of feeding may reflect this complex regulation since it indicates that at the onset of feeding reduced cleavage of peptides inhibiting food intake (e.g., allatostatin A, drosulfakinins) is a dominant factor. With ongoing feeding, accumulation of peptides promoting food intake (e.g., corazonin) may become decisive, thus restoring intake rates (Hallier, 2016).
In addition, Nep4 hydrolyzes numerous peptides that regulate dilp expression, including tachykinins, allatostatin A, and sNPF. However, AKH, a functional homolog of vertebrate glucagon that acts antagonistically to insulin, is also a substrate of Nep4. This finding indicates that the Nep4-mediated regulation of dilp expression and sugar homeostasis can also not be attributed to a single substrate or cleavage event. Rather, it is a result of the concerted hydrolysis of several critical peptides, including both, hemolymph circulating and CNS intrinsic factors. Taking into account that overexpression and knockdown of nep4 have discrete effects on dilp expression, but comparable effects on feeding, it furthermore appears likely that dysregulation of the Nep4-mediated peptide homeostasis affects both processes somewhat independently of each other. The fact that among the novel Nep4 substrates, peptides were identified that presumably affect either dilp signaling (e.g., DH31), or food intake (e.g., leucokinin, drosulfakinins) in a largely exclusive manner supports this indication (Hallier, 2016).
Because neprilysins and many of the novel substrates identified in this study are evolutionarily conserved factors, neprilysin-mediated regulation of insulin-like peptide expression and feeding behavior may be relevant not only to the energy metabolism in Drosophila, but also to corresponding processes in vertebrates, including humans. Interestingly, a critical function of murine Neprilysin in determining body mass has already been reported. The regulation depended primarily on the catalytic activity of peripheral NEP, while the CNS-bound enzyme was less important (Becker, 2010). However, until now, the underlying physiology has been obscure, essentially because no causative hydrolysis event had been identified. The finding that also in Drosophila mainly peripheral (muscle-bound) Nep4 activity affected body mass, while CNS-specific modulations had only minor effects on size or weight, indicates that the neprilysin-mediated regulation of food intake, body size and insulin expression involves similar physiological pathways in both species. Furthermore, the fact that altered catalytic activity and thus abnormal peptide hydrolysis is a critical factor in mice (Becker, 2010) and in Drosophila emphasizes the need to generate comprehensive, enzyme-specific lists of neprilysin in vivo substrates. In this context, the results of the current screen for novel Nep4 substrates may be a valuable resource in order to identify corresponding substrates in vertebrates and humans (Hallier, 2016).
Neprilysins are type II metalloproteinases known to degrade and inactivate a number of small peptides. Neprilysins in particular are the major amyloid-β peptide-degrading enzymes. In mouse models of Alzheimer's disease, neprilysin overexpression improves learning and memory deficits, whereas neprilysin deficiency aggravates the behavioral phenotypes. However, whether these enzymes are involved in memory in nonpathological conditions is an open question. Drosophila melanogaster is a well suited model system with which to address this issue. Several memory phases have been characterized in this organism and the neuronal circuits involved are well described. The fly genome contains five neprilysin-encoding genes, four of which are expressed in the adult. Using conditional RNA interference, this study shows that all four neprilysins are involved in middle-term and long-term memory. Strikingly, all four are required in a single pair of neurons, the dorsal paired medial (DPM) neurons that broadly innervate the mushroom bodies (MBs), the center of olfactory memory. Neprilysins are also required in the MB, reflecting the functional relationship between the DPM neurons and the MB, a circuit believed to stabilize memories. Together, these data establish a role for neprilysins in two specific memory phases and further show that DPM neurons play a critical role in the proper targeting of neuropeptides involved in these processes (Turrel, 2016).
Research on neprilysins has essentially focused on their role as the main Aβ-degrading enzymes in pathological situations and as biomarkers in heart failure. Using Drosophila this study has established that neprilysins are involved in specific types of memory. Disrupting the expression of any neprilysin impairs MTM and LTM, revealing that one or several neuropeptides need to be targeted to enable proper memory formation. Interestingly, all four neprilysins expressed in the fly are required in the MB and also in DPM neurons, a pair of large neurons that broadly innervates the MB and are involved in memory consolidation (Turrel, 2016).
Neprilysins have been described extensively as proteases acting on substrates of no more than 50 residues (Oefner, 2000), except Drosophila Nep4, which is involved in muscle integrity independently of its catalytic activity (Panz, 2012). There is a consensus that neprilysins function by turning off neuropeptide signals at the synapse (Turner, 2003). In addition, there is evidence to suggest that neprilysin processing could lead to the activation of neuromodulators. Therefore, in addition to their role in Aβ degradation, neprilysins also inactivate a large number of peptides and are thus equally involved in a large number of processes (Turrel, 2016).
In Drosophila, several small peptides have been linked to olfactory memory. Short neuropeptide F (sNPF) is highly expressed in the MB and has been described as a functional neuromodulator of appetitive memory. Drosophila neuropeptide F (dNPF) has been shown to provide a motivational switch in the MB that controls appetitive memory output. Interestingly, dNPF is an ortholog of mammalian NPY, a peptide identified as an hNEP substrate. hNEP can process NPY in transgenic mice to produce neuroactive fragments. Because components of dNPF/NPY signaling are conserved at both the functional and molecular levels, it is possible that dNPF is targeted by neprilysins. It remains to be determined whether such peptides are involved in aversive memory and, conversely, whether neprilysins are involved in appetitive memory (Turrel, 2016).
Although all four Drosophila neprilysins are involved in identical memory phases, they exhibit distinct features in terms of the neuronal circuits involved. Only Nep1 inhibition in α/β MB and DPM neurons alters both MTM and LTM. One hypothesis is that Nep1 expressed in DPM and MB neurons plays the same role, targeting a single substrate at synapses connecting the two structures. If so, the lifetime of such a substrate would need to be restricted strictly to limit its effect (Turrel, 2016).
Like the other neprilysins, Nep2 is involved in MTM and LTM, but it exhibits a peculiar characteristic: Nep2 inhibition in DPM neurons leads to MTM disruption, whereas it does not alter LTM. Although it cannot be ruled out that Nep2 expression in DPM neurons is required for LTM, but that its silencing does not reach a level critical for this process, the data suggest that Nep2 expression in DPM neurons is not required for LTM formation. It is noteworthy that neprilysins are synthesized as type II integral membrane proteins, whereas Nep2 is a soluble secreted endopeptidase (Thomas, 2005; Bland, 2007). Whether an endopeptidase is tethered or fully secreted will have important implications in terms of field of activity and enzyme concentration at the membrane surface. It is possible that Nep2 secreted from either DPM neurons or another structure in the vicinity, such as MB neurons, is able to play an identical role. Therefore, Nep2 reduction in either neuronal structure would not be sufficient to affect LTM. In contrast, Nep2 expression in such a structure would not be able to compensate Nep2 silencing in DPM neurons for MTM, pointing to a distinct requirement for MTM and LTM formation. Nep2 might be required at a distinct concentration and/or localization for MTM and LTM or it may target distinct substrates for these two processes (Turrel, 2016).
The data reveal functional redundancy among Nep2, Nep3, and Nep4 for LTM formation in the α/β neurons. Namely, concomitant silencing of Nep2 + Nep3 or Nep3 + Nep4 leads to altered LTM. It is possible that several neprilysins target a single neuropeptide. However, because concomitant silencing of Nep2 + Nep4 does not affect LTM, it seems more likely that several targets are involved in LTM (Turrel, 2016).
The memory phenotypes observed after each neprilysin reduction are reminiscent of the pattern in APPL mutants. Indeed, it was shown previously that expression of APPL, the APP fly ortholog, is required in the MB for MTM and LTM formation, but not for learning and ARM (Goguel, 2011; Bourdet, 2015). An attractive hypothesis is that Aβ peptide derived from physiological processing of APPL might play a role in memory and act as a substrate for one of the neprilysin peptidases. Nep2 would be a good candidate because several studies have shown that it is capable of degrading human Aβ. Supporting this hypothesis, several reports in mammals have implicated low physiological concentrations of Aβ peptide in memory formation (Turrel, 2016).
The memory phenotypes observed in this study are equally reminiscent of the pattern displayed by amnesiac (amn) mutants. The amn gene isolated through behavioral screening for memory mutants was later shown to encode a predicted neuropeptide precursor. Although the mature products of the amn gene have not been identified, sequence analyses suggested the existence of three potential peptides. One of them is homologous to mammalian pituitary adenylate cyclase-activating peptide (PACAP), a neuromodulator and neurotransmitter that regulates a variety of physiological processes through stimulation of cAMP production. In vitro studies have shown that hNEP can degrade PACAP and the analysis of the biological properties of the resulting fragments found that PACAP degradation by hNEP produces active metabolites selective for a particular receptor subtype. One of the major sites of PACAP cleavage by hNEP is conserved in the AMN peptide. It was shown that AMN is highly expressed in DPM neurons, where the four neprilysins are required for MTM. DPM output is required during the consolidation phase for MTM and it was suggested that DPM might release the AMN modulatory neuropeptide that alters the physiology of MB neurons to help stabilize or consolidate odor memories. The fact that neuropeptides are often coreleased with classical neurotransmitters, but generally have slower and longer-lasting postsynaptic effects, has prompted the hypothesis that AMN peptides may be released at the MB to produce relatively long-lasting, physiological changes. Given this context, it is tempting to speculate that AMN might be one of the Drosophila neprilysin's targets (Turrel, 2016).
Both the axons and dendrites of DPM are evenly distributed in different lobes of the MB, and it has been suggested that DPM neurons are presynaptic and postsynaptic to the MB neurons and are recurrent feedback neurons. Because neprilysins are necessary in the DPM, and also in the α/β neurons of the MB where MTM and LTM are stored, these proteins could be involved in maintaining a loop between the DPM and MB lobes by restricting the lifetime of neuromodulators. The DPM-α/β neurons circuit has been shown recently to also modulate egg-laying decision via the AMN neuropeptide (Wu, 2015). It would be interesting to learn whether neprilysins are involved in this process or if their function is restricted to memory formation (Turrel, 2016).
Despite the importance of the MB for olfactory memory, a functional neurotransmitter or coreleased peptidic neuromodulators produced by MB-intrinsic cells has long remained elusive. It was shown recently that acetylcholine is a Kenyon cell transmitter. The fact that several neprilysins are required for MTM and LTM suggests the involvement of at least one neuropeptide. It remains to be determined whether neprilysin targets are released from the DPM and/or MB and whether identical or distinct neuropeptide substrates support MTM and LTM processes. The sum of the work reported in this study highlights the critical role of the DPM in inactivating and/or processing neuropeptides involved in memory processes connected to the MB (Turrel, 2016).
Members of the M13 class of metalloproteases have been implicated in diseases and in reproductive fitness. Nevertheless, their physiological role remains poorly understood. To obtain a tractable model to analyze this protein family's function, the gene family was characterized in Drosophila melanogaster and focused on reproductive phenotypes. The D. melanogaster genome contains 24 M13 class protease homologs, some of which are orthologs of human proteases, including Neprilysin. Many are expressed in the reproductive tracts of either sex. Using RNAi the five Nep genes most closely related to vertebrate Neprilysin, Nep1-5, were individually targeted to investigate their roles in reproduction. A reduction in Nep1, Nep2, or Nep4 expression in females reduced egg-laying. Nep1 and Nep2 are required in the CNS and the spermathecae for wild-type fecundity. Females that are null for Nep2 also show defects as hosts of sperm competition as well as an increased rate of depletion for stored sperm. Furthermore, eggs laid by Nep2 mutant females are fertilized normally, but arrest early in embryonic development. In the male, only Nep1 was required to induce normal patterns of female egg-laying. Reduction in the expression of Nep2-5 in the male did not cause any dramatic effects on reproductive fitness, which suggests that these genes are either nonessential for male fertility or perform redundant functions. These results suggest that, consistent with the functions of neprilysins in mammals, these proteins are also required for reproduction in Drosophila, opening up this model system for further functional analysis of this protein class and their substrates (Sitnik, 2014).
Neprilysins (Neps) are membrane-bound M13 endopeptidases responsible for the activation and/or inactivation of peptide signalling events on cell surfaces. By hydrolysing their respective substrates, mammalian Neps are crucial to the metabolism of numerous bioactive peptides, especially in the nervous, immune, cardiovascular and inflammatory systems. On the basis of their involvement in essential physiological processes, proteins of the Nep family constitute putative therapeutic agents as well as targets in different diseases, including Alzheimer's disease. This study demonstrates that overexpression of Neprilysin 4 (Nep4) in Drosophila melanogaster leads to a severe muscle degeneration phenotype. This phenotype is observed for overexpression of full-length Nep4 in somatic muscles and is accompanied by severely impaired movement of larvae and lethality in late larval development. On the contrary, down-regulation of expression caused only the latter two effects. By expressing several mutated and truncated forms of Nep4 in transgenic animals, it was shown that the intracellular domain is responsible for the observed phenotypes while catalytic activity of the enzyme was apparently dispensable. A yeast two-hybrid screen identified a yet uncharacterised carbohydrate kinase as a first interaction partner of the intracellular domain of Nep4. These data demonstrate that the physiological significance of Nep4 is not limited to its function as an active peptidase but that the enzyme's intracellular N-terminus is affecting muscle integrity, independent of the protein's enzymatic activity. This is the first report of an intracellular Nep domain being involved in muscle integrity (Panz, 2012).
In the Drosophila embryonic CNS several subtypes of glial cells develop, which arrange themselves at characteristic positions and presumably fulfil specific functions. The mechanisms leading to the specification and differentiation of glial subtypes are largely unknown. By DiI labelling in glia-specific Gal4 lines the lineages of the lateral glia in the embryonic ventral nerve cord were clarified and each glial cell was linked to a specific stem cell. For the lineage of the longitudinal glioblast, it was shown to consist of 9 cells, which acquire at least four different identities. A large collection of molecular markers (many of them representing transcription factors and potential Gcm target genes) reveals that individual glial cells express specific combinations of markers. However, cluster analysis uncovers similar combinatorial codes for cells within, and significant differences between the categories of surface-associated, cortex-associated, and longitudinal glia. Glial cells derived from the same stem cell may be homogeneous (though not identical; stem cells NB1-1, NB5-6, NB6-4, LGB) or heterogeneous (NB7-4, NB1-3) with regard to gene expression. In addition to providing a powerful tool to analyse the fate of individual glial cells in different genetic backgrounds, each of these marker genes represents a candidate factor involved in glial specification or differentiation. This was demonstrated by the analysis of a castor loss of function mutation, which affects the number and migration of specific glial cells (Beckervordersandforth, 2008).
This report provides a comprehensive description of marker gene and enhancer trap expression in CNS glial cells of late Drosophila embryos. The markers include many transcription factors known to be involved in cell fate specification, as well as a number of still unknown factors. They were chosen for this analysis either because they were known to be expressed in subsets of glial cells or because they were known to be involved in cell fate determination in the nervous system. All together, more than 50 markers were tested, 39 of which showed expression in glial cells and hence were described in detail. Their specific expression patterns, though in many cases not restricted to glia, enable identification of groups of cells, as well as individual cells (Beckervordersandforth, 2008).
The lateral CNS glial cells have been assigned to three categories, according to their spatial distribution and morphology: the surface-, the cortex-, and the neuropile-associated glial cells. Categories were further divided into subgroups, as for example the surface-associated glial cells into subperineural glial cells and channel glia. Several of the molecular markers described in this study exhibit expression patterns, which correspond to the spatial/morphological definition of glial categories or subgroups. For example, moody or svp-lacZ are expressed in all surface-associated glial cells, whereas P101-lacZ is only expressed in the SPG-subgroup and engrailed only in the CG-subgroup. In addition, nearly each individual glial cell expresses a specific combination of markers indicating that they develop unique identities. Yet, nothing is known about how these identities are acquired. Comparing subtype affiliation or lineage ancestry of all glial cells with their respective marker gene expression patterns, it becomes obvious that glial cell specification is a process occurring on the level of individual cells. Cells might have a predisposition for a particular subtype laid down by lineage (e.g. NB5-6 derived cells to become subperineurial glia). In contrast, a temporal cascade within a lineage could determine individual cell identities (as it might be the case for the NB7-4 lineage) (Beckervordersandforth, 2008).
DiI labelling of the lineages of various progenitor cells in combination with cell-specific enhancer trap lines revealed that the composition of glial progeny within the lineages is invariant. Clonally related glia cells often express similar combinations of marker genes. The LGs, a prominent subgroup of the neuropile-associated glia and the only interface glia in the embryonic VNC, have been defined as the progeny of the LGB, which become aligned along the longitudinal connectives. However, there has been confusion about the size and composition of the LGB-lineage. By means of DiI labelling and marker gene expression, the size of this lineage was determined to be 9 cells. Although all cells of the LGB-lineage express a similar set of markers, a few markers are restricted to only parts of the lineage. Based on such markers, as well as positional criteria, the group of LGs was further subdivided. One of the cells, the LP-LG, is located slightly more lateral than the other LGs, and seems to be geared towards the ISN. Since it lies close to, and expresses a similar combination of markers as the M-ISNG, it would be justified to assign this cell to the group of nerve root glia; however, this was not done in order to avoid conflicts with the established nomenclature. Despite of their similarities, these two cells are of different origin: the LP-LG derives from the LGB, and the M-ISNG is generated by NB1-3 (Beckervordersandforth, 2008).
Most of the NBs that arise at corresponding positions and times in thoracic and abdominal segments (called serially homologous NBs) acquire the same fate, i.e. they generate the same lineages expressing corresponding sets of markers. However, some of these serially homologous lineages develop characteristic, tagma-specific differences with regard to cell number and/or cell types. Tagma-specific characteristics of these lineages have been shown to be under the control of Hox genes (Beckervordersandforth, 2008).
Although the total number of CNS glial cells is identical in thoracic and abdominal neuromeres, there are some differences in their origin and distribution of subtypes. This is due to tagma-specific differences among serially homologous lineages of NBs 1-1, 2-2, 5-6, and 6-4, which give rise to CBGs and SPGs. NB6-4A (A, abdominal) generates only two CBGs: MM-CBG and M-CBG, whereas the NB6-4T (T, thoracic) lineage comprises an additional MM-CBG2 and a neuronal sublineage. NB1-1T generates only neurons, whereas NB1-1A produces three SPGs (A-SP-G, B-SPG, and LV-SPG) in addition to neurons. In the thorax, the LV-SPG is presumably generated by NB5-6T, a cell at the position of A-SPG is produced by NB2-2T, and a cell at the B-SPG position is missing. Despite their different origin, the NB1-1A- and NB2-2T-derived SPGs specifically express hkb-lacZ and mirr-lacZ. Furthermore, the NB1-1A- and the NB2-2T-derived A-SPG appear to assume the same identity, as they express the same set of markers (including castor, which is not found in the abdominal B-SPG. Taken together, the differences between thorax and abdomen are restricted to only few glial cells, most of which acquire similar cell fates in thorax and abdomen (as judged by marker gene expression) irrespective of their progenitor (Beckervordersandforth, 2008).
The collection of marker genes and enhancer trap lines presented in this study provides powerful tools for the identification of specific glial cells in different genetic backgrounds. Each of markers also represents a candidate factor involved in glial subtype specification and/or differentiation. Many of these genes encode transcription factors known to be involved in cell fate specification, like fushi tarazu, mirror, and muscle segment homeobox. Other genes encode factors involved in cell signalling, such as moody and CG11910, or enzymes like CG7433 and CG6218 (Beckervordersandforth, 2008).
moody is expressed in all cells belonging to the surface-associated glia. At the end of embryogenesis, surface-associated glial cells form a thin layer ensheathing the entire CNS, thereby establishing the blood-brain barrier. Moody is a G-protein coupled receptor, which acts in a complex pathway to regulate the cortical actin, thereby stabilizing the extended morphology of the surface-glia. This is necessary for the formation of septate junctions to achieve proper sealing of the nerve cord. Moody therefore represents a protein, which is essential for establishing and maintaining a specific function of surface glia (Beckervordersandforth, 2008).
Two of the markers analyzed represent metalloproteases: Neprilysin4 (Nep4) and Invadolysin. Invadolysin has been described to play a role in mitotic progression and in migration of germ cells, but as for Nep4, its function in the nervous system is unknown. In vertebrates it has been shown that metalloproteases are involved in various processes in the CNS: they are associated with neurite outgrowth, migration of neurons and myelination of axons. For one matrix-metalloprotease, MMP-12, it has been shown that it is expressed in oligodendrocytes, where it functions in maturation and morphological differentiation of OL lineages. It has been postulated that LGs are analogous to vertebrate oligodendrocytes, as both groups of cells enwrap axonal projections in the CNS, although to different degrees (no myelination in Drosophila). The two metalloproteases analysed are exclusively expressed in neuropile- (LGs) and cortex-associated glial cells (CBGs). Thus, both Nep4 and Invadolysin may possibly be involved in the differentiation of LGs. In invadolysin loss of function mutants, the specification of lateral glial cells does not seem to be affected, but the LGs show a very subtle phenotype in their positioning (data not shown). An explanation for the subtle phenotype may be redundant function of both enzymes. Indeed, in vertebrates it has been shown that metalloproteases have many overlapping substrates in vitro, and redundancy and compensation has been shown for matrix-metalloproteases (MMPs) in double mutants. Furthermore, it has been shown for members of the neprilysin family of metalloendopeptidases in Caenorhabditis elegans and Drosophila melanogaster, that many of the enzymatic properties have been conserved during evolution (Beckervordersandforth, 2008).
Making use of the molecular markers, this study characterized the phenotype of a cas loss of function mutation. Cas is a transcription factor, which acts in temporal cell fate specification. Together with Pdm, Cas is involved in the determination of late progeny cells in CNS lineages. In late embryonic stages, cas is specifically expressed in four glial cells per hemisegment, the V-CG and D-CG, the A-SPG and the LV-SPG, as well as in many neurons. The A- and LV-SPG, which are late progeny of the NB1-1A, are not affected in cas mutants, whereas the NB7-4-derived CGs seem mislocalized, with the medial migration of both CGs being impaired in cas mutants. This points to different functions of Cas in distinct NB lineages. As can be deduced from Repo stainings, general aspects of glial differentiation do not seem to be affected in cas mutants. Further analysis will have to clarify whether the role of Cas in NB7-4 derived glial cells is on the level of cell fate determination and/or whether it directly acts on specific aspects of differentiation (migration, motility). It also remains to be shown whether Cas acts cell-autonomously in this process (Beckervordersandforth, 2008).
Recent studies have firmly established pigment dispersing factor (PDF), a C-terminally amidated octodecapeptide, as a key neurotransmitter regulating rhythmic circadian locomotory behaviours in adult Drosophila melanogaster. The mechanisms by which PDF functions as a circadian peptide transmitter are not fully understood, however; in particular, nothing is known about the role of extracellular peptidases in terminating PDF signalling at synapses. This study shows that PDF is susceptible to hydrolysis by neprilysin, an endopeptidase that is enriched in synaptic membranes of mammals and insects. Neprilysin cleaves PDF at the internal Ser7-Leu8 peptide bond to generate PDF1-7 and PDF8-18. Neither of these fragments were able to increase intracellular cAMP levels in HEK293 cells cotransfected with the Drosophila PDF receptor cDNA and a firefly luciferase reporter gene, confirming that such cleavage results in PDF inactivation. The Ser7-Leu8 peptide bond was also the principal cleavage site when PDF was incubated with membranes prepared from heads of adult Drosophila. This endopeptidase activity was inhibited by the neprilysin inhibitors phosphoramidon. It is proposed that cleavage by a member of the Drosophila neprilysin family of endopeptidases is the most likely mechanism for inactivating synaptic PDF and that neprilysin might have an important role in regulating PDF signals within circadian neural circuits (Isaac, 2007).
Amyloid β-peptide (Aβ), which plays a central role in Alzheimer's disease, is generated by presenilin-dependent γ-secretase cleavage of β-amyloid precursor protein (βAPP). The presenilins (PS1 and PS2) also regulate Aβ degradation. Presenilin-deficient cells fail to degrade Aβ and have drastic reductions in the transcription, expression, and activity of neprilysin, a key Aβ-degrading enzyme. Neprilysin activity and expression are also lowered by γ-secretase inhibitors and by PS1/PS2 deficiency in mouse brain. Neprilysin activity is restored by transient expression of PS1 or PS2 and by expression of the amyloid intracellular domain (AICD), which is cogenerated with Aβ, during γ-secretase cleavage of βAPP. Neprilysin gene promoters are transactivated by AICDs from APP-like proteins (APP, APLP1, and APLP2), but not by Aβ or by the γ-secretase cleavage products of Notch, N- or E- cadherins. The presenilin-dependent regulation of neprilysin, mediated by AICDs, provides a physiological means to modulate Aβ levels with varying levels of γ-secretase activity (Pardossi-Piquard, 2005).
If Aβ production and degradation are tightly linked, this raises the question of why Aβ accumulates in AD. The net accumulation of Aβ in AD pathology likely reflects the cumulative effect of multiple events acting on production, fibrillogenesis, and degradation. In many forms of AD, especially the late-onset sporadic forms, it has not been shown that there is increased β- and γ-secretase activity. In fact, some have suggested that these forms may reflect defective degradation of Aβ. Therefore, AICD levels are likely to be unchanged in these late-onset forms of AD, and as a result, the AICD-mediated ability to upregulate neprilysin activity would not be efficiently brought into play to protect the brain. In contrast, in those cases of AD arising from mutations in APP and PS1, which activate γ-secretase and AICD production, the principal effect is to produce longer Aβ isoforms such as Aβ42. However, although Aβ40 is efficiently degraded by NEP, Aβ42 is degraded by NEP both in vitro and in vivo at a 6-fold lower rate. As a result, the upregulation of AICD (and thus, NEP expression), which would be anticipated in subjects with presenilin mutations, would not completely abolish the accumulation of Aβ42 in these cases. It should be noted that, in agreement with the above hypothesis, neprilysin expression and activity were higher only in brain tissues with familial Alzheimer’s disease linked to various presenilin-1 mutations, while sporadic AD cases displayed neprilysin levels similar to those exhibited by normal brain tissues. Interestingly, PS1 mutations selectively affect neprilysin and do not alter insulin-degrading enzyme expression (Pardossi-Piquard, 2005).
The above observations are also of direct practical interest because they indicate the possibility of new avenues for controlling Aβ levels without directly affecting γ-secretase. This latter concept is important because of the various developmental and postnatal side-effects associated with the inhibition of γ-secretase-mediated cleavage of other signaling molecules, including Notch. This work now suggests that Aβ levels might be modulated by directly increasing neprilysin expression, using AICD or small molecule mimics of AICD. Upregulation of neprilysin by transgenic overexpression, at least to modest levels, appears to be sufficient to reduce brain Aβ levels and to pose few toxic side effects. This strategy would also circumvent the other side effects of γ-secretase inhibitors, including the potentially self-defeating effect of reducing AICD and thus preventing NEP-mediated degradation of Aβ (Pardossi-Piquard, 2005).
In order to sustain lifelong production of gametes, many animals have evolved a stem cell-based gametogenic program. In the Drosophila ovary, germline stem cells (GSCs) arise from a pool of primordial germ cells (PGCs) that remain undifferentiated even after gametogenesis has initiated. The decision of PGCs to differentiate or remain undifferentiated is regulated by somatic stromal cells: specifically, epidermal growth factor receptor (EGFR) signaling activated in the stromal cells determines the fraction of germ cells that remain undifferentiated by shaping a Decapentaplegic (Dpp) gradient that represses PGC differentiation. However, little is known about the contribution of germ cells to this process. This study shows that a novel germline factor, Gone early (Goe; CG9634), limits the fraction of PGCs that initiate gametogenesis. goe encodes a non-peptidase homologue of the Neprilysin family metalloendopeptidases. At the onset of gametogenesis, Goe was localized on the germ cell membrane in the ovary, suggesting that it functions in a peptidase-independent manner in cell-cell communication at the cell surface. Overexpression of Goe in the germline decreased the number of PGCs that enter the gametogenic pathway, thereby increasing the proportion of undifferentiated PGCs. Inversely, depletion of Goe increased the number of PGCs initiating differentiation. Excess PGC differentiation in the goe mutant was augmented by halving the dose of argos, a somatically expressed inhibitor of EGFR signaling. This increase in PGC differentiation resulted in a massive decrease in the number of undifferentiated PGCs, and ultimately led to insufficient formation of GSCs. Thus, acting cooperatively with a somatic regulator of EGFR signaling, the germline factor goe plays a critical role in securing the proper size of the GSC precursor pool. Because goe can suppress EGFR signaling activity and is expressed in EGF-producing cells in various tissues, goe may function by attenuating EGFR signaling, and thereby affecting the stromal environment (Matsuoka, 2014).
Members of the neprilysin family of neutral endopeptidases (M13) are typically membrane-bound enzymes known to be involved in the extra-cellular metabolism of signalling peptides and have important roles during mammalian embryogenesis. This study shows that membranes prepared from embryos of Drosophila melanogaster possess neprilysin-like activity that is inhibited by phosphoramidon and thiorphan, both inhibitors of mammalian neprilysin. Unexpectedly, strong neprilysin-like neutral endopeptidase activity was found in a soluble embryo fraction that was identified as NEP2 by Western blot and immunoprecipitation experiments using NEP2 specific antibodies. NEP2 is a soluble secreted member of the neprilysin family that has been shown previously to be expressed in larval and adult Malpighian tubules and in the testes of adult males. In situ hybridization studies reveal expression at stage 10-11 in a pattern similar to that previously described for stellate cell progenitors of the caudal visceral mesoderm. In later stages of embryogenesis, some of these cells appear to migrate into the growing Malpighian tubule. Recombinant NEP2 protein is N-glycosylated and displays optimum endopeptidase activity at neutral pH, consistent with a role as an extracellular peptidase. The recombinant enzyme hydrolyses Drosophila tachykinin peptides (DTK) at peptide bonds N-terminal to hydrophobic residues. DTK2, like Locusta tachykinin-1, was cleaved at the penultimate peptide bond (Gly7-Leu8), whereas the other Drosophila peptides were cleaved centrally at Xxx-Phe bonds. However, the rates of hydrolysis of the latter substrates were much slower than the hydrolysis rates of DTK2 and Locusta tachykinin-1, suggesting that the interaction of the bulky side-chain of phenylalanine at the S'1 sub-site is less favorable for peptide bond hydrolysis. The secretion of NEP2 from tissues during embryogenesis suggests a possible developmental role for this endopeptidase in peptide signalling in D. melanogaster (Bland, 2007).
According to the World Health Organization there is a pandemic of obesity with approximately 300 million people being obese. Typically, human obesity has a polygenetic causation. Neutral endopeptidase (NEP), also known as neprilysin, is considered to be one of the key enzymes in the metabolism of many active peptide hormones. An incidental observation in NEP-deficient mice was a late-onset excessive gain in body weight exclusively from a ubiquitous accumulation of fat tissue. In accord with polygenetic human obesity, mice were characterized by deregulation of lipid metabolism, higher blood glucose levels, with impaired glucose tolerance. The key role of NEP in determining body mass was confirmed by the use of the NEP inhibitor candoxatril in wild-type mice that increased body weight due to increased food intake. This is a peripheral and not a central NEP action on the switch for appetite control, since candoxatril cannot cross the blood-brain barrier. Furthermore, inhibition of NEP in mice with cachexia delayed rapid body weight loss. Thus, lack in NEP activity, genetically or pharmacologically, leads to a gain in body fat. This study has identified NEP to be a crucial player in the development of obesity. NEP-deficient mice start to become obese under a normocaloric diet in an age of 6-7 months and thus are an ideal model for the typical human late-onset obesity. Therefore, the described obesity model is an ideal tool for research on development, molecular mechanisms, diagnosis, and therapy of the pandemic obesity (Becker, 2010).
Population density-dependent dispersal is a well-characterized strategy of animal behavior in which dispersal rate increases when population density is higher. Caenorhabditis elegans shows positive chemotaxis to a set of odorants, but the chemotaxis switches from attraction to dispersal after prolonged exposure to the odorants. This plasticity of olfactory behavior was shown to be dependent on population density; this regulation is mediated by pheromonal signaling. A peptide, suppressor of NEP-2 (SNET-1), negatively regulates olfactory plasticity, and its expression is down-regulated by the pheromone. NEP-2, a homolog of the extracellular peptidase neprilysin, antagonizes SNET-1, and this function is essential for olfactory plasticity. These results suggest that population density information is transmitted through the external pheromone and endogenous peptide signaling to modulate chemotactic behavior (Yamada, 2010).
Search PubMed for articles about Drosophila Neprilysin
Becker, M., Siems, W. E., Kluge, R., Gembardt, F., Schultheiss, H. P., Schirner, M. and Walther, T. (2010). New function for an old enzyme: NEP deficient mice develop late-onset obesity. PLoS One 5(9). PubMed ID: 20862277
Beckervordersandforth, R. M., Rickert, C., Altenhein, B. and Technau, C. M. (2008). Subtypes of glial cells in the Drosophila embryonic ventral nerve cord as related to lineage and gene expression. Mech. Dev. 125: 542-557. PubMed ID: 18296030
Bland, N. D., Thomas, J. E., Audsley, N., Shirras, A. D., Turner, A. J. and Isaac, R. E. (2007). Expression of NEP2, a soluble neprilysin-like endopeptidase, during embryogenesis in Drosophila melanogaster. Peptides 28(1): 127-135. PubMed ID: 17157960
Bourdet, I., Preat, T. and Goguel, V. (2015). The full-length form of the Drosophila amyloid precursor protein is involved in memory formation. J Neurosci 35(3): 1043-1051. PubMed ID: 25609621
Goguel, V., Belair, A. L., Ayaz, D., Lampin-Saint-Amaux, A., Scaplehorn, N., Hassan, B. A. and Preat, T. (2011). Drosophila amyloid precursor protein-like is required for long-term memory. J Neurosci 31(3): 1032-1037. PubMed ID: 21248128
Hallier, B., Schiemann, R., Cordes, E., Vitos-Faleato, J., Walter, S., Heinisch, J. J., Malmendal, A., Paululat, A. and Meyer, H. (2016). Drosophila neprilysins control insulin signaling and food intake via cleavage of regulatory peptides. Elife 5. PubMed ID: 27919317
Isaac, R. E., Johnson, E. C., Audsley, N. and Shirras, A. D. (2007). Metabolic inactivation of the circadian transmitter, pigment dispersing factor (PDF), by neprilysin-like peptidases in Drosophila. J Exp Biol 210(Pt 24): 4465-4470. PubMed ID: 18055635
Matsuoka, S., Gupta, S., Suzuki, E., Hiromi, Y. and Asaoka, M. (2014). gone early, a novel germline factor, ensures the proper size of the stem cell precursor pool in the Drosophila ovary. PLoS One 9: e113423. PubMed ID: 25420147
Meyer, H., Panz, M., Zmojdzian, M., Jagla, K. and Paululat, A. (2009). Neprilysin 4, a novel endopeptidase from Drosophila melanogaster, displays distinct substrate specificities and exceptional solubility states. J Exp Biol 212(Pt 22): 3673-3683. PubMed ID: 19880729
Meyer, H., Panz, M., Albrecht, S., Drechsler, M., Wang, S., Husken, M., Lehmacher, C. and Paululat, A. (2011). Drosophila metalloproteases in development and differentiation: the role of ADAM proteins and their relatives. Eur J Cell Biol 90(9): 770-778. PubMed ID: 21684629
Nassel, D. R. and Winther, A. M. (2010). Drosophila neuropeptides in regulation of physiology and behavior. Prog Neurobiol 92(1): 42-104. PubMed ID: 20447440
Oefner, C., D'Arcy, A., Hennig, M., Winkler, F. K. and Dale, G. E. (2000). Structure of human neutral endopeptidase (Neprilysin) complexed with phosphoramidon. J Mol Biol 296(2): 341-349. PubMed ID: 10669592
Panz, M., Vitos-Faleato, J., Jendretzki, A., Heinisch, J. J., Paululat, A. and Meyer, H. (2012). A novel role for the non-catalytic intracellular domain of Neprilysins in muscle physiology. Biol Cell 104(9): 553-568. PubMed ID: 22583317
Pardossi-Piquard, R., et al. (2005). Presenilin-dependent transcriptional control of the Aß-degrading enzyme Neprilysin by intracellular domains of ßAPP and APLP. Neuron 46: 541-554. 15944124
Sitnik, J. L., Francis, C., Hens, K., Huybrechts, R., Wolfner, M. F. and Callaerts, P. (2014). Neprilysins: An evolutionarily conserved family of metalloproteases that play important roles in reproduction in Drosophila. Genetics 96(3): 781-97. PubMed ID: 24395329
Sofola-Adesakin, O., Khericha, M., Snoeren, I., Tsuda, L. and Partridge, L. (2016). pGluAβ increases accumulation of Aβ in vivo and exacerbates its toxicity. Acta Neuropathol Commun 4: 109. PubMed ID: 27717375
Thomas, J. E., Rylett, C. M., Carhan, A., Bland, N. D., Bingham, R. J., Shirras, A. D., Turner, A. J. and Isaac, R. E. (2005). Drosophila melanogaster NEP2 is a new soluble member of the neprilysin family of endopeptidases with implications for reproduction and renal function. Biochem J 386(Pt 2): 357-366. PubMed ID: 15554877
Turner, A. J., Isaac, R. E. and Coates, D. (2001). The neprilysin (NEP) family of zinc metalloendopeptidases: genomics and function. Bioessays 23(3): 261-269. PubMed ID: 11223883
Turrel, O., Lampin-Saint-Amaux, A., Préat, T. and Goguel, V. (2016). Drosophila neprilysins are involved in middle-term and long-term memory. J Neurosci 36: 9535-9546. PubMed ID: 27629706
Wu, C. L., Fu, T. F., Chou, Y. Y. and Yeh, S. R. (2015). A single pair of neurons modulates egg-laying decisions in Drosophila. PLoS One 10(3): e0121335. PubMed ID: 25781933
Yamada, K., Hirotsu, T., Matsuki, M., Butcher, R. A., Tomioka, M., Ishihara, T., Clardy, J., Kunitomo, H. and Iino, Y. (2010). Olfactory plasticity is regulated by pheromonal signaling in Caenorhabditis elegans. Science 329(5999): 1647-1650. PubMed ID: 20929849
date revised: 22 June 2017
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