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spichthyin: Biological Overview | References

BIOLOGICAL OVERVIEW

To understand the functions of NIPA1, mutated in the neurodegenerative disease hereditary spastic paraplegia, and of ichthyin, mutated in autosomal recessive congenital ichthyosis, their Drosophila melanogaster ortholog was studied. Spichthyin (Spict) is found on early endosomes. Loss of Spict leads to upregulation of bone morphogenetic protein (BMP) signaling and expansion of the neuromuscular junction. BMP signaling is also necessary for a normal microtubule cytoskeleton and axonal transport; analysis of loss- and gain-of-function phenotypes indicate that Spict may antagonize this function of BMP signaling. Spict interacts with BMP receptors and promotes their internalization from the plasma membrane, implying that it inhibits BMP signaling by regulating BMP receptor traffic. This is the first demonstration of a role for a hereditary spastic paraplegia protein or ichthyin family member in a specific signaling pathway, and implies disease mechanisms for hereditary spastic paraplegia that involve dependence of the microtubule cytoskeleton on BMP signaling (Wang, 2007).

Axonal abnormalities, including impairment of transport, are a hallmark of many neurological and neurodegenerative diseases. These include the hereditary spastic paraplegias (HSPs), a heterogeneous set of diseases characterized by degeneration of corticospinal tract axons and spasticity of the lower extremities. Different forms of the disease are termed either pure or complicated, depending on whether other mainly neurological symptoms are present. The mechanisms of degeneration in HSPs are unknown, but over twenty causative loci (SPG loci) have been mapped and thirteen cloned. Some SPG products are implicated in microtubule function or transport, including the microtubule motor protein kinesin, and the microtubule-severing protein spastin. Since microtubules are the route for fast axonal transport, the most distal portions of axons are likely to be most sensitive to impairments of microtubule function. A second class of SPG products are mitochondrial proteins, but it is not known how mutations in these cause axonal degeneration. A third class of SPG products are apparently associated with endosomes, judged by immunolocalization or the presence of domains such as MIT or FYVE. HSP is also caused by some mutations in the amyotrophic lateral sclerosis gene ALS2, which encodes alsin, a guanine-nucleotide-exchange-factor for the early endosomal GTPase Rab5. However, the mechanism by which impairment of endosomal membrane traffic might cause axonal degeneration is unknown. (Wang, 2007).

One membrane protein encoded by an SPG gene is SPG6, mutations in which cause a dominant pure form of HSP, and which is widely expressed, although enriched in brain tissue (Rainier, 2003; Chen, 2005; Reed, 2005; Kaneko, 2006). SPG6 is a member of a protein family (Pfam: DUF803) predicted to have between seven and nine transmembrane (TM) domains. Three different amino-acid substitutions are known, one of which is found in ethnically disparate families and another caused by different nucleotide substitutions in the same codon, suggesting a dominant gain-of-function disease mechanism that can be mediated by only a few mutations in the protein. This protein family includes another human disease protein, ichthyin, mutations in which cause autosomal recessive congenital ichthyosis (ARCI), a skin disorder whose cellular basis is not understood. Ichthyin is widely expressed, although with high expression in keratinocytes, and little or no expression in brain, and at least six recessive alleles are known that cause substitutions of mainly conserved amino acids in different parts of the protein. In summary, little is known of the cellular roles of the SPG6 and ichthyin family (Wang, 2007).

To understand the normal role of the SPG6 and ichthyin protein family, and how changes in their function might lead to cellular defects, their Drosophila homolog, spichthyin (Spict) was have studied. Spict shows preferential localization on early endosomes. It regulates growth of the neuromuscular junction (NMJ) presynaptically, by inhibition of BMP (Bone Morphogenic Protein)/TGF-β (Transforming Growth Factor-β) signaling. BMP signaling regulates synaptic growth, function and stabilization at the NMJ. This study shows a novel role for BMP signaling in maintenance of microtubules and axonal transport, and that this function is also inhibited by Spict. These data suggest that Spict inhibits BMP signaling by regulating BMP receptor traffic. These findings provide a cellular role for the Spict family of proteins, and suggest potential mechanisms for the pathology of HSPs and ARCI that include dependence of microtubules on BMP signaling (Wang, 2007).

A BLASTP search using human SPG6 identified one Drosophila homolog, CG12292. A search using CG12292 identified four predicted human proteins that were 40-50% identical to it: SPG6 (NIPA1), NIPA2, ichthyin and NPAL1. Two more distantly related human proteins, NPAL2 and NPAL3 are more closely related to plant and fungal homologs than to CG12292, and probably represent a subfamily lost from the Drosophila lineage. Since Drosophila CG12292 appears orthologous to both SPG6 and ichthyin, it was designated spichthyin (spict).

To generate spict mutant flies, transposase-mediated imprecise excision was used of a P element, EP(2)2202, inserted in the spict 5' untranslated region. One imprecise excision, spict^mut, had lost the entire coding region, and was therefore a null allele of spict. Several precise excision events were recovered; one of these was used as a wild-type control in most subsequent experiments, and is referred to as spict⁺. Homozygous spict^mut flies were viable and fertile, and took about a day longer than spict⁺ flies to reach adulthood (Wang, 2007).

To determine where Spict might act, its expression pattern and subcellular localization was examined. spict mRNA was found ubiquitously during embryogenesis, with elevated expression in some tissues, including CNS and muscles. EGFP-Spict and Spict-EGFP fusions both showed punctate distributions in Drosophila S2 cells, that overlapped substantially with the early endosome compartment detected using anti-Rab5, but showed no striking overlap with the late endosomal/multivesicular body marker Hook, the recycling endosomal marker Rab11, or the late endosomal/lysosomal markers Spinster and LysoTracker. A Spict-mRFP fusion protein also showed a punctate cytoplasmic distribution in wild-type and spict^mut third instar larvae, which also overlapped substantially with Rab5, but not with late endosomal/lysosomal markers, in muscles and NMJs. Trypsin digestion of N-terminally and C-terminally tagged Spict, redistibuted to the plasma membrane by blockage of endocytosis, suggested that the N-terminus of Spict is in the endosome lumen, and its C-terminus in the cytosol. This result is consistent with previous suggestions that Spict family members might either have nine transmembrane domains, or be divergent members of the 7-TM superfamily. Attempts to raise an antibody that recognized endogenous Spict in immunomicroscopy were unsuccessful. However, since Spict-EGFP and EGFP-Spict fusions had apparently identical localizations in S2 cells, the Spict-mRFP fusion could rescue a spict^mut phenotype and cause the same overexpression phenotypes as wild type Spict, these fusions are likely to have the same localization as endogenous Spict (Wang, 2007).

Since tagged Spict proteins localized with Rab5, tests were performed to see whether Rab5 staining is normal when Spict is lacking. Rab5 staining was less intense in spict^mut NMJ boutons compared to wild-type; these phenotypes were rescued by ubiquitous expression of UAS-spict. Rab5 staining was also reduced in muscles but not obviously affected in neuronal cell bodies and axons of spict^mut larvae, or in S2 cells treated by spict RNAi. Therefore, Spict is essential for a normal Rab5 compartment at the NMJ, but not in all situations (Wang, 2007).

One of the signaling pathways with the largest effects on synaptic size at the Drosophila NMJ is the BMP pathway, which stimulates synaptic growth. The expanded NMJ phenotype of spict^mut is similar to that of spinster (also known as benchwarmer), which also shows defects in endosomal-lysosomal trafficking and requires an active BMP/TGF-β signaling pathway for NMJ expansion. It is also similar to the increase in bouton number of highwire NMJs. Highwire encodes a putative E3 ubiquitin ligase that appears to affect multiple signaling pathways including JNK and BMP. To determine whether the synaptic overgrowth of spict^mut larvae requires BMP signaling, key BMP signaling components were genetically removed from spict^mut larvae. Mutations affecting the type I receptor subunits Tkv (Thickvein) and Sax (Saxophone), the type II receptor subunit Wit (Wishful Thinking), the type II receptor ligand Gbb (Glass Bottom Boat), or the co-Smad Med (Medea) all suppressed the NMJ overgrowth of spict^mut larvae. In all cases, the synaptic undergrowth in larvae that were doubly homozygous for spict^mut and BMP pathway mutations was indistinguishable from that of homozygous BMP pathway mutations alone. In addition, all heterozygous BMP pathway mutations tested partly suppressed the NMJ expansion of spict^mut larvae, but had no effect on NMJ bouton number in a wild type background. Therefore, BMP signaling is essential for the excessive NMJ growth of spict^mut larvae (Wang, 2007).

The contrasting phenotypes of spict^mut and loss of BMP signaling, and the genetic interactions between spict and BMP signaling mutants, suggest that Spict antagonizes BMP signaling in the control of NMJ growth. Nevertheless, alternative models are possible: for example, highwire mutations interact with BMP signaling mutations, but Highwire affects synaptic size primarily through a MAPK signaling pathway. However, evidence strongly supports a direct effect of Spict on BMP signaling. During BMP signaling in neurons, the R-Smad protein Mad is phosphorylated by active BMP receptors, and phosphorylated Mad (PMad) is then translocated to the nucleus and acts as a transcription factor. At the NMJ, PMad overlaps mainly with the presynaptic marker cysteine string protein (CSP), but also with the largely postsynaptic marker Discs-large (Dlg). PMad is also found in cell body nuclei in the larval CNS. PMad levels were significantly higher in spict^mut than in spict⁺ larvae, both at the NMJ and in CNS cell bodies, and this phenotype was fully rescued by neuronal expression of UAS-spict. Therefore, BMP signaling is upregulated at spict^mut neurons, in contrast to highwire neurons. Next the possibility of upregulation of BMP receptors at spict^mut NMJs was tested. HA-tagged Tkv was found mainly in a punctate distribution in the periphery of synaptic boutons, at or close to the plasma membrane, and at higher levels in spict^mut than in spict⁺ boutons. Wit was barely detectable in spict⁺ boutons, but was present at higher levels in spict^mut boutons, also in a punctate pattern mainly at or close to the plasma membrane. The effect of spict^mut on Tkv-HA and Wit levels was rescued by neuronal expression of UAS-spict. No effect was found of spict^mut on levels of other neuronal membrane proteins (Fasciclin II, Syntaxin), or on the neuronal surface antigen recognized by anti-Horseradish Peroxidase (HRP) at the NMJ. Therefore, Spict action specifically lowers the levels of BMP receptors at the presynaptic NMJ (Wang, 2007).

The opposing effects of Spict and BMP signaling on NMJ and neuronal microtubules suggest that Spict is a novel antagonist of BMP signaling. BMP signaling acts both presynaptically and postsynaptically at the NMJ; rescue experiments show that Spict acts presynaptically to regulate NMJ expansion. The data suggest a direct effect of Spict on the presynaptic BMP signaling machinery. First, elevated levels of PMad and BMP receptors are seen at spict^mut NMJs. Second, Spict can be co-immunoprecipitated with Wit. Third, Spict shows partial colocalization with the BMP receptors Tkv-HA or Wit at NMJ boutons. Fourth, Spict promotes relocalization of Wit from the surface of S2 cells to the Rab5 early endosomal compartment. Therefore, the data suggest strongly that Spict antagonizes BMP signaling by regulating its receptor traffic. This is in contrast to Highwire - while synaptic overgrowth in highwire mutants can be suppressed by BMP signaling mutants, the highwire phenotype is more completely suppressed by loss of the Wallenda MAP kinase kinase kinase, and there is no apparent upregulation of PMad in highwire mutants (Wang, 2007).

The posterior crossveinless phenotype in some spict^mut adult wings is also typical of reduced BMP signaling in pupal wing discs. At first sight a crossveinless phenotype is inconsistent with Spict being an antagonist of BMP signaling. However, lowered BMP signaling in the posterior crossvein primordium could be due not only to direct downregulation of signaling, but also to upregulation of receptors that reduces diffusion of BMP ligands. No changes were detected in the level of BMP signaling about the time when the posterior crossvein primordium develops, but this could be due to either the partial penetrance of the phenotype, or the robustness of the regulatory and feedback mechanisms that translate smooth gradients of BMP ligands into more sharply defined developmental features (Wang, 2007).

How might an endosomal protein regulate BMP signaling? Membrane trafficking from the plasma membrane to lysosomes regulates many signaling pathways including BMP/TGF-β. For example, mutations that impair endosome to lysosome traffic cause an increase in BMP signaling, in at least some cases accompanied by increased levels of Tkv. However, the predominant localization of Spict on early endosomes, and its ability to internalize Wit to this compartment suggest that Spict functions at some step of plasma membrane to endosome traffic. (1) Rab5 compartments fail to accumulate at spict^mut NMJs, rather than enlarge as in Hrs mutants. (2) Spict overexpression in S2 cells redistributes Wit mainly to early endosomes, rather than to late endosomes or lysosomes. (3) There is no obvious degradation of Wit in Spict-overexpressing cells that internalize Wit, suggesting that Spict does not directly target Wit for degradation, at least in S2 cells. While levels of BMP receptors are elevated locally in NMJ boutons that lack Spict, this could be either to altered trafficking or degradation, and BMP signaling in S2 cells can be affected by Spict, without detectable changes in levels of BMP receptors. Therefore, Spict might inhibit BMP signaling by internalizing vacant receptors and thus preventing them from responding to ligand; since clathrin RNAi treatment redistributes Spict to the plasma membrane, Spict probably appears at least transiently at the plasma membrane. However, more complex models are possible. For example, Spict might sequester BMP receptors in a compartment from which they cannot signal; Notch receptors apparently have to reach a specific endosomal compartment before they can signal (Wang, 2007).

By studying Spict, this study has identified a role for BMP signaling in maintenance of axonal microtubules. Notably, local loss of presynaptic microtubules has also been seen in loss of BMP signaling at the NMJ, and apical microtubule arrays are eliminated in tkv mutant clones in wing imaginal discs. Since BMP signaling promotes synaptic growth and synaptic strength at the NMJ, it would be logical for it also to stimulate the additional transport of materials and organelles that a larger more active synapse requires (Wang, 2007).

If human SPG6 alleles are dominant gain-of-function, then the HSP that they cause would resemble the situation of Spict overexpression in Drosophila, and axonal degeneration in HSP could then be caused by inhibition of BMP signaling, loss of axonal microtubules, and impaired axonal transport. Given the effect of BMP signaling on axonal microtubules, other HSP gene products apart from SPG6 may affect BMP signaling and thus maintenance of axonal microtubules. (Wang, 2007).

In contrast to SPG6, ARCI appears to be caused by loss of ichthyin function (Lefevre, 2004). Identification of a role for the ichthyin ortholog Spict in inhibiting BMP signaling suggests upregulation of BMP signaling as a possible disease mechanism in ARCI. Indeed, the BMP-like ligand TGF-β1 has complex roles in maintenance of skin, and its overexpression can cause psoriasis, a condition that bears some resemblance to ichthyosis. Inhibitors of BMP signaling may therefore be candidates for therapeutic purposes in ARCI or similar conditions. (Wang, 2007).

In conclusion, this study has established a cellular role for the SPG6 and ichthyin family of proteins, thus identifying a novel group of players in BMP signaling, and providing a framework for future understanding of diseases caused by mutations that affect these proteins (Wang, 2007).

Search PubMed for articles about Drosophila Spichthyin

Chen, S., et al. (2005). Distinct novel mutations affecting the same base in the NIPA1 gene cause autosomal dominant hereditary spastic paraplegia in two Chinese families. Human Mutation. 25: 135-141. PubMed ID: 15643603

Kaneko, S., et al. (2006). Novel SPG6 mutation p.A100T in a Japanese family with autosomal dominant form of hereditary spastic paraplegia. Mov. Disord. 21: 1531-1533. PubMed ID: 16795073

Lefevre, C., et al. (2004). Mutations in ichthyin a new gene on chromosome 5q33 in a new form of autosomal recessive congenital ichthyosis. Hum. Mol. Gen. 13: 2473-2482. PubMed ID: 15317751

Rainier, S., et al. (2003). NIPA1 Gene mutations cause autosomal dominant hereditary spastic paraplegia (SPG6). Am. J. Hum. Genet. 73: 967-971. PubMed ID: 14508710

Reed, J. A., et al. (2005). A novel NIPA1 mutation associated with a pure form of autosomal dominant hereditary spastic paraplegia. Neurogenetics 6: 79-84. PubMed ID: 15711826

Wang, X., Shaw, W. R., Tsang, H. T., Reid, E. and O'Kane, C. J. (2007). Drosophila spichthyin inhibits BMP signaling and regulates synaptic growth and axonal microtubules. Nat. Neurosci. 10(2): 177-85. PubMed ID: 17220882

date revised: 25 February 2009

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