Advances in Head and Neck Cancer Pain.

Head and neck cancer (HNC) affects over 890,000 people annually worldwide and has a mortality rate of 50%. Aside from poor survival, HNC pain impairs eating, drinking, and talking in patients, severely reducing quality of life. Different pain phenotype in patients (allodynia, hyperalgesia, and spontaneous pain) results from a combination of anatomical, histopathological, and molecular differences between cancers. Poor pathologic features (e.g., perineural invasion, lymph node metastasis) are associated with increased pain. The use of syngeneic/immunocompetent animal models, as well as a new mouse model of perineural invasion, provides novel insights into the pathobiology of HNC pain. Glial and immune modulation of the tumor microenvironment affect not only cancer progression but also pain signaling. For example, Schwann cells promote cancer cell proliferation, migration, and secretion of nociceptive mediators, whereas neutrophils are implicated in sex differences in pain in animal models of HNC. Emerging evidence supports the existence of a functional loop of cross-activation between the tumor microenvironment and peripheral nerves, mediated by a molecular exchange of bioactive contents (pronociceptive and protumorigenic) via paracrine and autocrine signaling. Brain-derived neurotrophic factor, tumor necrosis factor α, legumain, cathepsin S, and A disintegrin and metalloprotease 17 expressed in the HNC microenvironment have recently been shown to promote HNC pain, further highlighting the importance of proinflammatory cytokines, neurotrophic factors, and proteases in mediating HNC-associated pain. Pronociceptive mediators, together with nerve injury, cause nociceptor hypersensitivity. Oncogenic, pronociceptive mediators packaged in cancer cell-derived exosomes also induce nociception in mice. In addition to increased production of pronociceptive mediators, HNC is accompanied by a dampened endogenous antinociception system (e.g., downregulation of resolvins and µ-opioid receptor expression). Resolvin treatment or gene delivery of µ-opioid receptors provides pain relief in preclinical HNC models. Collectively, recent studies suggest that pain and HNC progression share converging mechanisms that can be targeted for cancer treatment and pain management.

Effects of levobupivacaine and ropivacaine on rat sciatic nerve blood flow.

BACKGROUND: Ischaemia is one of the causative mechanisms of peripheral nerve injury, a documented complication of regional anaesthesia. Local anaesthetics per se and/or vasopressor adjuvants may account for changes in peripheral nerve blood flow. The aim of this study was to test the effects of levobupivacaine and ropivacaine in a rat sciatic nerve model with respect to local blood flow and histopathological changes. METHODS: Forty-eight female Sprague-Dawley rats were anaesthetized for left sciatic nerve exposure. After baseline nerve blood flow measurement with a laser Doppler flowmeter, 0.2 ml of one of the following solutions was applied topically to the nerve in a random fashion: saline 0.9%; lidocaine 10 mg ml(-1); levobupivacaine 2.5 mg ml(-1); levobupivacaine 5 mg ml(-1); levobupivacaine 7.5 mg ml(-1); ropivacaine 2 mg ml(-1); ropivacaine 7.5 mg ml(-1); and ropivacaine 7.5 mg ml(-1) plus epinephrine 5 microg ml(-1); all in saline 0.9%. Nerve blood flow was evaluated at 5-min intervals up to 30 min after local application of anaesthetic solution. Three animals per group were killed for histological evaluation 48 h later. Multiple one-way analyses of variance followed by Scheffé's post hoc test was used for statistical analysis. P<0.05 was considered significant. RESULTS: Local anaesthetics at all concentrations tested caused significant reduction in nerve blood flow. The combination of ropivacaine 7.5 mg ml(-1) plus epinephrine did not reduce nerve blood flow to a greater extent than ropivacaine 7.5 mg ml(-1) alone. Low concentrations of levobupivacaine (2.5 and 5 mg ml(-1)) reduced nerve blood flow to the same extent as lidocaine 10 mg ml(-1). No significant histological changes were observed at 48 h. CONCLUSION: Despite acute reductions in peripheral nerve blood flow, significant histopathological changes were not observed in this rat sciatic nerve model after topical application of levobupivacaine and ropivacaine at concentrations relevant to clinical practice.

A retro-inverso Prosaptide D5 promotes a myelination process in developing rats.

The myelinotrophic action of Prosaptide D5 was investigated in developing rats. Sulfatide concentrations in brain and sciatic nerve were determined to assess the development of myelination. Subcutaneous D5-injection significantly increased sulfatide concentrations in both brain and sciatic nerve by 250 and 150% over controls, respectively. D5 promoted ERK phosphorylation in iSC Schwann cells similar to prosaposin. The results showed that D5 treatment stimulated a myelination process in developing rat.

Prosaposin is immunolocalized to muscle and prosaptides promote myoblast fusion and attenuate loss of muscle mass after nerve injury.

Prosaposin is the precursor of the saposins and has both neurotrophic and myelinotrophic activity in vitro and in vivo. Using an antibody specific for the holoprotein, an immunocytochemical survey demonstrated intense staining of adult rat skeletal, cardiac, and smooth muscle cells. Prosaposin immunoreactivity in muscle appears dependent on innervation, as denervated adult rat skeletal muscles showed decreased immunostaining that returned to normal levels after reinnervation. TX14(A), a peptide derived from the neurotrophic sequence of prosaposin, attenuated the decline in muscle mass loss following nerve injury induced by a constricting ligature. In vitro, both L6 myoblasts and primary chick-embryo myoblasts showed similar prosaposin immunopositivity, mainly in myotubes. TX14(A) induced a threefold increase in L6 myoblast fusion during early stages of differentiation without affecting cell proliferation. The fusion process was decreased in vitro in a dose-dependent fashion by addition of a neutralizing anti-prosaposin antibody. These data suggest that, in addition to neurotrophic and myelinotrophic activities, prosaposin has myotrophic properties.

Prosaposin treatment induces PC12 entry in the S phase of the cell cycle and prevents apoptosis: activation of ERKs and sphingosine kinase.

We report that prosaposin treatment induced extracellular signal-regulated kinases (ERKs) and sphingosine kinase activity, increased DNA synthesis, and prevented cell apoptosis. Prosaposin treatment induced pheochromocytoma cells (PC12) to enter the S phase of the cell cycle; this effect was inhibited by the MEK inhibitor PD98059, indicating that prosaposin-induced ERK phosphorylation is required for stimulation of DNA synthesis. The prosaposin effect was also inhibited by pertussis toxin, indicating that the prosaposin receptor is a G-protein-coupled receptor. Prosaposin rescued PC12 cells from apoptosis induced by staurosporine or ceramide. Sphingosine kinase activity was increased by prosaposin treatment. We propose that this effect is a mechanism underlying the proliferative and anti-apoptotic functions of prosaposin. Prosaposin appears to be a key regulatory factor in the ceramide-S-1-P rheostat, which regulates cell fate.

Prosaposin-derived peptides enhanced sprouting of sensory neurons in vitro and induced sprouting at motor endplates in vivo.

Prosaposin exhibits neurotrophic factor properties that are localized to a 12-amino acid sequence located in the amino terminal portion of the saposin C domain. Prosaptides are peptides derived from the neurotrophic portion of prosaposin; these have been previously reported to be bioactive in neuroblastoma cell lines in vitro. We report that prosaptides were also bioactive in explants of adult primary sensory neurons by dose-dependently increasing both the number (3- to 4-fold) and elongation of these neurites by 50%. Local injection of prosaptides into the gluteus muscle of adult mice also induced sprouting at the motor endplate. Our results indicate that prosaptides are potent neuritogenic factors for both sensory and motor neurons of adult peripheral nerve.

Transforming growth factor-beta1 and glial growth factor 2 reduce neurotrophin-3 mRNA expression in cultured Schwann cells via a cAMP-dependent pathway.

The aim of the study was to determine which factors regulated the expression of neurotrophin-3 (NT-3) mRNA in cultured primary Schwann cells derived from sciatic nerve of neonatal rats. Treatment of primary Schwann cells with the adenylate cyclase activator, forskolin, or the cAMP agonist, 8-Br-cAMP, induced a significant reduction in NT-3 transcript levels. Transforming growth factor-beta1 (TGF-beta1) and glial growth factor 2 (GGF(2)) also reduced the levels of NT-3 mRNA in a dose and time-dependent manner. Treatment with nerve growth factor, brain-derived neurotrophic factor, NT-3, ciliary neurotrophic factor or interleukin-1beta was without effect. The TGF-beta1, GGF(2) and forskolin dependent reduction in NT-3 mRNA levels involved a destabilization of transcripts which was antagonised by co-treatment with cycloheximide. The cAMP-dependent protein kinase A (PKA) inhibitor, H-89, blocked the reduction in levels of NT-3 mRNA induced by TGF-beta1, GGF(2) and forskolin. The data show that the effects of TGF-beta1, GGF(2) and forskolin on the downregulation of NT-3 mRNA, at least in part, were due to a post-transcriptional event involving a labile protein intermediate under the control of PKA. The results suggest that the down-regulation of NT-3 mRNA in Schwann cells at a site of peripheral nerve damage may be mediated via a cAMP-dependent pathway and possibly involve neuroma-related elevations in TGF-beta1 and GGF(2).

Phosphatidylinositol 3-kinase and Akt protein kinase mediate IGF-I- and prosaptide-induced survival in Schwann cells.

Withdrawal of trophic factors necessary for Schwann cell survival regulates Schwann cell number during development and after nerve injury. In the present study, we identified signaling pathways involved in Schwann cell survival by prosaposin, prosaptides (peptides incorporating the neurotrophic sequence of prosaposin), and insulinlike growth factor-I (IGF-I). When postnatal Schwann cells were placed in low serum medium, cells underwent abrupt shrinkage, condensation of nuclei occurred, and smooth rounded apoptotic bodies appeared. Dose-response studies of cell death, measured by lactate dehydrogenase (LDH) release, demonstrated that both prosaptide TX14(A) and IGF-I dose dependently reduced cell death in primary Schwann cells. Histone-associated DNA fragmentation enzyme-linked immunosorbent assay, showed a 10- and 14-fold increase in apoptosis after 4 and 24 hr in low serum medium, respectively, that was reduced by prosaposin, TX14(A), or IGF-I. Phosphatidylinositol 3-kinase (PI3K) inhibitors, wortmannin or LY294002, blocked the survival effects of both TX14(A) and IGF-I. In contrast, only TX14(A) anti-apoptotic activity was blocked by the MEK inhbitor, PD98059, although TX14(A) and IGF-I are potent activators of extracellular regulated kinases in Schwann cells. Phosphorylation of the PI3K signaling target, Akt, was measured; TX14(A) and IGF-I increased Akt activity by 12-fold and 22-fold, respectively, that was inhibited by LY294002. These findings indicate that prosaposin and IGF-I use the PI3K/Akt pathway to induce survival of Schwann cells.

Secretion of prosaposin, a multifunctional protein, by breast cancer cells.

Western blotting and immunodetection with three antibodies were used to probe conditioned media of breast cancer cells (MDA231, MDA435, MCF-7) for prosaposin, a lysosomal protein that occurs in milk. It was readily detected in media from these cells, and from that of an sv40-transformed mammary epithelial cell, HBL100, but not from medium of human neural tumor cells (SK-N-MC). In cultures of MCF-7 cells, the prosaposin pattern of secretion over time closely resembled that of procathepsin D, another lysosomal protein occurring in milk. Supplementing medium with 17beta-estradiol (0. 1-100 nM) dose dependently increased secretion of both proteins after 48 h without changes in cell viability. The influence of 17beta-estradiol on secretion could play a role in the trophic activity of prosaposin in cellular differentiation and cell death protection. In concert with other lysosomal proteins in the tumor environment, such as procathepsin D, prosaposin may be a factor in eliminating barriers to tumor metastasis by facilitating hydrolysis of membrane glycolipids. The number of milk proteins known to be secreted by breast cancer cells is growing. There is evidence that at least some of these may be secreted in an endocrine manner in the normal, non-lactating breast.

Prosaposin gene expression and the efficacy of a prosaposin-derived peptide in preventing structural and functional disorders of peripheral nerve in diabetic rats.

We have recently demonstrated that prosaposin is a neurotrophic and myelinotrophic factor with the active trophic sequence located at the N-terminal region of the saposin C domain. There are also reports that prosaposin mRNA is increased distal to a physical nerve injury and that exogenous prosaposin treatment induces subsequent neuronal sprouting, suggesting involvement in repair processes. In the present study, we show that prosaposin mRNA is significantly (p < 0.05) elevated in the peripheral nerve of streptozotocin-diabetic rats, a model of insulin-deficient diabetes in which nerve injury arises from the metabolic trauma of hyperglycemia and its consequences. A 14 amino acid peptide derived from the neurotrophic region of prosaposin prevented the development of deficits in both large and small fiber function caused by diabetes in rats. The dose-dependent prevention of nerve conduction slowing by TX 14(A) was accompanied by preservation of axonal caliber and sodium-potassium ATPase activity, while prevention of thermal hypoalgesia was associated with attenuation of the decline in nerve substance P levels. It is concluded that nerve subject to the metabolic injury of uncontrolled diabetes responds by increasing prosaposin gene expression, and that prosaposin-derived neurotrophic peptides may provide a novel therapeutic approach to treatment of diabetic and other peripheral neuropathies.

Prosaposin: a myelinotrophic protein that promotes expression of myelin constituents and is secreted after nerve injury.

Recently, we demonstrated that prosaposin and prosaptides (peptides encompassing the neurotrophic sequence in prosaposin) prevent cell death and increase extracellular regulated kinase (ERK) phosphorylation and sulfatide content in primary Schwann cells or oligodendrocytes (Hiraiwa et al., 1997a). Here, we examine the effect of prosaptide on other myelin constituents, on Schwann cell morphology and proliferation, and characterize the time course of expression of prosaposin protein after sciatic nerve injury. After 24 h of treatment with 10 nM TX14(A), a 14-mer prosaptide, the specific activity of UDP-galactose:ceramide galactosyltransferase (GalT) in primary Schwann cells was increased by 150% over controls. Under the same conditions, the maximum content of sulfatide increased 3-fold over controls after 48 h of treatment. Northern blot analysis, probed with oligonucleotide sequences from the GalT and P0 cDNAs, revealed that the mRNA levels of GalT and P0 protein were elevated about 30 and 200%, respectively, over controls after 24 h of treatment with TX14(A). Treatment of primary Schwann cells with TX14(A) also induced a morphological change at 10 nM; the peptide-treated cells had a bipolar (spindle-shaped) appearance after 48 h of treatment, compared to control cells which were irregular and multipolar. TX14(A) did not induce cell proliferation, indicating that TX14(A), unlike IGF-I, is not mitogenic. After sciatic nerve transection, Western blot analysis demonstrated the presence of intact prosaposin in tubular fluid in a silicon chamber into which the proximal and distal nerve stumps were sutured. The concentration of prosaposin in the fluid was maximum after 9 days post-surgery and returned to normal after 28 days post-surgery. In uninjured and injured nerve, prosaposin immunolocalized to the smooth muscle of epineurial and endoneurial vessels. These findings indicated that sciatic nerve secreted prosaposin after injury and that prosaposin is a naturally occurring injury-repair protein which acts to prevent degeneration and to promote regeneration of peripheral nerves.

Necrotizing myopathy induced by overexpression of interferon-gamma in transgenic mice.

A transgenic mouse model has been established in which the cytokine interferon-gamma (IFN-gamma) is overexpressed through the action of the acetylcholine receptor epsilon promoter acting at the neuromuscular junction. While originally developed as a model for the study of the pathogenesis of myasthenia gravis, there are important differences from both human myasthenia gravis and its animal model, experimental autoimmune myasthenia gravis. By 4 months of age there was a well-established inflammatory, predominantly necrotizing myopathy, with marked dystrophic calcification. Dystrophic and degenerative changes in terminal axons and adjacent Schwann cells were also apparent. The acetylcholine receptor was not the primary target of the inflammatory response, since at 10 weeks of age the receptor content was not decreased and antibodies were not detected bound to the receptor. The IFNgamma transgenic mouse model may provide a clinically relevant model of necrotizing myopathy for investigation of the pathological changes associated with, and presumably precipitated by, overexpression of the pro-inflammatory cytokine interferon-gamma on the neuromuscular junction, intramuscular nerves and myofibers.

Colocalization and complex formation between prosaposin and monosialoganglioside GM3 in neural cells.

Prosaposin, the precursor of saposins A, B, C, and D, was recently identified as a neurotrophic factor in vitro as well as in vivo. Its neurotrophic activity has been localized to a linear 12-amino acid sequence located in the NH2-terminal portion of the saposin C domain. In this study, we show the colocalization of prosaposin and ganglioside GM3 on NS20Y cell plasma membrane by scanning confocal microscopy. Also, TLC and western blot analyses showed that GM3 was specifically associated with prosaposin in immunoprecipitates; this binding was Ca2+-independent and not disassociated during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The association of prosaposin-GM3 complexes on the cell surface appeared to be functionally important, as determined by differentiation assays. Neurite sprouting, induced by GM3, was inhibited by antibodies raised against a 22-mer peptide, prosaptide 769, containing the neurotrophic sequence of prosaposin. In addition, pertussis toxin inhibited prosaptide-induced neurite outgrowth, as well as prosaptide-enhanced ganglioside concentrations in NS20Y cells, suggesting that prosaposin acted via a G protein-mediated pathway, affecting both ganglioside content and neuronal differentiation. Our findings revealed a direct and tight GM3-prosaposin association on NS20Y plasma membranes. We suggest that ganglioside-protein complexes are structural components of the prosaposin receptor involved in cell differentiation.

Identification of a neurotrophic sequence in erythropoietin.

Erythropoietin (Epo) is a hematopoietic factor that facilitates erythroid progenitor cell proliferation and differentiation. Recently, trophic effects of Epo have been observed in central cholinergic neurons. We have confirmed the neurotrophic factor activity of Epo and moreover, demonstrated sprouting and signaling by Epo in neural cells. Further, we have identified a 17-mer peptide sequence (epopeptide AB) in Epo (AEHCSLNENITVPDTKV) with activity similar to that of the holoprotein. This peptide induces differentiation and prevents cell death in both murine NS20Y and human SK-N-MC neuroblastoma cell lines. However, epopeptide AB does not promote the proliferation of erythropoietic cell lines or mouse primary spleen cells. The biological activities in neural cells were blocked by the addition of an antibody to the extracellular domain of the Epo receptor, indicating that the bioactive effects of epo-peptide AB in neural cells are Epo receptor mediated. Both epopeptide AB and Epo stimulated phosphorylation of ERKs in PC12 cells. When epopeptide AB or Epo was locally injected into mice, the frequency of motor end plate sprouting in adjacent muscles increased in a manner similar to that induced by CNTF. These findings indicate that neural cells and not hematological cells respond to a peptide sequence within erythropoietin and suggests that Epo may have separate domains for neurotrophic and hematotrophic function.

Prosaptide prevents paclitaxel neurotoxicity.

Paclitaxel (Taxol), a chemotherapeutic agent used to treat breast and ovarian tumors, has been reported to induce a predominantly sensory neuropathy. Co-treatment with neurotrophic factors and paclitaxel has been proposed for preventing or reversing paclitaxel-induced peripheral neuropathy. Prosaposin, the precursor of saposins A, B, C and D was recently identified as a neurotrophic factor and was reported to facilitate nerve regeneration in vivo. Peptides (prosaptides) encompassing the neurotrophic sequence located in the saposin C domain, have neurotrophic activity similar to the holoprotein (O'Brien et al. 1995). In the present study, we investigated the effect of a 14-mer prosaptide, TX14(A), or a 22-mer prosaptide, 769P, on paclitaxel-induced neutrotoxicity in vitro and in vivo. Paclitaxel treatment (1 microM) decreased cell viability of both PC12 and Schwann cells. TX14(A) (10 nM) prevented paclitaxel-induced loss of cell viability in PC12 cells but not in Schwann cells. Systemic injections (i.p.) of paclitaxel (1.2 mg/kg/day) given five times per week for three weeks (cumulative dose 18 mg/kg) or given every third day (25, 12.5 and 12.5 mg/kg) for 10 ten days (cumulative dose 50 mg/kg) in adult rats induced thermal hypoalgesia that was not accompanied by morphological changes in the sciatic nerve or changes of nerve conduction velocity. Co-administration of paclitaxel with prosaptides (cumulative dose 3 or 10 mg/kg) prevented paclitaxel-induced thermal hypoalgesia. In the short-term high dose study, paclitaxel treated rats lost 10% of their body weight, had reduced erythrocyte counts, hematocrit and hemoglobin concentrations which were not prevented by treatment with prosaptide. TX14(A) did not diminish paclitaxel cytotoxicity of breast cancer cells in vitro. These findings suggest that prosaptide prevents the neurotoxic effects of paclitaxel while not interfering with its anti-tumor activity.

Prosaptide activates the MAPK pathway by a G-protein-dependent mechanism essential for enhanced sulfatide synthesis by Schwann cells.

Prosaposin, the precursor of saposins A, B, C, and D, was recently reported to be a neurotrophic factor in vivo and in vitro. The neurotrophic region of prosaposin has been localized to a 12-amino acid sequence within the saposin C domain and has been used to derive biologically active synthetic peptides (14-22 residues), called prosaptides. Treatment of primary Schwann cells and an immortalized Schwann cell line, iSC, with a 14-mer prosaptide, TX14(A) (10 nM), enhanced phosphorylation of mitogen-activated kinases ERK1 (p44 MAPK) and ERK2 (p42 MAPK) within 5 min, which was blocked by 4 h pretreatment with pertussis toxin. Furthermore, incubation of Schwann cells with the nonhydrolyzable GDP analog GDP-betaS inhibited TX14(A)-induced ERK phosphorylation. TX14(A) enhanced the sulfatide content of primary Schwann cells by 2.5-fold, which was inhibited by pretreatment with pertussis toxin or the synthetic MAP kinase kinase inhibitor PD098059. In addition, TX14(A) increased the tyrosine phosphorylation of all three isoforms of the adapter molecule, Shc, which coincided with the association of p60Src and PI(3)K. Inhibition of PI3(K) by wortmannin blocked TX14(A)-induced ERK phosphorylation. These data demonstrate that TX14(A) uses a pertussis toxin-sensitive G-protein pathway to activate ERKs, which is essential for enhanced sulfatide synthesis in Schwann cells.

Prosaposin receptor: evidence for a G-protein-associated receptor.

Prosaposin, the precursor of sphingolipid activator protein (saposins A-D), has been identified as a neurotrophic factor capable of inducing neural differentiation and preventing cell death. The putative prosaposin receptor was partially purified from baboon brain membranes by affinity chromatography using a saposin C-column. The purified preparation gave a single major protein band with an apparent molecular weight of 54 kDa on SDS-PAGE. Affinity cross-linking of 11 kDa 125I-saposin C demonstrated the presence of a 66 kDa product, indicative of an apparent molecular weight of 55 kDa for the receptor. A GTP gamma S-binding assay using cell membranes from SHSY5Y neural cells demonstrated agonist stimulated binding of [35S]-GTP gamma S upon treatment with prosaptide TX14(A) a peptide from the neurotrophic region; maximal binding was obtained at 2 nM. TX14(A) stimulated binding was abolished by prior treatment of SHSY5Y cells with pertussis toxin and by a scrambled and an all D-amino acid-derivative of the 14-mer. A 14-mer mutant prosaptide (6N-->6D) competed with TX14(A) with a Ki of 0.7 nM. Immunoblot analysis using an antibody against the G0 alpha subunit demonstrated that the purified receptor preparation contained a 40 kDa reactive band consistent with association of G0 alpha and the receptor. These findings indicate that the signaling induced by prosaposin and TX14(A) is generated by binding to a G0-protein associated receptor.

Cell death prevention, mitogen-activated protein kinase stimulation, and increased sulfatide concentrations in Schwann cells and oligodendrocytes by prosaposin and prosaptides.

Prosaposin, the precursor of saposins A, B, C, and D, was recently identified as a neurotrophic factor. Herein prosaposin was found to increase sulfatide concentrations in primary and transformed Schwann cells (iSC) and oligodendrocytes (differentiated CG4 cells). Of the four mature saposins, only saposin C was found to increase sulfatide concentrations in these cell types. A similar result was obtained by using peptides (prosaptides) encompassing the neurotrophic sequence located in the saposin C domain. Dose-response curves demonstrated maximal enhancement by saposin C and prosaptides at low nanomolar concentrations (5-10 nM). The increase in sulfatide concentration by a 14-mer prosaptide, TX14(A), in CG4 oligodendrocytes was about 3-fold greater than in primary Schwann cells. A mutant prosaptide with a single amino acid replacement of Asn --> Asp was inactive. Prosaptides did not induce cell proliferation of primary Schwann cells, iSC cells, or CG4 oligodendrocytes but nanomolar concentrations of prosaptides prevented cell death of iSC cells and CG4 oligodendrocytes. Immunoblot analysis demonstrated that phosphorylation of both mitogen-activated protein kinase p-42 and p-44 isoforms were enhanced 3- to 5-fold after 5 min of treatment with prosaptides at concentrations of 1-5 nM. These findings suggest that prosaposin and prosaptides bind to a receptor that initiates signal transduction to promote myelin lipid synthesis and prolong cell survival in both Schwann cells and oligodendrocytes. Prosaposin may function as a myelinotrophic factor in vivo during development and repair of myelinated nerves explaining the deficiency of myelin observed in prosaposin-deficient mice and humans.

Induction of MAPK phosphorylation by prosaposin and prosaptide in PC12 cells.

Prosaposin is a 66 kDa glycoprotein which has neurotrophic activity in vitro and in vivo. The neurotrophic sequence (8CEFLVKEVTKLIDNNKTEKEI29L) within prosaposin has been located to the amino terminal portion of the saposin C domain. This 22-mer peptide, prosaptide, has neurotrophic activity equivalent to prosaposin. We present binding studies using 125I-prosaposin and 125I-prosaptide which revealed a single class of specific binding sites with a Kd of 2.5 nM and 18.3 nM, respectively. Both prosaposin and prosaptide rapidly stimulated protein tyrosine phosphorylation in PC12 cells and increased phosphorylation of MAPK 20-fold especially of p44 MAPK which peaked at 5 minutes of stimulation and then rapidly declined. Treatment of PC12 cells with a mutant 22-mer prosaptide (21Asn to 21Asp) did not induce phosphorylation. These findings suggest a role for MAPK in signal transduction by prosaposin.