TX14(A), a prosaposin-derived peptide, reverses established nerve disorders in streptozotocin-diabetic rats and prevents them in galactose-fed rats.

Recently, TX14(A), a prosaposin-derived neurotrophic peptide, was shown to prevent both large and small fiber deficits in streptozotocin diabetes. Here, the efficacy of TX14(A) in reversing established nerve conduction disorders in streptozotocin diabetes, a model of insulin deficiency, and preventing them in galactose feeding, an insulin-replete model of polyol pathway flux, was investigated. Following streptozotocin injection (50 mg/kg ip), TX14(A) treatment (1 mg/kg ip thrice weekly) was initiated in half of the animals. After 8 wk, treatment was begun in half of the untreated animals and discontinued in half of the treated animals, and the experiment continued for 6 wk. TX14(A) reversed established motor and sensory nerve conduction deficits in streptozotocin-diabetic rats and the impact of previous treatment was still evident 3 wk after withdrawal. With the onset of 40% galactose feeding, the same dose of TX14(A) was given to half of the control and half of the galactose-fed animals for 16 wk. TX14(A) was without effect in control animals but it attenuated motor and sensory nerve conduction deficits in galactose-fed rats, an effect associated with amelioration of axonal dwindling in the sciatic nerve. These observations extend the therapeutic utility of TX14(A) and highlight its potential in treating established diabetic neuropathy.

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.

A fragment of prosaposin (SGP-1) from rooster sperm promotes sperm-egg binding and improves fertility in chickens.

A protein isolated from the supernatant of cryopreserved rooster sperm was found to increase the capability of cryopreserved rooster sperm to bind in vitro to the perivitelline membrane of a chicken egg and substantially raise fertility after artificial insemination (AI). That activity was partially purified and termed universal primary sperm-egg binding protein (UPSEBP). Insufficient protein remained from 6 x 10(11) sperm, despite retention of bioactivity, to allow sequencing. We deduced that the protein may be related to prosaposin (also termed SGP-1, for sulfated glycoprotein-1), and we used published amino acid sequences of prosaposin as a guide for synthesis of peptides. Certain peptides were found to increase in vitro sperm-egg binding and increase fertility of frozen-thawed or fresh rooster sperm, in a manner similar to semipurified UPSEBP. Active epitopes were in a 60 amino acid sequence, reflecting the intervening sequence between saposins A and B, plus short extensions into saposins A and B. Highest activity was found when this synthetic peptide was oxidized to form a disulfide bond between terminal cysteines. Antibody against a synthetic peptide consisting of 58 of these 60 amino acids bound to a 7-9 kilodalton protein in UPSEBP. Collectively, the data support the conclusion that UPSEBP is a fragment of prosaposin. Because prosaposin is in semen in humans and animals, these observations have broad implications for possible cause and therapy of one type of subfertility.

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.

Protection of sensory function and antihyperalgesic properties of a prosaposin-derived peptide in diabetic rats.

BACKGROUND: Short-term diabetes causes sensory disorders in rats ranging from thermal hypoalgesia to exaggerated behavioral responses to other sensory stimuli. As impaired neurotrophic support may promote sensory nerve disorders during diabetes, the authors investigated whether TX14(A), a neurotrophic peptide derived from prosaposin, was able to ameliorate nerve disorders in diabetic rats. METHODS: TX14(A) was delivered by intraperitoneal or intrathecal injection to control or streptozotocin-diabetic rats in either single or multiple (three times weekly) dose regimens. Efficacy was measured against diabetes-induced disorders of sensory nerve conduction velocity, paw withdrawal latency to radiant heat, tactile response thresholds to von Frey filaments, and flinching after paw formalin injection. RESULTS: Prolonged TX14(A) treatment of diabetic rats prevented the progressive decline in large sensory fiber conduction velocity in the sciatic nerve, development of paw thermal hypoalgesia, and increased flinching after paw formalin injection. The effect on formalin hyperalgesia persisted for 48 h but not 72 h after injection. No effects were noted in control rats. A single injection of TX14(A) 30 min before testing did not alter thermal response latencies in control or diabetic rats but prevented formalin hyperalgesia in diabetic rats. Tactile allodynia and the prolonged paw thermal hyperalgesia to radiant heat after intrathecal delivery of substance P were also dose-dependently ameliorated in diabetic rats by a single injection of TX14(A), whereas no effects were observed on the responses to these tests in control rats. CONCLUSIONS: TX14(A) exhibits both neuroprotective and acute antihyperalgesic properties in diabetic rats without altering normal nociceptive function.

Retro-inverso prosaptide peptides retain bioactivity, are stable In vivo, and are blood-brain barrier permeable.

Prosaptide (trademark of Myelos Corporation, San Diego, CA) peptides are based on the 14-amino-acid neurotrophic sequence of human prosaposin and, like the parent protein, have potent neurotrophic and neuroprotective properties. We previously examined the in vivo stability of a series of bioactive Prosaptide peptides and designed peptides with increased enzymatic stability in the central and peripheral nervous systems. In this article, we examined the stability, biological activity, and permeability of the blood-brain barrier to retro-inverso Prosaptide peptidomimetics. Retro-inversion both reverses the primary sequence and replaces L-amino acids with D-amino acids. We examined the bioactivity of five peptidomimetics, Prosaptides D1-D5. Prosaptide D1, a peptide containing all D-amino acids with the primary sequence intact, was inactive. However, four retro-inverso peptidomimetics, Prosaptides D2-D5 retained bioactivity in neurite outgrowth and [(35)S]GTPgammaS binding assays. We focused on Prosaptide D4 as a prototypical retro-inverso Prosaptide peptidomimetic for further study. (125)I-Prosaptide D4 remained intact in brain or serum for 60 min after i.v. administration and was transported across the blood-brain barrier with a unidirectional influx constant of 2.5 x 10(-4) ml. g(-1). min(-1). We conclude that retro-inverso Prosaptide peptidomimetics are excellent candidates for development as therapeutics for central nervous system neurodegeneration.

Neuroprotective effect of retro-inverso Prosaptide D5 on focal cerebral ischemia in rat.

Prosaposin (the precursor of saposins A-D) has been identified as a neurotrophic factor in vitro and in vivo. In this study, a novel 11-mer retro-inverso peptidomimetic, Prosaptide D5, was injected i.m. to assess its effectiveness in a rat ischemic model produced by reversible total occlusion of the left middle cerebral artery (MCA). Prosaptide (300 microg/kg, i.m.) injected 3 h after reversible occlusion reduced brain infarct area by 56% compared with a saline group (p < 0.01) at 21 h of reperfusion. A similar injection of D5 6h after occlusion produced a 32% decrease.

Designing stable blood-brain barrier-permeable prosaptide peptides for treatment of central nervous system neurodegeneration.

Prosaposin-derived peptides have been proposed as potential therapeutics for neurodegenerative diseases. Previously, we reported that the minimal length for bioactivity was 12 amino acids, and key amino acids were described based on interspecies conservation. In this article, we have further investigated the sequence requirements for bioactive Prosaptide (Myelos Corporation) peptides in terms of length and amino acid identity. The use of Prosaptide peptides for treatment of central nervous system (CNS) disorders requires that they are stable in vivo. Although robust effects of our prototypical peptide Prosaptide TX14(A) have been shown in the peripheral nervous system, minimal success has been achieved when treating the CNS in rats and this may be due to instability of Prosaptide TX14(A) in brain. Herein, we demonstrate that, indeed, Prosaptide TX14(A) is rapidly degraded in the brain and we have attempted to design prosaptides with increased CNS stability. One peptide, Prosaptide TX15-2, shows increased stability in brain and may be of use in the treatment of CNS disorders. With the aim of designing Prosaptide peptides that may be systemically administered for CNS treatment, we have investigated the blood-brain barrier permeability of Prosaptide TX14(A) and TX15-2. Both of these peptides cross the blood-brain barrier via a nonspecific mechanism.

Prosaptide D5 reverses hyperalgesia: inhibition of calcium channels through a pertussis toxin-sensitive G-protein mechanism in the rat.

A retro-inverso 11-mer peptidomimetic of prosaposin, Prosaptide D5, induced neurite outgrowth in NS20Y neuroblastoma cells and enhanced [35S]GTPgammaS binding to rat synaptosomal membrane at low nanomolar concentrations similar to prosaposin. Intramuscular injection of D5 ameliorated thermal hyperalgesia in the Seltzer rat model of neuropathic pain, returning paw withdrawal latency to control levels within 3 h after treatment. The effect was sustained for at least 48 h after injection. Prosaposin and D5 inhibited K+-stimulated synaptosomal 45Ca2+ uptake similar to omega-conotoxin MVIIC, demonstrating that both effectors modulated voltage-dependent calcium channels (VDCC); inhibition was largely abolished by pretreatment with pertussis toxin before D5 treatment. The results suggest a mechanism whereby VDCC are modulated by a pertussis toxin-sensitive G-protein coupled receptor; D5 binds to this receptor and thereby ameliorates hyperalgesia in the Seltzer model of neuropathic pain.

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.

Intracellular transport of acid beta-glucosidase and lysosome-associated membrane proteins is affected in Gaucher's disease (G202R mutation).

Gaucher's disease (GD) is caused by an inherited deficiency of acid beta-glucosidase with storage of glucosylceramides in the lysosomes of macrophages. This study identifies a G202R mutation in the acid beta-glucosidase gene in an infant with severe neuronopathic (type 2) GD and only slightly reduced acid beta-glucosidase activity. Western blot analysis, pulse chase experiments, and the thin frozen section immunogold method were used to analyse the implications of this mutation on the pathogenesis, clinical heterogeneity and diagnostic evaluation of GD. The results show that acid beta-glucosidase persists in the patient's fibroblasts as a mannose-rich polypeptide in the endoplasmic reticulum and is not transported to the lysosomes. By contrast, high expression of the lysosome-associated membrane proteins LAMP-1 and LAMP-2, saposin C, and cathepsin D was observed in the patient's lysosomes. Immunogold labelling of the integral membrane proteins LAMP-1 and LAMP-2 increases significantly at the cell surface of Kupffer cells and fibroblasts as well as at the apical membrane of hepatocytes. In addition, LAMP-1 and LAMP-2 associate with the bilayer of stored glucosylceramide. It is concluded that defective intracellular transport of mutant acid beta-glucosidase from the endoplasmic reticulum to lysosomes leads to a more severe clinical phenotype than the residual enzyme activity may indicate. Furthermore, the detection of LAMP in the tubular bundles of undigested glucosylceramides, as well as their increased concentration at the surfaces of the affected cells, suggests that these proteins play a role in the storage or removal of substrate in GD. Intracellular targeting of acid beta-glucosidase and LAMP contributes to the broad phenotypic heterogeneity of GD.

Reversal of thermal hyperalgesia in a rat partial sciatic nerve ligation model by Prosaptide TX14(A).

We used the partial sciatic nerve ligation (PSL) model of Seltzer to assess thermal hyperalgesia after administration of Prosaptide 14-mer, TX14(A). At a dose of 200 microg/kg in Wistar rats, subcutaneous delivery of TX14(A) reversed thermal hyperalgesia at 3 and 24 h. Values declined at 48 h and returned to baseline at 72 h. A dosing study of TX(14)A gave a dependent response with 100 microg/kg having a similar potency to the 200 microg/kg study with 50 and 10 microg/kg responding somewhat lower. When TX(14)A was administered every fourth day for 12 days at 100 microg/kg, 24 h post injection values returned to baseline each time. Our results suggest that Prosaptide may have potential for therapeutic use in neuropathic pain syndromes in humans.

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.

Administration of prosaposin ameliorates spatial learning disturbance and reduces cavity formation following stab wounds in rat brain.

The effectiveness of prosaposin as a neurotrophic factor was investigated using rats with bilateral stab wounds, injecting 240 ng per day of prosaposin for 3 days. In Morris water maze task, after 3 weeks postoperation, the stab-wounds rats show significant impairment in acquisition compared with the sham-operated rats. In the transfer test the mean number of crossings of the platform place in stab-wounds was significantly lower than that in sham-operated rats (P < 0.01). The stab-wounds rats treated with prosaposin showed significant improvement (P < 0.05). The cavities following stab wounds in the rats treated with prosaposin were significantly smaller than those in the rats treated with (P < 0.05). Our data support that prosaposin is likely to be a new agent for brain injury.

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.