Rearrangement of substituted propionanilides.

The rearrangements of substituted anilides of propionic acid to amino ketones in a solvent medium were studied, employing bismuth chloride as a catalyst. Rearranged compounds were then characterized by qualitative tests and spectral data.

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.

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.

Focally administered nerve growth factor suppresses molecular regenerative responses of axotomized peripheral afferents in rats.

Effects of delivery of nerve growth factor, from a catheterized osmotic mini-pump to the proximal stump of a transected sciatic nerve, were compared with the effects of normal saline. A pilot measured retrograde axonal transport of nerve growth factor to determine a pump concentration which raised axonal transport ipsilaterally, but not contralaterally. The effects of this delivery over 12 days were then determined on expression of growth-associated protein-43, trkA, p75NTR and preprotachykinin A ipsilateral and contralateral to the pump in dorsal root ganglia at L4 and L5 (pooled). Ganglionic expression was measured both as messenger RNA and protein. Axotomy (saline pumps) increased growth-associated protein-43 messenger RNA (318 +/- 14%: all changes are percent of contralateral, non-axotomized ganglia with saline pumps) and immunoreactivity (431 +/- 43%). The increase was significantly less (P < 0.001) ipsilateral to nerve growth factor pumps (191 +/- 45%). Axotomy reduced expression of p75NTR (messenger RNA: 52 +/- 17%, P < 0.01; immunoreactivity: 74 +/- 3%, P < 0.05). These decreases were converted to increases by nerve growth factor delivery (respectively 143 +/- 40% and 281 +/- 67%; both P < 0.01). With trkA, axotomy decreased the expression of the messenger RNA (68 +/- 40%, P < 0.01) and of the primary translation product--110,000 mol. wt protein (55 +/- 12%, P < 0.01)--but not the fully glycosylated trkA protein (mol. wt 145,000). Nerve growth factor delivery did not affect trkA expression. Axotomy reduced messenger RNA for the substance P precursor, preprotachykinin A, to 42 +/- 17% (P < 0.01) and this reduction was prevented by nerve growth factor treatment. We suggest that the primary effect of nerve growth factor on axotomized C-fibres is not to promote regeneration, although that may be its secondary effect via an action on Schwann cells. It is possible that reduced neuronal sensitivity to nerve growth factor during regeneration is advantageous in suppressing nociception.

Impaired molecular regenerative responses in sensory neurones of diabetic rats: gene expression changes in dorsal root ganglia after sciatic nerve crush.

This study investigated changes in gene expression in lumbar dorsal root ganglia (DRG), contralateral and ipsilateral to a sciatic nerve crush in control and streptozotocin (STZ)-induced diabetic rats. After 10 weeks of diabetes, the left sciatic nerves of all rats were crushed at mid-thigh level, and the rats were maintained for a further 2 weeks. Northern blots, with internal standards, were made from L4 and L5 (pooled) DRG on each side to compare RNA hybrids from ganglia attached to crushed nerves with those attached to intact nerves. The expression of growth-associated proteins, GAP-43 and Talpha1 alpha-tubulin mRNA in DRG, was stimulated (all P < 0.05) by crush injury in control and diabetic rats. Steady-state expression of transcripts for neurofilament (NF) proteins (NF-L, NF-H) and the high-affinity NGF receptor, trkA was decreased by diabetes in the contralateral ganglia to the crush (all P < 0.05). Crush injury further decreased expression of these transcripts in both control and diabetic rats (all P < 0.05). This reduced expression of mRNA coding for both growth-associated proteins, and neurofilament proteins in ganglia of diabetic rats could participate in the reduced competence of the regenerative response to nerve crush.

Acidic fibroblast growth factor enhances neurite outgrowth and stimulates expression of GAP-43 and T alpha 1 alpha-tubulin in cultured neurones from adult rat dorsal root ganglia.

The affects of acidic fibroblast growth factor (FGF-1) on neurite outgrowth were investigated and related to the levels of mRNA encoding the growth associated protein genes GAP-43 and the neuron specific alpha-tubulin, T alpha 1, in primary cultures of dorsal root ganglia (DRG) neurones from adult rats. In response to FGF-1, sensory neurones produced a high level of neurite outgrowth which was highest (2.70 +/- 0.09-fold over control) at 50 ng/ml of FGF-1. Treatment with 250 ng/ml of a monoclonal anti-nerve growth factor (NGF) antibody, which can block the similar increase in neurite outgrowth produced by 0.1 ng/ml of NGF, did not antagonise the response to 50 ng/ml of FGF-1, showing that FGF-1 has effects on regeneration in adult sensory neurones not mediated via NGF. The estimated ED50 value for FGF-1 was 20 ng/ml for induction of neurite outgrowth. Treatment with FGF-1 elevated the level of mRNA for GAP-43 to 2.9-fold at 50 ng/ml and T alpha 1 alpha-tubulin by 9.2-fold at 10 ng/m, though the FGF-1 concentration/effect range for stimulation of these two mRNA was different from that for neurite outgrowth.

Human recombinant nerve growth factor replaces deficient neurotrophic support in the diabetic rat.

Manipulation of neurotrophic support is a developing strategy for new therapy aimed at neurodegenerative diseases. This study demonstrates reduced content and retrograde transport of endogenous nerve growth factor (NGF) in sciatic nerve of diabetic rats. There were also reductions in the diabetic rats in NGF protein and mRNA in skin and muscle of the hindlimb. These deficits correlated with reductions in substance P and calcitonin gene-related peptide--both products of NGF-influenced genes in primary afferents. These manifestations of deficient neurotrophic support were corrected by intensive insulin treatment and surmounted by administration of exogenous human recombinant NGF in a dose-related manner. Impaired neurotrophic support may, therefore, participate in the pathogenesis of diabetic and other peripheral neuropathies.

Changes in nerve growth factor and preprotachykinin messenger RNA levels in the iris and trigeminal ganglion in diabetic rats; effects of treatment with insulin or nerve growth factor.

This study was designed to explore effects of experimental diabetes mellitus on expression of substance P in the trigeminal ganglion and of nerve growth factor (NGF) in the iris. Rats with streptozotocin-diabetes showed an increase in NGF mRNA in the iris (P < 0.01) which was corrected by insulin treatment. There was also an increase in the levels of mRNA for the substance P precursor, preprotachykinin (PPT), in the trigeminal ganglion of these animals, despite there being no change in GAP-43 mRNA. Since expression of both of these peptides is sensitive to NGF in vitro, we examined the effect of treatment of diabetic rats with NGF at three different doses (0.2, 0.5 and 1.0 mg/kg body weight). There was a dose-dependent increase in both gamma-PPT and GAP-43 mRNA in the trigeminal ganglia of NGF-treated diabetic rats. The findings indicate that increased NGF may be responsible for raised substance P levels in the iris.

Nerve growth factor and neurotrophin-3 enhance neurite outgrowth and up-regulate the levels of messenger RNA for growth-associated protein GAP-43 and T alpha 1 alpha-tubulin in cultured adult rat sensory neurones.

The effect of nerve growth factor (NGF) and neurotrophin-3 (NT-3) on neurite outgrowth was related to the level of mRNA for the growth-associated protein GAP-43 and the neurone specific alpha-tubulin, T alpha 1, in dissociated cultures of adult rat sensory neurones. Treatment with NGF or NT-3 for 3 days enhanced neurite outgrowth in a dose-dependent manner and by 7-fold and 5-fold, respectively, over control at the highest concentrations used. NGF and NT-3 elevated the level of mRNA encoding GAP-43 by 2.3-fold and T alpha 1 alpha-tubulin by 3.2-3.5-fold. The estimated ED50 values were 0.1-0.3 ng/ml for NGF and 2 ng/ml for NT-3 for both neurite outgrowth and mRNA up-regulation.

Reduced levels of mRNA encoding endoskeletal and growth-associated proteins in sensory ganglia in experimental diabetes.

This study investigated changes in levels of mRNAs encoding the three neurofilament (NF) proteins NF-L (low), NF-M (medium), and NF-H (high) and two growth-associated proteins, GAP-43 and T alpha 1 alpha-tubulin, in lumbar dorsal root ganglia of control and streptozocin-induced diabetic rats. After 8 weeks of diabetes the animals were killed, and total RNA was isolated from the L4 and L5 dorsal root ganglia and subjected to Northern blotting, with constant amounts of total RNA loaded onto each lane. A truncated sense RNA for GAP-43 was included as an internal standard during RNA isolation to enable accurate quantification of mRNA levels. The filters were probed sequentially with 32P-labeled cDNAs encoding NF-L, NF-M, NF-H, GAP-43, T alpha 1 alpha-tubulin, and citrate synthase. Hybridizing RNAs were detected by autoradiography and quantified by image analysis. Hybridization signals were normalized to those of the internal standard. In diabetes, NF-L mRNA levels (2.5- and 4-kilobase [kb] transcripts) were decreased by 35 (P = 0.002) and 34% (P < 0.001), respectively, the NF-H mRNA level was decreased by 65% (P < 0.001), but the NF-M mRNA remained unchanged. T alpha 1 alpha-tubulin and GAP-43 mRNA levels were reduced by 56 (P < 0.001) and 30% (P < 0.05), respectively. Levels of citrate synthase mRNA were unchanged. These data indicate a selective defect of expression of growth-associated and endoskeletal proteins in experimentally induced diabetes.

Diabetic neuropathy, nerve growth factor and other neurotrophic factors.

Diabetic neuropathy typically presents as an insidious symmetrical distal degenerative disease of peripheral nerves. A failure of neurotrophic factors to regulate neuronal phenotype might be expected to result in such a clinical picture. Experimentally, diabetic rats show reduced expression of target-derived nerve growth factor as well as reduced expression of neuronal genes that are responsive to nerve growth factor. The latter is corrected by administration of exogenous nerve growth factor. Thus, deficient neurotrophic support might contribute to the pathogenesis of diabetic neuropathy, and any successful treatment might include exogenous neurotrophins or other strategies to correct their deficiency of action.