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.

From differentiation to proliferation: the secretory amyloid precursor protein as a local mediator of growth in thyroid epithelial cells.

In various species, thyrotropin (TSH) is known to stimulate both differentiation and proliferation of thyroid follicle cells. This cell type has also been shown to express members of the Alzheimer amyloid precursor (APP) protein family and to release the secretory N-terminal domain of APP (sAPP) in a TSH-dependent fashion. In this study on binding to the cell surfaces, exogenously added recombinant sAPP stimulated phosphorylation mediated by mitogen-activated protein kinase and effectively evoked proliferation in the rat thyroid epithelial cell line FRTL-5. To see whether this proliverative effect of sAPP is of physiological relevance, we used antisense techniques to selectively inhibit the expression of APP and the proteolytic release of sAPP by cells grown in the presence of TSH. The antisense-induced inhibition was detected by immunoblot, immunoprecipitation, and immunocytochemical analyses. After the reduced APP expression and sAPP secretion, we observed a strong suppression of the TSH-induced cell proliferation down to 35%. Recombinant sAPP but not TSH was able to overcome this antisense effect and to completely restore cell proliferation, indicating that sAPP acts downstream of TSH, in that it is released from thyroid epithelial cells during TSH-induced differentiation. We propose that sAPP operates as an autocrine growth factor mediating the proliferative effect of TSH on neighboring thyroid epithelial cells.

Reduction of neurological damage by a peptide segment of the amyloid beta/A4 protein precursor in a rabbit spinal cord ischemia model.

Although found as a precursor of Alzheimer amyloid, substantial evidence suggests that beta/A4 protein precursor (APP) is involved in regulation of neuronal growth and survival. Recently, we have obtained evidence that the trophic properties of APP are fully preserved in a 17-amino acid sequence. If APP is neurotrophic, then it would be anticipated that administration of the growth-promoting segment of the APP 17-mer peptide might attenuate the neuronal dysfunction or loss or behavioral deficits associated with neuronal injury, such as that accompanying central nervous system ischemia. We evaluated this 17-mer peptide in a rabbit spinal cord ischemia model and found that this peptide alleviates paraplegia resulting from ischemia/reperfusion. Ischemia of the distal lumbar cord was produced by temporary occlusion of the abdominal aorta. Saline, 17-mer APP peptide, or a control peptide (200, 500, or 1000 nM) was administered intrathecally 20 min prior to ischemia and once daily for 3 days thereafter. The neurologic and morphologic outcomes were evaluated after 4 days. Durations of ischemia encompassing all grades of neurologic function were included. The 500 nM dose of 17-mer APP peptide significantly reduced neurologic damage. The average ischemia duration necessary to produce permanent neurologic damage increased from 27.9 +/- 1.9 min in saline-injected controls and 27.7 +/- 2.0 in scrambled sequence peptide-injected controls to 40.2 +/- 4.0 min in the 500 nM 17-mer APP-injected group. The 200 nM dose produced a nonsignificant trend toward reduced neurologic damage.(ABSTRACT TRUNCATED AT 250 WORDS)

Peptides containing the RERMS sequence of amyloid beta/A4 protein precursor bind cell surface and promote neurite extension.

Amyloid beta/A4 protein precursor (APP) is secreted into medium by most cultured cells and can function as an autocrine factor. To study the biological function of secreted forms of APP (sAPP) on neurons, we used a clonal CNS neuronal line, B103, which does not synthesize detectable levels of APP. B103 cells transfected with APP construct developed neurites faster than the parent B103 cells when plated in a serum-free defined medium. Neurite outgrowth of B103 cells was promoted by the conditioned medium of APP-695-over-producing cells or by the bacteria-produced sAPP-695 (named KB75). A series of peptides having sequences between Ala-319 and Met-335 of APP-695 also stimulated neurite outgrowth of B103 cells. The sequence of five amino acids, RERMS (APP 328-332), within this stretch of sequence, was the shortest active peptide, although the concentration required for the neuritotropic activity was higher than that of KB75. Binding assay using 125I-labeled APP 17-mer peptide corresponding to Ala-319 to Met-335 of APP-695 as a ligand demonstrated specific and saturable cell-surface binding sites. The predicted KD value was 20 +/- 5 nM and the Bmax value was 80 +/- 8 fmol/10(6) cells. The binding could be displaced with KB75. A 17-mer peptide with reverse sequence neither induced neurite outgrowth nor competed for the binding. A bacteria-produced sAPP fragment lacking the active 17-mer sequence (named KB75 delta) did not compete with 125I-labeled 17-mer for binding or stimulate neurite extension. A peptide of sequence RMSQ (APP 330-333), which partially overlaps the active sequence RERMS, could block the neuritotropic effects of both KB75 and the 17-mer at higher concentrations. APP 17-mer was also found to induce the accumulation of inositol polyphosphates, suggesting that the APP 17-mer effects involve activation of inositol phospholipid signal transduction systems. These data indicate that sAPP induces neurite extension through cell-surface binding and that the domain containing the RERMS sequence (APP 328-332) represents the active site responsible for this function.

Molecular cloning of cDNA encoding an unrecognized component of amyloid in Alzheimer disease.

A neuropathological hallmark of Alzheimer disease (AD) is a widespread amyloid deposition. We analyzed the entire amino acid sequences in an amyloid preparation and found, in addition to the major beta/A4-protein (A beta) fragment, two unknown peptides. We raised antibodies against synthetic peptides using subsequences of these peptides. These antibodies immunostained amyloid in neuritic and diffuse plaques as well as vascular amyloid. Electron microscopic analysis demonstrated that the immunostaining was localized on amyloid fibrils. We have isolated an apparently full-length cDNA encoding a 140-amino-acid protein within which two previously unreported amyloid sequences are encoded in tandem in the most hydrophobic domain. We tentatively named this 35-amino acid peptide NAC (non-A beta component of AD amyloid) and its precursor NACP. NAC is the second component, after A beta, identified chemically in the purified AD amyloid preparation. Secondary structure predictions indicate that the NAC peptide sequence has a strong tendency to form beta-structures consistent with its association with amyloid. NACP is detected as a M(r) 19,000 protein in the cytosolic fraction of brain homogenates and comigrates on immunoblots with NACP synthesized in Escherichia coli from NACP cDNA. NACP mRNA is expressed principally in brain but is also expressed in low concentrations in all tissues examined except in liver, suggesting its ubiquitous and brain-specific functions. The availability of the cDNA encoding full-length NACP should help to elucidate the mechanisms of amyloidosis in AD.