ABSTRACT
1Inst. of Cell Biology and Neurobiology, CNR, Rome, Italy, 2Dept. of Human Physiology and Pharmacology, University of Rome “La Sapienza”, 3Dept. of Neuroscience, University of Rome “Tor Vergata”, 4European Brain Research Institute, Rome, Italy.
The tachykinin endecapeptide Substance P (SP) has been demonstrated to exert a functional role in neurodegenerative disorders, including Alzheimer’s disease (AD). Indeed, altered levels of SP have been observed in cortical regions of post-mortem brain tissues from patients with AD and a consistent SP reduction in other brain areas and in the cerebrospinal fluid was reported, indicating the possible involvement of SP in this pathology (Raffa, 1998, Jiménez-Corral et al., 2006).
One of the hallmarks of AD is the abnormal extracellular deposition of neurotoxic beta amyloid (Aβ ) peptides, which are derived from the proteolytic processing of amyloid precursor protein (APP). APP can be processed via two alternative pathways: amyloidogenic and non-amyloidogenic. The non-amyloidogenic processing of APP by α -secretases leads to proteolytic cleavage within the Aβ peptide sequence and generates a large extracellular soluble fragment (sAPPα ) (Selkoe, 2001). The sAPPα fragment appears to have no pathological significance and might even have neuroprotective and trophic properties.
Since in different neuronal systems it has been demonstrated the neuroprotective activity of SP, these observations prompted us to investigate, in cultured rat cerebellar granule cells (CGCs), the potential effect of SP on APP proteolytic processing with a focus on its action on α -secretase pathway.
We found, by live/death cell assay, that SP (200nM) protected neurons from death and significantly attenuated the neuronal fibrillogenesis, as demonstrated by the Thioflavin T fluorometric assay. Elisa studies conducted on CGCs extracts demonstrated that the released structures correlate with an increased amount of Aβ 1-42 secreted by CGCs in apoptotic conditions and that SP significantly lowered this amyloidogenic increase, by promoting activation of the nonamyloidogenic α -secretase pathway. Therefore, sAPPα secretion was markedly increased in these cells after SP treatment, effect correlated with an enhanced α -secretase cleavage activity, as demonstrated by a specific fluorimetric assay.
Α -secretase activity depends on three members of the ADAM family: ADAM9, ADAM10 and ADAM17 (TACE) (Endres and Fahrenholz, 2010). Western blot analysis showed that SP treatment was able to modify ADAM9 expression levels, by increasing both the expression of the proenzyme form and by promoting its maturation (cleavage of the prodomain motif).
In conclusion, the results of our studies demonstrate that SP provides substantial protection from apoptosis caused by low potassium in cultured rat CGCs, modulating release of the neurotrophic/neuroprotective sAPPα , through a selective ADAM9 modulation. This effect could be of clinical value toward accelerating non-amyloidogenic APP processing, thereby reducing the possibility of generation of the toxic β -amyloid peptides.
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Jiménez-Corral et al. (2006). Rev Neurol. 42, 354-9.
Selkoe (2001). Physiol Rev. 81, 741–766.
Endres and Fahrenholz (2010). FEBS J. 277, 1585-96.