| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 21, 2002 as doi:10.1096/fj.02-0018fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
* Department of Human Physiology and Pharmacology, University of Rome ‘La Sapienza’, 00185, Rome;
I.N.M. Neuromed, Pozzilli; and
Department of Pharmaceutical Sciences, University of Catania, Italy
2Correspondence: Institute of Human Physiology and Pharmacology ‘Vittorio Erspamer’, University of Rome ‘La Sapienza’, Piazzale Aldo Moro 5, 00185 Rome, Italy. E-mail: andrea.caricasole{at}uniroma1.it
SPECIFIC AIMS
To study the biology of Humanin-related genes in vivo, we identified and cloned a homologue of Humanin in the rat, an animal model widely used for pharmacological studies. The cDNA, which we termed Rattin, encodes a peptide of 38 amino acids (14 residues longer than Humanin). Full-length Rattin and its short 1–24 fragment shared with Humanin the ability to protect neurons against beta-amyloid (Aß) toxicity. However, as opposed to Humanin, Rattin could also display protective activity against excitotoxic neuronal death. Rattin provides the first example of a Humanin-related peptide endowed with a broad neuroprotective activity.
PRINCIPAL FINDINGS
1. Identification and characterization of Rattin
We used the published Humanin DNA sequence to identify homologous rat ESTs, which we assembled to yield a predicted Rattin cDNA of 1591 bp displaying 88% identity to the Humanin cDNA sequence. Upon translation, the predicted Rattin cDNA sequence (Fig. 1
) yielded an ORF of 38 amino acids (14 residues longer than Humanin) displaying a high degree of similarity (81% identity, 85% homology) to the published Humanin peptide sequence. In silico analysis using NNSSP revealed that the Rattin ORF is predicted to encode a soluble protein, consistent with the soluble nature of Humanin, with a calculated theoretical pI of 9.86 and a mol wt of 4338.11. As previously reported for Humanin, no homology was found between known proteins and Rattin. Secondary structure prediction using GORIV postulated similar overall characteristics for the Rattin and Humanin peptides. Compared to Humanin, the Rattin ORF encodes a peptide displaying a conserved hydrophobic core region GFNCLLLSISEIDL preceded by a conserved amino-terminal sequence MAKR and followed by a more divergent carboxyl-terminal polar region PVKRLESPNKTRRPYGASIY. Therefore, the Rattin peptide differs from its human counterpart in the length of the carboxyl-terminal sequence (20 amino acids, instead of 6) and in the presence of four substitutions (of which one is conservative) in the rest of the molecule. Based on the predicted Rattin cDNA sequence, specific primers were designed and full-length cDNA was amplified by RT-PCR from rat tissues using a proofreading thermostable polymerase. DNA sequencing of the cloned product confirmed the predicted Rattin cDNA sequence. The expression pattern of Rattin was examined in a panel of adult rat tissues by RT-PCR (standard and semiquantitative) and Northern blotting. The results indicate a pattern of expression essentially superimposable to that reported for Humanin, with significant expression in cardiac and skeletal muscles and in CNS, and relatively lower expression in other organs. Some areas within the brain (e.g., the cerebellum) expressed higher Rattin levels than others (such as the hippocampus), in accord with that reported for Humanin.
|
2. Neuroprotective activity of Rattin
We next tested the neuroprotective properties of Rattin in three in vitro paradigms of neurodegeneration representative of pathologically relevant neurotoxic insults (excitotoxicity, deprivation of trophic support, and ß-amyloid-induced neurodegeneration). Analysis of the predicted Rattin peptide suggested that Rattin and Humanin should present comparable neuroprotective properties, despite their different size. To undertake a comparative approach of the neuroprotective properties of Rattin an Humanin, synthetic peptides corresponding to Rattin, Humanin, or a truncated Rattin peptide lacking the carboxyl-terminal, 14 residue rat-specific domain were used in the studies. In pure cultures of rat cortical neurons challenged with the toxic fragment of Aß (Aß25–35, 25 µM, applied in the presence of ionotropic glutamate receptor antagonists), all three peptides displayed substantial neuroprotection against Aß neurotoxicity at 1-10 µM (Fig. 1B
). To examine whether a protective activity of RNcould still be observed in a more physiological context, we used mouse mixed cortical cultures containing neurons and glial cells. In these experiments, neurotoxicity was induced by the longer fragment Aß 1–40 (25 µM) preincubated in aqueous solution for 1 wk at 37°C to allow the formation of aggregates. Similar to what was observed in pure neuronal cultures, RN, RNS, or HN (all at 1 µM) produced neuroprotection against Aß toxicity in mixed cultures (Fig. 1C
). We next extended the analysis to other paradigms of neuronal degeneration. Excitotoxic neuronal death was examined in mixed cultures of cortical cells challenged with NMDA, a widely used model to assess excitotoxicity. Cultures were challenged with 100 µM NMDA for 10 min and peptides were applied either 16 h before NMDA, then removed after the toxic pulse (‘preexposure’), or during the 20 h after the pulse (‘postexposure’). Under both conditions, both RN and RNS produced substantial neuroprotection whereas HN was virtually inactive (Fig. 1A
). Apoptosis by trophic deprivation was instead induced in cultured cerebellar granule cells by switching the growth medium containing 25 mM K+ (K25) into a conditioned medium containing 10 mM K+ (K10). The conditioned medium was collected from sister cultures grown in K10 since the time of plating. Apoptosis developed within 48 h after switching the medium, as assessed by Hoechst staining (% of apoptotic neurons: K25 cultures = 6.4 ± 3.8; cultures switched from K25 into K10 = 42 ± 5.3; n=6). No protection was induced by RN, RNS, or HN (1 µM) against ‘low- K+’-induced apoptosis of cultured cerebellar granule cells (Fig. 2
B).
|
CONCLUSIONS AND SIGNIFICANCE
Humanin was recently identified as a novel endogenous, secreted peptide capable of selectively protecting neurons against ßAP and Familial Alzheimer’s disease mutations. To permit pharmacological studies of Humanin gene expression, we have cloned and identified a homologous gene in the rat. The high degree of sequence homology between Rattin and Humanin and the comparable expression profile are consistent with Rattin being a Humanin-related peptide. However, an important difference exists in the spectrum of neuroprotection displayed by these two peptides, with only Rattin being able to protect neurons against excitotoxic death. This is a relevant finding because excitotoxic neuronal death is implicated in a variety of CNS disorders including stroke, head trauma, amyotrophic lateral sclerosis, and Huntington’s chorea. The data presented in the present report should aid the development of novel neuroprotective strategies aimed at modulating the expression of endogenous neuroprotective factors.
|
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0018fje; to cite this article, use FASEB J. (June 21, 2002) 10.1096/fj.02-0018fje 
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
