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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 6, 2005 as doi:10.1096/fj.05-3786fje. |
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u
niewska*
gorzata Ber
sewicz*
,1ocka*,1,2
* Molecular Biology Unit and
Department of Neurorepair, Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland;
Department of Anatomy,
Department of Biochemistry and Molecular Biology, and
Molecular Tissue Repair Unit, Department of Surgery, Royal Free and University College Medical School, London, England, UK; and
¶ Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, Portsmouth, England, UK
2 Correspondence: D.C.G.: IBBS, St. Michael’s Building, White Swan Rd., Portsmouth PO1 2DT, UK. E-mail: darek.gorecki{at}port.ac.uk; B.Z.: Medical Research Centre, Polish Academy of Sciences ul. Pawi
skiego 5 02-106 Warsaw, Poland E-mail: zablocka{at}cmdik.pan.pl
SPECIFIC AIMS
Ischemic stroke is a common and devastating disease and its frequency is increasing in aging populations. Unfortunately, no treatment is available and the human and economic costs of this disease are enormous. The insulin-like growth factor 1 (IGF-1), among its many functions, also has a role in neuronal maintenance and can prevent neuronal damage. In this study we examine the neuroprotective potential of the alternatively spliced form of IGF-1 (MGF) and investigate whether the synthetic, stabilized C-terminal peptide of MGF can act autonomously to prevent or at least decrease acute ischemia-evoked neurodegeneration.
PRINCIPAL FINDINGS
1. The sequence of IGF-1 splice variants is highly conserved
After sequence database analysis, we found that the alternative isoforms of IGF-1 show high conservation in mammals, which indicates functional importance of these isoforms. Using PCR amplification of gerbil postischemic brain mRNAs with IGF-1 primers and cloning we obtained a number of clones of gerbil IGF-1 and confirmed the occurrence of the specific splicing producing the so-called liver type (termed class 1 IGF-1Ea in human and rodents) and the mechano growth factor (MGF or IGF-1Ec in human, IGF-1Eb in rodents) forms of transcripts. In gerbil, as in other species, splicing in the IGF-1Ec produces an open reading frame shift and the resulting mRNA translates into an isoform with a specific C-terminal E domain.
2. Transient brain ischemia induces endogenous expression of MGF
PCR analyses showing the MGF mRNA in post-ischemic gerbil brain prompted us to test whether ischemia induces increased expression of endogenous MGF. Western blot analysis of protein extracts from the hippocampal regions dissected after ischemia showed clear increase in MGF levels in the CA2-3 and DG parts (resistant to ischemia). This increase was evident as early as 3 h after the insult and MGF levels remained elevated for up to 72 h postischemia. In the CA1 region (vulnerable to ischemia), there was no MGF expression at any time. Likewise, there was no significant expression of MGF in the control brains.
To analyze the cellular source and distribution of MGF expression in the brain, we performed immunolocalization with two available MGF antibodies in gerbil brain sections. We established that MGF is not expressed in the normal brain, in agreement with the Western blot data. This was in keeping with the in situ hybridization data showing only very low levels of the MGF transcript in normal brain. However, expression rapidly increased after ischemia and was particularly prominent in the CA3 and DG regions and in the dentate gyrus. The confocal analyses of the CA3 region revealed that MGF was concentrated in the perinuclear region in the pyramidal neurones where it had a well-isolated granular appearance.
3. The synthetic C-terminal MGF peptide can prevent ischemia-evoked neurodegeneration in vivo
The expression analyses suggested that the specific IGF-1 splice variant IGF-1Eb/Ec (or MGF) could be a novel neuroprotective agent in brain ischemia. As we have shown earlier that the terminal peptide of MGF can act on muscle cells independently from the rest of the molecule, we tested here its neuroprotective potential. Based on the high degree of homology we have generated a consensus sequence for the mammalian C-terminal MGF domains, which was used to prepare a synthetic, 24 amino acid long MGF C-terminal peptide (NH2-YQPPSTNKNTKSQ(d)R(d)RKGSTFEEHK-NH2). To increase its stability (peptide was found to be rapidly degraded in serum or tissue fluids) the D form of arginine was used for synthesis and the N terminus of the peptide was PEGylated.
In a gerbil model of brain ischemia, transient bilateral ligation of the common carotid arteries produces specific hippocampal lesions: in the CA1 region, pyramidal neurones start to die and at 7 days after the ischemic episode only 
12% (15±5, n=7, mean±SD) of neurones remained intact compared with 121 ± 12 (n=5) morphologically intact neurones per 300 µm length of CA1 region found in control animals. In contrast, single bolus injection of 25 µg of the MGF C-terminal peptide into the left carotid artery immediately after reperfusion, provided very significant neuroprotection: in some animals, the protective effect was noticeable bilaterally, while in some gerbils it was mostly evident on the injected side. On average, 74.5% of the CA1 hippocampal neurones on the injected side (P<0.001) and 54% on the contralateral side (P<0.001) survived the insult [i.e., 83±25 (n=10) and 66±30 (n=10) neurons, respectively]. In contrast, analogous injection of the full-length IGF-1 peptide had no significant effect on the postischemic survival of CA1 neurones: only 15.8% of neurones (19±7, n=5) survived (Fig. 1
A).
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4. The C-terminal MGF peptide acts via an IGF-1 receptor-independent mechanism
The synthetic C-terminal MGF peptide is able to function independently from the rest of the IGF-1 molecule and can prevent the acute ischemia-evoked neurodegeneration in vivo. However, this peptide is lacking the domain present in the full-length peptide, which is responsible for the IGF-1 receptor binding. We have therefore used the well-characterized systems of NMDA- and oxidative stress-induced neurodegeneration in rat organotypic hippocampal slices to gain some insight into the mechanism of action of this autonomous domain. In the excitotoxicity model we observed 55% ± 7 of cell damage 24 h after addition of NMDA (n=6) expressed as a percentage of the maximum fluorescence produced by glutamate toxicity (Fig. 1B
). In contrast, there was a 44% decrease in the extent of cell death (to 31%±1) in the presence of 100 ng/mL of the C-terminal MGF peptide (P<0.001). In the free radical paradigm, 100 ng/mL of MGF C-terminal peptide effectively prevented hippocampal neurone degeneration and produced up to 85% of protection (P<0.001). In comparison, the same concentration of the full-length IGF-1 produced 70% of neuronal protection. However, the protective effect of IGF-1 was only noticeable up to 24 h, whereas the MGF C-terminal peptide functioned significantly longer as its neuroprotective effect was still clearly observed after 48 h (P<0.001) (Fig. 1B
). This effect could be due to the increased stability of this C-terminal peptide of MGF or a different mode of action of these two peptides.
The MGF C-terminal peptide function was completely independent from the IGF-1 receptor. Blockade of this receptor with a specific antibody abolished the neuroprotective effect of IGF-1 but had no effect on protection exerted by MGF C-terminal peptide (Fig. 1B
). This result indicates that both peptides, despite a common origin, act independently and have specific modes of action. Moreover, the C-terminal peptide of MGF showed additive effect with the full-length IGF-1 in this model: both peptides used together providing stronger neuroprotection than each compound alone (Fig. 1B
).
CONCLUSIONS AND SIGNIFICANCE
IGF-1 transcript shows elaborate alternative splicing patterns that result in the production of distinct circulating and tissue-specific isoforms. Alternative splicing of the 3'-exons results in IGF-1s with specific C-terminal domains. We found that these alternative C-terminal isoforms show high conservation in mammals. Such evolutionary conservation indicates functional importance of these isoforms, which all appear to have specific roles, including involvement in neuronal maintenance and neuroprotection. We show here that brain ischemia induces changes in endogenous expression of the specific alternatively spliced IGF-1 variant (called MGF or IGF-1Ec in human, IGF-1Eb in rodents). In the postischemic hippocampus the levels of MGF transcript and the peptide were increased selectively in neurones resistant to the ischemic insult. While MGF is known for its regenerative potential in skeletal muscle, we show here for the first time a role for this IGF-1 isoform in the brain. We demonstrate that the 24 amino acid C-terminal domain unique to this isoform can function as an autonomous biologically active peptide and is an extremely potent neuroprotective agent in vivo and in vitro. Such a small and biologically active molecule is a good candidate for development into a therapeutic modality. A single dose of chemically stabilized C-terminal MGF peptide prevented neurodegeneration of vulnerable CA1 neurones following global brain ischemia in vivo. The unilateral injection often produced the bilateral neuroprotection. Therefore, this C-terminal MGF peptide may not need to be targeted directly into its site of action. This is in contrast to the native IGF-1 (and many other compounds) showing neuroprotective potential only when targeted directly to the area of brain lesion and/or used at very high doses over time. This C-terminal MGF peptide was also highly protective in a hippocampal slice model of oxidative stress and NMDA-induced neurodegeneration in vitro. Protection provided by the C-terminal MGF was not inhibited by the blockade of the canonical ligand binding site on the IGF-1 receptor. These results indicate that in addition to the functional complexity of the IGF-1 isoforms, which stems from alternative splicing of the primary transcript, posttranslational modifications may produce further peptide domain(s) with autonomous functions and a new IGF-1 receptor-independent mode of action. This short C-terminal MGF peptide has a potential to be developed into a therapeutic modality for the prevention of neuronal damage. Several important questions remain to be answered: in our model the peptide was administered via injection into the carotid artery immediately after reperfusion. Neither the timing nor the mode of administration are applicable to clinical situations. We are working to establish the window of opportunity and alternative delivery routes for effective treatment of brain ischemia with this peptide.
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FOOTNOTES
1 These authors share senior authorship. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3786fje;
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