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Neuroprotective Effect of EDR Peptide in Mouse Model of Huntington's Disease

Huntington's disease (HD) is a fatal, inherited neurodegenerative disorder. HD is caused by expansion of a cytosine-adenine-guanine (CAG) repeat in the coding region of the huntingtin gene (HTT), located on chromosome 4p16.3. The CAG repeats in HTT are translated into a polyglutamine (polyQ) sequence in the N-terminal region of the huntingtin (Htt) protein. HD typically occurs in midlife, but extensive CAG expansion leads to a juvenile onset of the disease. An average number of repeats makes 19 CAG in the huntingtin gene of unaffected individuals, while HD patients reveal 36 to 121 CAG repeats. Patients with 36 to 39 repeats show reduced penetrance for the disease and can be asymptomatic for many years. The most HD affected appears to be striatum with subsequent atrophy of the cerebral cortex at the late stage. Mutant huntingtin (mHtt) is inclined to aggregation with its possible toxic effect on neurons. Mutant Htt induces neuronal cell death in several ways: gene transcription, formation of toxic aggregates, direct induction of apoptosis, disruption of key neuronal functions such as proteosomal or mitochondrial functions, ubiquitination pathways, axonal transport, endocytosis and synaptic transmission.

The mutant huntingtin protein was shown to directly and specifically bind to the inositol-1,4,5-triphosphate receptors type 1 (InsP3R1) and activate these receptors in lipid bilayers. An increased release of Ca2+ from intracellular storage - endoplasmic reticulum - in striatal medium spiny neurons (MSN) in primary cultures of transgenic mice (models of HD), obviously causes cell death. Expansion of polyQ sequence led to violation of Htt conformation thus resulting in Htt aggregation. It hindered the movement of vesicles containing neurotransmitters via the cytoskeleton, which disrupted signaling in neurons. The search for effective treatment of HD is particularly relevant. Neurons functioning and aging studies may prompt new opportunities for regulation of these processes. Short neuroprotective peptides employment opens great perspectives for the development of novel highly effective and side effects free neuroprotective drugs aimed at treatment of neurodegenerative diseases.

Among short peptides showing potential modulatory functions is EDR (Glu-Asp-Arg) with pronounced neuro-protective properties.

EDR influence on the functional activity of the central nervous system was studied in an experimental model of prenatal hyper-homocysteinemia in rats. The induction of oxidative stress in vivo is known to be associated with a high level of homocysteine in the blood of animals, reduction of cognitive abilities and impairment of glutamatergic systems in the brain. Intramuscular injection of peptide EDR to rats contributed to the improvement of spatial orientation and learning capacity in progeny during "Morris water maze" test. EDR protective effect might be related to its ability to inhibit the accumulation of reactive oxygen species (ROS) in the neurons, elevating their resistance to oxidative stress and preventing interaction between homocysteine and its derivatives with glutamate receptors. Peptide EDR administration in the model of hypoxia in the embryonic or prenatal period of rats contributed to the restoration of the ability to latent learning up to a normal level in 3-week-old rats. The rats, prenatally administered with peptide EDR simulating a prenatal stress showed a normalized behavioral sleep, eating and relaxed waking.

The effect of peptide EDR on activation of MAP-kinase was evaluated in cultures of cerebellar granule cells. Its temporary profile determined which genes were going to be expressed - adaptation or apoptosis. Addition of peptide EDR in cell culture increased lag-time of MAP-kinase activation that could be estimated as a protective effect against homocysteine toxic action. The influence of peptide EDR on oxidation process caused by ouabain or hydrogen peroxide in neurons was recently investigated. Peptide EDR reduced ROS in neurons.

There is a range of peptides known to penetrate cells and concentrate on the surface of cell nucleus. As a rule, they have an enhanced content of arginine and lysine residues in their sequence. Moreover, these peptides can not only penetrate into the cell, but also form complexes with DNA and RNA. An important experimental fact that confirms the ability of short peptides to penetrate into the cell consists in registering FITC-labeled di-, tri-, and tetrapeptides penetration not only into the cytoplasm, but also into the nucleus and nucleolus of HeLa cells. As an example, myelopeptide-4 has been shown to bind on the HL-60 cell surface, to penetrate into their cytoplasm, and finally to concentrate around the cell nucleus. Recently it was shown that peptide EDR linked with СNG-containing de-oxyribooligonucleotides (preferably CAG-containing structures), making these sites unavailable to DNA methyltransferases, whereby the promoter was unmethylated.

Neurons cell cultures are an important models to study different diseases, allowing not only to simulate complex molecular mechanisms of the disease, but also to control various effects and search for potential therapeutic agents [26]. The correct development, functioning, viability of the striatal MSN and formation of functional dendritic spines require cortical neurons in cell culture. Therefore, in this study we used a physiological cell model of HD - mixed cortical-striatal neurons.

Our investigation was aimed to study the effect of the peptide EDR on the formation of MSN spines in the cortical-striatal neuronal cultures obtained from HD-bearing mice and to validate the hypothesis of DNA-peptide interactions underlying this effect.

In this work the data collection of DNA-peptide interactions in solution showed that the character of peptide-DNA binding depended on peptide concentration in solution. With a sufficiently large concentration of peptide it inhibited the binding of the compounds on the major groove of DNA and had little effect on the binding components on the minor groove. Furthermore, the presence of peptide extinguishes the ethidium bromide luminescence in DNA solution. It can be connected, for example, with gradual formation of a double helix peptide coverage and local change of permittivity of the medium near the macromolecule.

The present simulations of DNA binding to the EDR peptide allow the understanding of short peptide properties in the cell. Combination of the data obtained from the present analysis of EDR–DNA complex and from experimental studies suggests a recognition mechanism by peptides. Based on the established biological activity of the peptide and our experimental data we can assume that small peptides (di-, tri- and tetrapeptides) revealed capability to interact with DNA in the area of specific binding site on the promoter segment of genes.

Discovery of the phenomenon of peptide activation of gene transcription points out the natural mechanism of organism to maintain physiologic functions, which is based on the interaction of the DNA and regulatory peptides. This process is fundamental for the development and functioning of the living substance. Thus, the proposed models may serve for studying mechanisms of biological activity of short peptides and quantitative assessment of their expected activity.

This work has demonstrated that the peptide EDR normalized spines morphology of neurons in a mouse model of HD and interacted with DNA in a solution. This effect points to the ability of EDR peptide to regulate homeostasis in neurons. According to the previous data and present study we propose that EDR peptide penetrates into neurons and regulates a number of dendritic spines through binding with nucleotides.

Thus, the peptide EDR as a representative of a pool of regulatory biologically active peptides, holds promise as one of neuroprotective agents that are encouraging for further study as a compound effective for the treatment of HD.
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