Molecular modelling of mitofusin 2 for a prediction for Charcot-Marie-Tooth 2A clinical severity.

Charcot-Marie-Tooth disease type 2A (CMT2A) is an autosomal dominant neuropathy caused by mutations in the mitofusin 2 gene (MFN2). More than 100 MFN2 gene mutations have been reported so far, with majority located within the GTPase domain encoding region. These domain-specific mutations present wide range of symptoms with differences associated with distinct amino acid substitutions in the same position. Due to the lack of conclusive phenotype-genotype correlation the predictive value of genetic results remains still limited. We have explored whether changes in the protein structure caused by MFN2 mutations can help to explain diseases phenotypes. Using a stable protein model, we evaluated the effect of 26 substitutions on the MFN2 structure and predicted the molecular consequences of such alterations. The observed changes were correlated with clinical features associated with a given mutation. Of all tested mutations positive correlation of molecular modelling with the clinical features reached 73%. Our analysis revealed that molecular modelling of mitofusin 2 mutations is a powerful tool, which predicts associated pathogenic impacts and that these correlate with clinical outcomes. This approach may aid an early diagnosis and prediction of symptoms severity in CMT2A patients.

Ischemia/Reperfusion-Induced Translocation of PKCßII to Mitochondria as an Important Mediator of a Protective Signaling Mechanism in an Ischemia-Resistant Region of the Hippocampus.

Emerging reports indicate that activated PKC isoforms that translocate to the mitochondria are pro- or anti-apoptotic to mitochondrial function. Here, we concentrate on the role of PKCß translocated to mitochondria in relation to the fate of neurons following cerebral ischemia. As we have demonstrated previously ischemia/reperfusion injury (I/R) results in translocation of PKCß from cytoplasm to mitochondria, but only in ischemia-resistant regions of the hippocampus (CA2-4, DG), we hypothesize that this translocation may be a mediator of a protective signaling mechanism in this region. We have therefore sought to demonstrate a possible relationship between PKCßII translocation and ischemic resistance of CA2-4, DG. Here, we reveal that I/R injury induces a marked elevation of PKCßII protein levels, and consequent enzymatic activity, in CA2-4, DG in the mitochondrial fraction. Moreover, the administration of an isozyme-selective PKCßII inhibitor showed inhibition of I/R-induced translocation of PKCßII to the mitochondria and an increase in neuronal death following I/R injury in CA1 and CA2-4, DG in both an in vivo and an in vitro model of ischemia. The present results suggest that PKCßII translocated to mitochondria is involved in providing ischemic resistance of CA2-4, DG. However, the exact mechanisms by which PKCßII-mediated neuroprotection is achieved are in need of further elucidation.

The Effect of a Novel c.820C>T (Arg274Trp) Mutation in the Mitofusin 2 Gene on Fibroblast Metabolism and Clinical Manifestation in a Patient.

Charcot-Marie-Tooth disease type 2A (CMT2A) is an autosomal dominant axonal peripheral neuropathy caused by mutations in the mitofusin 2 gene (MFN2). Mitofusin 2 is a GTPase protein present in the outer mitochondrial membrane and responsible for regulation of mitochondrial network architecture via the fusion of mitochondria. As that fusion process is known to be strongly dependent on the GTPase activity of mitofusin 2, it is postulated that the MFN2 mutation within the GTPase domain may lead to impaired GTPase activity, and in turn to mitochondrial dysfunction. The work described here has therefore sought to verify the effects of MFN2 mutation within its GTPase domain on mitochondrial and endoplasmic reticulum morphology, as well as the mtDNA content in a cultured primary fibroblast obtained from a CMT2A patient harboring a de novo Arg274Trp mutation. In fact, all the parameters studied were affected significantly by the presence of the mutant MFN2 protein. However, using the stable model for mitofusin 2 obtained by us, we were next able to determine that the Arg274Trp mutation does not impact directly upon GTP binding. Such results were also confirmed for GTP-hydrolysis activity of MFN2 protein in patient fibroblast. We therefore suggest that the biological malfunctions observable with the disease are not consequences of impaired GTPase activity, but rather reflect an impaired contribution of the GTPase domain to other MFN2 activities involving that region, for example protein-protein interactions.

[Mitofusin 2 and mitochondrial dynamics in norm and pathology]. / Mitofuzyna 2 i dynamika mitochondriów w normie i patologii.

Results of an intensive research performed during last 25 years have revealed that an understanding of biochemical and molecular principles of oxidative phosphorylation has not finished the streak of ground-breaking discoveries of newly identified mitochondrial functions in numerous cellular processes. Among other things it has been shown that mitochondria undergo reversible fission and fusion processes, and may form a complex network which functionally and structurally interacts with the endoplasmic reticulum membranes and probably also other organelles. An organization of mitochondrial network is closely controlled and is of high importance for numerous intracellular processes to occur properly. In this review, mitofusin 2 - one of a few proteins involved in a maintenance of an appropriate mitochondrial architecture, and in the consequence in the regulation of mitochondrial metabolism and calcium signalling, the controlling of the mitochondrial DNA level, and the regulation of cell proliferation and differentiation is the focus. Mutations within mitofusin 2-encoding gene are a cause of Charcot-Marie-Tooh 2A - type neuropathies while an affected expression of this protein seems to be related to neoplasia, type 2 diabetes, or vascular hyperplasia. Numerous experimental data confirm pleiotropic effects of mitofisin 2 in animal cells.

Mitofusin 2 Deficiency Affects Energy Metabolism and Mitochondrial Biogenesis in MEF Cells.

Mitofusin 2 (Mfn2), mitochondrial outer membrane protein which is involved in rearrangement of these organelles, was first described in pathology of hypertension and diabetes, and more recently much attention is paid to its functions in Charcot-Marie-Tooth type 2A neuropathy (CMT2A). Here, cellular energy metabolism was investigated in mouse embryonic fibroblasts (MEF) differing in the presence of the Mfn2 gene; control (MEFwt) and with Mfn2 gene depleted MEFMfn2-/-. These two cell lines were compared in terms of various parameters characterizing mitochondrial bioenergetics. Here, we have shown that relative rate of proliferation of MEFMfn2-/- cells versus control fibroblasts depend on serum supplementation of the growth media. Moreover, MEFMfn2-/- cells exhibited significantly increased respiration rate in comparison to MEFwt, regardless of serum supplementation of the medium. This effect was correlated with increased level of mitochondrial markers (TOM20 and NAO) as well as mitochondrial transcription factor A (TFAM) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) protein levels and unchanged total ATP content. Interestingly, mitochondrial DNA content in MEFMfn2-/- cells was not reduced. Fundamentally, these results are in contrast to a commonly accepted belief that mitofusin 2 deficiency inevitably results in debilitation of mitochondrial energy metabolism. However, we suggest a balance between negative metabolic consequences of mitofusin 2 deficiency and adaptive processes exemplified by increased level of PGC-1α and TFAM transcription factor which prevent an excessive depletion of mtDNA and severe impairment of cell metabolism.

Role of the blood-brain barrier in differential response to opioid peptides and morphine in mouse lines divergently bred for high and low swim stress-induced analgesia.

Over 20 years ago, the Sadowski group separated two mouse lines, one with high (HA) and the other with low (LA) sensitivity to swim stress-induced analgesia (SSIA). Recently, we proposed that increased leakage of the blood-brain barrier (BBB) in the HA line created the difference in the response to SSIA. To search for further evidence for this hypothesis, differences in the levels of the BBB proteins occludin and claudin-5 were analysed. In addition, we sought to evaluate practical differences in BBB permeability by examining the antinociceptive levels in HA and LA mouse lines after IV administration of peptides that have limited access to the CNS. Western blot was used to analyse the differences between occludin and claudin-5. To evaluate the functional differences between the BBB of HA and LA mice, the antinociception levels of endomorphin I, biphalin and AA2016 (peptides with limited BBB permeabilities) in the tail flick test were examined. The expression levels of occludin and claudin-5 in the HA mouse line were lower than in the LA and control mice. Central antinociception of the opioid peptides were significantly higher in the HA line than in the LA and control lines. Our data support the hypothesis that BBB leakage is responsible for the differences between the HA and LA mouse lines. Although SSIA confirmed BBB differences between both lines, it is not limited to the opioid system and could be a useful model for studying the role of the BBB in molecular communications between the periphery and CNS.

Mitofusin 2 expression dominates over mitofusin 1 exclusively in mouse dorsal root ganglia - a possible explanation for peripheral nervous system involvement in Charcot-Marie-Tooth 2A.

Mitofusin 2 (Mfn2), a protein of the mitochondrial outer membrane, is essential for mitochondrial fusion and contributes to the maintenance and operation of the mitochondrial network. Mutations in the mitofusin 2 gene cause axonal Charcot-Marie-Tooth type 2A (CMT2A), an inherited disease affecting peripheral nerve axons. The precise mechanism by which mutations in MFN2 selectively cause the degeneration of long peripheral axons is not known. There is a hypothesis suggesting the involvement of reduced expression of a homologous protein, mitofusin 1 (Mfn1), in the peripheral nervous system, and less effective compensation of defective mitofusin 2 by mitofusin 1. We therefore aimed to perform an analysis of the mitofusin 1 and mitofusin 2 mRNA and protein expression profiles in different mouse tissues, with special attention paid to dorsal root ganglia (DRGs), as parts of the peripheral nervous system. Quantitative measurement relating to mRNA revealed that expression of the Mfn2 gene dominates over Mfn1 mainly in mouse DRG, as opposed to other nervous system samples and other tissues studied. This result was further supported by Western blot evaluation. Both these sets of data confirm the hypothesis that the cellular consequences of mutations in the mitofusin 2 gene can mostly be manifested in the peripheral nervous system.

Protein kinase C beta in postischemic brain mitochondria.

PKC is implicated in the regulation of mitochondrial metabolism. We examined the association of PKCß with mitochondria and followed postischemic changes in its amount in mitochondria isolated from ischemia-vulnerable (CA1) and ischemia-resistant (CA2-4,DG) hippocampus in gerbil model of transient brain ischemia. Our observations suggest that transient ischemic episode induces a significant, rapid and long lasting increase of PKCß in mitochondria in CA2-4,DG, which may bespeak neuroprotection. In organotypic hippocampal culture (OHC) model of neurodegeneration, PKCß inhibition imposed over NMDA toxicity extended the death area beyond the CA1. These results suggest that PKCß might have a protective effect against excitotoxic damage in rat OHC. The pull-down method and LC-MS/MS analysis revealed mitochondrial proteins that can bind directly with PKCßΙ. The proteins were parts of i) mitochondrial redox carriers forming the electron transport chain including ATP synthase and ii) MPTP: ANT and creatine kinase. PKCß acting through mitochondrial proteins could play a role in protecting the cells from death by e.g. influencing ROS and ATP production after ischemia in CA2-4,DG region of the hippocampus.

Neuroprotective potential of biphalin, multireceptor opioid peptide, against excitotoxic injury in hippocampal organotypic culture.

UNLABELLED: Biphalin is a dimeric opioid peptide that exhibits affinity for three types of opioid receptors (MOP, DOP and KOP). Biphalin is undergoing intensive preclinical study. It was recognized that activation of δ-opioid receptor elicits neuroprotection against brain hypoxia and ischemia. We compare the effect of biphalin and morphine and the inhibition of opioid receptors by naltrexone on survival of neurons in rat organotypic hippocampal cultures challenged with NMDA. FINDINGS: (1) 0.025-0.1 µM biphalin reduces NMDA-induced neuronal damage; (2) biphalin neuroprotection is abolished by naltrexone; (3) reduced number of dead cells is shown even if biphalin is applied with delay after NMDA challenge.

Identification of a voltage-gated potassium channel in gerbil hippocampal mitochondria.

Transient cerebral ischemia is known to induce endogenous mechanisms that can prevent or delay neuronal injury, such as the activation of mitochondrial potassium channels. However, the molecular mechanism of this effect remains unclear. In this study, the single-channel activity was measured using the patch-clamp technique of the mitoplasts isolated from gerbil hippocampus. In 70% of all patches, a potassium-selective current with the properties of a voltage-gated Kv-type potassium channel was recorded with mean conductance 109+/-6pS in a symmetrical solution. The channel was blocked at negative voltages and irreversibly by margatoxin, a specific Kv1.3 channel inhibitor. The ATP/Mg(2+) complex and Ca(2+) ions had no effect on channel activity. Additionally, agitoxin-2, a potent inhibitor of voltage-gated potassium channels, had no effect on mitochondrial channel activity. This observation suggests that in contrast to surface membrane channels, the mitochondrial voltage-gated potassium channel could have a different molecular structure with no affinity to agitoxin-2. Western blots of gerbil hippocampal mitochondria and immunohistochemistry on gerbil brain sections confirmed the expression of the Kv1.3 protein in mitochondria. Our findings indicate that gerbil brain mitochondria contain a voltage-gated potassium channel that can influence the function of mitochondria in physiological and pathological conditions and that has properties similar to the surface membrane Kv1.3 channel.

Morgana/CHP-1 is a novel chaperone able to protect cells from stress.

Morgana/CHP-1 (CHORD containing protein-1) has been recently shown to be necessary for proper cell divisions. However, the presence of the protein in postmitotic tissues such as brain and striated muscle suggests that morgana/CHP-1 has additional cellular functions. Here we show that morgana/CHP-1 behaves like an HSP90 co-chaperone and possesses an independent molecular chaperone activity towards denatured proteins. The expression time profile of morgana/Chp-1 in NIH3T3 cells in response to heat stress is similar to that of Hsp70, a classical effector of Heat Shock Factor-1 mediated stress response. Moreover, overexpression of morgana/CHP-1 in NIH3T3 cells leads to the increased stress resistance of the cells. Interestingly, morgana/Chp-1 upregulation in response to transient global brain ischemia lasts longer in ischemia-resistant regions of the gerbil hippocampus than in vulnerable ones, suggesting the involvement of morgana/CHP-1 in natural protective mechanisms in vivo.

Changes in the expression of insulin-like growth factor 1 variants in the postnatal brain development and in neonatal hypoxia-ischaemia.

Insulin-like growth factor-1 (IGF-1) is a multifunctional peptide of which numerous isoforms exist. The predominant form, IGF-1Ea is involved in physiological processes while IGF-1Ec (mechano-growth factor, MGF) is expressed in response to a different set of stimuli. We have identified specific changes in the expression patterns of these IGF-1 variants in brain development in normal rats and following neonatal hypoxia-ischaemia (HI). Both IGF-1Ea and IGF-1Ec are expressed during normal postnatal brain development, albeit with highly specific temporal distributions. In contrast, HI produced increased and prolonged expression of the IGF-1Ec isoform only. Importantly, hypoxia alone stimulated the expression of IGF-1Ec as well. Thus, IGF-1Ec may play a role in HI pathology. Neonatal hypoxia-ischaemia occurs in approximately 1:4000-1:10,000 newborns and causes neurological deficits in approximately 75% of those affected. Unfortunately, no specific treatment is available. IGF-1 is known to have neuroprotective activity and its IGF-1Ec variant appears to be an endogenous protective factor in hypoxia-ischaemia. Therefore, IGF-1Ec could potentially be developed into a therapeutic modality for the attenuation or prevention of neuronal damage in this and related disorders.

Association of protein kinase C delta and phospholipid scramblase 3 in hippocampal mitochondria correlates with neuronal vulnerability to brain ischemia.

Recent findings support the idea that mitochondrial integrity plays an important role in the propagation of excitotoxic ischemic signal and PKC is implicated in the regulation of mitochondrial membranes properties. One of the targets of PKC delta is phospholipid scramblase 3 (PLSCR3), an enzyme responsible for cardiolipin translocation from the inner to outer mitochondrial membrane. To get an insight into in vivo mechanism by which PKC delta mediates ischemia/reperfusion injury of hippocampal neurons, we examined the effects of transient brain ischemia in gerbil on association of PKC delta with mitochondria isolated from ischemia-vulnerable (CA1) and ischemia-resistant regions, and interactions between PKC delta and PLSCR3. Postischemic, biphasic and brain region-specific translocation of PKC delta to mitochondria was observed. First peak was at 30-60 min of reperfusion and the second was observed after 72-96 h following ischemia. PKC delta was translocated to mitochondria only in CA1 region. The PLSCR3 mRNA and protein was detected in brain by RT-PCR and sequence analysis, Western blotting and immunocytochemistry in electron microscopy (EM). Co-immunoprecipitation and double-labeled immuno-EM showed association of PKC delta and PLSCR3 in postischemic CA1 mitochondria. Additionally, the amount of tBid associated with mitochondria was elevated 96 h following ischemia. Our data suggest that in the postischemic brain PKC delta co-localizes with PLSCR3 in mitochondria and this event might influence the mitochondrial membranes architecture and delayed neurons degeneration.

Diazepam neuroprotection in excitotoxic and oxidative stress involves a mitochondrial mechanism additional to the GABAAR and hypothermic effects.

The aim of the present investigation was to analyze the molecular mechanism(s) of diazepam neuroprotection in two models of selective neuronal death in CA1 sector of hippocampus: in vivo following transient gerbil brain ischemia and in vitro in rat hippocampal brain slices subjected to glutamatergic (100 microM NMDA) or oxidative (30 microM tertbutyl-hydroksyperoxide (TBH)) stress. In the in vivo model the diazepam treatment (two doses of 10mg/kg i.p. 30 and 90 min after the insult) resulted in more than 60% of CA1 hippocampal neurons surviving the insult comparing with 15% in untreated animals. To test whether the protective effect of diazepam was due to the postulated drug-induced hypothermia we followed the fluxes of body temperature during postischemic reperfusion: diazepam reduced temperature from 36.6+/-1 degrees C to 33.4+/-2 degrees C. Equivalent hypothermia induced and maintained in animals after ischemia did not prevent neuronal cell loss to the same extent as diazepam did (42.8+/-9.2% and 72.4+/-14.5% of live neurons, respectively). In vitro, under constant temperature conditions, diazepam exerted neuroprotective effects following a "U-shaped" dose-response curve, with concentration efficacy window of 0.5-10 microM. Five micro-molar diazepam showed significant protection by reducing over 50% the number of (dead) propidium iodide labeled cells even in the presence of GABA(A) receptor antagonist bicuculline. Next, we have shown that diazepam reduced the efflux of cytochrome c out of mitochondria both in compromised CA1 neurons in vitro and in isolated mitochondria treated with 30 microM THB. Our results suggest that the neuroprotective action of diazepam relies on additional mechanism(s) and not solely on its hypothermic effect. We suggest that diazepam evokes neuroprotection through its central receptors located on the GABA(A) receptor complex and, possibly, through its peripheral receptor, the translocator protein TSPO (previously called the peripheral benzodiazepine receptor) located in the outer mitochondrial membrane.

Kalirin-7, a protein enriched in postsynaptic density, is involved in ischemic signal transduction.

Regulators of mitogen activated protein kinases (MAPK) and c-Jun N-terminal/stress-activated kinase (JNK) include Rho-like small GTP-binding proteins and their regulators. SynGAP and kalirin-7 are postsynaptic density-enriched proteins identified through their interaction with Rho GTPases and PSD-95 scaffold protein. We examined immunoreactivity of SynGAP, kalirin-7, and PSD-95, phosphorylation of MAPK and JNK in control and postischemic hippocampus in gerbil model of transient forebrain ischemia. In normal brain higher amount of kalirin-7 but a lower amount of P-JNK was found in ischemia-resistant hippocampal area: CA2-3, DG than in ischemia-vulnerable CA1. After 5 min ischemia and 1 h reperfusion a decrease of P-ERK and increase of P-JNK were uniformly observed in the hippocampal parts. By contrast, the amount of kalirin-7 in CA2-3, DG reached 56% (P < 0.001) of control while was doubled in CA1. Oppositely, the immunoreactivity of SynGAP was increased in CA2-3, DG and reduced in CA1. Our data indicate that SynGAP and kalirin-7 take part in the regulation of ischemic signal transduction but the mechanism does not seem directly connected with the activation of MAPK and JNK.

[Scaffold proteins (MAGUK, Shank and Homer) in postsynaptic density in the central nervous system]. / Bialka kotwiczace i ich udzial w przekazywaniu sygnalów w zageszczeniu postsynaptycznym w osrodkowym ukladzie nerwowym.

The postsynaptic density (PSD) is a dynamic multi-protein complex attached to the postsynaptic membrane composed of several hundred proteins such as receptors and channels, scaffolding and adaptor proteins, cell-adhesion proteins, cytoskeletal proteins, G-proteins and their modulators and signaling molecules including kinases and phosphtases. This review focuses on the prominent PSD scaffolds proteins such as members of the MAGUK (membrane-associated guanylyl kinase), Shank (SH3 domain and ankyrin repeat-containing protein) and Homer families. These molecules interact simultaneously with different kinds of receptors and modulate their function by linking the receptors to downstream signaling events. For example PSD 95, a main member of MAGUK family, interacts directly with carboxyl termini of NMDA receptor subunits and clusters them to the postsynaptic membrane. In addition, PSD 95 is involved in binding and organizing proteins connected with NMDAR signaling. Based on the modular character and ability to form multiproteins interactions, MAGUK, Shank and Homer are perfectly suited to act as a major scaffold in postsynaptic density.

Neuroprotective effects of short peptides derived from the Insulin-like growth factor 1.

Insulin-like growth factor I (IGF-1) is a peptide synthesized in response to growth hormone stimulation. While most of the circulating IGF-1 comes from the liver, it can also be produced in other tissues and both its expression and processing undergo tissue-specific regulation. The predominant form, IGF-1Ea is a circulating factor while two others, IGF-1Eb and IGF-1Ec (MGF), are mostly expressed in different tissues or in response to various stimuli and show some preferences with respect to the signal transduction pathways they activate. In skeletal muscle specific forms of IGF-1 play a role in development and growth and in addition to these physiological roles IGF-1 functions in the damaged muscle. IGF-1 is also important for the developing and adult brain and can reduce neuronal death caused by different types of injuries. Like many other peptide hormones IGF-1 originates from a precursor pro-hormone that undergoes extensive post-translational modifications. Processing liberates the mature peptide, which acts via the specific IGF-1 receptor but additional short peptides can arise from both N- and C-termini of various IGF-1 isoforms. These derivatives function as autonomous biologically active peptides and extremely potent neuroprotective agents. Their biological effects are independent of the activation of the IGF-1 receptor. Unfortunately, little is known about their mechanism(s) of action. Likewise, the existence of the endogenous production and wider biological effects of these short peptides are uncertain. However, considering the difference in the modes of action it might be possible to dissociate the unwanted and potentially dangerous mitogenic activity of the full-length IGF-1 exerted via its receptor from the neuroprotective effects of short derivatives mediated through different pathways. Such small molecules show good penetration through the blood brain barrier, can be inexpensively manufactured and modified to increase their stability. Therefore, they are good candidates for development into a neuroprotective therapeutic modality.

Cytochrome c binds to inositol (1,4,5) trisphosphate and ryanodine receptors in vivo after transient brain ischemia in gerbils.

Previously we have shown that the biphasic efflux of mitochondrial protein cytochrome c to cytoplasm is one of the important events of the delayed postichemic neuronal death. We concluded that early and transient appearance of cytochrome c in cytoplasm of cells recovering after ischemia was decisive for initiation of the pathological signaling cascade leading to neuronal death, but the precise mechanism remained unknown. In vitro cytochrome c was identified as a messenger that coordinates mitochondrial-endoplasmatic reticulum interactions that drive apoptosis. Here we show that in vivo cytochrome c interacts with inositol (1,4,5) trisphosphate receptor type 1 in gerbil hippocampus subjected to transient brain ischemia and short reperfusion. Moreover, cytochrome c binds also to ryanodine receptor type 2, the role of which in postischemic neuronal death is suggested. The complexes could be coimmunoprecipitated by antibodies against any of the two proteins. Our data verified that the mechanism observed in vitro applies to the pathological in vivo situation.

A strong neuroprotective effect of the autonomous C-terminal peptide of IGF-1 Ec (MGF) in brain ischemia.

The ischemic stroke is the third leading cause of death in developed countries. The C-terminal peptide of mechano-growth factor (MGF), an alternatively spliced variant of insulin-like growth factor 1 (IGF-1), was found to function independently from the rest of the molecule and showed a neuroprotective effect in vivo and in vitro. In vivo, in a gerbil model of transient brain ischemia, treatment with the synthetic MGF C-terminal peptide provided very significant protection to the vulnerable neurons. In the same model, ischemia evoked increased expression of endogenous MGF in the ischemia-resistant hippocampal neurons, suggesting that the endogenous MGF might have an important neuroprotective function. In an in vitro organotypic hippocampal culture model of neurodegeneration, the synthetic peptide was as potent as the full-length IGF-1 while its effect lasted significantly longer than that of recombinant IGF-1. While two peptides showed an additive effect, the neuroprotective action of the C-terminal MGF was independent from the IGF-1 receptor, indicating a new mode of action for this molecule. Although MGF is known for its regenerative capability in skeletal muscle, our findings demonstrate for the first time a neuroprotective role against ischemia for this specific IGF-1 isoform. Therefore, the C-terminal MGF peptide has a potential to be developed into a therapeutic modality for the prevention of neuronal damage.

Transient cerebral ischemia induces delayed proapoptotic Bad translocation to mitochondria in CA1 sector of hippocampus.

Delayed ischemic brain damage is associated with mitochondrial dysfunction, but the underlying mechanisms are not known in detail. Recent data suggest that the process is associated with multidirectional changes in the activities of various proteins located in mitochondria. Of these, the stress-activated kinase JNK is delay-activated postischemia. We induced 5 min cerebral ischemia in gerbils followed by 3, 24, 48, 72 and 96 h of reperfusion. Here we show the postischemic translocation of proapoptotic protein Bad to mitochondria. Immunoelectron microscopic examination revealed the co-appearance of Bad and Bcl-2 proteins in postischemic mitochondria in ischemia-vulnerable CA1 sector of hippocampus as opposed to the ischemia-resistant DG region. Mitochondrial increase of Bad protein is coincident with a transient decrease of the active, phosphorylated form of prosurvival kinase, Raf-1, under conditions of long reperfusion. The above demonstrated sequence of events is likely to play a role in delayed postischemic nerve cell death.