DIABETIC NEUROPATHY: CLINICAL MANAGEMENT - PART 3 potx

6 Effectors—Sonic Hedgehog and p38 Mitogen-Activated Protein Kinase Sally A. Price, Rebecca C. Burnand, and David R. Tomlinson SUMMARY This chapter covers the identification of mitogen-activated protein kinases as early stage transducers of the damaging effects of glucose on peripheral nerves. They are activated by several metabolic consequences of hyperglycemia, in particular oxidative stress, osmotic stress, and advanced glycation end products. Inhibition of one group of mitogen-activated protein kinases––the p38 group—prevents the development of reduced nerve conduction velocity in experimental diabetes; such inhibition can also be achieved by an aldose reductase inhibitor, giving an explanation for the mechanism underlying the damaging effect of the polyol pathway. The effect of treatment is also described with sonic hedgehog in preventing reduced nerve con- duction velocity and normalising expression of genes coding for endoskeletal proteins, which may be instrumental in preserving the integrity of the distal axon. Key Words: Sonic hedgehog; p38 MAP kinase; nerve conduction; gene expression; axonal endoskeleton. INTRODUCTION The development of potential new therapies for diabetic neuropathy has been sporadic over the last 20 years. In general, the process has been boosted by a prospective aetio- logical mechanism reaching consensus among scientists together with the development of drugs to counteract it. The polyol pathway and aldose reductase inhibitors provide a classical example. As is shown in Fig. 1, interest in the polyol pathway rose dramatically in the 1980s, peaking at around 1990; thereafter there has been a steady decline as clin- ical findings indicated that the hypothesis was inapplicable to complications, at least as a sole explanation of pathogenesis. Subsequently, no hypothesis has reached such a con- sensus and the development of potential novel therapeutics has virtually stalled. This chapter attempts to revitalize the process by proposing two new hypotheses to explain the development of diabetic neuropathy. These are not mutually exclusive; indeed it is instrumental that more than one set of pathogenetic mechanisms coexist and act in concert. If these hypotheses are cogent, then new avenues for development of therapeutics open up. It has been obvious for many years that, if glucose itself is the damaging agent in the initial aetiology of neuropathy, then there must be some processes that are sensitive to From: Contemporary Diabetes: Diabetic Neuropathy: Clinical Management, Second Edition Edited by: A. Veves and R. Malik © Humana Press Inc., Totowa, NJ 91 glucose and are interpolated between hyperglycemia and the onset of neurodegeneration. We have made an extensive study of the way in which the mitogen-activated protein kinases (MAPKs), and especially p38 MAPK, are activated directly by glucose and indirectly by the osmotic and oxidative stresses that it induces in diabetes (1,2). In this chapter is presented and discussed evidence for involvement of p38 MAPK in func- tional changes and its inhibition as a therapeutic strategy considered. The influence of long-term trophic support and its defects in diabetes on the devel- opment of neuropathy have been examined (3). It is clear from this that more than one neurotrophic factor is defective in diabetes and reversal of this possibly requires a pleio- typic response characteristic of several factors. It is possible that agents that govern multiple developmental changes may exert just such a broad–based influence. Such a factor is sonic hedgehog (Shh) and this chapter begins with consideration of its poten- tial influence and the novel therapeutic opportunities that it might present (4). SONIC HEDGEHOG AND DIABETIC NEUROPATHY The hedgehog proteins are a highly homologous family of proteins that are widely expressed during development. There are three known mammalian homologues sonic (Shh), desert (Dhh), and indian (Ihh). Treatment of the streptozotocin (STZ) rat model of diabetes with a fusion protein containing human recombinant Shh and rat immunoglobin G (Shh–IgG) ameliorates a range of diabetes-induced functional and structural disorders of the peripheral nerve. For example, motor and sensory nerve conduction velocities in the lower limbs are both increased to values comparable to that of nondiabetic animals (4). In addition, deficits in nerve growth factor and the related peptide substance P, shown in diabetic rats (5), are not present in rats treated with Shh–IgG (4). 92 Price et al. Fig. 1. Publications per year on the sorbitol/polyol pathway as indexed by PubMed (http://www.ncbi.nlm.nih.gov/PubMed/). There is a clear disruption in the gene expression of hedgehog genes in the periph- eral nervous system of diabetic animals. The mRNA encoding Dhh is reduced in the sciatic nerve of the diabetic rat (4). In addition, shh was downregulated in the dorsal root ganglion (DRG) neurons of diabetic animals at 8 weeks duration of diabetes (Burnand et al., unpublished observations). The mechanism by which treatment with Shh–IgG restores functional deficits in the nerve is unknown. The Hedgehog Family of Proteins The name hedgehog comes from the spiky processes that cover the larval cuticle in hh homozygotes. The hedgehog proteins (Hh) are a family of morphogens that act in a dose dependent manner after being secreted from their tissue source; they exert their effect by altering gene expression. The hedgehog gene (Hh) was first identified in Drosophila embryos, as a gene encoding for a protein implicated in segment polarity (6). Since then, most studies in Drosophila have focused on the role of hedgehog in regulating the growth and patterning of the wing and other appendages (7). Three mammalian hedgehog homologues have been found and are named Shh, Dhh, and Ihh (8). Two homologues have been found in fish and are named echidna and tiggywinkle hedgehog (9,10). The multiple hh genes of vertebrates have presumably arisen by duplication and sub- sequent divergence of a single ancestral hh gene. Although shh, ihh, and dhh are highly homologous, shh is closer to ihh than dhh in sequence identity. Pathi et al. (11) have shown that the three proteins have the ability to function similarly, but with different potencies, hence the proteins can substitute for each other. They showed that the rank order of potencies in each of the contexts they tested was Shh > Ihh > Dhh. Shh is expressed in numerous tissues including the central nervous system, the peripheral nervous system, limbs, somites, the skeleton, and skin. It has numerous roles during mammalian development, directing pattern formation, and inducing cell proliferation. Humans or mice lacking Shh develop holoprosencephaly and cyclopia because of a failure of separation of the lobes of the forebrain (12). Shh organises the developing neural tube by establishing distinct regions of homeodomain transcription factor pro- duction along the dorsoventral axis (13). These transcription factors, including Nkx, Pax, and Dbx family members, specify neuronal identity. Shh acts directly on target cells and not through other secreted mediating factors, to specify neuronal cell fate (14). It also has important known patterning roles in the formation of other tissues including the brain (15) and the eye (9). In addition to the many functions of Shh in determining cell fate, it also has roles in controlling cell proliferation and differentiation in neuronal and nonneuronal cell types. The numerous responses to Shh are achieved by controlling the production, amount, and biochemical nature of the signal itself, including covalent modification of Shh. During development, the expression of Dhh mRNA is highly restricted. Its expres- sion has been shown in the Sertoli cells of the developing testes (16,17) and in the Schwann cells of the peripheral nerve (18). Male Dhh-null mice are sterile and fail to produce mature spermatozoa (16). The peripheral nerves of Dhh-null mice are also highly abnormal. The perineurial sheaths surrounding the nerve fascicles are abnormally Effectors—Sonic Hedgehog and p38 MAPK 93 thin and extensive microfasicles consisting of perineurial like cells are formed within the endoneurium. The nerve tissue barrier is permeable, and the tight junctional arrays between, adjacent perineurial cells are abnormal and incomplete (18). Ihh has two known roles in vertebrate development. The first is in the formation of the endoderm where Ihh is critical for the differentiation of the visceral endoderm (19). The second is in postnatal bone growth (20) where Ihh appears to coordinate growth and morphogenesis, a suggestion has also been made proposing a role for Ihh in healing long bone fractures (21). Until recently, it was thought that hedgehog proteins directly bind to a single recep- tor named Patched (Ptc). Ptc, located on the surface of responding cells, is a 1500 amino acid glycoprotein that constitutes 12 membrane-spanning domains (22,23). Two human homologues of Ptc have been identified named Ptc1 and Ptc2 (24). Ptc1 is the main receptor for Shh, Ihh, and Dhh, the function of Ptc2 is unknown. It has been shown that a number of isoforms of Ptc2 exist it is proposed that the expression of the different iso- forms is associated with the “fine-tuning” of the Hh response (25). Ptc is required for the repression of target genes in the absence of Hh. The Hh signal induces target gene expression by binding to and inactivating Ptc. Inactivation of Ptc allows smoothened to become active; Smo is a 115 kDa transmembrane protein that is essential for transducing the Shh signal, only one human homolog is known. It is not yet clear whether the inhibition of Smo by Ptc is the result of direct or indirect interaction. Either way, the binding of Hh to Ptc results in a change that allows smoothened to trans- duce the signal. In humans and mice, the loss of ptc function causes medulloblastomas, tumors of the cerebullum, and other developmental abnormalities resulting from the inappropriate expression of Shh target genes (26,27). In addition to repressing target gene transcription, Ptc also regulates the movement of Hh through tissues; the binding of Hh to Ptc limits the spread of Hh from its source. In Drosophila producing mutant Ptc, Hh can be detected at distances greater than those producing the wide-type protein (28). The binding of Shh to Ptc induces rapid internalization of Shh into endosomes, the fate of Shh after internalization is not yet known (29). In 2002 it was shown that Shh also directly binds to another protein called megalin (30). This single chain protein is approx 600 KDa and consists of a C-terminal cyto- plasmic domain, a single transmembrane domain and an extremely large ectodomain (31). Megalin functions as an endocytic receptor which mediates the endocytosis of lig- ands including insulin (32), the presence of functional motifs at the C-terminal cyto- plasmic domain suggest that this protein may also have a role in signal transduction (33). The phenotypes of megalin deficient mice are consistent with phenotypes of mice deficient in Shh and Smo (34,35). The signal transduction pathway downstream to Ptc and Smo is not well under- stood. Ultimately, it results in the nuclear translocation of the Gli proteins. The Gli genes encode transcription factors that share five highly conserved tandem C 2 –H 2 zinc fingers and a consensus histidine–cysteine linker sequence between the zinc fingers (36). The Drosophila homolog is called cubitus interruptus (Ci). Ci is regulated post-transcriptionally; the full length Ci protein consists of 155 amino acid residues (Ci-155) (37,38). 94 Price et al. In the absence of a Hh signal, Ci forms a tetrameric complex with proteins named: Costal-2, Fused, and Suppressor of fused at the microtubules (39,40). In this complex form Ci is cleaved to form a 75 amino acid residue (Ci [rep] ) (41) that retains the zinc finger domain and translocates to the nucleus to repress downstream target genes (42). In some cells, proteolysis of Ci seems to be dependent on protein kinase-A mediated phosphorylation (43). Transduction of the Hh signal inhibits proteolysis of Ci, result- ing in an accumulation of the full-length protein. On translocation to the nucleus this activator form stimulates transcription of target genes. In the absence of Hh signal not all full length Ci is cleaved, a residual amount escapes but is prevented from activating target genes by its retention in the cytoplasm and active nuclear export (41,44) thus, it seems likely that there are many levels of control over Ci activity that remain to be fully elucidated. There are three known Gli homologues in mammals: Gli1 (also referred to as Gli), Gli 2, and Gli 3. All three Gli homologues have been tested for separate functional domains. C-terminally truncated forms of both Gli2 and Gli3 that resemble the truncated form of Ci have been shown to repress reporter gene expression in cell lines or Shh targets in vivo (45,46). Gli1 does not seem to contain a represser domain, instead only functioning as a transcription activator (46). Effects on Indices of Diabetic Neuropathy As previously mentioned, treatment of the diabetic rat with Shh–IgG reverses a number of indices of diabetic neuropathy including, deficits in nerve conduction velocity. Figure 2 shows sensory and motor nerve conduction velocity values at 8 and 12 weeks duration of diabetes in the STZ model of diabetes. There are clear deficits in the dia- betic animals that are reversed by treatment with Shh–IgG. Shh–IgG treatment had no effect on body weight or glycemia in diabetic rats, implying that the severity of diabetes was unaffected by Shh–IgG. Shh–IgG administration had no effect on the concentration of polyol pathway components in peripheral nerve (Burnand et al., unpublished). Shh protein signaling ultimately leads to the translocation of the Gli proteins to the nucleus where they act as transcription factors. Therefore, the mechanism by which Shh–IgG exerts its effects is likely to be transcription based. In both human and exper- imental models of diabetes there are a wide range of structural changes in the periph- eral nerve. These changes include a loss in the number of myelinated fibres and paranodal demyelination (47,48). There is also a reduction in the capacity of peripheral nerves to regenerate following injury (49,50). Actin, tubulin, and the neurofilament proteins are the main cytoskeletal proteins essential for structural integrity of the axon. Other accessory proteins including numerous actin binding proteins are present in the peripheral nerve and produce a structure of extreme complexity and versatility. Abnormalities in the production and processing of structural proteins have been widely reported in diabetic neuropathy (5,51,52). Evidence gathered in our laboratory shows that treatment of the diabetic rat with Shh–IgG reverses abnormalities in the gene expression of a range of structural proteins as shown in Fig. 3. This restoration in gene expression may form part of the mechanism by which treatment with Shh–IgG corrects deficits in nerve conduction velocity in diabetic rats. Effectors—Sonic Hedgehog and p38 MAPK 95 96 Price et al. Fig. 2. Motor and sensory nerve conduction velocities in control (open columns), diabetic (gray columns) and sonic hedgehog (diagonal hatching)-treated diabetic rats. Diabetes caused significant (p < 0.01) slowing of both at 8 and 12 weeks, which was normalised by sonic hedge- hog at both durations. To date, the work conducted on the use of Shh–IgG as a potential therapeutic agent in the treatment of diabetic neuropathy has resulted in positive outcomes. No adverse side effects have been observed at 12 weeks duration of diabetes. A longer term study is now necessary to determine the longer term potential of this promising new therapy. MITOGEN-ACTIVATED PROTEIN KINASES MAPKs are a family of enzymes involved in transducing signals derived from the extracellular environment. There are three main subtypes of MAPKs: extracellular reg- ulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), and p38 MAPKs. All family members are activated by dual phosphorylation of a consensus sequence, Thr-Xxx-Tyr by MAPK kinases. Upstream of these are the MAPK kinase kinases, thereby forming a three kinase cascade. There are fewer different kinases at each subsequent level of the cascade, resulting in refinement of the signal. Specificity may be achieved by stimulus-selective pathways, distinct cellular pools of kinases, or the presence of scaf- fold proteins required for the interaction of certain kinases. Activated MAPKs can phosphorylate targets within the cytoplasm, such as cytoskeletal proteins and other kinases, or they may be translocated to the nucleus where they activate transcription factors and mediate gene expression. Extracellular Signal-Regulated Kinases ERK1 was identified as a kinase activated by insulin, having a pivotal role in transduc- ing mitogenic signals by converting tyrosine phosphorylation into the serine/threonine phosphorylations that regulate downstream events (53). ERK2 and ERK3 were subse- quently identified (54). ERK1 and ERK2 have 83% amino acid homology, are expressed in most tissues to varying degrees, and are activated by growth factors, phorbol esters and serum. ERK1/2 activation is typically triggered by receptor tyrosine kinases and G protein-coupled receptors at the cell surface. These activate the small GTP-binding protein Ras, allowing signaling through the Raf/MEK/ERK cascade. Downstream, ERK1/2 activates other kinases (e.g., RSKs, MSKs, and MNKs), membrane components (e.g., CD120a, Syk, and calnexin), cytoskeletal proteins (e.g., neurofilament) or nuclear targets (e.g., SRC1, Pax6, NF-AT, Elk1, MEF2). ERK3 displays ubiquitous expression and responds to various growth factors (54). It is only 42% identical to ERK1 and differs from ERK1/2 in that it is a constitutively active nuclear kinase and does not phosphory- late typical MAPK substrates (54,55). The fifth mammalian ERK kinase is designated ERK5 or big MAPK1 (BMK1) because it is twice the size of the other ERK family mem- bers and has a distinct C-terminal (56,57). Erk5 contributes to Ras/Raf signaling (56,58) and is activated in response to growth factors and stress (56,59). ERK6 is a protein kinase involved in myoblast differentiation (60) but is usually referred to as p38γ. ERK7 and ERK8 have also been cloned recently (61,62). Effectors—Sonic Hedgehog and p38 MAPK 97 Fig. 3. In dorsal root ganglia of rats with 12 weeks streptozotocin diabetes there was a gen- eral reduction in gene expression (mRNA levels) for endoskeletal proteins; some of these reduc- tions were normalised by treatment with sonic hedgehog. Coding: open circles—β-actin; filled squares—γ-actin; filled circles—NFL, neurofilament light subunit; open squares—NFM, neuro- filament medium subunit; half-filled circles—NFH, neurofilament heavy subunit; half-filled squares—α-tubulin. C-Jun N-Terminal Kinases JNK was identified as the kinase that phosphorylated c-Jun after exposure of cells to transforming oncogenes and ultraviolet light (63). It was thus recognized as an important signaling cascade for modulating the activity of distinct nuclear targets. There are 10 mammalian isoforms of JNK arising from alternate splicing of the 3 JNK genes. The JNK proteins are activated by MAP kinase kinases such as MKK4 and MKK7 and upstream of these MAP kinase kinase kinases including MLKs and ASK. Scaffold proteins such as JIP and β-arrestin 2 are also integral to the JNK signaling module, determining proximity and specificity. JNK proteins differ in their associations with scaffold proteins and also in their interaction with downstream targets. Defined substrates of JNK total at about 50–60 pro- tein and include cytoskeletal proteins (e.g., neurofilament, tau, and microtubule associated proteins), mitochondria (e.g., bim), and nuclear proteins (c-Jun, ATF-2, and Elk-1) (64). Roles for the different isoforms of JNK are gradually becoming elucidated. It is known that basal activity of JNK1 is far greater than that of JNK2 and JNK3. Coupled with the fact that JNK1 knockout mice are defective/embryonically lethal, this suggests a greater role for JNK1 under physiological conditions. JNK3 knockout mice are healthy and are resistant to excitotoxic brain insults (65), suggesting a greater pathological role for this isoform. In addition tissue specific effects of the role have been described. In most situa- tions, inhibition of JNK is detrimental, however in cells such as cardiac myocytes and sensory neurones inhibition of JNK may confer protection. Mitogen-Activated Protein Kinase p38 The p38 MAPK signal transduction pathway is activated by proinflammatory cytokines and environmental stresses such as osmotic shock, ultraviolet radiation, heat, and chemicals (see refs. 66–68 for reviews). There are four members of the p38 MAPK family: p38α (69,70), p38β (71), p38γ (72), and p38δ (73), each encoded by a different gene. The p38 MAPKs are phosphorylated and activated by MKK3 and MKK6 at thre- onine and tyrosine residues and can mediate signaling to the nucleus (74). A large num- ber of substrates have been described for p38, these include the transcription factors ATF-2, Elk-1, cAMP response element binding proteins (CREB), and cytoplasmic targets such as tau, MAPKAPK-2. p38 MAPKs are widely expressed, with at least 3 of the genes being expressed in the peripheral nervous system (S Price, personal observation). The effect of p38 activation in response to cellular stress is diverse, although the major- ity of reports favour a role in cell death rather than cell survival for neuronal cells. p38 signaling has been proposed to mediate apoptotic signaling in response to a variety of stimuli in neurons including oxidative stress in primary forebrain cultures (75), mesen- cephalic cells (76), and cortical neurons (77), and NGF withdrawal in PC12 cells (78). Conversely, p38 activation was not observed following NGF withdrawal in primary cul- tures of sympathetic neurons (79) and NGF has been shown to increase p38 activation in DRG in vivo (80). This suggests that activation of p38 alone does not predict a detri- mental outcome. High basal activity of p38 has been described in the adult rat brain (81), although the physiological roles of p38 activation have been sparsely investigated. Stress Kinases—Mechanism of Damage In 1993, the Diabetes Control and Complications Trial Research Group concluded that the incidence and severity of diabetic complications are increased by poor glycaemic 98 Price et al. control, indicating that hyperglycemia is likely to be the major causative factor. Several consequences are known to result from excess glucose these include hyperosmolarity, increased polyol pathway flux, oxidative stress, formation of advanced glycation end products (AGE), and activation of protein kinase C. These pathways are integrally linked with each other and with a variety of other cellular pathways. MAPK activation is impli- cated in all these pathways, suggesting a pivotal role in transducing the effects of high glucose in diabetic neuropathy. Uptake of extracellular glucose without the dependency for insulin is a common fea- ture of tissues affected by diabetic macrovascular complications. One major conse- quence is an increased flux through the polyol pathway (Fig. 4). In this pathway, aldose reductase converts glucose to sorbitol, and this is subsequently converted to fructose by sorbitol dehydrogenase. Excessive flux through the polyol pathway leads to accumula- tion of the poorly membrane permeable metabolites sorbitol and fructose in diabetic rats (82). One consequence is that cells may be subjected to osmotic stress. This mechanism is thought to account for the formation of sugar-induced cataractogenesis in diabetic rat lens (83). The contribution of osmotic stress resulting from increased polyol pathway flux in peripheral nerve is less well defined (84). Extracellular osmotic stress may also occur in diabetic nerves as these are subject to serum hyperosmolarity. Demonstrated a reduction in axonal size in myelinated fibres and suggested this was, at least in part, because of shrinkage as a result of increased tis- sue osmolarity (85). Hyperosmolarity activates MAPKs in a variety of cell types (69,86,87), and therefore it is plausible that hyperosmotic stress can activate MAPKs in diabetic neuropathy. In aortic smooth muscle cells from normal rats, glucose activates p38 by a PKC-δ isoform- dependent mechanism (88). However, at higher levels of glucose, p38 is activated by hyperosmolarity through a PKC independent pathway. This suggests that different path- ways may be activated simultaneously by high glucose. Furthermore, p38 has been shown to mediate the effects of hyperglycemia-induced osmotic stress in vivo in the rat mesenteric circulation (89). In recent years, oxidative stress has come to the forefront of hypotheses proposed to be causative of diabetic neuropathy. Numerous studies have shown that antioxidants such as vitamin E (90–92), DL-α-lipoic acid (93–95), and taurine (96,97) can prevent abnormalities in diabetic nerve. Oxidative stress results from an imbalance in the pro- duction of reactive oxygen species and cellular antioxidant defence mechanisms. The increased free radical production may then result in oxidization of various cellular com- ponents including lipids, proteins, and nucleic acids. Components that are modified by ROS may have decreased activity leading to widespread dysfunction including distur- bances in metabolism and defective signaling pathways. Oxidative stress in diabetic nerve may result from a variety of mechanisms including increased flux through the polyol pathway (Fig. 4), endoneurial hypoxia, hyperlipi- daemia, increases in free fatty acids, activation of PKC, activation of receptors for AGE, and glucose itself. The major source of oxidative stress in cells is the production of reac- tive oxygen species (ROS) and reactive nitrogen species (RNS). Naturally occurring ROS and RNS usually have oxygen or nitrogen based unpaired electrons resulting from enzymatic or nonenzymatic reactions. Examples include superoxide anion, hydroxyl radical, nitrogen oxide, and peroxynitrite. High glucose inhibits ATP synthase resulting Effectors—Sonic Hedgehog and p38 MAPK 99 in slowing of electron transfer in the mitochondria and increased production of super- oxide ions (98). Superoxide ions are normally converted to hydrogen peroxide and water by the enzyme superoxide dismutase. Hydrogen peroxide is also produced by enzymatic transfer of two electrons to molecular oxygen by enzymes such as monoamine oxidase and urate oxidase. Hydrogen peroxide is reduced by glutathione peroxidase, myeloperoxidase, and catalase or nonenzymatic decomposition occurs through the fen- ton reaction, producing the highly reactive hydroxyl radical (OH). The activity of both superoxide dismutase and catalase was found to be decreased (but not reaching statisti- cal significance) in peripheral nerve after 6 weeks of diabetes (99,100). Longer dura- tions of diabetes (3 or 12 months) failed to show a decrease in either gene expression or activity of either enzyme, an increase in catalase expression was reported at 12 months. These results suggest that changes in SOD and catalase may be dynamic in diabetic nerve. Superoxides can also react with NO, forming peroxynitrite (ONOO-), which rap- idly causes protein nitration or nitrosylation, lipid peroxidation, DNA damage, and cell death. In sciatic nerve of rats given a peroxynitrite decomposition catalyst, immunore- activity for nitrotyrosine and poly ADP-ribose (PARP) was present only in diabetic ani- mals (101), indicating that nitrosative stress is indeed present in animal models of diabetic neuropathy. Glutathione is another important cellular antioxidant that acts as a non-enzymatic reducing agent, helping to keep cysteine thiol side chains in a reduced state on the surface of proteins. The reduction of oxidized glutathione to reduced glutathione (GSH), catalysed by glutathione reductase is dependent on NADPH as a cofactor (Fig. 4). Increased flux through the polyol pathway can cause depletion of GSH (102,103), pos- sibly as a result of competition between aldose reductase and glutathione reductase for NADPH resulting in NADPH deficiency (104,105) but more likely because of a decrease in total glutathione (106,107). Increased polyol pathway flux can also create oxidative stress because of the reaction of NADH with NADH oxidase and mitochon- drial overloading with NADH. The significance of polyol-induced oxidative stress is 100 Price et al. Fig. 4. Interconnecting pathways for oxidative stress. The polyol pathway consumes NADPH, compromising the glutathione cycle, reducing levels of oxidized glutathione and impairing con- version of hydrogen peroxide to water by glutathione peroxidase. This favours the Fenton reac- tion generating super-hydroxyl radicals. [...]... machinery, Apaf-1, in the presence of dATP forms a complex with cytochrome-c and caspase-9 (the apoptosome) ( 13, 48,49) Selective binding of caspase-9 to Apaf-1 results in caspase-9-dependent hierarchical activation of caspases-2, -3 , -6 , -7 , -8 , and -1 0, resulting ultimately in DNA fragmentation seen in apoptosis (50– 53) In turn, caspase -3 activates caspase-2, -6 , -8 , and -1 0 and amplifies caspase-9 cleavage... p38 MAPK 1 03 Fig 5 Bar charts and Western blots showing the effects of Insulin, fidarestat and the p38 mitogen-activated protein kinases inhibitor, SB 239 0 63 on activation of mitogen-activated protein kinases p38 in dorsal root ganglia The Western blot shows the effect of diabetes (UD), compared with controls (C), and fidarestat-treated diabetes (DF) on total (p38-T) and phosphorylated p38 (p38-P) p38-P... cell culture model of diabetic neuropathy (98) In this model, inhibition of neuronal PCD is mediated by the Group II mGluR, mGluR3 2-PMPA neuroprotection is completely reversed by the mGluR3 antagonist, (S )- -ethylglutamic acid, but not by Group I and III mGluR inhibitors Other mGluR3 agonists, for example, (2R, 4R )-4 -aminopyrrolidine-2,4-dicarboxylate (APDC) and N-acetyl-aspartyl-glutamate provide protection... (32 34 ) BCL-2 and related proteins act directly on the outer Mt membrane to prevent permeabilization (3, 17 ,35 ,36 ), and suppress PCD (37 ,38 ) by regulating calcium fluxes through the Mt and endoplasmic reticulum (39 ) BCL-xL appears to function in a similar manner to BCL-2 Bag-1 shares no significant homology with BCL-2, but interacts functionally and additively with BCL-2 to prevent PCD (33 ) In contrast,... gp600/megalin Biochem J 2000 ;34 7Pt 3: 6 13 621 32 Orlando RA, Rader K, Authier F, et al Megalin is an endocytic receptor for insulin J Am Soc Nephrol 1998;9:1759–1766 33 Hjalm G, Murray E, Crumley G, et al Cloning and sequencing of human gp 330 , a Ca(2+)binding receptor with potential intracellular signaling properties Eur J Biochem 1996; 239 : 132 – 137 Effectors—Sonic Hedgehog and p38 MAPK 107 34 Chen W, Burgess... enhancement and caspase cleavage in thiol oxidant-induced neuronal apoptosis J Neurosci 2001;21 :33 03 33 11 76 Choi WS, Eom DS, Han BS, et al Phosphorylation of p38 MAPK induced by oxidative stress is linked to activation of both caspase- 8- and -9 -mediated apoptotic pathways in dopaminergic neurons J Biol Chem 2004;279:20,451–20,460 Effectors—Sonic Hedgehog and p38 MAPK 109 77 Wang JY, Shum AY, Ho YJ, Wang... 1996; 93: 435 5– 435 9 61 Abe MK, Kuo WL, Hershenson MB, Rosner MR Extracellular signal-regulated kinase 7 (ERK7), a novel ERK with a C-terminal domain that regulates its activity, its cellular localization, and cell growth Mol Cell Biol 1999;19: 130 1– 131 2 62 Abe MK, Saelzler MP, Espinosa R III, et al ERK8, a new member of the mitogen-activated protein kinase family J Biol Chem 2002;277:16, 733 –16,7 43 63 Hibi... glucose-induced apoptosis is by generation of AGEs In SC treated with different isoforms of AGE, a change in the ∆ΨM leading to depolarization and SC apoptosis was induced by AGE-2 and -3 , but not AGE-1 Apoptosis was ameliorated by the antioxidant α-lipoic acid and by inhibition of p38 signaling In addition, AGE-2 and -3 significantly Neuronal and Schwann Cell Death in Diabetic Neuropathy 125 Fig 3 Electron... divergent functions of hedgehog activity Development 2001:128: 238 5– 239 6 35 Litingtung Y, Chiang C Specification of ventral neuron types is mediated by an antagonistic interaction between Shh and Gli3 Nat Neurosci 2000 ;3: 979–985 36 Ruppert JM, Kinzler KW, Wong AJ, et al The GLI-Kruppel family of human genes Mol Cell Biol 1988;8 :31 04 31 13 37 Orenic TV, Slusarski DC, Kroll KL, Holmgren RA Cloning and characterization... nerve biopsies from diabetic patients there is an increase in both p38-T and p38-P (1) All changes that have been observed in diabetic animals could be reversed with insulin and also the aldose reductase inhibitor, fidarestat, indicating that activation is a consequence of hyperglycemia (Figs 5 and 6) Treatment of STZ -diabetic rats with the secondgeneration p38 inhibitor SB 239 0 63 (20 mg/kg per day) . members of the p38 MAPK family: p38α (69,70), p38β (71), p38γ (72), and p38δ ( 73) , each encoded by a different gene. 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