Mitogenactivated 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 regulated 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 scaffold proteins required for the interaction of certain kinases. Activated MAPKs can phosphorylate targets within the cytoplasm, such as cytoskeletal proteins and other

Fig. 3. In dorsal root ganglia of rats with 12 weeks streptozotocin diabetes there was a general reduction in gene expression (mRNA levels) for endoskeletal proteins; some of these reductions were normalised by treatment with sonic hedgehog. Coding: open circles—P-actin; filled squares—y-actin; filled circles—NFL, neurofilament light subunit; open squares—NFM, neurofilament medium subunit; half-filled circles—NFH, neurofilament heavy subunit; half-filled squares—a-tubulin.

Fig. 3. In dorsal root ganglia of rats with 12 weeks streptozotocin diabetes there was a general reduction in gene expression (mRNA levels) for endoskeletal proteins; some of these reductions were normalised by treatment with sonic hedgehog. Coding: open circles—P-actin; filled squares—y-actin; filled circles—NFL, neurofilament light subunit; open squares—NFM, neurofilament medium subunit; half-filled circles—NFH, neurofilament heavy subunit; half-filled squares—a-tubulin.

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 transducing mitogenic signals by converting tyrosine phosphorylation into the serine/threonine phosphorylations that regulate downstream events (53). ERK2 and ERK3 were subsequently 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 members 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 p38y. ERK7 and ERK8 have also been cloned recently (61,62).

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 P-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 protein 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 situations, 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: p38a (69,70), p38p (71), p38y (72), and p385 (73), each encoded by a different gene. The p38 MAPKs are phosphorylated and activated by MKK3 and MKK6 at threonine and tyrosine residues and can mediate signaling to the nucleus (74). A large number 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 majority 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), mesencephalic cells (76), and cortical neurons (77), and NGF withdrawal in PC12 cells (78). Conversely, p38 activation was not observed following NGF withdrawal in primary cultures 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 detrimental 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 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 implicated 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 feature of tissues affected by diabetic macrovascular complications. One major consequence 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 accumulation 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 tissue 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-5 isoform-dependent mechanism (88). However, at higher levels of glucose, p38 is activated by hyperosmolarity through a PKC independent pathway. This suggests that different pathways 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-a-lipoic acid (93-95), and taurine (96,97) can prevent abnormalities in diabetic nerve. Oxidative stress results from an imbalance in the production of reactive oxygen species and cellular antioxidant defence mechanisms. The increased free radical production may then result in oxidization of various cellular components including lipids, proteins, and nucleic acids. Components that are modified by ROS may have decreased activity leading to widespread dysfunction including disturbances 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 reactive 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

Fig. 4. Interconnecting pathways for oxidative stress. The polyol pathway consumes NADPH, compromising the glutathione cycle, reducing levels of oxidized glutathione and impairing conversion of hydrogen peroxide to water by glutathione peroxidase. This favours the Fenton reaction generating super-hydroxyl radicals.

Fig. 4. Interconnecting pathways for oxidative stress. The polyol pathway consumes NADPH, compromising the glutathione cycle, reducing levels of oxidized glutathione and impairing conversion of hydrogen peroxide to water by glutathione peroxidase. This favours the Fenton reaction generating super-hydroxyl radicals.

in slowing of electron transfer in the mitochondria and increased production of superoxide 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 fenton reaction, producing the highly reactive hydroxyl radical (OH). The activity of both superoxide dismutase and catalase was found to be decreased (but not reaching statistical significance) in peripheral nerve after 6 weeks of diabetes (99,100). Longer durations 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 rapidly 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 animals (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), possibly 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 highlighted by the fact that an aldose reductase inhibitor can prevent diabetes induced lipid peroxidation in peripheral nerve (107).

ROS and RNS such as hydrogen peroxide, superoxide, and peroxynitrite activate ERK, JNK, and p38 in a variety of in vitro models (108-112), whereas MAPK activation is now well documented in these in vitro models, there is a lack of evidence for MAPK activation by ROS and RNS in vivo. Recently, however (113), showed that ConA induced liver failure in mice resulted in TNF-a induced ROS production leading to sustained activation of JNK, which could be prevented by an antioxidant. Depleted GSH was also a consequence whereas there was less depletion in JNK1-/- mice, thus establishing a link between ROS activation and MAPK activation in vivo. P-adreno-receptor stimulation in cardiac tissues was also found to increase superoxide production and lipid peroxidation with concomitant activation of p38, JNK, and ERK. These changes could be prevented with the antioxidant Tempol (114). The relationship between high glucose, oxidative stress, and MAPK activation may only be apparent with more chronic hyperglycemia because acute (3h) glucose infusion in rats resulted in oxidative stress as measured by MDA and total glutathione but not in activation of ERK1/2 or p38 in liver (115). It will be of great significance to establish the existence of oxidative stress-induced MAPK activation in diabetic neuropathy.

AGE exert their cellular effects by interacting with cell surface receptors, the best characterized of these is the receptor for advanced glycation end products (RAGE). In rat pulmonary smooth muscle cells it was demonstrated that RAGE activation can induce ERK1/2 activated p21 (ras) and nuclear factor kB (NFkB) signaling (116). Subsequently a role for p38 in RAGE-induced NF-KB-dependent secretion of proinflammatory cytokines was established (117). RAGE-induced activation of JNK is not well documented but has been shown in RAGE-amphoterin induced tumour growth (118) and high-mobility group protein-1 (HMGB1—a novel inflammatory molecule) induced RAGE activation in human microvascular endothelial cells (119).

MAPK Activation in Sensory Neurones

In vitro models cannot replicate the chronic conditions of diabetes because primary cells slowly, but progressively die in culture. Furthermore, the interaction between neuronal and non-neuronal cells and the supply of nutrients cannot be mimicked directly. However cell culture models do provide a means of isolating components known to be important in diabetes and reduce the use of in vivo models. In primary cultures of dorsal root ganglia neurons, high glucose activated p38, and JNK but not ERK in a concentration-dependent manner (10-200 mM) following 16 hours treatment (1). Oxidative stress in the form of hydrogen peroxide or diethyl maleate resulted in activation of p38 and ERK but not JNK. Treatment with high glucose and oxidative stress had an additive effect on activation of p38, suggesting different mechanisms of activation.

Exposure of DRG neurones to high glucose and oxidative stress also resulted in a decrease in cell viability as indicated by lactate dehydrogenase and MTT assays, measurements of intact plasma membranes and mitochondrial function, respectively. Concomitant treatment with a specific ERK pathway inhibitor (U0126) or a specific p38 pathway inhibitor (SB20210) prevented activation of ERK or p38, respectively and prevented the decrease in cell viability. This suggests that activation of p38 and ERK by glucose or oxidative stress is detrimental in sensory neurones.

Commercially available specific inhibitors of the JNK pathway that are easily soluble have been lacking and therefore less is known about the role of the JNK pathway in sensory neurons. Treatment with the peptide inhibitor JNK inhibitor 1 (120) resulted in death of primary cultures of DRG neurons (121). This inhibitor appeared to be selective for JNK because c-Jun phosphorylation was prevented, whereas there was no effect on other MAPKs. The recent development of new and more soluble JNK inhibitors may help elucidate the effects of JNK signaling in sensory neurones in diabetic neuropathy.

MAPK Activation and Neuropathy in Diabetes

To investigate the effect of diabetes on MAPK activation, antibodies were used that either recognize an epitope found on all forms of a particular MAPK (total, -T) or an epitope specific to the phosphorylated (activate) form (phosphorylated, -P). Immuno-histochemical studies on normal rats showed that ERK was expressed in both neurones and satellite cells of the DRG, whereas ERK-P was found exclusively in satellite cells. In the sciatic nerve ERK-T and ERK-P immunoreactivity was seen in both axons and Schwann cells. Western blotting indicated that DRG from diabetic animals showed an increase in ERK-P relative to ERK-T for both the p42 (ERK2) and p44 (ERK1) iso-forms after 8, 10, or 12 weeks diabetes (1,122). The increase in ERK-P was because of activation in the satellite cells in the DRG. Activated p44 ERK was also found to be significantly increased in the sural nerve of 12 week STZ rats (123). However, no changes were found in ERK-P in the sural nerve in a separate study with the same duration of diabetes (122).

In control DRG, JNK-T staining was found predominantly in neurones. JNK-P showed a similar distribution; staining was observed in the cytoplasm of neurones, but was absent from the nuclei. In sciatic nerve, JNK immunohistochemistry was restricted to axons. JNK staining has also been documented in the ventral horn and motoneuron perikarya (122). In diabetic animals, Western blotting revealed increased JNK activation (p46 and p54/56) in the DRG (1,121,122). Increased levels of JNK-P in sciatic and sural nerve from 12 weeks STZ-rats were also observed (123). p54 JNK has also been shown to be elevated in the DRG and sural nerve in an alternative model of type 1 diabetes, the BB rat (122). Immunohistochemistry of diabetic DRG showed that JNK-P is translocated from the cytoplasm to the nucleus of neurones. In axons of the sciatic nerve, staining is increased in large myelinated fibers. In other studies carried out in STZ-rats in the same laboratory, only certain isoforms of JNK were shown to be activated (S. A. Price and D. R. Tomlinson, personal observations) or were shown to be increased but not reaching statistical significance. Activation of JNK was related to the duration of diabetes (increased activation with longer durations) and to the blood glucose levels (increased with higher blood glucose levels). Activated c-Jun, a transcription factor known to be downstream of JNK, displays a similar pattern of activation to that of JNK in diabetic rats (122).

Immunohistochemistry demonstrated that p38-T was also located in neuronal cells in the DRG of control rats (Fig. 5). Similar to JNK-T, immunoreactivity was largely restricted to the cytoplasm and appeared to be absent from the nuclei and satellite cells.

Control Diabetic untreated Diabetic-fidarestat

Control Diabetic untreated Diabetic-fidarestat

Diabetic-SB 239063 Diabetic minus primary Ab

Fig. 5. Bar charts and Western blots showing the effects of Insulin, fidarestat and the p38 mitogen-activated protein kinases inhibitor, SB239063 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).

Fig. 5. Bar charts and Western blots showing the effects of Insulin, fidarestat and the p38 mitogen-activated protein kinases inhibitor, SB239063 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 was present in the cytoplasm of neurones but more intense staining was also observed in the nuclei of some cells. In sciatic nerve p38-T was expressed in axons and Schwann cells and p-38-P was expressed in axons. Western blotting showed an increase in p38-P in the DRG of diabetic animals (1), accompanied by predominantly nuclear staining observed with immunohistochemistry. p38-P staining was also increased in the sciatic nerve of diabetic animals (124) and staining became visible in Schwann cells as well as axons. In the L1 spinal cord, diabetes also promoted p38 activation in motoneuron cell bodies, as identified by colocalization of choline acetyltransferase. There was also intense p38 activation in microglia and diffuse labeling in neuronal and non-neuronal cells of the gray matter. Interestingly, in sural 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 second-generation p38 inhibitor SB 239063 (20 mg/kg per day) prevents activation of p38 in DRG and sciatic nerve and also deficits in nerve conduction velocity observed in untreated diabetic rats (124). The inhibitor used specifically inhibits the a- and ß-isoforms of p38 that are the isoforms predominantly expressed in neuronal tissue (125) and has no effect on other MAP, tyrosine, or lipid kinases (126). Treatment of diabetic rats with the aldose reductase inhibitor fidarestat or insulin also prevented activation of p38 (Figs. 5 and 6)

Fig. 6. Immunocytochemistry showing activated (phosphorylated) p38 in cytoplasm and, especially, nuclei of both small and large neurone cell bodies in dorsal root ganglia. There was marked activation in diabetes, which was specific, as is shown by the sections exposed to secondary, but no primary antibody. Both fidarestat and the p38 inhibitor, SB239063 prevented the activation of p39 mitogen-activated protein kinase.

Fig. 6. Immunocytochemistry showing activated (phosphorylated) p38 in cytoplasm and, especially, nuclei of both small and large neurone cell bodies in dorsal root ganglia. There was marked activation in diabetes, which was specific, as is shown by the sections exposed to secondary, but no primary antibody. Both fidarestat and the p38 inhibitor, SB239063 prevented the activation of p39 mitogen-activated protein kinase.

suggesting that p38 activation is a consequence of hyperglycemia and lies downstream of the polyol pathway (124). This evidence supports the in vitro work by (1) and indicates that MAPK activation is detrimental in diabetic neuropathy. Elucidation of the down stream targets of p38 in sensory neurons may suggest therapeutic targets for diabetic neuropathy in the future.

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Peripheral Neuropathy Natural Treatment Options

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