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).

There is a clear disruption in the gene expression of hedgehog genes in the peripheral 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in the dorsal root ganglion (DRG) neurons of diabetic animals at 8 weeks duration of diabetes (Burnand et al., unpublished observations).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 subsequent 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 production 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 expression 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 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 receptor 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 Ptcl and Ptc2 (24). Ptcl 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 transduce 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 cytoplasmic 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 cytoplasmic 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 understood. Ultimately, it results in the nuclear translocation of the Gli proteins. The Gli genes encode transcription factors that share five highly conserved tandem C2-H2 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).

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, resulting 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: Glil (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). Glil 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 diabetic 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 experimental models of diabetes there are a wide range of structural changes in the peripheral 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.

Mechanism Diabetic Neuropathy

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 hedgehog at both durations.

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 hedgehog 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.

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