Myelinated axons fail to develop properly in a genetically authentic mouse model of Charcot-Marie-Tooth disease type 2E.

We have analyzed a mouse model of Charcot-Marie-Tooth disease 2E (CMT2E) harboring a heterozygous p.Asn98Ser (p.N98S) Nefl mutation, whose human counterpart results in a severe, early-onset neuropathy. Behavioral, electrophysiological, and pathological analyses were done on separate cohorts of NeflN98S/+ mutant mice and their wild type Nefl+/+ littermates between 8 and 48 weeks of age. The motor performance of NeflN98S/+ mice, as evidenced by altered balance and gait measures, was impaired at every age examined (from 6 to 25 weeks of age). At all times examined, myelinated axons were smaller and contained markedly fewer neurofilaments in NeflN98S/+ mice, in all examined aspects of the PNS, from the nerve roots to the distal ends of the sciatic and caudal nerves. Similarly, the myelinated axons in the various tracts of the spinal cord and in the optic nerves were smaller and contained fewer neurofilaments in mutant mice. The myelinated axons in both the PNS and the CNS of mutant mice had relatively thicker myelin sheaths. The amplitude and the nerve conduction velocity of the caudal nerves were reduced in proportion with the diminished sizes of myelinated axons. Conspicuous aggregations of neurofilaments were only seen in primary sensory and motor neurons, and were largely confined to the cell bodies and proximal axons. There was evidence of axonal degeneration and regeneration of myelinated axons, mostly in distal nerves. In summary, the p.N98S mutation causes a profound reduction of neurofilaments in the myelinated axons of the PNS and CNS, resulting in substantially reduced axonal diameters, particularly of large myelinated axons, and distal axon loss in the PNS.

PMP22 antisense oligonucleotides reverse Charcot-Marie-Tooth disease type 1A features in rodent models.

Charcot-Marie-Tooth disease type 1A (CMT1A) is caused by duplication of peripheral myelin protein 22 (PMP22) and is the most common hereditary peripheral neuropathy. CMT1A is characterized by demyelination and axonal loss, which underlie slowed motor nerve conduction velocity (MNCV) and reduced compound muscle action potentials (CMAP) in patients. There is currently no known treatment for this disease. Here, we show that antisense oligonucleotides (ASOs) effectively suppress PMP22 mRNA in affected nerves in 2 murine CMT1A models. Notably, initiation of ASO treatment after disease onset restored myelination, MNCV, and CMAP almost to levels seen in WT animals. In addition to disease-associated gene expression networks that were restored with ASO treatment, we also identified potential disease biomarkers through transcriptomic profiling. Furthermore, we demonstrated that reduction of PMP22 mRNA in skin biopsies from ASO-treated rats is a suitable biomarker for evaluating target engagement in response to ASO therapy. These results support the use of ASOs as a potential treatment for CMT1A and elucidate potential disease and target engagement biomarkers for use in future clinical trials.

Schwann cell overexpression of the GPR7 receptor in inflammatory and painful neuropathies.

The human 7-transmembrane receptor GPR7 has sequence similarity to opioid and somatostatin receptors, and can be activated by the recently discovered neuropeptides NPB and NPW. This receptor is highly expressed in the nervous system, with suggested roles in neuroendocrine events and pain signaling. In this study, we investigated whether the GPR7 receptor is expressed in the peripheral nervous system under normal and pathological conditions. A low level of GPR7 receptor was observed in myelin-forming Schwann cells in both normal human and rat nerve, and in primary rat Schwann cell cultures. Peripheral nerve samples taken from patients exhibiting inflammatory/immune-mediated neuropathies showed a dramatic increase of GPR7 receptor expression restricted to myelin-forming Schwann cells. Complementary animal models of immune-inflammatory and ligation-induced nerve injury and neuropathic pain similarly exhibited an increased myelin-associated expression of GPR7 receptor. These results suggest a relationship between the pathogenesis of inflammatory/immune-mediated neuropathies, GPR7 receptor expression, and pain transmission.

Selective expression of regulators of G-protein signaling (RGS) in the human central nervous system.

The human tissue distribution of the nineteen known human regulators of G-protein signaling (RGS) is described. Measurement of RGS mRNA levels in human brain and in nine peripheral tissues revealed striking tissue preferences in gene expression. Five RGS members were identified with enriched expression in brain. RGS4, RGS7, RGS8, RGS11 and RGS17 were all significantly expressed in striatal regions including the nucleus accumbens and putamen. RGS4 had the highest measured levels of mRNA expression and was highly enriched in the gyrus of the cortex and in the parahippocampus. RGS7 and RGS17 had overlapping distribution profiles and were both noticeably enriched in the cerebellum. Several RGS family members showed high expression in peripheral tissues. RGS5 was preferentially expressed in heart, and RGS1, RGS13, RGS18 and GAIP were predominately expressed in lymphocytes. RGS1 was also highly enriched in the lung, as was RGS2 and RGS16. Five family members, RGS3, RGS9, RGS10, RGS 12 and RGS14 had a broad and overlapping mRNA distribution. These results suggest roles of the individual RGS members in a diversity of functions in humans and support a role of several RGS members in the regulation of central nervous system function via modulation of signaling by G-protein coupled receptors.

Modification of nociception and morphine tolerance by the selective opiate receptor-like orphan receptor antagonist (-)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol (SB-612111).

(-)-cis-1-Methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol (SB-612111) is a novel human opiate receptor-like orphan receptor (ORL-1) antagonist that has high affinity for the clonal human ORL-1 receptor (hORL-1 K(i) = 0.33 nM), selectivity versus mu-(174-fold), delta-(6391-fold), and kappa (486-fold)-opioid receptors and is able to inhibit nociceptin signaling via hORL-1 in a whole cell gene reporter assay. SB-612111 has no measurable antinociceptive effects in vivo in the mouse hot-plate test after intravenous administration but is able to antagonize the antimorphine action of nociceptin [ED(50) = 0.69 mg/kg, 95% confidence limit (CL) = 0.34-1.21]. SB-62111 administration can also reverse tolerance to morphine in this model, established via repeated morphine administration. In addition, intravenous SB-612111 can antagonize nociceptin-induced thermal hyperalgesia in a dose-dependent manner (ED(50) = 0.62 mg/kg i.v., 95% CL = 0.22-1.89) and is effective per se at reversing thermal hyperalgesia in the rat carrageenan inflammatory pain model. These data show that an ORL-1 receptor antagonist may be a useful adjunct to chronic pain therapy with opioids and can be used to treat conditions in which thermal hyperalgesia is a significant component of the pain response.

4-Amino-3(2H)-pyridazinones bearing arylpiperazinylalkyl groups and related compounds: synthesis and antinociceptive activity.

A series of 4-amino-3(2H)-pyridazinones substituted at position 2 with arylpiperazinylalkyl groups and analogues were synthesized and their antinociceptive effect was evaluated in the mouse abdominal constriction model. Preliminary SARs studies were performed. Several of the novel compounds dosed at 100 mg/kg s.c. significantly reduced the number of writhes induced by the noxious stimulus. Compound 12e showed 100% inhibition of writhes and was able to protect all the treated animals from the effect of the chemical stimulus. Subsequent dose-response studies revealed 12e to be almost 40-fold more potent than the structurally related Emorfazone.

Evidence for a selective role of the delta-opioid agonist [8R-(4bS*,8aalpha,8abeta, 12bbeta)]7,10-Dimethyl-1-methoxy-11-(2-methylpropyl)oxycarbonyl 5,6,7,8,12,12b-hexahydro-(9H)-4,8-methanobenzofuro[3,2-e]pyrrolo[2,3-g]isoquinoline hydrochloride (SB-235863) in blocking hyperalgesia associated with inflammatory and neuropathic pain responses.

The specific involvement of the delta-opioid receptor in the control of nociception was explored by investigating the pharmacological activity in vivo of a selective, orally active, and centrally penetrant delta-opioid agonist. [8R-(4bS*,8aalpha,8abeta,12bbeta)]7,10-dimethyl-1-methoxy-11-(2-methylpropyl)oxycarbonyl 5,6,7,8,12,12b-hexahydro-(9H)-4,8-methanobenzofuro[3,2-e]pyrrolo[2,3-g]isoquinoline hydrochloride (SB-235863) is a new pyrrolomorphinan with high affinity (Ki = 4.81 +/- 0.39 nM) for the delta-opioid receptor, full agonist activity, and binding selectivity versus the mu- and kappa-opioid receptors of 189-fold and 52-fold, respectively. Perorally administered SB-236863 was inactive in the rat tail-flick and hot-plate tests of acute pain response, but potently reversed thermal hyperalgesia in rats resulting from a carrageenan-induced inflammatory response. This activity could be blocked by the delta-opioid antagonist naltrindole (3 mg/kg s.c.), but selective mu- and kappa-opioid antagonists were ineffective. Naltrindole (1 microg i.c.v.) also blocked the activity of 10 mg/kg (p.o.) SB-235863, showing that the compound activates delta-opioid receptor sites in the central nervous system. SB-235863 was additionally effective at reversing chronic hyperalgesia in the Seltzer rat model of partial sciatic nerve ligation after peroral administration. These data show that the delta-opioid receptor plays a selective role in regulating evoked and lasting changes in nociceptive pain signaling. Classical side effects of mu- and kappa-opioid receptor activation (slowing of gastrointestinal transit and motor incoordination, respectively) were not observed after administration of 70 mg/kg (p.o.) SB-235863, nor was evoked seizure activity affected. These results suggest a selective and limited role of delta-opioid receptors in the modulation of nociception.

PTH2 receptor-mediated inhibitory effect of parathyroid hormone and TIP39 on cell proliferation.

The parathyroid hormone (PTH) has dual mitogenic and inhibitory effects on cell proliferation, depending on the cell type and experimental conditions. PTH can signal via two different receptors, both positively coupled to the adenylyl cyclase/cyclic AMP pathway which can mimic some of the proliferative effects of PTH. We evaluated the role of the type-2 PTH (PTH2) receptor on cell proliferation in clonal human embryonic kidney HEK293 cells stably expressing the human PTH2 receptor. Using a cyclic AMP-responsive gene-reporter, we confirmed that the tuberoinfundibular peptide (TIP39) and various human (h) PTH fragments including hPTH-(1-34) were potent agonists (EC(50) in the range of 0.01-0.3 nM) whereas the bovine (b) PTH peptides b(Tyr(34))PTH-(7-34) and its tryptophan derivative b[D-Trp(12),Tyr(34)]PTH-(7-34) behaved as antagonists (IC(50)=117 and 249 nM, respectively). hPTH-(1-34) produced a dose-dependent inhibition of cell proliferation (EC(50)=8.5+/-0.4 nM) after 3 days and this effect was fully reversed by the tryptophan derivative antagonist. The same effect was observed with TIP39 which caused a 30% maximal inhibition. These findings reveal that PTH2 receptor activation can inhibit cell proliferation and might explain the dual functionality of PTH on cell proliferation.

Up-regulation of regulator of G protein signaling 4 expression in a model of neuropathic pain and insensitivity to morphine.

We hypothesized that the up-regulated expression of one or more members of the regulator of G protein signaling (RGS) family can cause an attenuation of signaling via Gi/Go-coupled opioid receptors, and thereby play a role in the development of hyperalgesia and accompanying insensitivity to morphine observed in animal models of neuropathic pain. Accordingly, we examined the mRNA expression of several RGS genes in a rat model of chronic neuropathic pain induced by partial ligation of the sciatic nerve. During the development of hyperalgesia, RGS4 was the only isoform examined whose mRNA levels increased significantly (up to 230%) in the lumbar spinal cord. In situ hybridization studies confirmed that RGS4 is present in the dorsal horn of the spinal cord where mu-opioid receptors (MORs) are also expressed. Overexpression of RGS4 in human embryonic kidney 293 cells stably expressing mu-opioid receptors predictably attenuated opioid agonist-induced inhibition of adenylyl cyclase. This inhibitory effect was overcome partially at high agonist concentrations, supporting the view that morphine insensitivity is promoted by RGS4 overexpression. These studies provide evidence that the up-regulation of RGS4 expression may contribute to changes in pain signal processing that lead to the development of hyperalgesia, and further affect its modulation by morphine.

The novel and systemically active metabotropic glutamate 1 (mGlu1) receptor antagonist 3-MATIDA reduces post-ischemic neuronal death.

We examined the pharmacological properties of 3-methyl-aminothiophene dicarboxylic acid (3-MATIDA) by measuring second messenger responses in baby hamster kidney cells stably transfected with mGlu1a, mGlu2, mGlu4a or mGlu5a receptors and ionotropic glutamate receptor agonist-induced depolarizations in mouse cortical wedges. 3-MATIDA was a potent (IC(50)=6.3 microM, 95% confidence limits 3-15) and relatively selective mGlu1 receptor antagonist. When tested on mGlu2, mGlu4 or mGlu5 receptors its IC(50) was >300 microM. When tested in cortical wedges, however, 3-MATIDA was also able to antagonize AMPA or NMDA responses with an IC(50) of 250 microM. When present in the incubation medium of cultured murine cortical cells, 3-MATIDA (1-100 microM) significantly reduced the death of neurons induced by 60 min of oxygen and glucose deprivation (OGD), even when added up to 60 min after OGD. A similar neuroprotective activity was observed when 3-MATIDA was present at 10-100 microM in the medium of rat organotypic hippocampal slice cultures exposed to 30 min OGD. Systemic administration of 3-MATIDA (3-10 mg/kg, immediately and 1 h after the onset of ischemia) reduced the volume of brain infarcts following permanent middle cerebral artery occlusion in rats. Our results show that 3-MATIDA is a potent and possibly selective mGlu 1 receptor antagonist that may be considered as a novel prototype neuroprotective agent.

Side-chain lactam-bridge conformational constraints differentiate the activities of salmon and human calcitonins and reveal a new design concept for potent calcitonin analogues.

We have recently reported the potent hypocalcemic effects of side-chain lactam-bridged analogues of human calcitonin (hCT) (Kapurniotu, A.; et al. Eur. J. Biochem. 1999, 265, 606-618). To extend these studies, we have now synthesized a new series of (Asp(17), Lys(21)) and (Asp(17), Orn(21)) side-chain bridged salmon calcitonin (sCT) and hCT analogues. The affinities of these analogues for the human calcitonin receptor, hCTR(I1)(-), and for rat-brain membrane receptors were assayed in competitive binding assays, and agonist potencies at the hCTR(I1)(-) receptors were assessed, using a cAMP-responsive gene-reporter assay. The bridged sCT analogues had activities similar to sCT itself. In contrast, an (Asp(17), Orn(21)) side-chain bridged hCT analogue, cyclo(17-21)-[Nle(8), Phe(12), Asp(17), Orn,(21) Tyr(22))-hCT, was 80 and 450 times more active than hCT in the hCTR(I1)(-) and rat-brain receptor binding assays, respectively, and was 90 times more potent than hCT and 16 times more potent than sCT in initiating receptor signaling. An uncyclized, isosteric analogue of this peptide was also more potent than hCT, demonstrating that the cyclization constraint and these single-residue substitutions enhance the activities of hCT in an additive fashion. This study demonstrates that the potency-enhancing effects of lactam-bridge constraints at hCT residues 17-21 are not transferable to sCT. We also show that, in comparison to the hCT analogues, sCT and its analogues are less potent agonists than expected from their hCTR(I1)(-) affinities. This suggests that it may be possible to preserve the efficient signal transduction of hCT while introducing additional receptor affinity-enhancing elements from sCT into our potent lactam-bridged hCT analogue, thereby creating new super-potent, hCT-based agonists.