Rabinder Prinjha

Inhibitor of neurite outgrowth in humans.

Axons are generally believed to be incapable of regeneration in the adult central nervous system. Inhibition results from physical barriers imposed by glial scars, a lack of neurotrophic factors, and growth-inhibitory molecules associated with myelin, the insulating axon sheath. These molecules include proteoglycans, myelin-associated glycoprotein and, in bovine brain, two proteins called Nogo. We have used this bovine sequence to identify the human Nogo gene and have isolated complementary DNA clones encoding three different Nogo isoforms that are potent inhibitors of neurite outgrowth and which may help block the regeneration of the central nervous system in adults.

Neuropilin-2 Is Required In Vivo for Selective Axon Guidance Responses to Secreted Semaphorins

Neuropilins are receptors for class 3 secreted semaphorins, most of which can function as potent repulsive axon guidance cues. We have generated mice with a targeted deletion in the neuropilin-2 (Npn-2) locus. Many Npn-2 mutant mice are viable into adulthood, allowing us to assess the role of Npn-2 in axon guidance events throughout neural development. Npn-2 is required for the organization and fasciculation of several cranial nerves and spinal nerves. In addition, several major fiber tracts in the brains of adult mutant mice are either severely disorganized or missing. Our results show that Npn-2 is a selective receptor for class 3 semaphorins in vivo and that Npn-1 and Npn-2 are required for development of an overlapping but distinct set of CNS and PNS projections.

Neurobiology: Inhibitor of neurite outgrowth in humans

Axons are generally believed to be incapable of regeneration in the adult central nervous system. Inhibition results from physical barriers imposed by glial scars, a lack of neurotrophic factors, and growth-inhibitory molecules associated with myelin, the insulating axon sheath. These molecules include proteoglycans, myelin-associated glycoprotein and, in bovine brain, two proteins called Nogo. We have used this bovine sequence to identify the human Nogo gene and have isolated complementary DNA clones encoding three different Nogo isoforms that are potent inhibitors of neurite outgrowth and which may help block the regeneration of the central nervous system in adults.

Nogo Provides a Molecular Marker for Diagnosis of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder characterized by the selective degeneration of upper and lower motor neurons. The lack of a molecular diagnostic marker is of increasing concern in view of the therapeutic strategies in development. Using an unbiased subtractive suppressive hybridization screen we have identified a clone encoding the neurite outgrowth inhibitor Nogo and shown that its isoforms display a characteristic altered expression in ALS. This was first confirmed by analyzing Nogo isoform expression in a transgenic ALS model at early asymptomatic stages where we found increased levels of Nogo-A and decreased Nogo-C and importantly, not following experimentally induced denervation. Furthermore, we confirmed these changes in both post-mortem and biopsy samples from diagnosed ALS patients but not control patients. Thus, the alteration in Nogo expression pattern, common to sporadic and familial ALS, represents a potential diagnosis tool and points strongly to Nogo having a central role in disease.

Characterization using FLIPR of rat vanilloid receptor (rVR1) pharmacology

The vanilloid receptor (VR1) is a ligand‐gated ion channel, which plays an important role in nociceptive processing. Therefore, a pharmacological characterization of the recently cloned rat VR1 (rVR1) was undertaken. HEK293 cells stable expressing rVR1 (rVR1‐HEK293) were loaded with Fluo‐3AM and then incubated at 25°C for 30 min with or without various antagonists or signal transduction modifying agents. Then intracellular calcium concentrations ([Ca2+]i) were monitored using FLIPR, before and after the addition of various agonists. The rank order of potency of agonists (resiniferatoxin (RTX)>capsaicin>olvanil>PPAHV) was as expected, and all were full agonists. The potencies of capsaicin and olvanil, but not RTX or PPAHV, were enhanced at pH 6.4 (pEC50 values of 7.47±0.06, 7.16±0.06, 8.19±0.06 and 6.02±0.03 respectively at pH 7.4 vs 7.71±0.05, 7.58±0.14, 8.10±0.05 and 6.04±0.08 at pH 6.4). Capsazepine, isovelleral and ruthenium red all inhibited the capsaicin (100 nM)‐induced Ca2+ response in rVR1‐HEK293 cells, with pKB values of 7.52±0.08, 6.92±0.11 and 8.09±0.12 respectively (n=6 each). The response to RTX and olvanil were also inhibited by these compounds. None displayed any agonist‐like activity. The removal of extracellular Ca2+ abolished, whilst inhibition of protein kinase C with chelerythrine chloride (10 μM) partially (∼20%) inhibited, the capsaicin (10 μM)‐induced Ca2+ response. However, tetrodotoxin (3 μM), nimodipine (10 μM), ω‐GVIA conotoxin (1 μM), thapsigargin (1 μM), U73122 (3 μM) or H‐89 (3 μM) had no effect on the capsaicin (100 nM)‐induced response. In conclusion, the recombinant rVR1 stably expressed in HEK293 cells acts as a ligand‐gated Ca2+ channel with the appropriate agonist and antagonist pharmacology, and therefore is a suitable model for studying the effects of drugs at this receptor.

PTEN couples Sema3A signalling to growth cone collapse

Distinct changes in glycogen synthase kinase-3 (GSK-3) signalling can regulate neuronal morphogenesis including the determination and maintenance of axonal identity, and are required for neurotrophin-mediated axon elongation. In addition, we have previously shown a dependency on GSK-3 activation in the semaphorin 3A (Sema3A)-mediated growth-cone-collapse response of sensory neurons. Regulation of GSK-3 activity involves the intermediate signalling lipid phosphatidylinositol 3,4,5-trisphosphate, which can be modulated by phosphatidylinositol 3-kinase (PI3K) and the tumour suppressor PTEN. We report here the involvement of PTEN in the Sema3A-mediated growth cone collapse. Sema3A suppresses PI3K signalling concomitant with the activation of GSK-3, which depends on the phosphatase activity of PTEN. PTEN is highly enriched in the axonal compartment and the central domain of sensory growth cones during axonal extension, where it colocalises with microtubules. Following exposure to Sema3A, PTEN accumulates rapidly at the growth cone membrane suggesting a mechanism by which PTEN couples Sema3A signalling to growth cone collapse. These findings demonstrate a dependency on PTEN to regulate GSK-3 signalling in response to Sema3A and highlight the importance of subcellular distributions of PTEN to control growth cone behaviour.

Proteomic identification and early validation of complement 1 inhibitor and pigment epithelium‐derived factor: Two novel biomarkers of Alzheimer's disease in human plasma

Emerging disease modifying therapeutic strategies for Alzheimers disease (AD) have generated a critical need for biomarkers of early stage disease. Here, we describe the identification and assessment of a number of candidate biomarkers in patients with mild to moderate probable AD. Plasma from 47 probable Alzheimers patients and 47 matched controls were analysed by proteomics to define a significant number of proteins whose expression appeared to be associated with AD. These were compared to a similar proteomic comparison of a mouse transgenic model of amyloidosis, which showed encouraging overlap with the human data. From these studies a prioritised list of 31 proteins were then analysed by immunoassay and/or functional assay in the same human cohort to verify the changes observed. Eight proteins continued to show significance by either immunoassay or functional assay in the human plasma and these were tested in a further set of 100 probable AD patients and 100 controls from the original cohort. From our data it appeared that two proteins, serpin F1 (pigment epithelium‐derived factor) and complement C1 inhibitor are down‐regulated in plasma from AD patients.

A complementary peptide approach applied to the design of novel semaphorin/neuropilin antagonists

Semaphorin 3A can inhibit axonal growth and induce neuronal apoptosis following binding to neuropilin‐1, with the membrane proximal MAM (meprin, A5, mu) domain in neuropilin‐1 playing a key role in the formation of a higher order receptor complex. If functional motifs on semaphorin 3A and/or the MAM domain can be identified, then small‐constrained peptides might be developed as antagonists. We have scored peptide pairs for complementary hydropathy and antisense homology to identify a candidate functional motif in the Ig domain of semaphorin 3A, and in the MAM domain of neuropilin‐1. Synthetic peptides corresponding to these sequences fully inhibit growth cone collapse induced by semaphorin 3A. A number of smaller peptides derived from the parental sequence also inhibited the response, particularly after they were constrained by a disulfide bond. Finally, we have used an algorithm to design a peptide that is a near‐perfect hydropathic complement of the candidate functional site in the MAM domain; this also inhibits the semaphorin 3A response. Thus, an algorithm‐driven methodology has led to the identification of three independent semaphorin 3A antagonists. Semaphorin 3F stimulates growth cone collapse following binding to the closest relative to neuropilin‐1 in the genome, neuropilin‐2. Where tested, the peptides that antagonise semaphorin 3A failed to inhibit the semaphorin 3F response.

Treatment with an antibody directed against Nogo-A delays disease progression in the SOD1G93A mouse model of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal, neurodegenerative disorder in which motor neurons in the spinal cord and motor cortex degenerate. Although the majority of ALS cases are sporadic, mutations in Cu-Zn superoxide dismutase-1 (SOD1) are causative for 10-20% of familial ALS (fALS), and recent findings show that a hexanucleotide repeat expansion in the C9ORF72 gene may account for >30% of fALS cases in Europe. SOD1(G93A) transgenic mice have a phenotype and pathology similar to human ALS. In both ALS patients and SOD1(G93A) mice, the first pathological features of disease manifest at the neuromuscular junction, where significant denervation occurs prior to motor neuron degeneration. Strategies aimed at preventing or delaying denervation may therefore be of benefit in ALS. In this study, we show that Nogo-A levels increase in muscle fibres of SOD1(G93A) mice along with the elevation of markers of neuromuscular dysfunction (CHRNA1/MUSK). Symptomatic treatment of SOD1(G93A) mice from 70 days of age with an anti-Nogo-A antibody (GSK577548) significantly improves hindlimb muscle innervation at 90 days, a late symptomatic stage of disease, resulting in increased muscle force and motor unit survival and a significant increase in motor neuron survival. However, not all aspects of this improvement in anti-Nogo-A antibody-treated SOD1(G93A) mice were maintained at end-stage disease. These results show that treatment with anti-Nogo-A antibody significantly improves neuromuscular function in the SOD1(G93A) mouse model of ALS, at least during the earlier stages of disease and suggest that pharmacological inhibition of Nogo-A may be a disease-modifying approach in ALS.

Non-clinical development of ozanezumab: a humanised antibody targeting the amino terminus of neurite outgrowth inhibitor A (Nogo-A)

Ozanezumab (GSK1223249) is a humanised, Fc-disabled, monoclonal antibody (mAb) which targets the amino terminus of Neurite Outgrowth Inhibitor A (Nogo-A) which is currently being developed for the treatment of amyotrophic lateral sclerosis (ALS). Here we report on the biochemical and structural characterisation of Ozanezumab together with an assessment of pharmacology and non-clinical safety. A minimal binding epitope was characterised and emerging biology and pre-clinical pharmacology provide confidence that targeting the amino terminus of the neurite outgrowth inhibitor A (Nogo-A) through passive immunization may offer a promising approach to treat various neurodegenerative diseases, including ALS. A comprehensive non-clinical assessment of safety has been completed based on a package of in vitro and in vivo studies in rodents, non-human primates and female rabbits (reproductive toxicology only). There was no evidence for toxicological, cardiovascular, respiratory, neurobehavioural, immunogenic, reproductive or delayed toxicity effects in any species following repeat dose treatment with Ozanezumab (biweekly up to doses of 500 mg kg−1 iv for 52 weeks in the non-human primate). Based on these studies there are no non-clinical safety findings that would preclude the development of Ozanezumab in patients.