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Speckled 140 KDa (SP140) is a nuclear body protein, mainly expressed in immune cells, which contains multiple domains suggestive of an epigenetic reader function; namely a bromodomain, a PHD domain and a SAND domain. Single nucleotide polymorphisms and epigenetic modifications in the SP140 locus have been linked to autoimmune and inflammatory diseases including Crohn’s disease (CD). However, little is known about the cellular function of SP140; this is due in part to the fact that, unlike for other many other epigenetic proteins, no small molecule inhibitors have been available to investigate the biological role of SP140. We report the discovery of the first small molecule SP140 inhibitor (GSK761) and utilize this to elucidate SP140 function in innate immune cells. We show that SP140 is highly expressed in CD68+ CD mucosal macrophages and in in vitro-generated inflammatory macrophages. SP140 inhibition through GSK761 reduced monocyte differentiation into inflammatory macrophages and lipopolysaccharide (LPS)-induced inflammatory activation, whilst inducing the generation of CD206+ regulatory macrophages that mark anti-TNF remission induction in CD patients. ChIP-seq analyses revealed that SP140 preferentially occupies transcriptional start sites (TSS) in inflammatory macrophages, with enrichment at gene loci encoding pro-inflammatory cytokines/chemokines and inflammatory pathways. GSK761 specifically reduced SP140 binding and thereby expression of SP140-dependent downstream inflammatory genes. Notably, in CD14+ macrophages isolated from CD intestinal-mucosa, GSK761 inhibited the spontaneous expression of cytokines, including TNF. Together, this study identifies SP140 as a druggable epigenetic reader and therapeutic target for CD.

Humoral immunity in mammals relies on the function of two developmentally and functionally distinct B cell subsets - B1 and B2 cells. While B2 cells are responsible for the adaptive response to environmental antigens, B1 cells regulate the production of polyreactive and low affinity antibodies for innate humoral immunity. The molecular mechanism of B cell specification into different subsets is understudied. In this study, we identified lysine methyltransferase NSD2 (MMSET/WHSC1) as a critical regulator of B1 cell development. In contrast to its minor impact on B2 cells, deletion of the catalytic domain of NSD2 in primary B cells impairs the generation of B1 lineage. Thus, NSD2, a histone H3 K36 dimethylase, is the first-in-class epigenetic regulator of a B cell lineage in mice.

Humoral immunity in mice and man relies on the function of two developmentally and functionally distinct B cell subsets - B1 and B2 cells. While B2 cells are responsible for most of the adaptive response to environmental antigens, B1 cells, which are comprised of phenotypically distinct B1a and B1b cells, are carriers of the innate humoral immunity that relies on production of poly-reactive and low affinity antibodies. The molecular mechanism of B cell specification into different subsets is not well established. Here we identified lysine methyltransferase MMSET/NSD2 as a critical regulator of the B1 cell population. We show that NSD2 deficiency in B cell precursors prevents generation of the B1 cell compartment, while having a minor impact on B2 cells. Our data revealed MMSET/NSD2, which catalyzes histone H3 lysine 36 di-methylation, as the first in class epigenetic master regulator of a major B cell lineage in mice.

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.

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.

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.

Emerging disease modifying therapeutic strategies for Alzheimer's 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 Alzheimer's 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.

Axons are generally believed to be incapable of regeneration in the adult central nervous system1,2. Inhibition results from physical barriers imposed by glial scars, a lack of neurotrophic factors, and growth-inhibitory molecules associated with myelin3,4,5, the insulating axon sheath. These molecules include proteoglycans6, myelin-associated glycoprotein3,5 and, in bovine brain, two proteins called Nogo7,8,9. 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.