An interim exploratory proteomics biomarker analysis of a phase 2 clinical trial to assess the impact of CT1812 in Alzheimer's disease.

CT1812 is a novel, brain penetrant small molecule modulator of the sigma-2 receptor (S2R) that is currently in clinical development for the treatment of Alzheimer's disease (AD). Preclinical and early clinical data show that, through S2R, CT1812 selectively prevents and displaces binding of amyloid beta (Aß) oligomers from neuronal synapses and improves cognitive function in animal models of AD. SHINE is an ongoing phase 2 randomized, double-blind, placebo-controlled clinical trial (COG0201) in participants with mild to moderate AD, designed to assess the safety and efficacy of 6 months of CT1812 treatment. To elucidate the mechanism of action in AD patients and pharmacodynamic biomarkers of CT1812, the present study reports exploratory cerebrospinal fluid (CSF) biomarker data from 18 participants in an interim analysis of the first set of patients in SHINE (part A). Untargeted mass spectrometry-based discovery proteomics detects >2000 proteins in patient CSF and has documented utility in accelerating the identification of novel AD biomarkers reflective of diverse pathophysiologies beyond amyloid and tau, and enabling identification of pharmacodynamic biomarkers in longitudinal interventional trials. We leveraged this technique to analyze CSF samples taken at baseline and after 6 months of CT1812 treatment. Proteome-wide protein levels were detected using tandem mass tag-mass spectrometry (TMT-MS), change from baseline was calculated for each participant, and differential abundance analysis by treatment group was performed. This analysis revealed a set of proteins significantly impacted by CT1812, including pathway engagement biomarkers (i.e., biomarkers tied to S2R biology) and disease modification biomarkers (i.e., biomarkers with altered levels in AD vs. healthy control CSF but normalized by CT1812, and biomarkers correlated with favorable trends in ADAS-Cog11 scores). Brain network mapping, Gene Ontology, and pathway analyses revealed an impact of CT1812 on synapses, lipoprotein and amyloid beta biology, and neuroinflammation. Collectively, the findings highlight the utility of this method in pharmacodynamic biomarker identification and providing mechanistic insights for CT1812, which may facilitate the clinical development of CT1812 and enable appropriate pre-specification of biomarkers in upcoming clinical trials of CT1812.

Prostaglandin E receptor subtypes in cultured rat microglia and their role in reducing lipopolysaccharide-induced interleukin-1beta production.

Prostaglandins (PGs) are potent modulators of brain function under normal and pathological conditions. The diverse effects of PGs are due to the various actions of specific receptor subtypes for these prostanoids. Recent work has shown that PGE2, while generally considered a proinflammatory molecule, reduces microglial activation and thus has an antiinflammatory effect on these cells. To gain further insight to the mechanisms by which PGE2 influences the activation of microglia, we investigated PGE receptor subtype, i.e., EP1, EP2, EP3, and EP4, expression and function in cultured rat microglia. RT-PCR showed the presence of the EP1 and EP2 but not EP3 and EP4 receptor subtypes. Sequencing confirmed their identity with previously published receptor subtypes. PGE2 and the EP1 agonist 17-phenyl trinor PGE2 but not the EP3 agonist sulprostone elicited reversible intracellular [Ca2+] increases in microglia as measured by fura-2. PGE2 and the EP2/EP4-specific agonists 11-deoxy-PGE1 and 19-hydroxy-PGE2 but not the EP4-selective agonist 1-hydroxy-PGE1 induced dose-dependent production of cyclic AMP (cAMP). Interleukin (IL)-1beta production, a marker of activated microglia, was also measured following lipopolysaccharide exposure in the presence or absence of the receptor subtype agonists. PGE2 and the EP2 agonists reduced IL-1beta production. IL-1beta production was unchanged by EP1, EP3, and EP4 agonists. The adenylyl cyclase activator forskolin and the cAMP analogue dibutyryl cAMP also reduced IL-1beta production. Thus, the inhibitory effects of PGE2 on microglia are mediated by the EP2 receptor subtype, and the signaling mechanism of this effect is likely via cAMP. These results show that the effects of PGE2 on microglia are receptor subtype-specific. Furthermore, they suggest that specific and selective manipulation of the effects of PGs on microglia and, as a result, brain function may be possible.

Prostaglandin E2 and 4-aminopyridine prevent the lipopolysaccharide-induced outwardly rectifying potassium current and interleukin-1beta production in cultured rat microglia.

Brain inflammation includes microglial activation and enhanced production of diffusible chemical mediators, including prostaglandin E2. Prostaglandin E2 is generally considered a proinflammatory molecule, but it also promotes neuronal survival and down-regulates some aspects of microglial activation. It remains unknown, however, if and how prostaglandin E2 prevents microglial activation. In primary culture, microglial activation is predicted by a characteristic pattern of whole-cell potassium currents and interleukin-1beta production. We investigated if prostaglandin E2 could alter these currents and, if so, whether these currents are necessary for microglial activation. Microglia were isolated from mixed cell cultures prepared from neonatal rat brains and exposed to 0-10 microM prostaglandin E2 and lipopolysaccharide for 24 h. Currents were elicited by using standard patch-clamp technique, and interleukin-1beta production was measured by ELISA. Peak outward current densities in microglia treated with lipopolysaccharide plus prostaglandin E2 (10 nM) were reduced significantly from those of cells treated with lipopolysaccharide alone. Prostaglandin E2 and 4-aminopyridine (a blocker of outward potassium currents) also significantly reduced interleukin-1beta production. Thus, although prostaglandin E2 is classified generally as a proinflammatory chemical, it has complex roles in brain inflammation that include preventing microglial activation, perhaps by reducing the outward potassium current.

Neuronal nitric oxide synthase expression is induced in neocortical astrocytes after spreading depression.

Spreading depression (SD) confers either increased susceptibility to ischemic injury or a delayed protection. Because nitric oxide modulates ischemic injury, we investigated if altered expression of nitric oxide synthase (NOS) by SD could account for the effect of SD on ischemia. Furthermore, the identity of cells expressing NOS after SD is important, since SD results in heterogeneous, cell type-specific changes in intracellular environment, which can control NOS activity. Immunohistochemical, computer-based image analyses and Western blotting show that the number of neuronal NOS (nNOS)-positive cells in the somatosensory cortex was significantly increased at 6 hours and 3 days after SD (P < 0.05 and 0.01, respectively), whereas inducible NOS expression remained unchanged. Double-labeling of nNOS and glial fibrillary acidic protein identified these nNOS-positive cells as astrocytes. The effect of altered NO production on induced nNOS expression was examined by treating rats with sodium nitroprusside or NA-nitro-L-arginine methyl ester (LNAM) during SD. Increased nNOS expression was prevented by sodium nitroprusside and phenylephrine or phenylephrine alone, but not LNAM. Because SD increased astrocytic nNOS expression at time points correlating with both ischemic hypersensitivity and ischemic tolerance, the ability of SD to modulate ischemic injury must be complex, perhaps involving NOS but other factors as well.

Long-term elevation of cyclooxygenase-2, but not lipoxygenase, in regions synaptically distant from spreading depression.

Eicosanoids, produced from arachidonic acid by cyclooxygenases (COXs) and lipoxygenases (LIPOXs), are involved in numerous brain processes. To explore if brief and noninjurious stimuli chronically alter expression of these enzymes, we examined the induction of COX-2 and LIPOX expression following unilateral neocortical spreading depression (SD). Expression was examined over time and in regions not experiencing SD (hippocampus) but synaptically connected to the site of stimulation (cortex). One hundred six male Wistar rats had SD induced via microinjection of 0.5 M KCl (0.5 M NaCl for sham) into left parietal cortex every 9 minutes for 1 or 3 hours. One hour before SD some animals received dexamethasone (Dex), mepacrine (Mep), indomethacin (Indo), nordihydroguaiaretic acid (Ndga), phenylephrine (Pe), sodium nitroprusside (Snp) with Pe, or N omega-nitro-L-arginine methyl ester (Lnam). Animals survived for 0, 3, or 6 hours, or 1, 2, 3, 7, 14, 21, or 28 days. Brains were processed immunohistochemically for COX-2 and LIPOX, and the optical density (OD) of the left and right cortex, dentate gyrus (DG), CA3, and CA1 immunoreactivity (IR) were measured. Induction was expressed as the log of left divided by right side OD for each region. COX-2 IR in the left cortex was elevated rapidly and was sustained for 21 days following SD. COX-2 IR was also elevated in the ipsilateral hippocampus not experiencing SD, with the rank order of induction as follows: DG > CA3 > CA1. Dex, Snp, and/or Pe significantly reduced the induction of COX-2. No changes in LIPOX IR were observed. These results show that long-term changes in COX-2 expression are induced by SD and these changes decrease with synaptic distance. Benign stimuli increase COX-2 expression and thus may influence brain function for extended periods and at distant locations.

Eicosanoids and nitric oxide influence induction of reactive gliosis from spreading depression in microglia but not astrocytes.

Microglia and astrocytes are transformed into reactive glia (RG) by brain disease and normal function. Eicosanoids and nitric oxide (NO), two intercellular mediators, may influence gliosis. We investigated how drugs that alter production of these paracrine signals effect induction of glial reactivity from spreading depression. Unilateral (left) neocortical spreading depression was induced in 95 halothane anesthetized rats by intracortical injections of 0.5 M KCl, with or without drug treatment (five animals/group). Immunohistochemical staining (IS) intensity using the OX-42 and anti-glial fibrillary acidic protein (GFAP) antibodies determined reactivity in microglia and astrocytes, respectively. After 3 days, brains were processed for OX-42 and GFAP-IS and mean optical densities (OD) of IS were measured. Average OD's (for OX-42) and the log ratio (left/right) of OD's (OX-42 and GFAP) were compared to normal animals. Spreading depression induced significant log ratios for both OX-42- and GFAP-IS (P's < 0.01). However, dexamethasone (a glucocorticoid), nordihydroguaiaretic acid (a lipoxygenase inhibitor), and nitroprusside (a NO donor) prevented significant left sided and log ratio OD values for microglia (P's > 0.05). L-Name, a NO synthase inhibitor, caused significant increases in left and right OD's for microglia (P's < 0.05). Mepacrine, a phospholipase A2 inhibitor, Indomethacin, a cyclooxygenase inhibitor, and phenylephrine, an adrenergic agonist, did not prevent induction of significant OX-42 log ratios (P's < 0.01, 0.05, 0.01), and resulted in increases in left side OD's (P's < 0.01, 0.05, 0.05). Significant GFAP log ratios occurred after spreading depression in all drug groups, P's < 0.01. Thus, induction of reactivity in microglia is more sensitive to eicosanoids and NO than in astrocytes.

Microglia and the developing olfactory bulb.

The developmental appearance of microglia in the rat olfactory bulb was investigated through the use of selective staining with the B4-isolectin from Griffonia simplicifolia. No changes in the density or distribution of either the spherical, macrophage "ameboid" form or the highly arborized "ramified" variety of microglia were observed in the superficial layers of the bulb between postnatal days 10 and 30. The subependymal zone exhibited the only substantial population of ameboid cells and the only developmental increases in ramified cell density during this time-period. External single naris closure, which enhances cell death in the ipsilateral bulb, did not affect microglia density, presumably due to the unusually high numbers of microglia normally present in the bulb. The olfactory bulb has a dense and relatively uniform population of microglial cells from very early stages of postnatal life, perhaps because of the constant turnover of cells and processes.

Unilateral olfactory deprivation: effects on succinate dehydrogenase histochemistry and [3H]leucine incorporation in the olfactory mucosa.

Surgically closing one external naris reduces airflow through one half of the nasal cavity, decreasing the access of odors to the receptor sheet. In rats, unilateral naris occlusion performed near birth results in large reductions in the size of the olfactory bulb, the primary central relay, when examined 30 days later. Previous research has demonstrated that there is a rapid reduction in [3H]2-deoxyglucose (2-DG) and [3H]leucine uptake in the bulb within hours after naris closure. The present study examined whether similar rapid changes could be observed in the sensory periphery. Pups occluded on P1 and examined on P3 with succinate dehydrogenase histochemistry exhibited reduced staining on the closed side of the nasal cavity, suggesting occlusion results in reductions in mucosal metabolism. Larger differences in staining were observed in pups examined at P6. [3H]Leucine incorporation was quite similar on both sides of the nasal septum as late as 30 days post occlusion, suggesting less dramatic changes in protein synthesis. The results suggest that naris closure does indeed have rapid effects on mucosal function, but indicate that the changes are different than those observed in the bulb.