Anindya Bhattacharya

Overexpression of NGF in mouse urothelium leads to neuronal hyperinnervation, pelvic sensitivity and changes in urinary bladder function

NGF has been suggested to play a role in urinary bladder dysfunction by mediating inflammation, as well as morphological and functional changes, in sensory and sympathetic neurons innervating the urinary bladder. To further explore the role of NGF in bladder sensory function, we generated a transgenic mouse model of chronic NGF overexpression in the bladder using the urothelium-specific uroplakin II (UPII) promoter. NGF mRNA and protein were expressed at higher levels in the bladders of NGF-overexpressing (NGF-OE) transgenic mice compared with wild-type littermate controls from postnatal day 7 through 12-16 wk of age. Overexpression of NGF led to urinary bladder enlargement characterized by marked nerve fiber hyperplasia in the submucosa and detrusor smooth muscle and elevated numbers of tissue mast cells. There was a marked increase in the density of CGRP- and substance P-positive C-fiber sensory afferents, neurofilament 200-positive myelinated sensory afferents, and tyrosine hydroxylase-positive sympathetic nerve fibers in the suburothelial nerve plexus. CGRP-positive ganglia were also present in the urinary bladders of transgenic mice. Transgenic mice had reduced urinary bladder capacity and an increase in the number and amplitude of nonvoiding bladder contractions under baseline conditions in conscious open-voiding cystometry. These changes in urinary bladder function were further associated with an increased referred somatic pelvic hypersensitivity. Thus, chronic urothelial NGF overexpression in transgenic mice leads to neuronal proliferation, focal increases in urinary bladder mast cells, increased urinary bladder reflex activity, and pelvic hypersensitivity. NGF-overexpressing mice may, therefore, provide a useful transgenic model for exploring the role of NGF in urinary bladder dysfunction.

The microglial ATP‐gated ion channel P2X7 as a CNS drug target

Based on promising preclinical evidence, microglial P2X7 has increasingly being recognized as a target for therapeutic intervention in neurological and psychiatric diseases. However, despite this knowledge no P2X7‐related drug has yet entered clinical trials with respect to CNS diseases. We here discuss the current literature on P2X7 being a drug target and identify unsolved issues and still open questions that have hampered the development of P2X7 dependent therapeutic approaches for CNS diseases. It is concluded here that the lack of brain penetrating P2X7 antagonists is a major obstacle in the field and that central P2X7 is a yet untested clinical drug target. In the CNS, microglial P2X7 activation causes neuroinflammation, which in turn plays a role in various CNS disorders. This has resulted in a surge of brain penetrant P2X7 antagonists. P2X7 is a viable, clinically untested CNS drug target. GLIA 2016;64:1772–1787

Role of neuro-immunological factors in the pathophysiology of mood disorders

Mood disorders, despite the widespread availability of monoamine-based antidepressant treatments, are associated with persistently high rates of disability, together with elevated rates of mortality due to suicide, cardiovascular disease, and other causes. The development of more effective treatments has been hindered by the lack of knowledge about the etiology and pathogenesis of mood disorders. An emerging area of science that promises novel pathways to antidepressant and mood stabilizing therapies surrounds evidence that immune cells and their signaling play a major role in the pathophysiology of major depressive disorder (MDD) and bipolar disorder (BD). Here, we review evidence that the release of neuroactive cytokines, particularly interleukins such as IL-1β, IL-6, and TNF-α, is altered in these disorders and discuss mechanisms such as the ATP-gated ion channel P2X7, through which cytokine signaling can influence neuro-glial interactions. Brain P2X7, an emerging target and antagonism of P2X7 holds promise as a novel mechanism for targeting treatment-resistant depression. We further discuss the role of microglia and astroglia in central neuroinflammation and their interaction with the peripheral immune system We present extant clinical evidence that bolsters the role of neuroinflammation and neuroactive cytokines in mood disorders. To that end, the role of clinical imaging by probing neuroinflammatory markers is also discussed briefly. Finally, we present data using preclinical neuroinflammation models that produce depression-like behaviors in experimental animals to identify neuroinflammatory mechanisms which may aid in novel neuroimmune target identification for the development of exciting pharmacological interventions in mood disorders.

Critical data‐based re‐evaluation of minocycline as a putative specific microglia inhibitor

Minocycline, a second generation broad‐spectrum antibiotic, has been frequently postulated to be a “microglia inhibitor.” A considerable number of publications have used minocycline as a tool and concluded, after achieving a pharmacological effect, that the effect must be due to “inhibition” of microglia. It is, however, unclear how this “inhibition” is achieved at the molecular and cellular levels. Here, we weigh the evidence whether minocycline is indeed a bona fide microglia inhibitor and discuss how data generated with minocycline should be interpreted. GLIA 2016;64:1788–1794

P2X7 Antagonists as Potential Therapeutic Agents for the Treatment of CNS Disorders

The use of P2X7 antagonists to treat inflammatory disorders has garnered considerable interest in recent years. An increasing number of literature reports support the role of P2X7 in inflammatory pathways of the peripheral and central nervous systems (CNSs). A number of CNS indications such as neuropsychiatric and neurodegenerative disorders and neuropathic pain have been linked to a neuroinflammatory response, and clinical studies have shown that inflammatory biomarkers can be mitigated by modulating P2X7. Recent scientific and patent literature describing novel P2X7 antagonists has indicated their use in CNS disorders. In addition, several reports have disclosed the results of administering P2X7 antagonists in pre-clinical models of CNS disease or investigating brain uptake. This review describes small molecule P2X7 antagonists that have first appeared in the literature since 2009 and have potential therapeutic utility in the CNS, or for which new data have emerged implicating their use in CNS indications.

Transient P2X7 Receptor Antagonism Produces Lasting Reductions in Spontaneous Seizures and Gliosis in Experimental Temporal Lobe Epilepsy.

Neuroinflammation is thought to contribute to the pathogenesis and maintenance of temporal lobe epilepsy, but the underlying cell and molecular mechanisms are not fully understood. The P2X7 receptor is an ionotropic receptor predominantly expressed on the surface of microglia, although neuronal expression has also been reported. The receptor is activated by the release of ATP from intracellular sources that occurs during neurodegeneration, leading to microglial activation and inflammasome-mediated interleukin 1β release that contributes to neuroinflammation. Using a reporter mouse in which green fluorescent protein is induced in response to the transcription of P2rx7, we show that expression of the receptor is selectively increased in CA1 pyramidal and dentate granule neurons, as well as in microglia in mice that developed epilepsy after intra-amygdala kainic acid-induced status epilepticus. P2X7 receptor levels were increased in hippocampal subfields in the mice and in resected hippocampus from patients with pharmacoresistant temporal lobe epilepsy. Cells transcribing P2rx7 in hippocampal slices from epileptic mice displayed enhanced agonist-evoked P2X7 receptor currents, and synaptosomes from these animals showed increased P2X7 receptor levels and altered calcium responses. A 5 d treatment of epileptic mice with systemic injections of the centrally available, potent, and specific P2X7 receptor antagonist JNJ-47965567 (30 mg/kg) significantly reduced spontaneous seizures during continuous video-EEG monitoring that persisted beyond the time of drug presence in the brain. Hippocampal sections from JNJ-47965567-treated animals obtained >5 d after treatment ceased displayed strongly reduced microgliosis and astrogliosis. The present study suggests that targeting the P2X7 receptor has anticonvulsant and possibly disease-modifying effects in experimental epilepsy. SIGNIFICANCE STATEMENT Temporal lobe epilepsy is the most common and drug-resistant form of epilepsy in adults. Neuroinflammation is implicated as a pathomechanism, but the upstream mechanisms driving gliosis and how important this is for seizures remain unclear. In our study, we show that the ATP-gated P2X7 receptor is upregulated in experimental epilepsy and resected hippocampus from epilepsy patients. Targeting the receptor with a new centrally available antagonist, JNJ-47965567, suppressed epileptic seizures well beyond the time of treatment and reduced underlying gliosis in the hippocampus. The findings suggest a potential disease-modifying treatment for epilepsy based on targeting the P2X7 receptor.

Preclinical Evaluation of a P2X7 Receptor–Selective Radiotracer: PET Studies in a Rat Model with Local Overexpression of the Human P2X7 Receptor and in Nonhuman Primates

The P2X7 receptor (P2X7R) orchestrates neuroinflammation, and this is the basis for an increased interest in the development of antagonists inhibiting P2X7R function in the brain. This study provides the preclinical evaluation of 11C-JNJ-54173717, a PET tracer for P2X7R in both rats and nonhuman primates. Methods: 11C-JNJ-54173717 is a high-affinity radiotracer for the human P2X7R (hP2X7R). Biodistribution and radiometabolite studies were performed. Viral vectors encoding either enhanced green fluorescent protein-hP2X7R or 3flag-hP2X7R were engineered and validated in cell culture. hP2X7R was regionally overexpressed in the rat striatum after stereotactic injection of viral vectors. Dynamic small-animal PET studies were performed in vector-injected rats and in healthy monkeys using 11C-JNJ-54173717. Results: The affinity of JNJ-54173717 was 1.6 ± 0.1 nM in a rat cortex P2X7R membrane binding assay. In a functional assay at the recombinant human and rat P2X7R orthologs, the half maximal inhibitory concentration (IC50) of JNJ-54173717 was 4.2 ± 0.01 nM and 7.6 ± 0.01 nM, respectively. The rat biodistribution study showed that 11C-JNJ-54173717 crossed the blood–brain barrier and was cleared from plasma mainly via the hepatobiliary pathway. A polar radiometabolite was found in rat plasma. No radiometabolites were detected in rat brain. Dynamic small-animal PET showed binding of 11C-JNJ-54173717 in the striatum expressing hP2X7R, with rapid washout from the noninjected control striatum and other brain regions. Likewise, 11C-JNJ-54173717 PET signal was blocked by a chemically distinct P2X7R ligand, indicating specific binding to P2X7R in the monkey brain. Conclusion: JNJ-54173717 is a high-affinity P2X7R antagonist. An animal rat model stably expressing hP2X7R was developed and validated, identifying favorable characteristics for 11C-JNJ-54173717 as a PET radioligand for in vivo visualization of hP2X7R. 11C-JNJ-54173717 selectively visualized P2X7R in the monkey brain, and this radioligand will be further evaluated in a clinical setting to study P2X7R expression levels in neurodegenerative disorders.

Recent advances in the biology and medicinal chemistry of TRPA1

The transient receptor potential cation channel, subfamily A, member 1 (TRPA1) is a nonselective cation channel that is highly expressed in small-diameter sensory neurons, where it functions as a polymodal receptor, responsible for detecting potentially harmful chemicals, mechanical forces and temperatures. TRPA1 is also activated and/or sensitized by multiple endogenous inflammatory mediators. As such, TRPA1 likely mediates the pain and neurogenic inflammation caused by exposure to reactive chemicals. In addition, it is also possible that this channel may mediate some of the symptoms of chronic inflammatory conditions such as asthma. We review recent advances in the biology of TRPA1 and summarize the evidence for TRPA1 as a therapeutic drug target. In addition, we provide an update on TRPA1 medicinal chemistry and the progress in the search for novel TRPA1 antagonists.

Pharmacology of a Novel Central Nervous System–Penetrant P2X7 Antagonist JNJ-42253432

In the central nervous system, the ATP-gated Purinergic receptor P2X ligand-gated ion channel 7 (P2X7) is expressed in glial cells and modulates neurophysiology via release of gliotransmitters, including the proinflammatory cytokine interleukin (IL)-1β. In this study, we characterized JNJ-42253432 [2-methyl-N-([1-(4-phenylpiperazin-1-yl)cyclohexyl]methyl)-1,2,3,4-tetrahydroisoquinoline-5-carboxamide] as a centrally permeable (brain-to-plasma ratio of 1), high-affinity P2X7 antagonist with desirable pharmacokinetic and pharmacodynamic properties for in vivo testing in rodents. JNJ-42253432 is a high-affinity antagonist for the rat (pKi 9.1 ± 0.07) and human (pKi 7.9 ± 0.08) P2X7 channel. The compound blocked the ATP-induced current and Bz-ATP [2′(3′)-O-(4-benzoylbenzoyl)adenosine-5′-triphosphate tri(triethylammonium)]–induced release of IL-1β in a concentration-dependent manner. When dosed in rats, JNJ-42253432 occupied the brain P2X7 channel with an ED50 of 0.3 mg/kg, corresponding to a mean plasma concentration of 42 ng/ml. The compound blocked the release of IL-1β induced by Bz-ATP in freely moving rat brain. At higher doses/exposure, JNJ-42253432 also increased serotonin levels in the rat brain, which is due to antagonism of the serotonin transporter (SERT) resulting in an ED50 of 10 mg/kg for SERT occupancy. JNJ-42253432 reduced electroencephalography spectral power in the α-1 band in a dose-dependent manner; the compound also attenuated amphetamine-induced hyperactivity. JNJ-42253432 significantly increased both overall social interaction and social preference, an effect that was independent of stress induced by foot-shock. Surprisingly, there was no effect of the compound on either neuropathic pain or inflammatory pain behaviors. In summary, in this study, we characterize JNJ-42253432 as a novel brain-penetrant P2X7 antagonist with high affinity and selectivity for the P2X7 channel.

Uropathic Observations in Mice Expressing a Constitutively Active Point Mutation in the 5-HT3A Receptor Subunit

Mutant mice with a hypersensitive serotonin (5-HT)3A receptor were generated through targeted exon replacement. A valine to serine mutation (V13′S) in the channel-lining M2 domain of the 5-HT3A receptor subunit rendered the 5-HT3 receptor ∼70-fold more sensitive to serotonin and produced constitutive activity when combined with the 5-HT3B subunit. Mice homozygous for the mutant allele (5-HT3Avs/vs) had decreased levels of 5-HT3A mRNA. Measurements on sympathetic ganglion cells in these mice showed that whole-cell serotonin responses were reduced, and that the remaining 5-HT3 receptors were hypersensitive. Male 5-HT3Avs/vs mice died at 2-3 months of age, and heterozygous (5-HT3Avs/+) males and homozygous mutant females died at 4-6 months of age from an obstructive uropathy. Both male and female 5-HT3A mutant mice had urinary bladder mucosal and smooth muscle hyperplasia and hypertrophy, whereas male mutant mice had additional prostatic smooth muscle and urethral hyperplasia. 5-HT3A mutant mice had marked voiding dysfunction characterized by a loss of micturition contractions with overflow incontinence. Detrusor strips from 5-HT3Avs/vs mice failed to contract to neurogenic stimulation, despite overall normal responses to a cholinergic agonist, suggestive of altered neuronal signaling in mutant mouse bladders. Consistent with this hypothesis, decreased nerve fiber immunoreactivity was observed in the urinary bladders of 5-HT3Avs/vs compared with 5-HT3A wild-type (5-HT3A+/+) mice. These data suggest that persistent activation of the hypersensitive and constitutively active 5-HT3A receptor in vivo may lead to excitotoxic neuronal cell death and functional changes in the urinary bladder, resulting in bladder hyperdistension, urinary retention, and overflow incontinence.