Eryn L. Werry

Selective cell death in neurodegeneration: why are some neurons spared in vulnerable regions?

Neurodegenerative diseases exhibit varying and characteristic patterns of regional brain cell death, yet in each disorder there are distinct variations in the relative vulnerability of neurons within targeted brain regions. For example, in Parkinsons disease (PD) up to 90% of dopaminergic neurons of the ventral tier of the substantia nigra pars compacta are lost at post-mortem, while as few as 25% of dopaminergic neurons in the dorsal tier of this nucleus succumb to the disease process. To date the reasons underlying differential vulnerability of similar neurons within a defined cytoarchitectural region has received little attention. We suggest variations in protein distribution underlies this differential vulnerability. Within the substantia nigra pars compacta the more vulnerable neurons exhibit an increased expression of factors that may contribute to vulnerability (D(2) dopamine autoreceptors, GIRK-2 potassium channels, lactotransferrin and the dopamine transporter) while also expressing a relative lack of neuroprotective elements (dopamine vesicle transport protein and a number of trophic and growth factors). Differential distribution of key proteins expressed by individual cells within the same cytoarchitectural brain region may influence the severity and likelihood of common neurodegenerative mechanisms, such as protein aggregation, oxidative stress, neuroinflammation and apoptosis, and thus the eventual fate of individual cells in the disease process. An understanding of how variable protein expression can influence cell survival within the diseased human brain in a range of neurodegenerative disorders may provide avenues for the development of novel strategies to improve the survival of targeted neurons in vivo.

Glutamate-stimulated ATP release from spinal cord astrocytes is potentiated by substance P

ATP has recently emerged as a key molecule mediating pathological pain. The aim of this study was to examine whether spinal cord astrocytes could be a source of ATP in response to the nociceptive neurotransmitters glutamate and substance P. Glutamate stimulated ATP release from these astrocytes and this release was greatly potentiated by substance P, even though substance P alone did not elicit ATP release. Substance P also potentiated glutamate‐induced inward currents, but did not cause such currents alone. When glutamate was applied alone it acted exclusively through α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐proprionate receptors to stimulate Ca2+ influx‐dependent ATP release. However, when substance P was co‐applied with glutamate, ATP release could be elicited by activation of NMDA and metabotropic glutamate receptors. Activation of neurokinin receptor subtypes, protein kinase C and phospholipases A2, C and D were needed for substance P to bring about its effects. These results suggest that astrocytes may be a major source of ATP in the spinal cord on activation of nerve fibres that release substance P and glutamate.

Secretion of ATP from Schwann cells in response to uridine triphosphate

The mechanisms by which uridine triphosphate (UTP) stimulates ATP release from Schwann cells cultured from the sciatic nerve were investigated using online bioluminescence techniques. UTP, a P2Y2 and P2Y4 receptor agonist, stimulated ATP release from Schwann cells in a dose‐dependent manner with an ED50 of 0.24 µm. UTP‐stimulated ATP release occurs through P2Y2 receptors as it was blocked by suramin which inhibits P2Y2 but not P2Y4 receptors. Furthermore, positive immunostaining of P2Y2 receptors on Schwann cells was revealed and GTP, an equipotent agonist with UTP at rat P2Y4 receptors, did not significantly stimulate ATP release. UTP‐stimulated ATP release involved second messenger pathways as it was attenuated by the phospholipase C inhibitor U73122, the protein kinase C inhibitor chelerytherine chloride, the IP3 formation inhibitor lithium chloride, the cell membrane‐permeable Ca2+ chelator BAPTA‐AM and the endoplasmic reticulum Ca2+‐dependent ATPase inhibitor thapsigargin. Evidence that ATP may be stored in vesicles that must be transported to the cell membrane for exocytosis was found as release was significantly reduced by the Golgi‐complex inhibitor brefeldin A, microtubule disruption with nocodazole, F‐actin disruption with cytochalasin D and the specific exocytosis inhibitor botulinum toxin A. ATP release from Schwann cells also involves anion transport as it was significantly reduced by cystic fibrosis transmembrane conductance regulator inhibitor glibencamide and anion transporter inhibitor furosemide. We suggest that UTP‐stimulated ATP release is mediated by activation of P2Y2 receptors that initiate an IP3–Ca2+ cascade and protein kinase C which promote exocytosis of ATP from vesicles as well as anion transport of ATP across the cell membrane.

Purine Release from Spinal Cord Microglia after Elevation of Calcium by Glutamate

The propagation of Ca2+ waves in a network of microglial cells, after its initiation by glutamate, is mediated by purinergic transmission. In this study, we investigated the mechanisms by which glutamate releases ATP from cultured spinal cord microglia. The 4-fold increase in ATP release from microglia in response to glutamate (0.5 mM) was blocked by α-aminohydroxy-5-methyl-isoxazole-4-proprionate (AMPA)/kainate receptor antagonist 6-cyano-7-nitroguinoxaline-2,3-dione and specific AMPA receptor antagonist 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466) but not by N-methyl-d-aspartic acid or metabotropic glutamate receptor antagonists. Glutamate acting on AMPA receptors evoked an ATP release that was blocked by antagonizing the rise in intracellular Ca2+ as a result of its release from internal stores as well as by antagonizing protein kinase C with chelerythrine. Glutamate-stimulated ATP release was significantly antagonized by the cystic fibrosis transmembrane conductance regulator (CFTR) blockers flufenamic acid and glibenclamide. A role for the CFTR was further confirmed using microglia from CFTR knockout mice, which released significantly less ATP than microglia from control wild-type mice in response to glutamate. Use of 6-methoxy-1-(3-sulfopropyl)quinolinium fluorescence assay revealed functional CFTR in microglia. These observations suggest that glutamate acted on microglial AMPA receptors to stimulate release of Ca2+ from intracellular stores as well as a Ca2+-dependent isoform of protein kinase C, which then acts to trigger release of ATP with the CFTR acting as a regulator of the ATP release process, perhaps through another channel or transporter.

Lipopolysaccharide-stimulated interleukin-10 release from neonatal spinal cord microglia is potentiated by glutamate.

Interleukin-10 (IL-10) is a cytokine with important endogenous and therapeutic anti-inflammatory effects. Given this, it is of interest to investigate factors that modulate IL-10 levels in the central nervous system. IL-10 is released after lipopolysaccharide (LPS) stimulation of microglia. Microglia also express functional glutamate receptors and in inflammatory conditions are exposed to increased levels of glutamate. The aim of this research, then, is to investigate whether glutamate can modulate lipopolysaccharide stimulation of IL-10 release from neonatal rat spinal cord microglia. Enzyme-linked immunosorbent assays (ELISAs) were used to quantify IL-10 release from cultured neonatal spinal cord microglia and reverse transcriptase-polymerase chain reaction (RT-PCR) was used to measure IL-10 mRNA expression. Glutamate (1 mM) significantly increased LPS (1 μg/ml)-stimulated IL-10 release from microglia by 172% (EC(50) of 103 μM) and significantly upregulated IL-10 mRNA levels. Glutamate potentiated LPS-stimulated IL-10 release by binding all subtypes of glutamate receptor. These results show that glutamate substantially increases the release of an anti-inflammatory cytokine from neonatal spinal cord microglia activated by a high concentration of LPS.

Effect of age on proliferation-regulating factors in human adult neurogenic regions

J. Neurochem. (2010) 115, 956–964.

TSPO as a target for glioblastoma therapeutics

The translocator protein (TSPO) is an 18-kDa five-transmembrane protein, which is primarily found in the outer mitochondrial membrane. Levels of this protein are up-regulated in the most aggressive and common glioma, glioblastoma multiforme (GM). Levels of TSPO also correlate with GM clinical outcome, suggesting that TSPO may be a novel GM diagnostic imaging agent. Therapeutically, targeting the TSPO may provide a mechanism to abrogate the apoptotic-resistant, invasive and aggressive nature of GM and may also provide a way of targeting other anti-cancer treatments to GM sites. This review highlights recent progress in research on TSPO-based diagnostic imaging and therapeutics for GM.

N-substituted 8-aminopentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecanes as σ receptor ligands with potential neuroprotective effects.

Several libraries of similarly N-substituted 8-aminopentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecanes (9), N-methyl-8-aminopentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecanes (14), and N-methyl-11-aminopentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecan-8-ones (13) were synthesised and screened against a panel of CNS targets in order to develop structure-affinity relationships for cage-modified trishomocubane σ receptor ligands based on the N-substituted 4-azahexacyclo[5.4.1.0(2,6).0(3,10).0(5,9).0(8,11)]dodecan-3-ol (8) scaffold. In general, compared to the corresponding 4-azahexacyclo[5.4.1.0(2,6).0(3,10).0(5,9).0(8,11)]dodecan-3-ols, compounds of type 9 were potent σ receptor ligands with low levels of subtype selectivity, while the corresponding N-methyl-8-aminopentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecanes showed reduced affinity but greater selectivity for σ2 receptors. The N-methyl-11-aminopentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecan-8-ones demonstrated the poorest σ receptor affinities, suggesting that 4-azahexacyclo[5.4.1.0(2,6).0(3,10).0(5,9).0(8,11)]dodecan-3-ols interact with σ receptors in the bridged hemiaminal form rather than as the non-transannular, aminoketone tautomers. Several compounds of type 8, 9, and 14 were assessed for their ability to inhibit nitric oxide release in vitro, and demonstrated comparable or greater efficacy than 4-phenyl-1-(4-phenylbutyl)piperidine (PPBP), an established neuroprotective σ ligand with NOS inhibitory activity.

Discovery and pharmacological evaluation of a novel series of adamantyl cyanoguanidines as P2X7 receptor antagonists

Here we report adamantyl cyanoguanidine compounds based on hybrids of the adamantyl amide scaffold reported by AstraZeneca and cyanoguanidine scaffold reported by Abbott Laboratories. Compound 27 displayed five-fold greater inhibitory potency than the lead compound 2 in both pore-formation and interleukin-1β release assays, while 35-treated mice displayed an antidepressant phenotype in behavioral studies. This SAR study provides a proof of concept for hybrid compounds, which will help in the further development of P2X7R antagonists.

First Demonstration of Positive Allosteric-like Modulation at the Human Wild Type Translocator Protein (TSPO)

We show that changing the number and position of nitrogen atoms in the heteroatomic core of a pyrazolopyrimidine acetamide is sufficient to induce complex binding to wild type human TSPO. Only compounds with this complex binding profile lacked intrinsic effect on glioblastoma proliferation but positively modulated the antiproliferative effects of a synthetic TSPO ligand. To the best of our knowledge this is the first demonstration of allosteric-like interaction at the wild type human TSPO.