1. Activation of caspase-3 and apoptosis in cerebellar granule cells
A Guidotti, M Saito, M J Berg, N Marks J Neurosci Res . 1998 May 1;52(3):334-41. doi: 10.1002/(SICI)1097-4547(19980501)52:33.0.CO;2-E.
Caspase-3 activity increased dramatically in cytosolic extracts of rat cerebellar granule cells exposed to apoptotic conditions (basal medium Eagle (BME) containing 5 mM K+ without serum) when assayed with Ac-DEVD-amc, but not with Ac-YVAD-afc, a preferred substrate for caspase-1. This provided a basis to examine relationships between enzyme activity and cell viability for purposes of selecting an optimal time for comparing neuroprotective agents or strategies. Exposure of neurons to an apoptotic medium containing 5 mM K+ in absence of serum led to a rapid 5- to 10-fold increase in caspase-3 within 2-4 hr but without significant cell loss, or morphological alterations. Exposure to apoptotic medium followed by replacement with maintenance medium containing 25 mM K+ and serum led to a rapid fall in caspase-3 and prevention of cell death. This strategy was not effective after 13 hr exposure despite a large fall in enzyme activity. These temporal changes infer systems for rapid enzyme turnover and/or activation of cytoplasmic components linked to later DNA degradation. The effects of cycloheximide point to requirements for protein synthesis, and those of Glu exclude a caspase-3 dependent pathway for necrotic cell damage. Brief treatment with 10 microM LIGA20, an anti-necrotic agent, also attenuated cell loss and caspase-3 activity, indicating a broad spectrum of neuroprotection. Rapid and long-lasting effects, together with its biophysical properties, suggest that this semisynthetic ganglioside acted upstream at or near a membrane site. As such, gangliosides provide useful agents to further probe pathways relevant to neuronal death in culture.
2. Calcium Pyrophosphate And Monosodium Urate Activate The NLRP3 Inflammasome Within Bladder Urothelium Via Reactive Oxygen Species And TXNIP
Patrick D Leidig, Shelby N Harper, Francis M Hughes Jr, Huixia Jin, J Todd Purves Res Rep Urol . 2019 Nov 20;11:319-325. doi: 10.2147/RRU.S225767.
Objective:To investigate the in vitro activation of the NLRP3 inflammasome within bladder urothelium by stone-forming components. Further, to describe the contributions of reactive oxygen species (ROS) and thioredoxin-interacting protein (TXNIP), an important structural component of the inflammasome, to this activation.Methods:Urothelial cells were harvested and incubated overnight. For agonist studies, cells were treated with varying concentrations of calcium pyrophosphate (CPPD) and monosodium urate (MSU). For inhibitor studies, cells were treated with either N-acetylcysteine (NAC) (1 hr) or Verapamil (4 hrs) prior to incubation with either CPPD (62.5 ug/mL) or MSU (1.25 ug/mL) for 24 hrs. Untreated controls were incubated with ATP (1.25 mM) for 1 hr to maximally stimulate NLRP3 inflammasome activity (measured as caspase-1 cleavage of the fluorogenic substrate Ac-YVAD-AFC). Results are reported as a percentage of maximum ATP response.Results:CPPD and MSU activate caspase-1 in urothelial cells in a dose-dependent manner, reaching ~50% and ~25% of the ATP response, respectively. Pre-treatment with the general ROS scavenger NAC reduces this activation in a dose-dependent manner. Additionally, activation was suppressed through treatment with Verapamil, a known downregulator of TXNIP expression.Conclusion:The stone components CPPD and MSU activate NLRP3 in an ROS and TXNIP-dependent manner in bladder urothelium. These findings demonstrate the importance of ROS and TXNIP, and suggest that targeting either may be a way to decrease stone-dependent NLRP3 inflammation within the bladder.
