Neuronal caspase 2 activity and function requires RAIDD, but not PIDD.

Caspase 2 was initially identified as a neuronally expressed developmentally down-regulated gene (HUGO gene nomenclature CASP2) and has been shown to be required for neuronal death induced by several stimuli, including NGF (nerve growth factor) deprivation and Aß (ß-amyloid). In non-neuronal cells the PIDDosome, composed of caspase 2 and two death adaptor proteins, PIDD (p53-inducible protein with a death domain) and RAIDD {RIP (receptor-interacting protein)-associated ICH-1 [ICE (interleukin-1ß-converting enzyme)/CED-3 (cell-death determining 3) homologue 1] protein with a death domain}, has been proposed as the caspase 2 activation complex, although the absolute requirement for the PIDDosome is not clear. To investigate the requirement for the PIDDosome in caspase-2-dependent neuronal death, we have examined the necessity for each component in induction of active caspase 2 and in execution of caspase-2-dependent neuronal death. We find that both NGF deprivation and Aß treatment of neurons induce active caspase 2 and that induction of this activity depends on expression of RAIDD, but is independent of PIDD expression. We show that treatment of wild-type or PIDD-null neurons with Aß or NGF deprivation induces formation of a complex of caspase 2 and RAIDD. We also show that caspase-2-dependent execution of neurons requires RAIDD, not PIDD. Caspase 2 activity can be induced in neurons from PIDD-null mice, and NGF deprivation or Aß use caspase 2 and RAIDD to execute death of these neurons.

Intronically encoded siRNAs improve dynamic range of mammalian gene regulation systems and toggle switch.

Applications of conditional gene expression, whether for therapeutic or basic research purposes, are increasingly requiring mammalian gene control systems that exhibit far tighter control properties. While numerous approaches have been used to improve the widely used Tet-regulatory system, many applications, particularly with respect to the engineering of synthetic gene networks, will require a broader range of tightly performing gene control systems. Here, a generically applicable approach is described that utilizes intronically encoded siRNA on the relevant transregulator construct, and siRNA sequence-specific tags on the reporter construct, to minimize basal gene activity in the off-state of a range of common gene control systems. To demonstrate tight control of residual expression the approach was successfully used to conditionally express the toxic proteins RipDD and Linamarase. The intronic siRNA concept was also extended to create a new generation of compact, single-vector, autoinducible siRNA vectors. Finally, using improved regulation systems a mammalian epigenetic toggle switch was engineered that exhibited superior in vitro and in vivo induction characteristics in mice compared to the equivalent non-intronic system.