No abstract

Necroptosis and pyroptosis are two forms of programmed cell death with a common feature of plasma membrane rupture. Here we studied the morphology and mechanism of pyroptosis in comparison with necroptosis. Different from necroptosis, pyroptosis undergoes membrane blebbing and produces apoptotic body-like cell protrusions (termed pyroptotic bodies) prior to plasma membrane rupture. The rupture in necroptosis is explosion-like, whereas in pyroptosis it leads to flattening of cells. It is known that the execution of necroptosis is mediated by mixed lineage kinase domain-like (MLKL) oligomers in the plasma membrane, whereas gasdermin-D (GSDMD) mediates pyroptosis after its cleavage by caspase-1 or caspase-11. We show that N-terminal fragment of GSDMD (GSDMD-N) generated by caspase cleavage also forms oligomer and migrates to the plasma membrane to kill cells. Both MLKL and GSDMD-N are lipophilic and the N-terminal sequences of both proteins are important for their oligomerization and plasma membrane translocation. Unlike MLKL which forms channels on the plasma membrane that induces influx of selected ions which osmotically swell the cells to burst, GSDMD-N forms non-selective pores and does not rely on increased osmolarity to disrupt cells. Our study reveals the pore-forming activity of GSDMD and channel-forming activity of MLKL determine different ways of plasma membrane rupture in pyroptosis and necroptosis.

Locomotor activity is a polygenic trait that varies widely among inbred strains of mice (). To characterize the role of D2 dopamine receptors in locomotion, we generated F2 hybrid (129/Sv x C57BL/6) D2 dopamine receptor (D2R)-deficient mice by gene targeting and investigated the contribution of genetic background to open-field activity and rotarod performance. Horizontal activity of D2R-/- mice was approximately half that of drug-naive, strain-matched controls but was significantly greater than haloperidol-treated controls, which were markedly hypokinetic. Wild-type 129/SvEv and C57BL/6 mice with functional D2 receptors had greater interstrain differences in spontaneous activity than those among the F2 hybrid mutants. Incipient congenic strains of D2R-deficient mice demonstrated an orderly gene dosage reduction in locomotion superimposed on both extremes of parental background locomotor activity. In contrast, F2 hybrid D2R-/- mice had impaired motor coordination on the rotarod that was corrected in the congenic C57BL/6 background. Wild-type 129/SvEv mice had the poorest rotarod ability of all groups tested, suggesting that linked substrain 129 alleles, not the absence of D2 receptors per se, were largely responsible for the reduced function of the F2 hybrid D2R-/- and D2R+/- mice. Neurochemical and pharmacological studies revealed unexpectedly normal tissue striatal monoamine levels and no evidence for supersensitive D1, D3, or D4 dopamine receptors in the D2R-/- mice. However, after acute monoamine depletion, akinetic D2R+/- mice had a significantly greater synergistic restoration of locomotion in response to SKF38393 and quinpirole compared with any group of D2R+/+ controls. We conclude that D2R-deficient mice are not a model of Parkinson's disease. Our studies highlight the interaction of multiple genetic factors in the analysis of complex behaviors in gene knock-out mice.

The human dopamine D4 receptor (D4R) has received considerable attention because of its high affinity for the atypical antipsychotic clozapine and the unusually polymorphic nature of its gene. To clarify the in vivo role of the D4R, we produced and analyzed mutant mice (D4R-/-) lacking this protein. Although less active in open field tests, D4R-/- mice outperformed wild-type mice on the rotarod and displayed locomotor supersensitivity to ethanol, cocaine, and methamphetamine. Biochemical analyses revealed that dopamine synthesis and its conversion to DOPAC were elevated in the dorsal striatum from D4R-/- mice. Based on these findings, we propose that the D4R modulates normal, coordinated and drug-stimulated motor behaviors as well as the activity of nigrostriatal dopamine neurons.