Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.

Background-Gene-environment interactions (GEI) are involved in the pathogenesis of mental diseases. We evaluated interaction between mutant human Disrupted-In-Schizophrenia-1 (mhDISC1) and maternal immune activation implicated in schizophrenia and mood disorders.

Strong genetic evidence implicates mutations and polymorphisms in the gene Disrupted-In-Schizophrenia-1 (DISC1) as risk factors for both schizophrenia and mood disorders. Recent studies have shown that DISC1 has important functions in both brain development and adult brain function. We have described earlier a transgenic mouse model of inducible expression of mutant human DISC1 (hDISC1) that acts in a dominant-negative manner to induce the marked neurobehavioral abnormalities. To gain insight into the roles of DISC1 at various stages of neurodevelopment, we examined the effects of mutant hDISC1 expressed during (1) only prenatal period, (2) only postnatal period, or (3) both periods. All periods of expression similarly led to decreased levels of cortical dopamine (DA) and fewer parvalbumin-positive neurons in the cortex. Combined prenatal and postnatal expression produced increased aggression and enhanced response to psychostimulants in male mice along with increased linear density of dendritic spines on neurons of the dentate gyrus of the hippocampus, and lower levels of endogenous DISC1 and LIS1. Prenatal expression only resulted in smaller brain volume, whereas selective postnatal expression gave rise to decreased social behavior in male mice and depression-like responses in female mice as well as enlarged lateral ventricles and decreased DA content in the hippocampus of female mice, and decreased level of endogenous DISC1. Our data show that mutant hDISC1 exerts differential effects on neurobehavioral phenotypes, depending on the stage of development at which the protein is expressed. The multiple and diverse abnormalities detected in mutant DISC1 mice are reminiscent of findings in major mental diseases.

The spatial organization of the cerebellar afferent map has remarkable correspondence to two aspects of intrinsic patterning within the cerebellum embodied by a series of lobules and Purkinje cell (PC)-striped gene expression. Using male and female mice, we tested whether the Engrailed (En) homeobox genes are a common genetic substrate regulating all three systems, since they are expressed in spatially restricted domains within the cerebellum and are critical for patterning PC gene expression and foliation. Indeed, we discovered that En1/2 are necessary for the precise targeting of mossy fibers to distinct lobules, as well as their subsequent resolution into discrete parasagittal bands. Moreover, each En gene coordinately regulates afferent targeting and the striped pattern of PC protein expression (e.g., ZebrinII/AldolaseC) independent of regulating foliation. We further found that En1/2, rather than the presence of a full complement of lobules, are critical for generating PC protein stripes and mossy fiber bands, and that PC striped gene expression is determined before afferent banding. Thus, the En transcription factors not only regulate cerebellum circuit topography, but they also link afferent and efferent neurons precisely enough that alterations in PC protein expression can be used as a read out for underlying defects in circuitry. In summary, our data suggest that En1/2 are master regulators of three-dimensional organization of the cerebellum and coordinately regulate morphology, patterned gene expression, and afferent topography.

The Purkinje cell is a primary organizer in the development of the cerebellum. Purkinje cells may provide positional information cues that regulate afferent innervation, and Purkinje cell target size controls the adult number of afferent olivary neurons and granule cells. While Purkinje cells are necessary for the survival of olivary neurons and granule cells during periods of programmed cell death, little is known about the survival requirements of Purkinje cells in vivo. To determine if Purkinje cells are subject to programmed cell death during development we have analyzed Purkinje cell numbers in two lines of transgenic mice that overexpress a human gene for bcl-2 (Hu-bcl-2). Bcl-2 is a protooncogene that inhibits apoptosis in many cell types. Overexpression of bcl-2 in vitro and in vivo rescues neurons from trophic factor deprivation or naturally occurring cell death. In the mice analyzed in this study, transgene expression is driven by the neuron-specific enolase promoter that is first expressed embryonically in most regions of the brain in one line and postnatally in the second line. We have counted Purkinje cells in three adult control mice, five early overexpressing transgenics, and three late expressing transgenics. The number of Purkinje cells in the Hu-bcl-2 transgenic mice is significantly increased above control numbers, with an increase of 43% in the embryonically overexpressing line and an increase of 27% in the postnatally overexpressing line. Because bcl-2 overexpression has been shown to rescue other neurons from programmed cell death, the increase in Purkinje cell numbers in overexpressing bcl-2 transgenics suggests that Purkinje cells undergo a period of cell death during normal development.

The regulator of G protein signaling (RGS) molecules are a class of proteins that modulate the signaling activity of G-protein coupled receptors. Regulator of G protein signaling 4 (RGS4) is of particular interest in schizophrenia since it is associated with the dopamine (DA) receptor, its expression is altered in affected CNS tissue, and polymorphisms in the RGS4 gene are being examined as risk factors for the disease (Morris et al.2004, Am J Med Genet B Neuropsychiatr Genet 125:50-53; Prasad et al.2005, Mol Psychiatry 10:213-219; Williams et al.2004, Biol Psychiatry 55:192-195). To further test for the involvement of RGS4 expression in schizophrenia, we examined a selection of different cortical and subcortical regions in human brain for alterations in RGS4 mRNA and protein expression. To evaluate the effect of antipsychotic medication on RGS4 expression levels, we compared a subset of treated and untreated cases that were off antipsychotic medication for at least 3 months prior to death. We report a significant decrease in RGS4 mRNA levels in the cingulate gyrus, superior frontal gyrus, and the insular cortex of all schizophrenia cases when compared with controls. A decrease in RGS4 mRNA was also observed in the caudate, but only in the medicated schizophrenia cases. Measurement of protein levels using Western blot demonstrated that RGS4 protein is decreased in the frontal cortex of schizophrenia cases.

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