cDKO Mice Research Articles

Enhanced oxidative stress is an early event during development of Alzheimer-like pathologies in presenilin conditional knock-out mice

Conditional double knock-out of presenilin-1 (PS1) and presenilin-2 (PS2) (PS cDKO) in forebrain of mice led to progressive memory dysfunction and forebrain degeneration. These changes in the brain recapitulated most of the neurodegenerative phenotypes of Alzheimer's disease (AD). Oxidative stress in brain tissues is intimately related to AD. In this report, we examined oxidative stress status in cerebral cortex in 2-, 4- and 7-month PS cDKO and the age- and gender-matched control mice (WT). Lipid peroxidation (MDA as the measure) and protein oxidation (protein carbonyl as the measure) were found to be significantly increased in PS cDKO mice over the age points examined, notably in those at 2-month, suggesting that oxidative stress is an early event in response to PS loss-of-function. The oxidative modification of cortical proteins was further confirmed by Oxyblot assay. The investigations into endogenous antioxidant defense (CAT, SOD and GSH-px as measures) revealed a compensatory defense against oxidative stress, particularly at the early age stage, in PS cDKO mice. The expression level of cortical glial fibrillary acidic protein (GFAP) increased in an age-related manner, in particular in 2-month PS cDKO mice, suggesting that the interaction relationship between oxidative stress and inflammatory response may be closely associated with the underlying loss-of-function pathogenesis of AD.

Presenilins are required for maintenance of neural stem cells in the developing brain.

The early embryonic lethality of mutant mice bearing germ-line deletions of both presenilin genes precluded the study of their functions in neural development. We therefore employed the Cre-loxP technology to generate presenilin conditional double knockout (PS cDKO) mice, in which expression of both presenilins is inactivated in neural progenitor cells (NPC) or neural stem cells and their derivative neurons and glia beginning at embryonic day 11 (E11). In PS cDKO mice, dividing NPCs labeled by BrdU are decreased in number beginning at E13.5. By E15.5, fewer than 20% of NPCs remain in PS cDKO mice. The depletion of NPCs is accompanied by severe morphological defects and hemorrhages in the PS cDKO embryonic brain. Interkinetic nuclear migration of NPCs is also disrupted in PS cDKO embryos, as evidenced by displacement of S-phase and M-phase nuclei in the ventricular zone of the telencephalon. Furthermore, the depletion of neural progenitor cells in PS cDKO embryos is due to NPCs exiting cell cycle and differentiating into neurons rather than reentering cell cycle between E13.5 and E14.5 following PS inactivation in most NPCs. The length of cell cycle, however, is unchanged in PS cDKO embryos. Expression of Notch target genes, Hes1 and Hes5, is significantly decreased in PS cDKO brains, whereas Dll1 expression is up-regulated, indicating that Notch signaling is effectively blocked by PS inactivation. These findings demonstrate that presenilins are essential for neural progenitor cells to re-enter cell cycle and thus ensure proper expansion of neural progenitor pool during embryonic neural development.

Wiskott–Aldrich syndrome protein (WASP) and N-WASP are critical for T cell development

Although T cell dysfunction and lymphopenia are key features of immunodeficient patients with the Wiskott-Aldrich syndrome and Wiskott-Aldrich syndrome protein (WASP)-deficient mice, T cell development appears relatively normal. We hypothesized that N-WASP, a ubiquitously expressed homologue of WASP, may serve a redundant function with WASP. To examine the unique and redundant activities of WASP and N-WASP, we generated ES cells devoid of WASP and N-WASP [double knockout (DKO)] and used the RAG-2-deficient blastocyst complementation system to generate DKO lymphocytes. Moreover, we mated WASP KO mice with mice containing a conditionally targeted N-WASP allele and used the Cre-loxP system to generate mice lacking WASP and N-WASP in T cells [conditional DKO (cDKO)]. In both systems, N-WASP-deficient cells were indistinguishable from WT cells. In contrast, T cell development in DKO and cDKO mice was markedly altered, as shown by thymic hypocellularity and reduced numbers of peripheral T cells. We found that the combined activity of WASP and N-WASP was important for CD4(-)CD8(-) double-negative (DN)-to-CD4(+)CD8(+) double-positive (DP) cell transition, and this may be partly explained by reduced cycling DN3 cells. In addition, decreased migratory responses of CD4(+)CD8(-) and CD4(-)CD8(+) single-positive (SP) cells and increased percentage of CD69(low)CD24(low) and CD62L(low) SP cells in cDKO cells imply retention of SP cells in the thymus. In summary, this study suggests that, although WASP serves a unique role for peripheral T cell function, T cell development depends on the combined activity of WASP and N-WASP.

Environment enrichment rescues the neurodegenerative phenotypes in presenilins‐deficient mice

Presenilin (PS) 1 and 2 conditional double knockout (cDKO) mice show progressive memory dysfunction and forebrain degeneration. Gene expression profiling results revealed a strong activation of immunity and inflammation responses in the brains of 10-month-old cDKO mice. As environmental enrichment (EE) has been shown to be able to improve memory and induce neurogenesis of the brain, we assessed the effects of EE on the memory performance and the neurodegeneration in cDKO mice. We found that EE effectively enhanced memory and partially rescued the forebrain atrophy of the cDKO mice. Our results suggest that immunity and inflammation could play important roles in the neurodegeneration of cDKO mice. Furthermore, the beneficial effects of EE may be associated with the inhibition of the expression of immunity and inflammation-related genes in the brain.

Calorie restriction ameliorates neurodegenerative phenotypes in forebrain-specific presenilin-1 and presenilin-2 double knockout mice

Conditional double knockout of presenilin-1 and presenilin-2 (cDKO) in forebrain of mice led to brain atrophy, tau hyperphosphorylation, synaptic dysfunction and cognitive deficit. These brain changes recapitulated most of the neurodegenerative phenotypes of Alzheimer's disease (AD). In this report, we have investigated the effects of 4-month calorie restriction (CR) regimen on different phenotypes in cDKO mice. We found that CR improved novel object recognition and contextual fear conditioning memory in the cDKO mice. Histological and biochemical analysis showed that CR attenuated ventricle enlargement, caspase-3 activation and astrogliosis. In addition, the induction of tau hyperphosphorylation in the cDKO mice was reduced by CR, possibly through reduction of p25 accumulation and aberrant CDK5 activation. Finally, DNA microarray analysis demonstrated that CR could increase the expression of neurogenesis related genes and decrease the expression of inflammation related genes in the hippocampus of cDKO mice. The possible molecular mechanisms of the CR effects on alleviating AD pathogenesis have been discussed.

Definition of Genes and Paths Involved in Alzheimers Disease: Using Gene Expression Profiles and Chemical Genetics at the Mouse Brain Level

Gene expression profiling of a number of distinct brain regions under different behavioral and biological states was analyzed using DNA microarray technology. These included hippocampal development, aging process, environ-mental enrichment, fear conditioning, and calorie restriction. Our results identified numerous genes and signal pathways that may play critical roles in learning and memory, brain aging and longevity. Furthermore, chemical genetic approach combined with gene expression profiling analysis was applied to study the molecular mechanisms that contribute to brain dysfunction including Alzheimers diseases, neuronal degeneration and brain aging. These studies not only revealed that a number of genes and signal pathways may have participated in the pathological processes of the brain diseases, but also contributed to the identification of therapeutic targets for drug screening and development. Keywords: cDKO mice, SNAP-25, hypothalamus, neuronal plasticity, NMDA receptor

Reduced β-Amyloid Production and Increased Inflammatory Responses in Presenilin Conditional Knock-out Mice

Mutations in presenilins (PS) 1 and 2 are the major cause of familial Alzheimer's disease. Conditional double knock-out mice lacking both presenilins in the postnatal forebrain (PS cDKO mice) exhibit memory and synaptic plasticity impairments followed by progressive neurodegeneration in the cerebral cortex. Here we further investigate the molecular events that may underlie the observed phenotypes and identify additional neuropathological markers in the PS cDKO brain. Enzyme-linked immunosorbent assay analysis showed reduced levels of the toxic beta-amyloid (Abeta) peptides in the cerebral cortex of PS cDKO mice. Interestingly, the reduction in Abeta40 and Abeta42 peptides is similar in PS1 conditional knock-out and PS cDKO mice. We further examined the gene expression profile by oligonucleotide microarrays in the PS cDKO cerebral cortex and found that a high number of genes are differentially expressed, most notably a group of up-regulated inflammatory genes. Quantitative real-time reverse transcription PCR and Western analyses confirmed the elevated levels of glial fibrillary acidic protein, complement component C1q, and cathepsin S, up-regulation of which has been associated with inflammatory responses in various neurodegenerative processes. Immunohistochemical analysis revealed that the increase in complement component C1q is confined to the hippocampal formation, whereas glial fibrillary acidic protein and cathepsin S are up-regulated throughout the entire neocortex and hippocampus. In addition, strong microglial activation occurs in the hippocampus and the deeper cortical layers of PS cDKO mice. These results indicate that the memory impairment and neurodegeneration in PS cDKO mice are not caused by Abeta accumulation and that loss of PS function leads to differential up-regulation of inflammatory markers in the cerebral cortex.