Abstract
Presenilins are essential for synaptic function, memory formation, and neuronal survival. Previously, we reported that expression of cAMP response element-binding protein (CREB) target genes is reduced in the cerebral cortex of presenilin (PS) conditional double knock-out (cDKO) mice. To determine whether the reduced expression of the CREB target genes in these mutant mice is due to loss of presenilin directly or secondary to the impaired neuronal activity, we established a sensitive luciferase reporter system to assess direct transcriptional regulation in cultured cells. We first used immortalized PS-deficient mouse embryonic fibroblasts (MEFs), and found that both CREB-mediated transcription and Notch-mediated HES1 transcription are decreased. However, the ubiquitin-C promoter-mediated transcription is also reduced, and among these three reporters, transfection of exogenous PS1 can rescue only the Notch-mediated HES1 transcription. Further Northern analysis revealed transcriptional alterations of Creb, ubiquitin-C, and other housekeeping genes in PS-deficient MEFs, indicating transcriptional dysregulation in these cells. We then used the Cre/loxP system to develop a postnatal PS-deficient cortical neuronal culture. Surprisingly, in these PS-null neurons, CREB-mediated transcription is not significantly decreased, and levels of total and phosphorylated CREB proteins are unchanged as well. Notch-mediated HES1 transcription is markedly reduced, and this reduction can be rescued by exogenous PS1. Together, our findings suggest that CREB-mediated transcription is regulated indirectly by PS in the adult cerebral cortex, and that attenuation of CREB target gene expression in PS cDKO mice is likely due to reduced neuronal activity in these mutant brains.
Highlights
Over 150 mutations in the presenilin-1 (PS1)2 gene have been identified in patients with early-onset familial Alzheimer disease [1]
Our findings indicate that the primary cortical neuronal culture system is more suitable for assessing PS-dependent gene expression, and that cAMP response element-binding protein (CREB)-mediated transcription is not regulated by PS directly; rather, the decreased CREB target gene transcription observed in PS conditional double knockout (cDKO) mice is likely secondary to reduced neuronal activity present in these mutant mice
Transcriptional Alterations in PS DKO mouse embryonic fibroblasts (MEFs)— we previously observed decreases in levels of CREB target gene transcripts at multiple ages in the cerebral cortex of PS cDKO mice [9], the concomitant presence of a number of synaptic functional defects makes it difficult to conclude whether PS regulates CREB-mediated gene expression directly or indirectly due to impaired synaptic function in these mutant mice
Summary
Plasmids—A 360-bp KpnI-BglII fragment of Hes-Luc, which contains the region from Ϫ194 to ϩ160 of the mouse Hes promoter [18], and a 1.28-kbp PacI-HindIII fragment of pFUGW-EGFP-NLS [19] were cloned into the multicloning site of pGL4.10 (Promega) to generate HES1-luc and Ubc-luc, respectively. Cortical neuronal culture was transfected with calcium phosphate (Clontech) at DIV8. Lentivirus Production and Infection—Production of recombinant lentiviruses is achieved by transfecting HEK293T cells with three plasmids. Titer of the lentivirus was estimated by measuring the GFP-positive cells with flow cytometry, following the infection. To determine infection efficiency of the lentivirus, separate cultures were prepared besides reporter assay, and stained with Hoechst 33258 at DIV10. Luciferase Assay—Firefly luciferase plasmid and pGL4.74 (Tk promoter-driven Renilla luciferase) were transduced to MEF or DIV8 neuronal culture. 1 or 2 days after transfection, cell lysates were collected with 1ϫ Passive lysis buffer (Promega). Cultures were washed in phosphate-buffered saline and incubated with appropriate Alexa Fluor (546 or 680)-conjugated secondary antibodies (1: 500, Molecular Probes) for 1 h at room temperature.
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