Young-Don Lee

Apolipoprotein E and Reelin ligands modulate tau phosphorylation through an Apolipoprotein E receptor/disabled-1/glycogen synthase kinase-3β cascade

Neurofibrillary tangles comprised of highly phosphorylated tau proteins are a key component of Alzheimers disease pathology. Mice lacking Reelin (Reln), double‐knockouts lacking the VLDL receptor (VLDLR) and ApoE receptor2 (ApoER2), and mice lacking disabled‐1 (Dab1) display increased levels of phosphorylated tau. Because Reln binds to recombinant ApoE receptors, assembly of a Reln/ApoE‐receptor/Dab1 (RAD) complex may initiate a signal transduction cascade that controls tau phosphorylation. Conversely, disruption of this RAD complex may increase tau phosphorylation and lead to neurodegeneration. To substantiate this concept, we mated Reln‐deficient mice to ApoE‐deficient mice and found that in the absence of Reln, tau phosphorylation increased as the amount of ApoE decreased. Paralleling the change in tau phosphorylation levels, we found that GSK‐3β activity increased in Reln‐deficient mice and further increased in mice lacking both Reln and ApoE. CDK‐5 activity was similar in mice lacking Reln, ApoE, or both. GSK‐3β and CDK‐5 activity increased in Dab1‐deficient mice, independent of ApoE levels. Further supporting the idea that increased tau phosphorylation results primarily from increased kinase activity, the activity of two phosphatases was similar in all conditions tested. These data support a novel, ligand‐mediated signal transduction cascade— initiated by the assembly of a RAD complex that suppresses kinase activity and controls tau phosphorylation.

Deregulation of CREB Signaling Pathway Induced by Chronic Hyperglycemia Downregulates NeuroD Transcription

CREB mediates the transcriptional effects of glucose and incretin hormones in insulin-target cells and insulin-producing β-cells. Although the inhibition of CREB activity is known to decrease the β-cell mass, it is still unknown what factors inversely alter the CREB signaling pathway in β-cells. Here, we show that β-cell dysfunctions occurring in chronic hyperglycemia are not caused by simple inhibition of CREB activity but rather by the persistent activation of CREB due to decreases in protein phophatase PP2A. When freshly isolated rat pancreatic islets were chronically exposed to 25 mM (high) glucose, the PP2A activity was reduced with a concomitant increase in active pCREB. Brief challenges with 15 mM glucose or 30 µM forskolin after 2 hour fasting further increased the level of pCREB and consequently induced the persistent expression of ICER. The excessively produced ICER was sufficient to repress the transcription of NeuroD, insulin, and SUR1 genes. In contrast, when islets were grown in 5 mM (low) glucose, CREB was transiently activated in response to glucose or forskolin stimuli. Thus, ICER expression was transient and insufficient to repress those target genes. Importantly, overexpression of PP2A reversed the adverse effects of chronic hyperglycemia and successfully restored the transient activation of CREB and ICER. Conversely, depletion of PP2A with siRNA was sufficient to disrupt the negative feedback regulation of CREB and induce hyperglycemic phenotypes even under low glucose conditions. Our findings suggest that the failure of the negative feedback regulation of CREB is the primary cause for β-cell dysfunctions under conditions of pathogenic hyperglycemia, and PP2A can be a novel target for future therapies aiming to protect β-cells mass in the late transitional phase of non-insulin dependent type 2 diabetes (NIDDM).

Simultaneous deletion of floxed genes mediated by CaMKIIα-Cre in the brain and in male germ cells: application to conditional and conventional disruption of Goα

The Cre/LoxP system is a well-established approach to spatially and temporally control genetic inactivation. The calcium/calmodulin-dependent protein kinase II alpha subunit (CaMKIIα) promoter limits expression to specific regions of the forebrain and thus has been utilized for the brain-specific inactivation of the genes. Here, we show that CaMKIIα-Cre can be utilized for simultaneous inactivation of genes in the adult brain and in male germ cells. Double transgenic Rosa26+/stop-lacZ::CaMKIIα-Cre+/Cre mice generated by crossing CaMKIIα-Cre+/Cre mice with floxed ROSA26 lacZ reporter (Rosa26+/stop-lacZ) mice exhibited lacZ expression in the brain and testis. When these mice were mated to wild-type females, about 27% of the offspring were whole body blue by X-gal staining without inheriting the Cre transgene. These results indicate that recombination can occur in the germ cells of male Rosa26+/stop-lacZ::CaMKIIα-Cre+/Cre mice. Similarly, when double transgenic Gnao+/f::CaMKIIα-Cre+/Cre mice carrying a floxed Go-alpha gene (Gnaof/f) were backcrossed to wild-type females, approximately 22% of the offspring carried the disrupted allele (GnaoΔ) without inheriting the Cre transgene. The GnaoΔ/Δ mice closely resembled conventional Go-alpha knockout mice (Gnao−/−) with respect to impairment of their behavior. Thus, we conclude that CaMKIIα-Cre mice afford recombination for both tissue- and time-controlled inactivation of floxed target genes in the brain and for their permanent disruption. This work also emphasizes that extra caution should be exercised in utilizing CaMKIIα-Cre mice as breeding pairs.

Retrovirus-mediated transduction of a cytosine deaminase gene preserves the stemness of mesenchymal stem cells

Human mesenchymal stem cells (MSCs) have emerged as attractive cellular vehicles to deliver therapeutic genes for ex-vivo therapy of diverse diseases; this is, in part, because they have the capability to migrate into tumor or lesion sites. Previously, we showed that MSCs could be utilized to deliver a bacterial cytosine deaminase (CD) suicide gene to brain tumors. Here we assessed whether transduction with a retroviral vector encoding CD gene altered the stem cell property of MSCs. MSCs were transduced at passage 1 and cultivated up to passage 11. We found that proliferation and differentiation potentials, chromosomal stability and surface antigenicity of MSCs were not altered by retroviral transduction. The results indicate that retroviral vectors can be safely utilized for delivery of suicide genes to MSCs for ex-vivo therapy. We also found that a single retroviral transduction was sufficient for sustainable expression up to passage 10. The persistent expression of the transduced gene indicates that transduced MSCs provide a tractable and manageable approach for potential use in allogeneic transplantation.

Fas-associated factor 1 promotes in neurofibrillary tangle-mediated cell death of basal forebrain cholinergic neurons in P301L transgenic mice.

Dysfunction of the cholinergic system of the basal forebrain complex is one of the major contributors toward cognitive impairment in Alzheimer’s disease (AD). In AD, degeneration of cholinergic fibers that project to the cerebral cortex and hippocampus may be attributed to the development of neurofibrillary tangles (NFT). Here, we evaluated the relationship between neurofibrillary changes and degeneration of septal cholinergic neurons in P301L tau transgenic mice. The number of cholinergic neurons in the medial septum and vertical limb of the diagonal band of Broca (MS/VDB) in the basal forebrain was significantly reduced in P301L mice compared with age-matched wild-type mice. The phospho-tau levels and NFT formation in these P301L mice were higher than those in wild-type mice. However, it is still unknown how the development of NFT is caused in AD and AD-like pathology. Our results show that Fas-associated factor 1 (FAF1) expression as well as caspase-3 activation were increased in AT8-positive MS/VDB cholinergic neurons. Furthermore, the formation of AT8-positive paired helical filaments increased in proportion to FAF1 expression and caspase-3 activation in MS/VDB cholinergic neurons. On the basis of the collective findings, we suggest that increased FAF1 expression triggers caspase-3 cleavage and activation, leading to hyperphosphorylated tau aggregation and subsequent NFT formation, in turn initiating apoptosis in MS/VDB cholinergic neurons.