Apolipoprotein E, especially apolipoprotein E4, increases the oligomerization of amyloid ß peptide.

Alzheimer's disease (AD) is the most common progressive neurodegenerative disorder causing dementia. Massive deposition of amyloid ß peptide (Aß) as senile plaques in the brain is the pathological hallmark of AD, but oligomeric, soluble forms of Aß have been implicated as the synaptotoxic component. The apolipoprotein E ε 4 (apoE ε4) allele is known to be a genetic risk factor for developing AD. However, it is still unknown how apoE impacts the process of Aß oligomerization. Here, we found that the level of Aß oligomers in APOE ε4/ε4 AD patient brains is 2.7 times higher than those in APOE ε3/ε3 AD patient brains, matched for total plaque burden, suggesting that apoE4 impacts the metabolism of Aß oligomers. To test this hypothesis, we examined the effect of apoE on Aß oligomer formation. Using both synthetic Aß and a split-luciferase method for monitoring Aß oligomers, we observed that apoE increased the level of Aß oligomers in an isoform-dependent manner (E2 < E3 < E4). This effect appears to be dependent on the ApoE C-terminal domain. Moreover, these results were confirmed using endogenous apoE isolated from the TBS-soluble fraction of human brain, which increased the formation of Aß oligomers. Together, these data show that lipidated apoE, especially apoE4, increases Aß oligomers in the brain. Higher levels of Aß oligomers in the brains of APOE ε4/ε4 carriers compared with APOE ε3/ε3 carriers may increase the loss of dendritic spines and accelerate memory impairments, leading to earlier cognitive decline in AD.

Characterization of oligomer formation of amyloid-beta peptide using a split-luciferase complementation assay.

Amyloid-ß peptide (Aß) is the amyloid component of senile plaques in Alzheimer disease (AD) brains. Recently a soluble oliomeric form of Aß in Aß precursor protein transgenic mouse brains and AD brains was identified as a potential causative molecule for memory impairment, suggesting that soluble Aß oligomers cause neurodegeneration in AD. Further characterization of this species has been hampered, however, because the concentrations are quite small and it is difficult to monitor Aß oligomers specifically. Here we developed a novel method for monitoring Aß oligomers using a split-luciferase complementation assay. In this assay, the N- and C-terminal fragments of Gaussia luciferase (Gluc) are fused separately to Aß. We found that conditioned media from both N- and C-terminal fragments of Gluc-tagged Aß1-42 doubly transfected HEK293 cells showed strong luminescence. We used gel filtration analyses to analyze the size of oligomers formed by the luciferase complementation assay, and found that it matched closely with oligomers formed by endogenous Aß in Tg2576 neurons. Large oligomers (24-36-mers), 8-mers, trimers, and dimers predominate. In both systems, Aß formed oligomers intracellularly, which then appear to be secreted as oligomers. We then evaluated several factors that might impact oligomer formation. The level of oligomerization of Aß1-40 was similar to that of Aß1-42. Homodimers formed more readily than heterodimers. The level of oligomerization of murine Aß1-42 was similar to that of human Aß1-42. As expected, the familial AD-linked Arctic mutation (E22G) significantly enhanced oligomer formation. These data suggest that Gluc-tagged Aß enables the analysis of Aß oligomers.

Oligomeric amyloid beta associates with postsynaptic densities and correlates with excitatory synapse loss near senile plaques.

Synapse loss correlates with a cognitive decline in Alzheimer's disease (AD), but whether this is caused by fibrillar deposits known as senile plaques or soluble oligomeric forms of amyloid beta (Abeta) is controversial. By using array tomography, a technique that combines ultrathin sectioning of tissue with immunofluorescence, allowing precise quantification of small structures, such as synapses, we have tested the hypothesis that oligomeric Abeta surrounding plaques contributes to synapse loss in a mouse model of AD. We find that senile plaques are surrounded by a halo of oligomeric Abeta. Analysis of >14,000 synapses (represented by PSD95-stained excitatory synapses) shows that there is a 60% loss of excitatory synapses in the halo of oligomeric Abeta surrounding plaques and that the density increases to reach almost control levels in volumes further than 50 microm from a plaque in an approximately linear fashion (linear regression, r(2) = 0.9; P < 0.0001). Further, in transgenic cortex, microdeposits of oligomeric Abeta associate with a subset of excitatory synapses, which are significantly smaller than those not in contact with oligomeric Abeta. The proportion of excitatory synapses associated with Abeta correlates with decreasing density (correlation, -0.588; P < 0.0001). These data show that senile plaques are a potential reservoir of oligomeric Abeta, which colocalizes with the postsynaptic density and is associated with spine collapse, reconciling the apparently competing schools of thought of "plaque" vs. "oligomeric Abeta" as the synaptotoxic species in the brain of AD patients.

Refining the nuclear localization signal within the Egr transcriptional coregulator NAB2.

NAB1 and 2 are coregulators for early growth response (Egr) transcription factors. The NAB1 nuclear localization signal (NLS) was previously described as a bipartite NLS of sequence R(X2 )K(X11 )KRXK. The sequence is conserved in NAB2 as K(X2 )R(X11 )KKXK; however, whether it functions as the NAB2 NLS has not been tested. We show that the KKXK motif in NAB2 is necessary and sufficient to mediate nuclear localization. Mutation of the KKXK motif to AAXA causes cytoplasmic localization of NAB2, while Lys/Arg-to-Ala mutations of the upstream K(X2 )R motif have no effect. Fusion of the KKXK motif to cytoplasmic protein eIF2Bε causes nuclear localization. Altogether, this study refines our knowledge of the NAB2 NLS, demonstrating that KKXK343-346 is necessary and sufficient for nuclear localization.

Phosphatidylinositol 3-kinase signaling in proliferating cells maintains an anti-apoptotic transcriptional program mediated by inhibition of FOXO and non-canonical activation of NFkappaB transcription factors.

BACKGROUND: Phosphatidylinositol (PI) 3-kinase is activated by a variety of growth factor receptors and the PI 3-kinase/Akt signaling pathway is a key regulator of cell proliferation and survival. The downstream targets of PI 3-kinase/Akt signaling include direct regulators of cell cycle progression and apoptosis as well as a number of transcription factors. Growth factor stimulation of quiescent cells leads to robust activation of PI 3-kinase, induction of immediate-early genes, and re-entry into the cell cycle. A lower level of PI 3-kinase signaling is also required for the proliferation and survival of cells maintained in the presence of growth factors, but the gene expression program controlled by PI 3-kinase signaling in proliferating cells has not been elucidated. RESULTS: We used microarray analyses to characterize the changes in gene expression resulting from inhibition of PI 3-kinase in proliferating cells. The genes regulated by inhibition of PI 3-kinase in proliferating cells were distinct from genes induced by growth factor stimulation of quiescent cells and highly enriched in genes that regulate programmed cell death. Computational analyses followed by chromatin immunoprecipitations demonstrated FOXO binding to both previously known and novel sites in promoter regions of approximately one-third of the up-regulated genes, consistent with activation of FOXO1 and FOXO3a in response to inhibition of PI 3-kinase. NFkappaB binding sites were similarly identified in promoter regions of over one-third of the down-regulated genes. RelB was constitutively bound to promoter regions in cells maintained in serum, however binding decreased following PI 3-kinase inhibition, indicating that PI 3-kinase signaling activates NFkappaB via the non-canonical pathway in proliferating cells. Approximately 70% of the genes targeted by FOXO and NFkappaB regulate cell proliferation and apoptosis, including several regulators of apoptosis that were not previously known to be targeted by these transcription factors. CONCLUSION: PI 3-kinase signaling in proliferating cells regulates a novel transcriptional program that is highly enriched in genes that regulate apoptosis. At least one-third of these genes are regulated either by FOXO transcription factors, which are activated following PI 3-kinase inhibition, or by RelB, which is activated by PI 3-kinase via the non-canonical pathway in proliferating cells.

The LDL receptor-related protein can form homo-dimers in neuronal cells.

The ability of the low density lipoprotein receptor-related protein (LRP) to form homo-dimers was studied in mouse neuroblastoma and human neuroglioma cells as well as in primary cortical cultures from adult mouse brain. Homo-dimerization of LRP light chain (LC) was shown by several methods including co-immunoprecipitation, fluorescence lifetime imaging microscopy, and bimolecular fluorescence complementation assay. The requirement of intact NPXY motifs of LRP LC for homo-dimerization was ruled out by co-immunoprecipitation assay.

Role for Egr1 in the Transcriptional Program Associated with Neuronal Differentiation of PC12 Cells.

PC12 cells are a well-established model to study how differences in signal transduction duration can elicit distinct cell behaviors. Epidermal growth factor (EGF) activates transient ERK signaling in PC12 cells that lasts 30-60 min, which in turn promotes proliferation; nerve growth factor (NGF) activates more sustained ERK signaling that lasts 4-6 h, which in turns induces neuronal differentiation. Data presented here extend a previous study by Mullenbrock et al. (2011) that demonstrated that sustained ERK signaling in response to NGF induces preferential expression of a 69-member gene set compared to transient ERK signaling in response to EGF and that the transcription factors AP-1 and CREB play a major role in the preferential expression of several genes within the set. Here, we examined whether the Egr family of transcription factors also contributes to the preferential expression of the gene set in response to NGF. Our data demonstrate that NGF causes transient induction of all Egr family member transcripts, but a corresponding induction of protein was detected for only Egr1 and 2. Chromatin immunoprecipitation experiments provided clearest evidence that, after induction, Egr1 binds 12 of the 69 genes that are preferentially expressed during sustained ERK signaling. In addition, Egr1 expression and binding upstream of its target genes were both sustained in response to NGF versus EGF within the same timeframe that its targets are preferentially expressed. These data thus provide evidence that Egr1 contributes to the transcriptional program activated by sustained ERK signaling in response to NGF, specifically by contributing to the preferential expression of its target genes identified here.

Voluntary wheel-running attenuates insulin and weight gain and affects anxiety-like behaviors in C57BL6/J mice exposed to a high-fat diet.

It is widely accepted that lifestyle plays a crucial role on the quality of life in individuals, particularly in western societies where poor diet is correlated to alterations in behavior and the increased possibility of developing type-2 diabetes. While exercising is known to produce improvements to overall health, there is conflicting evidence on how much of an effect exercise has staving off the development of type-2 diabetes or counteracting the effects of diet on anxiety. Thus, this study investigated the effects of voluntary wheel-running access on the progression of diabetes-like symptoms and open field and light-dark box behaviors in C57BL/6J mice fed a high-fat diet. C57BL/6J mice were placed into either running-wheel cages or cages without a running-wheel, given either regular chow or a high-fat diet, and their body mass, food consumption, glucose tolerance, insulin and c-peptide levels were measured. Mice were also exposed to the open field and light-dark box tests for anxiety-like behaviors. Access to a running-wheel partially attenuated the obesity and hyperinsulinemia associated with high-fat diet consumption in these mice, but did not affect glucose tolerance or c-peptide levels. Wheel-running strongly increased anxiety-like and decreased explorative-like behaviors in the open field and light-dark box, while high-fat diet consumption produced smaller increases in anxiety. These results suggest that voluntary wheel-running can assuage some, but not all, of the physiological problems associated with high-fat diet consumption, and can modify anxiety-like behaviors regardless of diet consumed.

Apolipoprotein E: isoform specific differences in tertiary structure and interaction with amyloid-ß in human Alzheimer brain.

We applied a novel application of FLIM-FRET to in situ measurement and quantification of protein interactions to explore isoform specific differences in Aß-ApoE interaction and ApoE tertiary conformation in senile plaques in human Alzheimer brain. ApoE3 interacts more closely with Aß than ApoE4, but a greater proportion of Aß molecules within plaques are decorated with ApoE4 than ApoE3, lending strong support to the hypothesis that isoform specific differences in ApoE are linked with Aß deposition. We found an increased number of ApoE N-terminal fragments in ApoE4 plaques, consistent with the observation that ApoE4 is more easily cleaved than ApoE3. In addition, we measured a small but significant isoform specific difference in ApoE domain interaction. Based on our in situ data, supported by traditional biochemical data, we propose a pathway by which isoform specific conformational differences increase the level of cleavage at the hinge region of ApoE4, leading to a loss of ApoE function to mediate clearance of Aß and thereby increase the risk of AD for carriers of the APOEε4 allele.

Rapid turnover of mcl-1 couples translation to cell survival and apoptosis.

Inhibition of translation plays a role in apoptosis induced by a variety of stimuli, but the mechanism by which it promotes apoptosis has not been established. We have investigated the hypothesis that selective degradation of anti-apoptotic regulatory protein(s) is responsible for apoptosis resulting from translation inhibition. Induction of apoptosis by cycloheximide was detected within 2-4 h and blocked by proteasome inhibitors, indicating that degradation of short-lived protein(s) was required. Caspase inhibition and overexpression of Bcl-x(L) blocked cycloheximide-induced apoptosis. In addition, cycloheximide induced rapid activation of Bak and Bax, which required proteasome activity. Mcl-1 was degraded by the proteasome with a half-life of approximately 30 min following inhibition of protein synthesis, preceding Bak/Bax activation and the onset of apoptosis. Overexpression of Mcl-1 blocked apoptosis induced by cycloheximide, whereas RNA interference knockdown of Mcl-1 induced apoptosis. Knockdown of Bim and Bak, downstream targets of Mcl-1, inhibited cycloheximide-induced apoptosis, as did knockdown of Bax. Apoptosis resulting from inhibition of translation thus involves the rapid degradation of Mcl-1, leading to activation of Bim, Bak, and Bax. Because of its rapid turnover, Mcl-1 may serve as a convergence point for signals that affect global translation, coupling translation to cell survival and the apoptotic machinery.