Ellen Kilger

Exogenous induction of cerebral beta-amyloidogenesis is governed by agent and host

Protein aggregation is an established pathogenic mechanism in Alzheimers disease, but little is known about the initiation of this process in vivo. Intracerebral injection of dilute, amyloid-β (Aβ)–containing brain extracts from humans with Alzheimers disease or β-amyloid precursor protein (APP) transgenic mice induced cerebral β-amyloidosis and associated pathology in APP transgenic mice in a time- and concentration-dependent manner. The seeding activity of brain extracts was reduced or abolished by Aβ immunodepletion, protein denaturation, or by Aβ immunization of the host. The phenotype of the exogenously induced amyloidosis depended on both the host and the source of the agent, suggesting the existence of polymorphic Aβ strains with varying biological activities reminiscent of prion strains.

Epstein–Barr virus‐mediated B‐cell proliferation is dependent upon latent membrane protein 1, which simulates an activated CD40 receptor

The Epstein–Barr virus (EBV) latent membrane protein 1 (LMP1) is essential for the immortalization of human B cells and is linked etiologically to several human tumors. LMP1 is an integral membrane protein which acts like a constitutively active receptor. It binds tumor necrosis factor (TNF)‐receptor‐associated factors (TRAFs), activates NF‐κB and triggers the transcription factor AP‐1 via the c‐Jun N‐terminal kinase (JNK) cascade, but its specific contribution to B‐cell immortalization has not been elucidated fully. To address the function of LMP1, we established B cell lines with a novel mini‐EBV plasmid in which the LMP1 gene can be regulated at will without affecting the expression of other latent EBV genes. We demonstrate here that continuous expression of LMP1 is essential for the proliferation of EBV‐immortalized B cells in vitro. Re‐induction of LMP1 expression or activation of the cellular CD40 receptor both induce the JNK signaling cascade, activate the transcription factor NF‐κB and stimulate proliferation of these B cells. Our findings strongly suggest that LMP1 mimics B‐cell activation processes which are physiologically triggered by CD40–CD40 ligand signals. Since LMP1 acts in a ligand‐independent manner, it replaces the T cell‐derived activation signal to sustain indefinite B‐cell proliferation.

Latent membrane protein 1 of Epstein-Barr virus interacts with JAK3 and activates STAT proteins.

Latent membrane protein 1 (LMP1) acts like a permanently activated receptor of the tumor necrosis factor (TNF)‐receptor superfamily and is absolutely required for B cell immortalization by Epstein–Barr virus. Molecular and biochemical approaches demonstrated that LMP1 usurps cellular signaling pathways resulting in the induction of NF‐κB and AP‐1 via two C‐terminal activating regions. We demonstrate here that a third region encompassing a proline rich sequence within the 33 bp repetitive stretch of LMP1s C‐terminus is required for the activation of Janus kinase 3 (JAK3). The interaction of LMP1 and JAK3 leads to the enhanced tyrosine auto/transphosphorylation of JAK3 within minutes after crosslinking of a conditional NGF‐R:LMP1 chimera and is a prerequisite for the activation of STAT transcription factors. These results reveal a novel activating region in the LMP1 C‐terminus and identify the JAK/STAT pathway as a target of this viral integral membrane protein in B cells.

Epstein-Barr virus latent membrane protein-1 triggers AP-1 activity via the c-Jun N-terminal kinase cascade.

The Epstein–Barr virus latent membrane protein‐1 (LMP‐1) is an integral membrane protein which transforms fibroblasts and is essential for EBV‐mediated B‐cell immortalization. LMP‐1 has been shown to trigger cellular NF‐κB activity which, however, cannot fully explain the oncogenic potential of LMP‐1. Here we show that LMP‐1 induces the activity of the AP‐1 transcription factor, a dimer of Jun/Jun or Jun/Fos proteins. LMP‐1 effects on AP‐1 are mediated through activation of the c‐Jun N‐terminal kinase (JNK) cascade, but not the extracellular signal‐regulated kinase (Erk) pathway. Consequently, LMP‐1 triggers the activity of the c‐Jun N‐terminal transactivation domain which is known to be activated upon JNK‐mediated phosphorylation. Deletion analysis indicates that the 55 C‐terminal amino acids of the LMP‐1 molecule, but not its TRAF interaction domain, are essential for AP‐1 activation. JNK‐mediated transcriptional activation of AP‐1 is the direct output of LMP‐1‐triggered signaling, as shown by an inducible LMP‐1 mutant. Using a tetracycline‐regulated LMP‐1 allele, we demonstrate that JNK is also an effector of non‐cytotoxic LMP‐1 signaling in B cells, the physiological target cells of EBV. In summary, our data reveal a novel effector of LMP‐1, the SEK/JNK/c‐Jun/AP‐1 pathway, which contributes to our understanding of the immortalizing and transforming potential of LMP‐1.

Cystatin C modulates cerebral |[beta]|-amyloidosis

The CST3 Thr25 allele of CST3, which encodes cystatin C, leads to reduced cystatin C secretion and conveys susceptibility to Alzheimers disease. Here we show that overexpression of human cystatin C in brains of APP-transgenic mice reduces cerebral amyloid-β deposition and that cystatin C binds amyloid-β and inhibits its fibril formation. Our results suggest that cystatin C concentrations modulate cerebral amyloidosis risk and provide an opportunity for genetic risk assessment and therapeutic interventions.

Latent membrane protein 1 of Epstein–Barr virus coordinately regulates proliferation with control of apoptosis

Latent membrane protein 1 (LMP1), an oncoprotein encoded by Epstein–Barr virus (EBV), is an integral membrane protein, which acts like a constitutively active receptor. LMP1 is critical for some facet of EBVs induction and maintenance of proliferation of infected B cells. It, in part, mimics signaling by the CD40 receptor and has been implicated in regulating proliferation, survival, or both properties of EBV-infected cells. We established a conditional LMP1 allele in the context of the intact EBV genome to define the immediate-early cellular target genes regulated by LMP1 in order to assess its contributions to infected human B cells. The functional analysis of this conditional system indicated that LMP1 specifically induces mitogenic B-cell activation through c-myc and Jun/AP1 family members and confirms its direct role in upregulating expression of multiple genes with opposing activities involved in cell survival. LMP1s signals were found to be essential for the G1/S transition in human B cells; cells lacking LMP1s signals are cell cycle arrested and survive quiescently. LMP1s activities are therefore not required to maintain survival in nonproliferating cells. LMP1 does induce both pro- and antiapoptotic genes whose balance seems to permit survival during LMP1s induction and maintenance of proliferation.

Amyloid precursor protein intracellular domain modulates cellular calcium homeostasis and ATP content

Consecutive cleavages of amyloid precursor protein (APP) generate APP intracellular domain (AICD). Its cellular function is still unclear. In this study, we investigated the functional role of AICD in cellular Ca2+ homeostasis. We could confirm previous observations that endoplasmic reticulum Ca2+ stores contain less calcium in cells with reduced APP γ‐secretase cleavage products, increased AICD degradation, reduced AICD expression or in cells lacking APP. In addition, we observed an enhanced resting cytosolic calcium concentration under conditions where AICD is decreased or missing. In view of the reciprocal effects of Ca2+ on mitochondria and of mitochondria on Ca2+ homeostasis, we analysed further the cellular ATP content and the mitochondrial membrane potential. We observed a reduced ATP content and a mitochondrial hyperpolarisation in cells with reduced amounts of AICD. Blockade of mitochondrial oxidative phosphorylation chain in control cells lead to similar alterations as in cells lacking AICD. On the other hand, substrates of Complex II rescued the alteration in Ca2+ homeostasis in cells lacking AICD. Based on these observations, our findings indicate that alterations observed in endoplasmic reticulum Ca2+ storage in cells with reduced amounts of AICD are reciprocally linked to mitochondrial bioenergetic function.

Modeling familial Danish dementia in mice supports the concept of the amyloid hypothesis of Alzheimer's disease

Familial Danish dementia (FDD) is a progressive neurodegenerative disease with cerebral deposition of Dan-amyloid (ADan), neuroinflammation, and neurofibrillary tangles, hallmark characteristics remarkably similar to those in Alzheimers disease (AD). We have generated transgenic (tg) mouse models of familial Danish dementia that exhibit the age-dependent deposition of ADan throughout the brain with associated amyloid angiopathy, microhemorrhage, neuritic dystrophy, and neuroinflammation. Tg mice are impaired in the Morris water maze and exhibit increased anxiety in the open field. When crossed with TauP301S tg mice, ADan accumulation promotes neurofibrillary lesions, in all aspects similar to the Tau lesions observed in crosses between β-amyloid (Aβ)-depositing tg mice and TauP301S tg mice. Although these observations argue for shared mechanisms of downstream pathophysiology for the sequence-unrelated ADan and Aβ peptides, the lack of codeposition of the two peptides in crosses between ADan- and Aβ-depositing mice points also to distinguishing properties of the peptides. Our results support the concept of the amyloid hypothesis for AD and related dementias, and suggest that different proteins prone to amyloid formation can drive strikingly similar pathogenic pathways in the brain.

BRI2 Protein Regulates β-Amyloid Degradation by Increasing Levels of Secreted Insulin-degrading Enzyme (IDE)

Background: The British precursor protein (BRI2) influences amyloid precursor protein metabolism. Results: BRI2 lowers β-amyloid peptide levels by increasing levels of secreted insulin-degrading enzyme (IDE) in both cells and mice. Conclusion: BRI2 as a receptor protein regulates IDE levels and in turn promotes β-amyloid degradation. Significance: Targeting the regulation of IDE may lead to new approaches to therapeutically address sporadic Alzheimer disease. The amyloid precursor protein (APP) is one of the major proteins involved in Alzheimer disease (AD). Proteolytic cleavage of APP gives rise to amyloid-β (Aβ) peptides that aggregate and deposit extensively in the brain of AD patients. Although the increase in levels of aberrantly folded Aβ peptide is considered to be important to disease pathogenesis, the regulation of APP processing and Aβ metabolism is not fully understood. Recently, the British precursor protein (BRI2, ITM2B) has been implicated in influencing APP processing in cells and Aβ deposition in vivo. Here, we show that the wild type BRI2 protein reduces plaque load in an AD mouse model, similar to its disease-associated mutant form, ADan precursor protein (ADanPP), and analyze in more detail the mechanism of how BRI2 and ADanPP influence APP processing and Aβ metabolism. We find that overexpression of either BRI2 or ADanPP reduces extracellular Aβ by increasing levels of secreted insulin-degrading enzyme (IDE), a major Aβ-degrading protease. This effect is also observed with BRI2 lacking its C-terminal 23-amino acid peptide sequence. Our results suggest that BRI2 might act as a receptor protein that regulates IDE levels that in turn influences APP metabolism in a previously unrecognized way. Targeting the regulation of IDE may be a promising therapeutic approach to sporadic AD.

Expression of mucin (MUC-1) from a mini-Epstein-Barr virus in immortalized B-cells to generate tumor antigen specific cytotoxic T cells.

EBV immortalized B‐cells can be used as antigen presenting cells (APC) to stimulate specific T‐cell responses. Mini‐Epstein‐Barr virus (mini‐EBV) plasmids contain all functional elements of Epstein‐Barr virus (EBV) necessary to immortalize B‐cells in vitro. These immortalized B‐cells are incapable of releasing infectious virus in contrast to cells immortalized by wildtype EBV. In addition, mini‐EBVs can be modified in E. coli to alter their genetic composition or adopt new genes.