Direct visualization of the gamma secretase-generated carboxyl-terminal domain of the amyloid precursor protein: association with Fe65 and translocation to the nucleus

Amyloid-beta, the peptide that deposits as senile plaques in Alzheimer's disease, is derived from the amyloid precursor protein (APP) by a gamma secretase-mediated intramembranous cleavage. In addition to amyloid-beta, this cleavage produces a carboxyl-terminal intracellular fragment which has an unknown function. The carboxyl-terminal domain of APP interacts in the cytoplasm with an adapter protein, Fe65. We demonstrate by laser scanning confocal microscopy that a gamma secretase generated APP carboxyl-terminal domain, tagged with green fluorescent protein (GFP), translocates to the nucleus in a manner dependent upon stabilization by the adapter protein Fe65; APP which has been mutated to block interactions with Fe65 cannot be detected in the nucleus. The APP-CT domain continues to interact with Fe65 in the nucleus, as determined by both colocalization and fluorescence resonance energy transfer (FRET). Visualization of the APP-CT-Fe65 complex in the nucleus may serve as a readout for processes that modify gamma secretase release of APP-CT.     

The FATC domains of PIKK proteins are functionally equivalent and participate in the Tip60-dependent activation of DNA-PKcs and ATM

Members of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, including the ATM, DNA-PKcs, Atr, and Trrap proteins, function in signal transduction pathways that activate the DNA damage response. PIKK proteins contain a conserved C-terminal FAT/kinase domain/FATC domain structure. The FATC domain of ATM mediates the interaction between ATM and Tip60, a histone acetyltransferase that regulates activation of ATM. Here, we examined whether the FATC domains of DNA-PKcs, Atr, and Trrap were also able to interact with Tip60. Deletion of the FATC domain of ATM blocked the interaction between ATM and Tip60 and suppressed the activation of ATM kinase activity by DNA damage. Replacement of the FATC domain of ATM with the FATC domains of DNA-PKcs, Atr, or Trrap restored the activation of ATM and its association with Tip60. These results indicate that the FATC domains of DNA-PKcs, Atr, Trrap, and ATM are functionally equivalent. Immunoprecipitation experiments demonstrated that Tip60 is constitutively associated with DNA-PKcs and that the histone acetyltransferase activity associated with DNA-PKcs is up-regulated by DNA damage. When Tip60 expression was suppressed by small interfering RNA, the activation of DNA-PKcs (measured by autophosphorylation of DNA-PKcs at serine 2056 and threonine 2609) was inhibited, demonstrating a key role for Tip60 in the activation of DNA-PKcs by DNA damage. The conserved FATC domain of PIKK proteins may therefore function as a binding domain for the Tip60 histone acetyltransferase. Further, the ability of Tip60 to regulate the activation of both ATM and DNA-PKcs in response to DNA damage demonstrates that Tip60 is a key component of the DNA damage-signaling network.     

Epigenetic Regulation of Axonal Growth of Drosophila Pacemaker Cells by Histone Acetyltransferase Tip60 Controls Sleep

Tip60 is a histone acetyltransferase (HAT) enzyme that epigenetically regulates genes enriched for neuronal functions through interaction with the amyloid precursor protein (APP) intracellular domain. However, whether Tip60-mediated epigenetic dysregulation affects specific neuronal processes in vivo and contributes to neurodegeneration remains unclear. Here, we show that Tip60 HAT activity mediates axonal growth of the Drosophila pacemaker cells, termed “small ventrolateral neurons” (sLNvs), and their production of the neuropeptide pigment-dispersing factor (PDF) that functions to stabilize Drosophila sleep–wake cycles. Using genetic approaches, we show that loss of Tip60 HAT activity in the presence of the Alzheimer’s disease-associated APP affects PDF expression and causes retraction of the sLNv synaptic arbor required for presynaptic release of PDF. Functional consequence of these effects is evidenced by disruption of the sleep–wake cycle in these flies. Notably, overexpression of Tip60 in conjunction with APP rescues these sleep–wake disturbances by inducing overelaboration of the sLNv synaptic terminals and increasing PDF levels, supporting a neuroprotective role for dTip60 in sLNv growth and function under APP-induced neurodegenerative conditions. Our findings reveal a novel mechanism for Tip60 mediated sleep–wake regulation via control of axonal growth and PDF levels within the sLNv-encompassing neural network and provide insight into epigenetic-based regulation of sleep disturbances observed in neurodegenerative diseases like Alzheimer’s disease.     

NSE-Controlled Carboxyl-Terminus of APP Gene Over-Expressing in Transgenic Mice Induces Altered Expressions in Behavior, Aβ-42, and GSK3β Binding Proteins

Summary The amyloid protein precursor (APP) is cleaved in its intramembranous domain by γ-secrease to generate amyloid β and a free carboxyl-terminal intracellular fragment. The carboxyl-terminal of 105 amino acids of APP (APP-C105) plays a crucial role in the neuropathology of Alzheimer’s disease (AD), but it is incompletely understand how APP-C105 overexpression interacts and regulates the brain function and Aβ-42 levels, and whether or not it is associated with the expressions of GSK3β-binding proteins. To test this, transgenic mice expressing NSE-controlled APP-C105 were produced and tested for their above phenotypes. A behavioral deficit was observed in the 9 months old transgenic mice, and western blot indicated that there was a predominant expression of APP-C105 in transgenic brains compared with those of non-transgenic brains. In parallel, APP-C105 overexpression resulted in the modulation of the Aβ-42 level, γ-secretase activity, GSK3β-binding proteins including PS1, tau, and β-catenin in the brains of the transgenic mice relative to the non-transgenic mice. Thus, altered expressions of these neuropathological phenotypes in APP-C105 transgenic mice could be useful targets in developing new therapeutic treatments.     

ADAM10 is the physiologically relevant, constitutive ��-secretase of the amyloid precursor protein in primary neurons

The amyloid precursor protein (APP) undergoes constitutive shedding by a protease activity called α‐secretase. This is considered an important mechanism preventing the generation of the Alzheimer's disease amyloid‐β peptide (Aβ). α‐Secretase appears to be a metalloprotease of the ADAM family, but its identity remains to be established. Using a novel α‐secretase‐cleavage site‐specific antibody, we found that RNAi‐mediated knockdown of ADAM10, but surprisingly not of ADAM9 or 17, completely suppressed APP α‐secretase cleavage in different cell lines and in primary murine neurons. Other proteases were not able to compensate for this loss of α‐cleavage. This finding was further confirmed by mass‐spectrometric detection of APP‐cleavage fragments. Surprisingly, in different cell lines, the reduction of α‐secretase cleavage was not paralleled by a corresponding increase in the Aβ‐generating β‐secretase cleavage, revealing that both proteases do not always compete for APP as a substrate. Instead, our data suggest a novel pathway for APP processing, in which ADAM10 can partially compete with γ‐secretase for the cleavage of a C‐terminal APP fragment generated by β‐secretase. We conclude that ADAM10 is the physiologically relevant, constitutive α‐secretase of APP.     

A HAT for sleep?: Epigenetic regulation of sleep by Tip60 in Drosophila

Sleep disturbances are common in neurodegenerative diseases such as Alzheimer disease (AD). Unfortunately, how AD is mechanistically linked with interference of the body’s natural sleep rhythms remains unclear. Our recent findings provide insight into this question by demonstrating that sleep disruption associated with AD is driven by epigenetic changes mediated by the histone acetyltransferase (HAT) Tip60. In this study, we show that Tip60 functionally interacts with the AD associated amyloid precursor protein (APP) to regulate axonal growth of Drosophila small ventrolateral neuronal (sLNv) pacemaker cells, and their production of neuropeptide pigment dispersing factor (PDF) that stabilizes appropriate sleep-wake patterns in the fly. Loss of Tip60 HAT activity under APP neurodegenerative conditions causes decreased PDF production, retraction of the sLNv synaptic arbor required for PDF release and disruption of sleep-wake cycles in these flies. Remarkably, excess Tip60 in conjunction with APP fully rescues these sleep-wake disturbances by inducing overelaboration of the sLNv synaptic terminals and increasing PDF levels, supporting a neuroprotective role for Tip60 in these processes. Our studies highlight the importance of epigenetic based mechanisms underlying sleep disturbances in neurodegenerative diseases like AD.     

Role of 14-3-3gamma in FE65-dependent gene transactivation mediated by the amyloid beta-protein precursor cytoplasmic fragment

The amyloid beta-protein precursor intracellular domain fragment (AICD) is generated from amyloid beta-protein precursor by consecutive cleavages. AICD is thought to activate FE65-dependent gene expression, but the molecular mechanism remains under consideration. We found that dimeric 14-3-3gamma bound both AICD and FE65 simultaneously, and this binding facilitated FE65-dependent gene transactivation by enhancing the association of AICD with FE65. 14-3-3gamma bound to the 667VTPEER672 motif of AICD and, most interestingly, the phosphorylation of AICD at Thr-668 in this motif inhibited the interaction with 14-3-3gamma and blocked gene transactivation. 14-3-3gamma required a sequence between the WW domain and the first phosphotyrosine interaction domain of FE65 for association with FE65. Deletion of this region blocked 14-3-3gamma binding to FE65 and suppressed AICD-mediated FE65-dependent gene transactivation, although the deletion mutant FE65 was still able to bind Tip60, a histone acetyltransferase that forms a complex with FE65 in the nucleus. Taken together, these data demonstrate that 14-3-3gamma facilitates FE65-dependent gene transactivation by forming a complex containing AICD and FE65, and phosphorylation of AICD down-regulates FE65-dependent gene transactivation through the dissociation of 14-3-3gamma and/or FE65 from AICD. Our findings suggest that multiple interactions of AICD with FE65 and 14-3-3gamma modulate FE65-dependent gene transactivation.