An intracellular threonine of amyloid-ß precursor protein mediates synaptic plasticity deficits and memory loss.

Mutations in Amyloid-ß Precursor Protein (APP) and BRI2/ITM2b genes cause Familial Alzheimer and Danish Dementias (FAD/FDD), respectively. APP processing by BACE1, which is inhibited by BRI2, yields sAPPß and ß-CTF. ß-CTF is cleaved by gamma-secretase to produce Aß. A knock-in mouse model of FDD, called FDDKI, shows deficits in memory and synaptic plasticity, which can be attributed to sAPPß/ß-CTF but not Aß. We have investigated further the pathogenic function of ß-CTF focusing on Thr(668) of ß-CTF because phosphorylation of Thr(668) is increased in AD cases. We created a knock-in mouse bearing a Thr(668)Ala mutation (APP(TA) mice) that prevents phosphorylation at this site. This mutation prevents the development of memory and synaptic plasticity deficits in FDDKI mice. These data are consistent with a role for the carboxyl-terminal APP domain in the pathogenesis of dementia and suggest that averting the noxious role of Thr(668) is a viable therapeutic strategy for human dementias.

Caspase-9 mediates synaptic plasticity and memory deficits of Danish dementia knock-in mice: caspase-9 inhibition provides therapeutic protection.

BACKGROUND: Mutations in either Aß Precursor protein (APP) or genes that regulate APP processing, such as BRI2/ITM2B and PSEN1/PSEN2, cause familial dementias. Although dementias due to APP/PSEN1/PSEN2 mutations are classified as familial Alzheimer disease (FAD) and those due to mutations in BRI2/ITM2B as British and Danish dementias (FBD, FDD), data suggest that these diseases have a common pathogenesis involving toxic APP metabolites. It was previously shown that FAD mutations in APP and PSENs promote activation of caspases leading to the hypothesis that aberrant caspase activation could participate in AD pathogenesis. RESULTS: Here, we tested whether a similar mechanism applies to the Danish BRI2/ITM2B mutation. We have generated a genetically congruous mouse model of FDD, called FDD(KI), which presents memory and synaptic plasticity deficits. We found that caspase-9 is activated in hippocampal synaptic fractions of FDD(KI) mice and inhibition of caspase-9 activity rescues both synaptic plasticity and memory deficits. CONCLUSION: These data directly implicate caspase-9 in the pathogenesis of Danish dementia and suggest that reducing caspase-9 activity is a valid therapeutic approach to treating human dementias.

Tyr682 in the Aß-precursor protein intracellular domain regulates synaptic connectivity, cholinergic function, and cognitive performance.

Processing of Aß-precursor protein (APP) plays an important role in Alzheimer's disease (AD) pathogenesis. The APP intracellular domain contains residues important in regulating APP function and processing, in particular the 682YENPTY687 motif. To dissect the functions of this sequence in vivo, we created an APP knock-in allele mutating Y682 to Gly (APP(YG/YG) mice). This mutation alters the processing of APP and TrkA signaling and leads to postnatal lethality and neuromuscular synapse defects when expressed on an APP-like protein 2 KO background. This evidence prompted us to characterize further the APP(YG/YG) mice. Here, we show that APP(YG/YG) mice develop aging-dependent decline in cognitive and neuromuscular functions, a progressive reduction in dendritic spines, cholinergic tone, and TrkA levels in brain regions governing cognitive and motor functions. These data are consistent with our previous findings linking NGF and APP signaling and suggest a causal relationship between altered synaptic connectivity, cholinergic tone depression and TrkA signaling deficit, and cognitive and neuromuscular decline in APP(YG/YG) mice. The profound deficits caused by the Y682 mutation underscore the biological importance of APP and indicate that APP(YG/YG) are a valuable mouse model to study APP functions in physiological and pathological processes.

Memory deficits of British dementia knock-in mice are prevented by Aß-precursor protein haploinsufficiency.

Familial British Dementia (FBD) is caused by an autosomal dominant mutation in the BRI2/ITM2B gene (Vidal et al., 1999). FBD(KI) mice are a model of FBD that is genetically congruous to the human disease, because they carry one mutant and one wild-type Bri2/Itm2b allele. Analysis of these mice has shown that the British mutation causes memory impairments due to loss of Bri2 function (Tamayev et al., 2010b). BRI2 is a physiologic inhibitor of processing of the Aß-precursor protein (APP; Matsuda et al., 2008), a gene associated with Alzheimer's disease (Bertram et al., 2010). Here we show that APP haploinsufficiency prevents memory dysfunctions seen in FBD(KI) mice. This genetic suppression is consistent with a role for APP in the pathogenesis of memory deficits. Moreover, it provides compelling evidence that the memory dysfunctions caused by the British BRI2 mutant are dependent on endogenous APP and that BRI2 and APP functionally interact. This evidence establishes a mechanistic connection between Familial British and Alzheimer's dementias.

Inhibition of γ-secretase worsens memory deficits in a genetically congruous mouse model of Danish dementia.

BACKGROUND: A mutation in the BRI2/ITM2b gene causes familial Danish dementia (FDD). BRI2 is an inhibitor of amyloid-ß precursor protein (APP) processing, which is genetically linked to Alzheimer's disease (AD) pathogenesis. The FDD mutation leads to a loss of BRI2 protein and to increased APP processing. APP haplodeficiency and inhibition of APP cleavage by ß-secretase rescue synaptic/memory deficits of a genetically congruous mouse model of FDD (FDDKI). ß-cleavage of APP yields the ß-carboxyl-terminal (ß-CTF) and the amino-terminal-soluble APPß (sAPPß) fragments. γ-secretase processing of ß-CTF generates Aß, which is considered the main cause of AD. However, inhibiting Aß production did not rescue the deficits of FDDKI mice, suggesting that sAPPß/ß-CTF, and not Aß, are the toxic species causing memory loss. RESULTS: Here, we have further analyzed the effect of γ-secretase inhibition. We show that treatment with a γ-secretase inhibitor (GSI) results in a worsening of the memory deficits of FDDKI mice. This deleterious effect on memory correlates with increased levels of the ß/α-CTFs APP fragments in synaptic fractions isolated from hippocampi of FDDKI mice, which is consistent with inhibition of γ-secretase activity. CONCLUSION: This harmful effect of the GSI is in sharp contrast with a pathogenic role for Aß, and suggests that the worsening of memory deficits may be due to accumulation of synaptic-toxic ß/α-CTFs caused by GSI treatment. However, γ-secretase cleaves more than 40 proteins; thus, the noxious effect of GSI on memory may be dependent on inhibition of cleavage of one or more of these other γ-secretase substrates. These two possibilities do not need to be mutually exclusive. Our results are consistent with the outcome of a clinical trial with the GSI Semagacestat, which caused a worsening of cognition, and advise against targeting γ-secretase in the therapy of AD. Overall, the data also indicate that FDDKI is a valuable mouse model to study AD pathogenesis and predict the clinical outcome of therapeutic agents for AD.

ß- but not γ-secretase proteolysis of APP causes synaptic and memory deficits in a mouse model of dementia.

A mutation in the BRI2/ITM2b gene causes loss of BRI2 protein leading to familial Danish dementia (FDD). BRI2 deficiency of FDD provokes an increase in amyloid-ß precursor protein (APP) processing since BRI2 is an inhibitor of APP proteolysis, and APP mediates the synaptic/memory deficits in FDD. APP processing is linked to Alzheimer disease (AD) pathogenesis, which is consistent with a common mechanism involving toxic APP metabolites in both dementias. We show that inhibition of APP cleavage by ß-secretase rescues synaptic/memory deficits in a mouse model of FDD. ß-cleavage of APP yields amino-terminal-soluble APPß (sAPPß) and ß-carboxyl-terminal fragments (ß-CTF). Processing of ß-CTF by γ-secretase releases amyloid-ß (Aß), which is assumed to cause AD. However, inhibition of γ-secretase did not ameliorate synaptic/memory deficits of FDD mice. These results suggest that sAPPß and/or ß-CTF, rather than Aß, are the toxic species causing dementia, and indicate that reducing ß-cleavage of APP is an appropriate therapeutic approach to treating human dementias. Our data and the failures of anti-Aß therapies in humans advise against targeting γ-secretase cleavage of APP and/or Aß.

Increased AßPP processing in familial Danish dementia patients.

An autosomal dominant mutation in the BRI2/ITM2B gene causes Familial Danish Dementia (FDD). We have generated a mouse model of FDD, called FDDKI, genetically congruous to the human disease. These mice carry one mutant and one wild type Bri2/Itm2b allele, like FDD patients. Analysis of FDDKI mice and samples from human patients has shown that the Danish mutation causes loss of Bri2 protein. FDDKI mice show synaptic plasticity and memory impairments. BRI2 is a physiological interactor of amyloid-ß protein precursor (AßPP), a gene associated with Alzheimer's disease, which inhibits processing of AßPP. AßPP/Bri2 complexes are reduced in synaptic membranes of FDDKI mice. Consequently, AßPP metabolites derived from processing of AßPP by ß-, α-, and γ-secretases are increased in Danish dementia mice. AßPP haplodeficiency prevents memory and synaptic dysfunctions, consistent with a role for AßPP-metabolites in the pathogenesis of memory and synaptic deficits. This genetic suppression provides compelling evidence that AßPP and BRI2 functionally interact. Here, we have investigated whether AßPP processing is altered in FDD patients' brain samples. We find that the levels of several AßPP metabolites, including Aß, are significantly increased in the brain sample derived from an FDD patient. Our data are consistent with the findings in FDDKI mice, and support the hypothesis that the neurological effects of the Danish form of BRI2 are caused by toxic AßPP metabolites, suggesting that Familial Danish and Alzheimer's dementias share common pathogenic mechanisms.

APP heterozygosity averts memory deficit in knockin mice expressing the Danish dementia BRI2 mutant.

An autosomal dominant mutation in the BRI2/ITM2B gene causes familial Danish dementia (FDD). Analysis of FDD(KI) mice, a mouse model of FDD genetically congruous to the human disease since they carry one mutant and one wild-type Bri2/Itm2b allele, has shown that the Danish mutation causes loss of Bri2 protein, synaptic plasticity and memory impairments. BRI2 is a physiological interactor of Aß-precursor protein (APP), a gene associated with Alzheimer disease, which inhibits processing of APP. Here, we show that APP/Bri2 complexes are reduced in synaptic membranes of FDD(KI) mice. Consequently, APP metabolites derived from processing of APP by ß-, α- and γ-secretases are increased in Danish dementia mice. APP haplodeficiency prevents memory and synaptic dysfunctions, consistent with a role for APP metabolites in the pathogenesis of memory and synaptic deficits. This genetic suppression provides compelling evidence that APP and BRI2 functionally interact, and that the neurological effects of the Danish form of BRI2 only occur when sufficient levels of APP are supplied by two alleles. This evidence establishes a pathogenic sameness between familial Danish and Alzheimer's dementias.

Interactions of amyloid Aß(1-42) peptide with self-assembled peptide nanospheres.

In this work we have probed the interactions of the amyloid Aß(1-42) peptide with self-assembled nanospheres. The nanospheres were formed by self-assembly of a newly developed bolaamphiphile bis(N-alpha-amido-methionine)-1,8 octane dicarboxylate under aqueous conditions. It was found that the interactions of the Aß(1-42) peptide with the nanospheres were concentration as well as pH dependent and the peptide largely adopts a random coil structure upon interacting with the nanospheres. Further, upon incorporation with the nanospheres, we observed a relative diminution in the aggregation of Aß(1-42) at low concentrations of Aß(1-42). The interactions between the nanospheres and the Aß(1-42) peptide were investigated by atomic force microscopy, transmission electron microscopy, circular dichroism, FTIR and fluorescence spectroscopy, and the degree of fibrillation in the presence and absence of nanospheres was monitored by the Thioflavine T assay. We believe that the outcome from this work will help further elucidate the binding properties of Aß peptide as well as designing nanostructures as templates for further investigating the nucleation and fibrillation process of Aß-like peptides.

Memory deficits due to familial British dementia BRI2 mutation are caused by loss of BRI2 function rather than amyloidosis.

Familial dementias, which include Alzheimer disease (AD), familial British dementia (FBD), and familial Danish dementia (FDD), are caused by dominantly inherited autosomal mutations and are characterized by the production of amyloidogenic peptides, neurofibrillary tangles (NFTs) and neurodegeneration (St George-Hyslop and Petit, 2005; Garringer et al., 2009). The prevailing pathogenic theory, the "amyloid cascade hypothesis" (Hardy and Selkoe, 2002), posits that the accumulation of amyloidogenic peptides triggers tauopathy, neurodegeneration, and cognitive and behavioral changes. However, this hypothesis is yet to be validated, and causes of dementia may be multifaceted and involve other mechanisms, such as loss of function due to pathogenic mutations. Mouse models of human dementia invariably use transgenic expression systems (LaFerla and Oddo, 2005; McGowan et al., 2006; Vidal et al., 2009; Coomaraswamy et al., 2010) that do not reflect the genotypes of human disease and cannot replicate loss of function. Therefore, we generated a knock-in (KI) mouse model of FBD (FBD(KI)) genetically congruous with the human disease. FBD is caused by a missense mutation at the stop codon of the BRI2 gene (Vidal et al., 1999) and, like FBD patients, FBD(KI) mice carry this mutation in one of the two murine Bri2 alleles. We report that the British mutation drastically reduces expression of mature BRI2 in both KI mice and human FBD brains. This deficit is associated with severe hippocampal memory deficits in FBD(KI) mice. Remarkably, these animals showed no cerebral amyloidosis and tauopathy. Bri2(+/-) mice present memory deficits similar to those in FBD(KI) animals. Collectively, these results indicate that the British BRI2 mutation underlies abnormal memory due to loss of BRI2 function and independently of histopathological alterations typically evident in advanced neurodegenerative disease.

Tyr(682) in the intracellular domain of APP regulates amyloidogenic APP processing in vivo.

BACKGROUND: The pathogenesis of Alzheimer's disease is attributed to misfolding of Amyloid-ß (Aß) peptides. Aß is generated during amyloidogenic processing of Aß-precursor protein (APP). Another characteristic of the AD brain is increased phosphorylation of APP amino acid Tyr(682). Tyr(682) is part of the Y(682)ENPTY(687) motif, a docking site for interaction with cytosolic proteins that regulate APP metabolism and signaling. For example, normal Aß generation and secretion are dependent upon Tyr(682) in vitro. However, physiological functions of Tyr(682) are unknown. METHODOLOGY/PRINCIPAL FINDINGS: To this end, we have generated an APP Y682G knock-in (KI) mouse to help dissect the role of APP Tyr(682) in vivo. We have analyzed proteolytic products from both the amyloidogenic and non-amyloidogenic processing of APP and measure a profound shift towards non-amyloidogenic processing in APP KI mice. In addition, we demonstrate the essential nature of amino acid Tyr(682) for the APP/Fe65 interaction in vivo. CONCLUSIONS/SIGNIFICANCE: Together, these observations point to an essential role of APP intracellular domain for normal APP processing and function in vivo, and provide rationale for further studies into physiological functions associated with this important phosphorylation site.

Danish dementia mice suggest that loss of function and not the amyloid cascade causes synaptic plasticity and memory deficits.

According to the prevailing "amyloid cascade hypothesis," genetic dementias such as Alzheimer's disease and familial Danish dementia (FDD) are caused by amyloid deposits that trigger tauopathy, neurodegeneration, and behavioral/cognitive alterations. To efficiently reproduce amyloid lesions, murine models of human dementias invariably use transgenic expression systems. However, recent FDD transgenic models showed that Danish amyloidosis does not cause memory defects, suggesting that other mechanisms cause Danish dementia. We studied an animal knock-in model of FDD (FDD(KI/+)) genetically congruous with human cases. FDD(KI/+) mice present reduced Bri2 levels, impaired synaptic plasticity and severe hippocampal memory deficits. These animals show no cerebral lesions that are reputed characteristics of human dementia, such as tangles or amyloid plaques. Bri2(+/-) mice exhibit synaptic and memory deficits similar to FDD(KI/+) mice, and memory loss of FDD(KI/+) mice is prevented by expression of WT BRI2, indicating that Danish dementia is caused by loss of BRI2 function. Together, the data suggest that clinical dementia in Danish patients occurs via a loss of function mechanism and not as a result of amyloidosis and tauopathy.

The interactome of the amyloid beta precursor protein family members is shaped by phosphorylation of their intracellular domains.

BACKGROUND: Brain tissue from patients with Alzheimer's disease has shown an increase of phosphorylation of Tyr-682, located on the conserved Y682ENPTY motif, and Thr-668 residues, both in the intracellular domain (AID) of amyloid beta precursor protein (APP), although the role of these two residues is not yet known. RESULTS: Here, we report that the phosphorylation status of Tyr-682, and in some cases Thr-668, shapes the APP interactome. It creates a docking site for SH2-domain containing proteins, such as ShcA, ShcB, ShcC, Grb7, Grb2, as well as adapter proteins, such as Crk and Nck, that regulate important biological processes, cytosolic tyrosine kinases, such as Abl, Lyn and Src, which regulate signal transduction pathways, and enzymes that control phosphatidylinositols levels and signaling, such as PLC-gamma. At the same time, it either reduces (like for JIP1, NUMB, NUMBL and ARH) or abolishes (like for Fe65, Fe65L1 and Fe65L2) binding of other APP interactors. Phosphorylation of Thr-668, unlike Tyr-682, does not seem to affect APP's ability to interact with the various proteins, with Pin1 and X11 being the exclusions. We also found that there are some differences between the interactions to AID and to ALID1 and ALID2, its two homologues. CONCLUSION: Our data indicates that APP can regulate diverse cellular processes and that, vice versa, a network of signaling events can impact APP processing. Our results also suggest that phosphorylation of the APP Intracellular Domain will dramatically shape the APP interactome and, consequently, will regulate APP processing, APP transport and APP/AID-mediated functions.

Templated growth of calcium phosphate on tyrosine derived microtubules and their biocompatibility.

Microtubular structures were self-assembled in aqueous media from a newly synthesized bolaamphiphile, bis(N-alpha-amido-tyrosyl-tyrosyl-tyrosine)-1,5-pentane dicarboxylate. In order to increase the biocompatibility of the microtubules, they were functionalized with the peptide sequence GRGDSP. Further, calcium phosphate nanocrystals were grown on the microtubules. In some cases, collagen was added in order to mimic the components of natural bone tissue. The biomaterials obtained were characterized via transmission electron microscopy (TEM), atomic force microscopy (AFM), IR, and energy dispersive X-ray spectroscopy (EDX) analyses. The biocompatibility of the calcium phosphate-coated microtubules was studied by conducting in vitro cell-attachment, cell-proliferation and cytotoxicity studies using mouse embryonic fibroblast (MEF) cells. The studies revealed that the biomaterials were found to be non-toxic and biocompatible. The functionalized tubular assemblies coated with calcium phosphate nanocrystals mimic the nanoscale composition of natural bone and may potentially support bone in-growth and osseointegration when used in orthopaedic or dental applications.