Amyloid Precursor Protein Knock-in Research Articles

An impaired intrinsic microglial clock system induces neuroinflammatory alterations in the early stage of amyloid precursor protein knock-in mouse brain

BackgroundDisturbances in clock genes affect almost all patients with Alzheimer’s disease (AD), as evidenced by their altered sleep/wake cycle, thermoregulation, and exacerbation of cognitive impairment. As microglia-mediated neuroinflammation proved to be a driver of AD rather than a result of the disease, in this study, we evaluated the relationship between clock gene disturbance and neuroinflammation in microglia and their contribution to the onset of AD.MethodsIn this study, the expression of clock genes and inflammatory-related genes was examined in MACS microglia isolated from 2-month-old amyloid precursor protein knock-in (APP-KI) and wild-type (WT) mice using cap analysis gene expression (CAGE) deep sequencing and RT-PCR. The effects of clock gene disturbance on neuroinflammation and relevant memory changes were examined in 2-month-old APP-KI and WT mice after injection with SR9009 (a synthetic agonist for REV-ERB). The microglia morphology was studied by staining, neuroinflammation was examined by Western blotting, and cognitive changes were examined by Y-maze and novel object recognition tests.ResultsCLOCK/BMAL1-driven transcriptional negative feedback loops were impaired in the microglia from 2-month-old APP-KI mice. Pro-inflammatory genes in microglia isolated from APP-KI mice were significantly higher than those isolated from WT mice at Zeitgeber time 14. The expression of pro-inflammatory genes was positively associated with NF-κB activation and negatively associated with the BMAL1 expression. SR9009 induced the activation of microglia, the increased expression of pro-inflammatory genes, and cognitive decline in 2-month-old APP-KI mice.ConclusionClock gene disturbance in microglia is involved in the early onset of AD through the induction of chronic neuroinflammation, which may be a new target for preventing or slowing AD.

T-type calcium channel enhancer SAK3 promotes dopamine and serotonin releases in the hippocampus in naive and amyloid precursor protein knock-in mice.

T-type calcium channels in the brain mediate the pathophysiology of epilepsy, pain, and sleep. Recently, we developed a novel therapeutic candidate, SAK3 (ethyl 8'-methyl-2',4-dioxo-2-(piperidin-1-yl)-2'H-spiro[cyclopentane-1,3'-imidazo[1,2-a] pyridine]-2-ene-3-carboxylate), for Alzheimer’s disease (AD). The cognitive improvement by SAK3 is closely associated with enhanced acetylcholine (ACh) release in the hippocampus. Since monoamines such as dopamine (DA), noradrenaline (NA), and serotonin (5-HT) are also involved in hippocampus-dependent learning and psychomotor behaviors in mice, we investigated the effects of SAK3 on these monoamine releases in the mouse brain. Oral administration of SAK3 (0.5 mg/kg, p.o.) significantly promoted DA and 5-HT releases in the naive mouse hippocampal CA1 region but not in the medial prefrontal cortex (mPFC), while SAK3 did not affect NA release in either brain region. The T-type calcium channel-specific inhibitor, NNC 55–0396 (1 μM) significantly antagonized SAK3-enhanced DA and 5-HT releases in the hippocampus. Interestingly, the α7 nicotinic ACh receptor (nAChR) antagonist, methyllycaconitine (1 nM) significantly inhibited DA release, and the α4 nAChR antagonist, dihydro-β-erythroidine (100 μM) significantly blocked both DA and 5-HT releases following SAK3 (0.5 mg/kg, p.o.) administration in the hippocampus. SAK3 did not alter basal monoamine contents both in the mPFC and hippocampus. SAK3 (0.5 mg/kg, p.o.) administration also significantly elevated DA and 5-HT releases in the hippocampal CA1 region of amyloid-precursor protein (APP)NL-GF knock-in (KI) mice. Moreover, hippocampal DA and 5-HT contents were significantly decreased in APPNL-GF KI mice. Taken together, our data suggest that SAK3 promotes monoamine DA and 5-HT releases by enhancing the T-type calcium channel and nAChR in the mouse hippocampus.

Age-dependent behavioral and biochemical characterization of single APP knock-in mouse (APPNL-G-F/NL-G-F) model of Alzheimer's disease.

Saito etal developed a novel amyloid precursor protein (APP) knock-in mouse model (APPNL-G-F) for Alzheimer's disease (AD) to overcome the problem of overexpression of APP in available transgenic mouse models. However, this new mouse model for AD is not fully characterized age-dependently with respect to behavioral and biochemical changes. Therefore, in the present study, we performed an age-dependent behavioral and biochemical characterization of this newly developed mouse model. Here, we used 3-, 6-, 9-, and 12-month-old APPNL-G-F and C57BL/6J mice. We used a separate cohort of animals at each age point. Morris water maze, object recognition, and fear-conditioning tests were used for the assessment of learning and memory functions and open-field test to measure the general locomotor activity of mice. After each testing point, we perfused the mice and collected the brain for immunostaining. We performed the immunostaining for amyloid burden (4G8), glial fibrillary acidic protein, choline acetyltransferase, and tyrosine hydroxylase. The results of the present study indicate that APPNL-G-F mice showed age-dependent memory impairments with maximum impairment at the age of 12months. These mice showed memory impairment in Morris water maze and fear conditioning tests when they were 6months old, whereas, in object recognition test, memory deficit was found in 9-month-old mice. APPNL-G-F mice age dependently showed an increase in amyloid load in different brain regions. However, no amyloid pathology was found in 3-month-old APPNL-G-F mice. Choline acetyltransferase neurons in medial septum-diagonal band complex and tyrosine hydroxylase neurons in locus coeruleus were decreased significantly in APPNL-G-F mice. This mouse model also indicated an age-dependent increase in glial fibrillary acidic protein load. It can be concluded from the results that the APPNL-G-F mouse model may be used to explore the Aβ hypothesis, molecular, and cellular mechanisms involved in AD pathology and to screen the therapeutic potential compounds for the treatment of AD.

Increased Insoluble Amyloid-β Induces Negligible Cognitive Deficits in Old AppNL/NL Knock-In Mice.

Commonly used Alzheimer’s disease mouse models are based on the ectopic overexpression of the human amyloid precursor protein (APP) gene, together with a mutant presenilin gene. Surprisingly, humanized APP knock-in mouse models carrying a single APP Swedish mutation (AppNL), failed to develop amyloid plaque aggregation or cognitive deficits. Here we characterized the effect of this mutation in more advanced ages. We show that 24-month-old AppNL/NL mice, despite presenting an age dependent increase in insoluble amyloid-β oligomers in the prefrontal cortex, they do not develop amyloid plaque deposition, reactive gliosis, or cognitive deficits.

Impaired In Vivo Gamma Oscillations in the Medial Entorhinal Cortex of Knock-in Alzheimer Model.

The entorhinal cortex (EC) has bidirectional connections with the hippocampus and plays a critical role in memory formation and retrieval. EC is one of the most vulnerable regions in the brain in early stages of Alzheimer’s disease (AD), a neurodegenerative disease with progressive memory impairments. Accumulating evidence from healthy behaving animals indicates gamma oscillations (30–100 Hz) as critical for mediating interactions in the circuit between EC and hippocampus. However, it is still unclear whether gamma oscillations have causal relationship with memory impairment in AD. Here we provide the first evidence that in vivo gamma oscillations in the EC are impaired in an AD mouse model. Cross-frequency coupling of gamma (30–100 Hz) oscillations to theta oscillations was reduced in the medial EC of anesthetized amyloid precursor protein knock-in (APP-KI) mice. Phase locking of spiking activity of layer II/III pyramidal cells to the gamma oscillations was significantly impaired. These data indicate that the neural circuit activities organized by gamma oscillations were disrupted in the medial EC of AD mouse model, and point to gamma oscillations as one of possible mechanisms for cognitive dysfunction in AD patients.

Neuronal Store-Operated Calcium Entry and Mushroom Spine Loss in Amyloid Precursor Protein Knock-In Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common reason for elderly dementia in the world. We proposed that memory loss in AD is related to destabilization of mushroom postsynaptic spines involved in long-term memory storage. We demonstrated previously that stromal interaction molecule 2 (STIM2)-regulated neuronal store-operated calcium entry (nSOC) in postsynaptic spines play a key role in stability of mushroom spines by maintaining activity of synaptic Ca(2+)/calmodulin kinase II (CaMKII). Furthermore, we demonstrated previously that the STIM2-nSOC-CaMKII pathway is downregulated in presenilin 1 M146V knock-in (PS1-M146V KI) mouse model of AD, leading to loss of hippocampal mushroom spines in this model. In the present study, we demonstrate that hippocampal mushroom postsynaptic spines are also lost in amyloid precursor protein knock-in (APPKI) mouse model of AD. We demonstrated that loss of mushroom spines occurs as a result of accumulation of extracellular β-amyloid 42 in APPKI culture media. Our results indicate that extracellular Aβ42 acts by overactivating mGluR5 receptor in APPKI neurons, leading to elevated Ca(2+) levels in endoplasmic reticulum, compensatory downregulation of STIM2 expression, impaired synaptic nSOC, and reduced CaMKII activity. Pharmacological inhibition of mGluR5 or overexpression of STIM2 rescued synaptic nSOC and prevented mushroom spine loss in APPKI hippocampal neurons. Our results indicate that downregulation of synaptic STIM2-nSOC-CaMKII pathway causes loss of mushroom synaptic spines in both presenilin and APPKI mouse models of AD. We propose that modulators/activators of this pathway may have a potential therapeutic value for treatment of memory loss in AD. Significance statement: A direct connection between amyloid-induced synaptic mushroom spine loss and neuronal store-operated calcium entry pathway is shown. These results provide strong support for the calcium hypothesis of neurodegeneration and further validate the synaptic store-operated calcium entry pathway as a potential therapeutic target for Alzheimer's disease.

Vascular and parenchymal amyloid pathology in an Alzheimer disease knock-in mouse model: interplay with cerebral blood flow.

BackgroundAccumulation and deposition of β-amyloid peptides (Aβ) in the brain is a central event in the pathogenesis of Alzheimer’s disease (AD). Besides the parenchymal pathology, Aβ is known to undergo active transport across the blood–brain barrier and cerebral amyloid angiopathy (CAA) is a prominent feature in the majority of AD. Although impaired cerebral blood flow (CBF) has been implicated in faulty Aβ transport and clearance, and cerebral hypoperfusion can exist in the pre-clinical phase of Alzheimer’s disease (AD), it is still unclear whether it is one of the causal factors for AD pathogenesis, or an early consequence of a multi-factor condition that would lead to AD at late stage. To study the potential interaction between faulty CBF and amyloid accumulation in clinical-relevant situation, we generated a new amyloid precursor protein (APP) knock-in allele that expresses humanized Aβ and a Dutch mutation in addition to Swedish/London mutations and compared this line with an equivalent knock-in line but in the absence of the Dutch mutation, both crossed onto the PS1M146V knock-in background.ResultsIntroduction of the Dutch mutation results in robust CAA and parenchymal Aβ pathology, age-dependent reduction of spatial learning and memory deficits, and CBF reduction as detected by fMRI. Direct manipulation of CBF by transverse aortic constriction surgery on the left common carotid artery caused differential changes in CBF in the anterior and middle region of the cortex, where it is reduced on the left side and increased on the right side. However these perturbations in CBF resulted in the same effect: both significantly exacerbate CAA and amyloid pathology.ConclusionsOur study reveals a direct and positive link between vascular and parenchymal Aβ; both can be modulated by CBF. The new APP knock-in mouse model recapitulates many symptoms of AD including progressive vascular and parenchymal Aβ pathology and behavioral deficits in the absence of APP overexpression.

Modeling Alzheimer's disease in mouse without mutant protein overexpression: cooperative and independent effects of Aβ and tau.

BackgroundAlzheimer’s disease (AD), the most common cause of dementia in the elderly, has two pathological hallmarks: Aβ plaques and aggregation of hyperphosphorylated tau (p-tau). Aβ is a cleavage product of Amyloid Precursor Protein (APP). Presenilin 1 (PS1) and presenilin 2 (PS2) are the catalytic subunit of γ-secretase, which cleaves APP and mediates Aβ production. Genetic mutations in APP, PSEN1 or PSEN2 can lead to early onset of familial AD (FAD). Although mutations in the tau encoding gene MAPT leads to a subtype of frontotemporal dementia and these mutations have been used to model AD tauopathy, no MAPT mutations have been found to be associated with AD. ResultsTo model AD pathophysiology in mice without the gross overexpression of mutant transgenes, we created a humanized AD mouse model by crossing the APP and PSEN1 FAD knock-in mice with the htau mice which express wildtype human MAPT genomic DNA on mouse MAPT null background (APP/PS1/htau). The APP/PS1/htau mice displayed mild, age-dependent, Aβ plaques and tau hyperphosphorylation, thus successfully recapitulating the late-onset AD pathological hallmarks. Selected biochemical analyses, including p-tau western blot, γ-secretase activity assay, and Aβ ELISA, were performed to study the interaction between Aβ and p-tau. Subsequent behavioral studies revealed that the APP/PS1/htau mice showed reduced mobility in old ages and exaggerated fear response. Genetic analysis suggested that the fear phenotype is due to a synergic interaction between Aβ and p-tau, and it can be completely abolished by tau deletion.ConclusionThe APP/PS1/htau model represents a valuable and disease-relevant late-onset pre-clinical AD animal model because it incorporates human AD genetics without mutant protein overexpression. Analysis of the mice revealed both cooperative and independent effects of Aβ and p-tau.

The –YENPTY– domain of the amyloid precursor protein: Much more than just endocytosis? (Comment on DOI 10.1002/bies.201300041)

The fascination in amyloid precursor protein (APP), a type I transmembrane protein enriched in neurons, rests primarily on one of its proteolytic products, amyloid β-protein (Aβ), which is the principal component of senile plaques found in brains of Alzheimer's disease (AD) individuals. To date, research in AD pathogenesis has largely been driven by the amyloid cascade hypothesis, which posits that Aβ is the initiator of cellular changes resulting in AD. While supported by a large body of literature, this hypothesis has skeptics, as it fails to address a number of important questions, such as the lack of correlation between senile plaques and cognition, and the failure of APP transgenic animal models to recapitulate the full disease spectrum. Not surprisingly, a number of laboratories have asked whether perturbations of normal physiological functions of APP – beyond Aβ generation – may contribute to AD pathophysiology. Single knockout (KO) studies of APP and its orthologs in various species have implicated APP in facilitating transcriptional activities, cell adhesion, axonal transport, neuronal migration, and synaptogenesis 1. Double and triple KO studies in mice of APP and the other two members of the gene family, APLP1 and APLP2, yielded early postnatal lethality phenotypes, suggesting that although functional redundancy exists among APP family members, certain specificity in APLP2 is revealed when APP or APLP1 is absent 2. Of note, sequence similarities between the APP family members are in the N- and C-termini. And within the C-terminus lies the –YENPTY– motif that is both a signal for clathrin-mediated endocytosis and a binding site for numerous cytosolic adaptor proteins. The roles of this motif in normal or pathological functions are still unclear, but clues can be gleaned by comparing KO animals and single mutation of the Tyr682 residue. In particular, APP single KO animals showed diminished brain and body weight, reduced locomotor functions, depressed long-term potentiation, and loss of synapses, which are associated with neurobehavioral deficits (2). However, APP knockin (KI) animals deleted in the last 15 amino acid residues – which encompass the YENPTY domain – were able to rescue a variety of deficits seen in APP KO mice, suggesting that the YENPTY domain is dispensable for certain physiological functions of APP. On the other hand, the Y682G KI mice showed decreased synaptic density and impaired neuromuscular functions. Curiously, when this Y682G mutation was introduced into an APLP2−/− background, the resultant mouse exhibited early postnatal lethality highly reminiscent of the APP/APLP2 double-KO mice. This interesting finding thus places primary importance on the Y682 residue in APP functions, and conflicts with the aforementioned result obtained from a knockin mouse line missing the C-terminal 15 amino acids of APP. The existence of Tyr682 allows the interaction of various adaptor proteins through Tyr phosphorylation and dephosphorylation, creating many possibilities for the system to be modulated. Further, some of the APP binding proteins are phosphorylation state-independent, but their binding is nonetheless affected by other adaptor molecules that are phosphorylation-dependent. This suggests that the YENPTY domain might regulate APP signal transduction functions that are variably dependent on the phosphorylation state of Tyr682. Given this, one explanation of the divergent results from the two knockin mice lines may be that Tyr682 can act as a “biochemical switch,” as proposed by Matrone, and accordingly, complete loss of the APP C-terminus might disrupt multiple pathways that cancel each other's cellular effects 3. Nonetheless, the findings from the Y682G mice are both intriguing and noteworthy, as they point to the physiological importance of the YENPTY domain. Hence the versatile functions of APP continue to provide fodder for interesting studies. However, whether APP plays a role in AD pathophysiology in an Aβ-independent manner is a crucial question that remains to be answered.

Intracellular APP Domain Regulates Serine-Palmitoyl-CoA Transferase Expression and Is Affected in Alzheimer's Disease.

Lipids play an important role as risk or protective factors in Alzheimer's disease (AD), a disease biochemically characterized by the accumulation of amyloid beta peptides (Aβ), released by proteolytic processing of the amyloid precursor protein (APP). Changes in sphingolipid metabolism have been associated to the development of AD. The key enzyme in sphingolipid de novo synthesis is serine-palmitoyl-CoA transferase (SPT). In the present study we identified a new physiological function of APP in sphingolipid synthesis. The APP intracellular domain (AICD) was found to decrease the expression of the SPT subunit SPTLC2, the catalytic subunit of the SPT heterodimer, resulting in that decreased SPT activity. AICD function was dependent on Fe65 and SPTLC2 levels are increased in APP knock-in mice missing a functional AICD domain. SPTLC2 levels are also increased in familial and sporadic AD postmortem brains, suggesting that SPT is involved in AD pathology.

Genetic Dissection of the Amyloid Precursor Protein in Developmental Function and Amyloid Pathogenesis

Proteolytic processing of the amyloid precursor protein (APP) generates large soluble APP derivatives, β-amyloid (Aβ) peptides, and APP intracellular domain. Expression of the extracellular sequences of APP or its Caenorhabditis elegans counterpart has been shown to be sufficient in partially rescuing the CNS phenotypes of the APP-deficient mice and the lethality of the apl-1 null C. elegans, respectively, leaving open the question as what is the role of the highly conserved APP intracellular domain? To address this question, we created an APP knock-in allele in which the mouse Aβ sequence was replaced by the human Aβ. A frameshift mutation was introduced that replaced the last 39 residues of the APP sequence. We demonstrate that the C-terminal mutation does not overtly affect APP processing and amyloid pathology. In contrast, crossing the mutant allele with APP-like protein 2 (APLP2)-null mice results in similar neuromuscular synapse defects and early postnatal lethality as compared with mice doubly deficient in APP and APLP2, demonstrating an indispensable role of the APP C-terminal domain in these development activities. Our results establish an essential function of the conserved APP intracellular domain in developmental regulation, and this activity can be genetically uncoupled from APP processing and Aβ pathogenesis.

APP ΔNL695 expression in murine tissue downregulates CNBP expression

The cellular nucleic acid binding protein (CNBP) is a ubiquitously expressed protein involved in regulation of transcription and translation. CNBP, and its encoding gene ZNF9, have been shown to be involved in type 2 myotonic dystrophy. Both Alzheimer's disease (AD) and sporadic inclusion body myositis (sIBM) are age-related degenerative diseases associated with the accumulation of β-amyloid. Overexpression of amyloid precursor protein (APP) in mice has been used to generate models of both diseases. We show here that overexpression of APP in skeletal muscle from a mouse model of sIBM reduces the expression of CNBP significantly. We examined CNBP expression in a brain-specific APP-overexpressing strain, and a whole body APP knock-in strain, and found that there was a reduction in CNBP expression in tissue expressing APP Swe. We conclude that expression of APP Swe in murine tissue induces a decrease in CNBP expression. This effect does not appear to be due to alterations in CNBP transcription. APP Swe expression may provide a tool for the study of CNBP regulation and clues to the roles of both proteins in disease.

Analysis of Neurons Created from Wild-Type and Alzheimer's Mutation Knock-In Embryonic Stem Cells by a Highly Efficient Differentiation Protocol

It is impossible to obtain and amplify live neurons from Alzheimer's disease (AD) patients. To establish the neurons harboring AD abnormality, we constructed mouse embryonic stem (ES) cells, in which the AD-causative V642I mutation was introduced to the endogenous amyloid precursor protein (APP) gene, in combination with a protocol to efficiently differentiate ES cells into postmitotic neurons without using a cell sorter. By this protocol, ES cells differentiated into >90% of the central type of adult postmitotic neurons. Neurons derived from V642I-APP knock-in ES cells were indistinguishable from wild-type ES-derived neurons, as determined by the expression of various markers for neuronal differentiation. Notably, V642I-APP knock-in ES cell-derived neurons exhibited significantly increased secretion of Abeta42 without AD-related hyperphosphorylation of tau, indicating that the direct output of the AD-causative mutation is increased Abeta42 secretion. In this study, we analyze created neurons with wild-type and AD genotypes and propose a new strategy for generating neurons for any dominantly inherited neurodegenerative diseases. The strategy can be applied to create human neurons with AD or any other neurodegenerative disease by using human ES cells.

β-amyloid plaques in amyloid precursor protein knock-in mice

β-amyloid plaques in amyloid precursor protein knock-in mice