Amyloid Precursor Protein Knock-in Mouse Research Articles

Early disruption of entorhinal dopamine in a knock-in model of Alzheimer's disease.

The entorhinal cortex (EC) is a critical brain area for memory formation, while also the region exhibiting the earliest histological and functional alterations in Alzheimer's disease (AD). The EC thus has been long hypothesized as one of the originating brain areas of AD pathophysiology, although circuit mechanisms causing its selective vulnerability remain poorly understood. We found that dopamine neurons projecting their axons to the lateral EC (LEC), critical for memory formation in healthy brains, become dysfunctional and cause memory impairments in early AD brains. In amyloid precursor protein knock-in mice with associative memory impairment, LEC dopamine activity and associative memory encoding of LEC layer 2/3 neurons were disrupted in parallel from the early pathological stage. Optogenetic reactivation of LEC dopamine fibers, as well as L- DOPA treatment, rescued associative learning behavior. These results suggest that dysfunction of LEC-projecting dopamine neurons underlies memory impairment in AD from early stages, pointing to a need for clinical investigation of LEC dopamine in AD patients.

In vivo safety and efficacy of a CRISPR‐based gene therapy for Alzheimer’s disease

AbstractBackgroundOur lab has developed a gene‐editing strategy to target the extreme C‐terminus (C‐term) of APP (amyloid precursor protein) – a gene with a central and indisputable role in AD. In physiologic states, the APP protein is cleaved by α‐secretases to generate neuroprotective sAPPα; but in AD (both sporadic and familial), the alternative β‐cleavage pathway is active, generating pathologic fragments (including Aβ). Specifically, our approach attenuates pathologic β‐cleavage products by disrupting a pentapeptide “YENPTY” domain at the APP C‐term, and this retains the edited APP (APP‐ΔC) at the cell‐surface, augmenting neuroprotective and neuroregenerative α‐cleavage.MethodIn order to assess the safety and efficacy of our approach in vivo, we used two different CRISPR‐editing approaches. First, we generated germline edits in Wt and APP knockin (APPNL‐G‐F) mice by injecting embryos with APP C‐term targeting CRISPRs. We then selected founders to produce stable strains of WT and APP‐KI in which the APP C‐term was genomically deleted (referred to as WtΔC and KI‐ΔC mice respectively). In another set of experiments, we packaged APP C‐term CRISPRs into AAV vectors and systemically injected AAVs into APP‐KI mice.ResultWtΔC showed no cognitive deficits or histological abnormalities compared to Wt controls. KI‐ΔC showed a dramatic reduction of amyloid beta plaques and associated neuroinflammatory markers and similar results were observed in AAV‐treated KI mice. Moreover, both germline and somatic APP C‐term editing produced an increase in neuroprotective sAPPα.ConclusionIn total, these data demonstrate that our approach is safe and efficacious in vivo and validate the feasibility of our APP C‐terminus editing approach as a therapeutic for AD.

Proteomic alterations in the brain and blood–brain barrier during brain Aβ accumulation in an APP knock-in mouse model of Alzheimer’s disease

BackgroundBlood–brain barrier (BBB) dysfunction is supposed to be an early event in the development of Alzheimer’s disease (AD). This study aimed to investigate the relationship between BBB alterations and AD progression in terms of amyloid-β peptide (Aβ) accumulation in the brains of humanized amyloid precursor protein knock-in (APP-KI) mice.MethodsBrain Aβ accumulation was examined using immunohistochemical analysis. Alterations in differentially expressed proteins were determined using sequential window acquisition of all theoretical fragment ion mass spectroscopy (SWATH-MS)-based quantitative proteomics, and Metascape, STRING, Gene Ontology, and KEGG were used for network analyses of altered biological pathways and processes. Statistical significance was determined using the unpaired two-tailed Student’s t-test and Welch’s t-test for two groups and one-way analysis of variance followed by Tukey’s test for more than two groups. Correlations between two groups were determined using Pearson’s correlation analysis.ResultsBrain Aβ accumulation in APP-KI mice was detectable at 2 months, increased significantly at 5 months, and remained elevated at 12 months of age. The levels of differentially expressed proteins in isolated brain capillaries were higher in younger mice, whereas those in the brain were higher in older mice. Network analyses indicated changes in basement membrane-associated and ribosomal proteins in the brain capillaries. There were no significant changes in key proteins involved in drug or Aβ transport at the BBB. In contrast, solute carrier transporter levels in astrocytes, microglia, and neurons were altered in the brain of older mice. Moreover, the levels of the lipid transporters Apoe and Apoj were upregulated in both the brain and isolated brain capillaries after Aβ accumulation.ConclusionsOur results suggest that changes in the brain occurred after advanced Aβ accumulation, whereas initial Aβ accumulation was sufficient to cause alterations in the BBB. These findings may help elucidate the role of BBB alterations in AD progression and predict the distribution of drugs across the BBB in the brain of patients with AD.

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

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

Translational potential of synaptic alterations in Alzheimer's disease patients and amyloid precursor protein knock-in mice.

Synaptic dysfunction is an early event in Alzheimer's disease. Post-mortem studies suggest that alterations in synaptic proteins are associated with cognitive decline in Alzheimer's disease. We measured the concentration of three synaptic proteins, zinc transporter protein 3, dynamin1 and AMPA glutamate receptor 3 in cerebrospinal fluid of subjects with mild cognitive impairment (n = 18) and Alzheimer's disease (n = 18) and compared the levels to cognitively and neurologically healthy controls (n = 18) by using ELISA assay. In addition, we aimed to assess the translational potential of these synaptic proteins in two established amyloid precursor protein knock-in Alzheimer's disease mouse models by assessing the cerebrospinal fluid, hippocampal and cortical synaptic protein concentrations. Using ELISA, we measured in parallel these three proteins in cerebrospinal fluid and/or brain of 12- and 24-month-old AppNL-F and AppNL-G-F knock-in mice and AppWt control mice. The regional distribution and expression of these proteins were explored upon aging of the App knock-in models by quantitative immunofluorescence microscopy. Notably, we found a significant increase in concentrations of zinc transporter protein 3 and AMPA glutamate receptor 3 in cerebrospinal fluid of both patient groups compared with cognitively healthy controls. Dynamin1 concentration was significantly higher in Alzheimer's disease patients. Remarkably, patients with mild cognitive impairment who converted to Alzheimer's disease (n = 7) within 2 years exhibited elevated baseline cerebrospinal fluid zinc transporter protein 3 concentrations compared with mild cognitive impairment patients who did not convert (n = 11). Interestingly, similar to the alterations in Alzheimer's disease subjects, cerebrospinal fluid AMPA glutamate receptor 3 concentration was significantly higher in AppNL-G-F knock-in mice when compared with wild-type controls. Furthermore, we have detected age and brain regional specific changes of the three synaptic proteins in the hippocampus and prefrontal cortex of both AppNL-F and AppNL-G-F knock-in mice. Notably, all the three cerebrospinal fluid synaptic protein concentrations correlated negatively with concentrations in hippocampal lysates. The elevated zinc transporter protein 3 concentrations in the cerebrospinal fluid of converter versus non-converter mild cognitive impairment patients suggests a prospective role of zinc transporter 3 in differentiating dementia patients of the biological continuum of Alzheimer's disease. The increased cerebrospinal fluid concentrations of synaptic proteins in both patient groups, potentially reflecting synaptic alterations in the brain, were similarly observed in the amyloid precursor protein knock-in mouse models highlighting the translational potential of these proteins as markers for synaptic alterations. These synaptic markers could potentially help reduce the current disparities between human and animal model-based studies aiding the translation of preclinical discoveries of pathophysiological changes into clinical research.

Tooth Loss Induces Memory Impairment and Glial Activation in Young Wild-Type Mice.

Tooth loss is closely associated with Alzheimer's disease (AD). Previously, we reported that tooth loss induced memory impairment in amyloid precursor protein knock-in mice by decreasing neuronal activity and synaptic protein levels and increasing glial activation, neuroinflammation, and pyramidal neuronal cell loss without altering amyloid-β levels in the hippocampus. However, the effects of tooth loss in young wild-type mice have not been explored yet. We investigated the effects of tooth loss on memory impairment, neuronal activity, synaptic protein levels, glial activation, and pyramidal neuronal cell loss in young wild-type mice. Two-month-old wild-type mice were randomly divided into control and tooth loss groups. In the tooth loss group, maxillary molar teeth on both sides were extracted, whereas no teeth were extracted in the control group. Two months after tooth extraction, we performed a novel object recognition test to evaluate memory function. Glial activation, neuronal activity, synaptic protein levels, and the number of pyramidal neurons were evaluated using immunofluorescence staining, immunohistochemistry, and western blotting. The tooth loss group exhibited memory impairment and decreased neuronal activity and the levels of synaptic proteins in both the hippocampus and cortex. Moreover, tooth loss increased the activation of phosphorylated c-Jun N-terminal kinase (JNK), heat shock protein 90 (HSP90), and glial activation and reduced the number of pyramidal neurons in the hippocampus. Tooth loss in the young wild-type mice will attenuate neuronal activity, decrease synaptic protein levels, and induce pyramidal neuronal loss, and eventually lead to memory impairment.

Impairment of ciliary dynamics in an APP knock-in mouse model of Alzheimer's disease

The primary cilium is a specialized microtubule-based sensory organelle that extends from the cell body of nearly all cell types. Neuronal primary cilia, which have their own unique signaling repertoire, are crucial for neuronal integrity and the maintenance of neuronal connectivity throughout adulthood. Dysfunction of cilia structure and ciliary signaling is associated with a variety of genetic syndromes, termed ciliopathies. One of the characteristic features of human ciliopathies is impairment of memory and cognition, which is also observed in Alzheimer's disease (AD). Amyloid β peptide (Aβ) is produced through the proteolytic processing of amyloid precursor protein (APP), and Aβ accumulation in the brain is proposed to be an early toxic event in the pathogenesis of AD. To evaluate the effect of increased Aβ level on primary cilia, we assessed ciliary dynamics in hippocampal neurons in an APP knock-in AD model (AppNL-G-F mice) compared to that in wild-type mice. Neuronal cilia length in the CA1, CA3, and dentate gyrus (DG) of wild-type mice increased significantly with age. In AppNL-G-F mice, such elongation was detected in the DG but not in the CA1 and CA3, where more Aβ accumulation was observed. We further demonstrated that Aβ1-42 treatment decreased cilia length both in hTERT-RPE1 cells and dissociated rat hippocampal neurons. There is growing evidence that reduced cilia length is associated with perturbations of synaptic connectivity and dendrite complexity. Thus, our observations raise the important possibility that structural alterations in neuronal cilia might have a role in AD development.

Endothelial expression of human amyloid precursor protein leads to amyloid β in the blood and induces cerebral amyloid angiopathy in knock-in mice

The deposition of amyloid β (Aβ) in blood vessels of the brain, known as cerebral amyloid angiopathy (CAA), is observed in most patients with Alzheimer’s disease (AD). Compared with the pathology of CAA in humans, the pathology in most mouse models of AD is not as evident, making it difficult to examine the contribution of CAA to the pathogenesis of AD. On the basis of biochemical analyses that showed blood levels of soluble amyloid precursor protein (APP) in rats and mice were markedly lower than those measured in human samples, we hypothesized that endothelial APP expression would be markedly lower in rodents and subsequently generated mice that specifically express human WT APP (APP770) in endothelial cells (ECs). The resulting EC-APP770+ mice exhibited increased levels of serum Aβ and soluble APP, indicating that endothelial APP makes a critical contribution to blood Aβ levels. Even though aged EC-APP770+ mice did not exhibit Aβ deposition in the cortical blood vessels, crossing these animals with APP knock-in mice (AppNL-F/NL-F) led to an expanded CAA pathology, as evidenced by increased amounts of amyloid accumulated in the cortical blood vessels. These results highlight an overlooked interplay between neuronal and endothelial APP in brain vascular Aβ deposition. We propose that these EC-APP770+:AppNL-F/NL-F mice may be useful to study the basic molecular mechanisms behind the possible breakdown of the blood–brain barrier upon administration of anti-Aβ antibodies.

Microglial cathepsin E plays a role in neuroinflammation and amyloid β production in Alzheimer's disease.

Regulation of neuroinflammation and β‐amyloid (Aβ) production are critical factors in the pathogenesis of Alzheimer's disease (AD). Cathepsin E (CatE), an aspartic protease, is widely studied as an inducer of growth arrest and apoptosis in several types of cancer cells. However, the function of CatE in AD is unknown. In this study, we demonstrated that the ablation of CatE in human amyloid precursor protein knock‐in mice, called APPNL−G−F mice, significantly reduced Aβ accumulation, neuroinflammation, and cognitive impairments. Mechanistically, microglial CatE is involved in the secretion of soluble TNF‐related apoptosis‐inducing ligand, which plays an important role in microglia‐mediated NF‐κB‐dependent neuroinflammation and neuronal Aβ production by beta‐site APP cleaving enzyme 1. Furthermore, cannula‐delivered CatE inhibitors improved memory function and reduced Aβ accumulation and neuroinflammation in AD mice. Our findings reveal that CatE as a modulator of microglial activation and neurodegeneration in AD and suggest CatE as a therapeutic target for AD by targeting neuroinflammation and Aβ pathology.

Lack of occlusal support did not impact amyloid β deposition in APP knock-in mice.

The lack of occlusal support is an epidemiological risk factor linked to Alzheimer's disease. This study sought to assess the relationship between amyloid β (Aβ) deposition and the lack of occlusal support in amyloid precursor protein (APP) knock-in mice. Sixteen experimental animals were divided into two groups. The upper molars were extracted in the extraction group (group E), and a sham operation was performed in the control group (group C). The Morris water maze test was performed 4 months after the tooth extraction. Aβ immunohistochemical staining and Nissl staining of the hippocampus were performed. Hippocampal plasma corticosterone and Aβ protein levels were measured. In the maze task, the escape latency was significantly longer in group E than in group C. In the probe trials, the time elapsed in the target quadrant was significantly shorter in group E than in group C. The number of hippocampal neurons decreased in group E. There was no significant difference in the plasma corticosterone levels between the two groups, indicating that there was no effect of chronic stress on the behavioral results. Hippocampal Aβ40 and Aβ42 protein levels and Aβ deposition areas by immunohistochemical staining were not significantly different between the two groups. Aβ deposition was not increased in the hippocampus of molarless APP knock-in mice. As such, it appears that cognitive impairment due to a lack of occlusal support was not related to Aβ deposition.

Levetiracetam Treatment Normalizes Levels of Presynaptic Endocytosis Machinery and Restores Nonamyloidogenic APP Processing in App Knock-in Mice.

Toxic amyloid-beta (Aβ) peptides, produced by sequential proteolytic cleavage of the amyloid precursor protein (APP), play a key role in the initial stage of Alzheimer’s disease (AD). Increasing evidence indicates that Aβ42 induces neuronal circuit hyperexcitability in the early stages of AD pathology. As a result, researchers have investigated treatments that modulate the excitatory/inhibitory imbalance as potential AD therapies. For example, levetiracetam, an atypical antiepileptic drug used to quell hyperexcitability, has garnered recent interest in the AD field, even though its exact mechanism(s) of action remains elusive. Here, we show that in APP knock-in mouse models of amyloid pathology, chronic levetiracetam administration decreases cortical Aβ42 levels and lowers the amyloid plaque burden. In addition, using multiplexed tandem mass tag-quantitative mass spectrometry-based proteomic analysis, we determined that chronic levetiracetam administration selectively normalizes levels of presynaptic endocytic proteins. Finally, we found that levetiracetam treatment selectively lowers beta carboxyl-terminal fragment levels, while the abundance of full-length APP remains unchanged. In summary, this work reports that chronic treatment with levetiracetam serves as a useful therapeutic in AD by normalizing levels of presynaptic endocytic proteins and altering APP cleavage preference, leading to a decrease in both Aβ42 levels and the amyloid plaque burden. These novel findings provide novel evidence for the previously documented therapeutic value of levetiracetam to mitigate AD pathology.

T-Type Ca2+ Enhancer SAK3 Activates CaMKII and Proteasome Activities in Lewy Body Dementia Mice Model.

Lewy bodies are pathological characteristics of Lewy body dementia (LBD) and are composed of α-synuclein (α-Syn), which is mostly degraded via the ubiquitin–proteasome system. More importantly, 26S proteasomal activity decreases in the brain of LBD patients. We recently introduced a T-type calcium channel enhancer SAK3 (ethyl-8-methyl-2,4-dioxo-2-(piperidin-1-yl)- 2H-spiro[cyclopentane-1,3-imidazo [1,2-a]pyridin]-2-ene-3-carboxylate) for Alzheimer’s disease therapeutics. SAK3 enhanced the proteasome activity via CaMKII activation in amyloid precursor protein knock-in mice, promoting the degradation of amyloid-β plaques to improve cognition. At this point, we addressed whether SAK3 promotes the degradation of misfolded α-Syn and the aggregates in α-Syn preformed fibril (PFF)-injected mice. The mice were injected with α-Syn PFF in the dorsal striatum, and SAK3 (0.5 or 1.0 mg/kg) was administered orally for three months, either immediately or during the last month after injection. SAK3 significantly inhibited the accumulation of fibrilized phosphorylated-α-Syn in the substantia nigra. Accordingly, SAK3 significantly recovered mesencephalic dopamine neurons from cell death. Decreased α-Syn accumulation was closely associated with increased proteasome activity. Elevated CaMKII/Rpt-6 signaling possibly mediates the enhanced proteasome activity after SAK3 administration in the cortex and hippocampus. CaMKII/Rpt-6 activation also accounted for improved memory and cognition in α-Syn PFF-injected mice. These findings indicate that CaMKII/Rpt-6-dependent proteasomal activation by SAK3 recovers from α-Syn pathology in LBD.

Disrupted Place Cell Remapping and Impaired Grid Cells in a Knockin Model of Alzheimer's Disease

Patients with Alzheimer's disease (AD) suffer from spatial memory impairment and wandering behavior, but the brain circuit mechanisms causing such symptoms remain largely unclear. In healthy brains, spatially tuned hippocampal place cells and entorhinal grid cells exhibit distinct spike patterns in different environments, a circuit function called "remapping." We tested remapping in amyloid precursor protein knockin (APP-KI) mice with impaired spatial memory. CA1 neurons, including place cells, showed disrupted remapping, although their spatial tuning was only mildly diminished. Medial entorhinal cortex (MEC) neurons severely lost their spatial tuning and grid cells were almost absent. Fast gamma oscillatory coupling between the MEC and CA1 was also impaired. Mild disruption of MEC grid cells emerged in younger APP-KI mice, although the spatial memory and CA1 remapping of the animals remained intact. These results point to remapping impairment in the hippocampus, possibly linked to grid cell disruption, as circuit mechanisms underlying spatial memory impairment in AD.

Neuroprotection of SAK3 on scopolamine-induced cholinergic dysfunction in human neuroblastoma SH-SY5Y cells.

Alzheimer's disease (AD) is the most common type of senile dementia. A number of factors have been proposed regarding pathology of AD, such as presence of β-amyloid, and cholinergic and oxidative stress. SAK3 (ethyl 8'-methyl-2',5-dioxo-2-piperidin-1-ylspiro[cyclopentene-3,3'-imidazo[1,2-a]pyridine]-1-carboxylate) reduces β-amyloid deposition and improves cognitive functions in amyloid precursor protein knock-in mice. Scopolamine is used to induce in cell lines a cholinergic deficit that mimics AD. In order to evaluate the possible neuroprotective properties of SAK3, human neuroblastoma SH-SY5Y cells were pretreated with the compound (25-100nM) and further incubated in the presence of scopolamine (2mM). SAK3 inhibited scopolamine-induced cellular apoptosis (morphologically and by determination of pro- and anti-apoptotic factor levels), increase in ROS levels, decrease in choline acetyltransferase level, phosphorylation of NF-κB, activation of Akt, JNK and p38 intracellular signaling pathways, and elevation of proinflammatory cytokines IL-1β and IL-6, but not enhanced level of β-site amyloid precursor protein cleaving enzyme 1 (BACE1). These results indicate SAK3 possessed protective properties against cholinergic deficit associated with anti-oxidant, anti-apoptotic and anti-inflammatory activities, suggesting that SAK3 might be a potential agent in the development of AD drug therapeutics.

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.

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.