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A previous epidemiological study in Northern Europe showed that the A673T mutation (Icelandic mutation) in the amyloid precursor protein gene ( APP ) can protect against Alzheimer's disease (AD). While the effect of the A673T mutation on APP processing has been investigated primarily in vitro, its in vivo impact has not been evaluated. This is mainly because most existing AD mouse models carry the Swedish mutation. The Swedish and Icelandic mutations are both located near the β-cleavage site, and each mutation is presumed to have the opposite effect on β-cleavage. Therefore, in the AD mouse models with the Swedish mutation, its effects could compete with the effects of the Icelandic mutation. Here, we introduced the A673T mutation into App knock-in mice devoid of the Swedish mutation ( App G-F mice) to avoid potential deleterious effects of the Swedish mutation and generated App G-F-A673T mice. APP-A673T significantly downregulated β-cleavage and attenuated the production of Aβ and amyloid pathology in the brains of these animals. The Icelandic mutation also reduced neuroinflammation and neuritic alterations. Both sexes were studied. This is the first successful demonstration of the protective effect of the Icelandic mutation on amyloid pathology in vivo. Our findings indicate that specific inhibition of the APP-BACE1 interaction could be a promising therapeutic approach. Alternatively, the introduction of the disease-protective mutation such as APP-A673T using in vivo genome editing technology could be a novel treatment for individuals at high risk for AD, such as familial AD gene mutation carriers and APOE ε4 carriers., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 the authors.)

Systemic study of pathogenic pathways and interrelationships underlying genes associated with Alzheimer's disease (AD) facilitates the identification of new targets for effective treatments. Recently available large-scale multiomics datasets provide opportunities to use computational approaches for such studies. Here, we devised a novel di sease g ene id entification (digID) computational framework that consists of a semi-supervised deep learning classifier to predict AD-associated genes and a protein-protein interaction (PPI) network-based analysis to prioritize the importance of these predicted genes in AD. digID predicted 1,529 AD-associated genes and revealed potentially new AD molecular mechanisms and therapeutic targets including GNAI1 and GNB1, two G-protein subunits that regulate cell signaling, and KNG1, an upstream modulator of CDC42 small G-protein signaling and mediator of inflammation and candidate coregulator of amyloid precursor protein (APP). Analysis of mRNA expression validated their dysregulation in AD brains but further revealed the significant spatial patterns in different brain regions as well as among different subregions of the frontal cortex and hippocampi. Super-resolution STochastic Optical Reconstruction Microscopy (STORM) further demonstrated their subcellular colocalization and molecular interactions with APP in a transgenic mouse model of both sexes with AD-like mutations. These studies support the predictions made by digID while highlighting the importance of concurrent biological validation of computationally identified gene clusters as potential new AD therapeutic targets., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 the authors.)

Studies on genetic robustness recently revealed transcriptional adaptation (TA) as a mechanism by which an organism can compensate for genetic mutations through activation of homologous genes. Here, we discovered that genetic mutations, introducing a premature termination codon (PTC) in the amyloid precursor protein-b ( appb ) gene, activated TA of two other app family members, appa and amyloid precursor-like protein-2 ( aplp2 ), in zebrafish. The observed transcriptional response of appa and aplp2 required degradation of mutant mRNA and did not depend on Appb protein level. Furthermore, TA between amyloid precursor protein (APP) family members was observed in human neuronal progenitor cells; however, compensation was only present during early neuronal differentiation and could not be detected in a more differentiated neuronal stage or adult zebrafish brain. Using knockdown and chemical inhibition, we showed that nonsense-mediated mRNA decay (NMD) is involved in degradation of mutant mRNA and that Upf1 and Upf2, key proteins in the NMD pathway, regulate the endogenous transcript levels of appa , appb , aplp1 , and aplp2 In conclusion, our results suggest that the expression level of App family members is regulated by the NMD pathway and that mutations destabilizing app / APP mRNA can induce genetic compensation by other family members through TA in both zebrafish and human neuronal progenitors., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 Rahmati et al.)

Alzheimer's disease (AD) is the most common form of dementia and results in neurodegeneration and cognitive impairment. White matter (WM) is affected in AD and has implications for neural circuitry and cognitive function. The trajectory of these changes across age, however, is still not well understood, especially at earlier stages in life. To address this, we used the App NL-G-F/NL-G-F knock-in (APPKI) mouse model that harbors a single copy knock-in of the human amyloid precursor protein ( APP ) gene with three familial AD mutations. We performed in vivo diffusion tensor imaging (DTI) to study how the structural properties of the brain change across age in the context of AD. In late age APPKI mice, we observed reduced fractional anisotropy (FA), a proxy of WM integrity, in multiple brain regions, including the hippocampus, anterior commissure (AC), neocortex, and hypothalamus. At the cellular level, we observed greater numbers of oligodendrocytes in middle age (prior to observations in DTI) in both the AC, a major interhemispheric WM tract, and the hippocampus, which is involved in memory and heavily affected in AD, prior to observations in DTI. Proteomics analysis of the hippocampus also revealed altered expression of oligodendrocyte-related proteins with age and in APPKI mice. Together, these results help to improve our understanding of the development of AD pathology with age, and imply that middle age may be an important temporal window for potential therapeutic intervention., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 Morrissey et al.)

Genome wide association study (GWAS) uncovered Alzheimer's disease (AD) risk genes linked to the endo-lysosomal pathway. This pathway seems to be the gateway of protein aggregates, such as tau and α-synuclein, to the cytoplasm. Furthermore, we and others reported that the amyloid precursor protein (APP) C99 is predominantly processed by γ-secretase in the endo-lysosomal compartments, and β-amyloid (Aβ) peptides are enriched in the same subcellular loci. While the role(s) of APP/Aβ in the endo-lysosomal pathway has not been fully established, a recent study reported that Aβ, in particular Aβ42, inhibits cathepsin D (CTSD) activity. Here, we show using a cell-free in vitro assay that Aβ42 also blocks cathepsin B (CTSB) activity. Furthermore, we uncovered that the autocatalytic processing (i.e., conversion of single chain to heavy/light chains) of CTSB and CTSD is accelerated in APP-deficient cells compared with wild-type controls. Taken together, our findings further support the negative regulation of cathepsins by Aβ., (Copyright © 2024 Lundin et al.)

Mutations in amyloidβ precursor protein (APP) gene alter APP processing, either causing familial Alzheimer's disease (AD) or protecting against dementia. Under normal conditions, β-siteAPPcleaving enzyme 1 (BACE1) cleavesAPPat minor Asp1 site to generate C99 for amyloid β protein (Aβ) production, and predominantly at major Glu11 site to generate C89, resulting in truncated Aβ production. We discovered that A673V mutation, the only recessive AD-associated APP mutation, shifted the preferential β-cleavage site of BACE1 in APP from the Glu11 site to the Asp1 site both in male and female transgenic mice in vivo and in cell lines and primary neuronal culture derived from timed pregnant rats in vitro, resulting in a much higher C99 level and C99/C89 ratio. All other mutations at this site, including the protective Icelandic A673T mutation, reduced C99 generation, and decreased the C99/C89 ratio. Furthermore, A673V mutation caused stronger dimerization between mutant and wild-type APP, enhanced the lysosomal degradation of the mutant APP, and inhibited γ-secretase cleavage of the mutant C99 to generate Aβ, leading to recessively inherited AD. The results demonstrate that APP673 regulates APP processing and the BACE1 cleavage site selection is critical for amyloidogenesis inADpathogenesis, and implicate a pharmaceutical potential for targeting the APP673 site for AD drug development. [ABSTRACT FROM AUTHOR]

High levels of amyloid-β peptide (Aβ) have been related to Alzheimer's disease pathogenesis. However, in the healthy brain, low physiologically relevant concentrations of Aβ are necessary for long-term potentiation (LTP) and memory. Because cGMP plays a key role in these processes, here we investigated whether the cyclic nucleotide cGMP influences Aβ levels and function during LTP and memory. We demonstrate that the increase of cGMP levels by the phosphodiesterase-5 inhibitors sildenafil and vardenafil induces a parallel release of Aβ due to a change in the approximation of amyloid precursor protein (APP) and the β-site APP cleaving enzyme 1. Moreover, electrophysiological and behavioral studies performed on animals of both sexes showed that blocking Aβ function, by using anti-murine Aβ antibodies or APP knock-out mice, prevents the cGMP-dependent enhancement of LTP and memory. Our data suggest that cGMP positively regulates Aβ levels in the healthy brain which, in turn, boosts synaptic plasticity and memory. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) increases the risk for seizures and sleep disorders. We show here that germline deletion of β-site amyloid precursor protein (APP) cleaving enzyme-1 (BACE1) in neurons, but not in astrocytes, increased epileptiform activity. However, Bace1 deletion at adult ages did not alter the normal EEG waveform, indicating less concern for BACE1 inhibition in patients. Moreover, we showed that deletion of Bace1 in the adult was able to reverse epileptiform activity in 5xFAD mice. Intriguingly, treating 5xFAD and APP NL-G-F/NL-G-F (APP KI) mice of either sex with one BACE1 inhibitor Lanabecestat (AZD3293) dramatically increased epileptiform spiking, likely resulting from an off-target effect. We also monitored sleep-wake pathologies in these mice and showed increased wakefulness, decreased non-rapid eye movement sleep, and rapid eye movement sleep in both 5xFAD and APP KI mice; BACE1 inhibition in the adult 5xFAD mice reversed plaque load and sleep disturbances, but this was not seen in APP KI mice. Further studies with and without BACE1 inhibitor treatment showed different levels of plaque-associated microgliosis and activated microglial proteins in 5xFAD mice compared with APP KI mice. Together, BACE1 inhibition should be developed to avoid off-target effect for achieving benefits in reducing epileptic activity and sleep disturbance in Alzheimer's patients. SIGNIFICANCE STATEMENT BACE1 is widely recognized as a therapeutic target for treating Alzheimer's disease patients. However, BACE1 inhibitors failed in clinical trials because of inability to show cognitive improvement in patients. Here we show that BACE1 inhibition actually reduces sleep disturbances and epileptic seizures; both are seen in AD patients. We further showed that one of clinically tested BACE1 inhibitors does have off-target effects, and development of safer BACE1 inhibitors will be beneficial to AD patients. Results from this study will provide useful guidance for additional drug development., Competing Interests: The authors declare no competing financial interests., (Copyright © 2023 Yao et al.)

The perforant path provides the primary cortical excitatory input to the hippocampus. Because of its important role in information processing and coding, entorhinal projections to the dentate gyrus have been studied in considerable detail. Nevertheless, synaptic transmission between individual connected pairs of entorhinal stellate cells and dentate granule cells remains to be characterized. Here, we have used mouse organotypic entorhino-hippocampal tissue cultures of either sex, in which the entorhinal cortex (EC) to dentate granule cell (GC; EC-GC) projection is present, and EC-GC pairs can be studied using whole-cell patch-clamp recordings. By using cultures of wild-type mice, the properties of EC-GC synapses formed by afferents from the lateral and medial entorhinal cortex were compared, and differences in short-term plasticity were identified. As the perforant path is severely affected in Alzheimer's disease, we used tissue cultures of amyloid precursor protein (APP)-deficient mice to examine the role of APP at this synapse. APP deficiency altered excitatory neurotransmission at medial perforant path synapses, which was accompanied by transcriptomic and ultrastructural changes. Moreover, presynaptic but not postsynaptic APP deletion through the local injection of Cre-expressing adeno-associated viruses in conditional APP flox/flox tissue cultures increased the neurotransmission efficacy at perforant path synapses. In summary, these data suggest a physiological role for presynaptic APP at medial perforant path synapses that may be adversely affected under altered APP processing conditions. SIGNIFICANCE STATEMENT The hippocampus receives input from the entorhinal cortex via the perforant path. These projections to hippocampal dentate granule cells are of utmost importance for learning and memory formation. Although there is detailed knowledge about perforant path projections, the functional synaptic properties at the level of individual connected pairs of neurons are not well understood. In this study, we investigated the role of APP in mediating functional properties and transmission rules in individually connected neurons using paired whole-cell patch-clamp recordings and genetic tools in organotypic tissue cultures. Our results show that presynaptic APP expression limits excitatory neurotransmission via the perforant path, which could be compromised in pathologic conditions such as Alzheimer's disease., (Copyright © 2023 the authors.)

The key pathologic entities driving the destruction of synaptic function and integrity during the evolution of Alzheimer's disease (AD) remain elusive. Astrocytes are structurally and functionally integrated within synaptic and vascular circuitry and use calcium-based physiology to modulate basal synaptic transmission, vascular dynamics, and neurovascular coupling, which are central to AD pathogenesis. We used high-resolution multiphoton imaging to quantify all endogenous calcium signaling arising spontaneously throughout astrocytic somata, primary processes, fine processes, and capillary endfeet in the brain of awake APP/PS1 transgenic mice (11 male and 6 female mice). Endogenous calcium signaling within capillary endfeet, while surprisingly as active as astrocytic fine processes, was reduced ∼50% in the brain of awake APP/PS1 mice. Cortical astrocytes, in the presence of amyloid plaques in awake APP/PS1 mice, had a cell-wide increase in intracellular calcium associated with an increased frequency, amplitude, and duration of spontaneous calcium signaling. The cell-wide astrocytic calcium dysregulation was not directly related to distance to amyloid plaques. We could re-create the cell-wide intracellular calcium dysregulation in the absence of amyloid plaques following acute exposure to neuronally derived soluble Abeta from Tg2576 transgenic mice, in the living brain of male C57/Bl6 mice. Our findings highlight a role for astrocytic calcium pathophysiology in soluble-Abeta mediated neurodegenerative processes in AD. Additionally, therapeutic strategies aiming to protect astrocytic calcium physiology from soluble Abeta-mediated toxicity may need to pharmacologically enhance calcium signaling within the hypoactive capillary endfeet while reducing the hyperactivity of spontaneous calcium signaling throughout the rest of the astrocyte. SIGNIFICANCE STATEMENT Astrocytic calcium signaling is functionally involved in central pathologic processes of Alzheimer's disease. We quantified endogenous calcium signaling arising spontaneously in the brain of awake APP/PS1 mice, as general anesthesia suppressed astrocytic calcium signaling. Cell-wide astrocytic calcium dysregulation was not related to distance to amyloid plaques but mediated in part by neuronally derived soluble Abeta, supporting a role for astrocytes in soluble-Abeta mediated neurodegeneration. Spontaneous calcium signaling is largely compartmentalized and capillary endfeet were as active as fine processes but hypoactive in the presence of amyloid plaques, while the rest of the astrocyte became hyperactive. The cell-wide calcium pathophysiology in astrocytes may require a combination therapeutic strategy for hypoactive endfeet and astrocytic hyperactivity., (Copyright © 2023 the authors.)

The amyloid precursor protein (APP) is linked to the genetics and pathogenesis of Alzheimer's disease (AD). It is the parent protein of the β-amyloid (Aβ) peptide, the main constituent of the amyloid plaques found in an AD brain. The pathways from APP to Aβ are intensively studied, yet the normal functions of APP itself have generated less interest. We report here that glutamate stimulation of neuronal activity leads to a rapid increase in App gene expression. In mouse and human neurons, elevated APP protein changes the structure of the axon initial segment (AIS) where action potentials are initiated. The AIS is shortened in length and shifts away from the cell body. The GCaMP8f Ca 2+ reporter confirms the predicted decrease in neuronal activity. NMDA antagonists or knockdown of App block the glutamate effects. The actions of APP on the AIS are cell-autonomous; exogenous Aβ, either fibrillar or oligomeric, has no effect. In culture, APP Swe (a familial AD mutation) induces larger AIS changes than wild type APP. Ankyrin G and βIV-spectrin, scaffolding proteins of the AIS, both physically associate with APP, more so in AD brains. Finally, in humans with sporadic AD or in the R1.40 AD mouse model, both females and males, neurons have elevated levels of APP protein that invade the AIS. In vivo as in vitro , this increased APP is associated with a significant shortening of the AIS. The findings outline a new role for the APP and encourage a reconsideration of its relationship to AD. SIGNIFICANCE STATEMENT While the amyloid precursor protein (APP) has long been associated with Alzheimer's disease (AD), the normal functions of the full-length Type I membrane protein have been largely unexplored. We report here that the levels of APP protein increase with neuronal activity. In vivo and in vitro , modest amounts of excess APP alter the properties of the axon initial segment. The β-amyloid peptide derived from APP is without effect. Consistent with the observed changes in the axon initial segment which would be expected to decrease action potential firing, we show that APP expression depresses neuronal activity. In mouse AD models and human sporadic AD, APP physically associates with the scaffolding proteins of the axon initial segment, suggesting a relationship with AD dementia., (Copyright © 2023 the authors.)

Cognitive-behavioral testing in preclinical models of Alzheimer's disease has failed to capture deficits in goal-directed action control. Here, we provide the first comprehensive investigation of goal-directed action in a transgenic mouse model of Alzheimer's disease. Specifically, we tested outcome devaluation performance in male and female human amyloid precursor protein (hAPP)-J20 mice. Mice were first trained to press left and right levers for pellet and sucrose outcomes, respectively (counterbalanced), over 4 d. On test, mice were prefed one of the outcomes to satiety and given a choice between levers. Devaluation performance was intact for 36-week-old wild-types of both sexes, who responded more on the valued relative to the devalued lever (Valued > Devalued). By contrast, devaluation was impaired (Valued = Devalued) for J20 mice of both sexes, and for 52-week-old male mice regardless of genotype. After additional lever press training (i.e., 8-d lever pressing in total), devaluation was intact for all mice, demonstrating that the initial deficits were not a result of a nonspecific impairment in reward processing, depression, or locomotor activity in J20 or aging mice. Follow-up analyses revealed that microglial expression in the dorsal CA1 region of the hippocampus was associated with poorer outcome devaluation performance on initial, but not later tests. Together, these data demonstrate that goal-directed action is initially impaired in J20 mice of both sexes and in aging male mice regardless of genotype, and that this impairment is related to neuroinflammation in the dorsal CA1 hippocampal region., Competing Interests: The authors declare no competing financial interests., (Copyright © 2023 Dhungana et al.)

Alzheimer's Disease (AD) is characterized by the pathologic assembly of amyloid β (Aβ) peptide, which deposits into extracellular plaques, and tau, which accumulates in intraneuronal inclusions. To investigate the link between Aβ and tau pathologies, experimental models featuring both pathologies are needed. We developed a mouse model featuring both tau and Aβ pathologies by knocking the P290S mutation into murine Mapt and crossing these Mapt P290S knock-in (KI) mice with the App NL-G-F KI line. Mapt P290S KI mice developed a small number of tau inclusions, which increased with age. The amount of tau pathology was significantly larger in App NL-G-F xMapt P290S KI mice from 18 months of age onward. Tau pathology was higher in limbic areas, including hippocampus, amygdala, and piriform/entorhinal cortex. We also observed AT100-positive and Gallyas-Braak-silver-positive dystrophic neurites containing assembled filamentous tau, as visualized by in situ electron microscopy. Using a cell-based tau seeding assay, we showed that Sarkosyl-insoluble brain extracts from both 18-month-old Mapt P290S KI and App NL-G-F xMapt P290S KI mice were seed competent, with brain extracts from double-KI mice seeding significantly more than those from the Mapt P290S KI mice. Finally, we showed that App NL-G-F xMapt P290S KI mice had neurodegeneration in the piriform cortex from 18 months of age. We suggest that App NL-G-F xMapt P290S KI mice provide a good model for studying the interactions of aggregation-prone tau, Aβ, neuritic plaques, neurodegeneration, and aging., Competing Interests: The authors declare no competing financial interests., (Copyright © 2022 Huang et al.)

Kainate receptors (KARs) form a family of ionotropic glutamate receptors that regulate the activity of neuronal networks by both presynaptic and postsynaptic mechanisms. Their implication in pathologies is well documented for epilepsy. The higher prevalence of epileptic symptoms in Alzheimer's disease (AD) patients questions the role of KARs in AD. Here we investigated whether the synaptic expression and function of KARs was impaired in mouse models of AD. We addressed this question by immunostaining and electrophysiology at synapses between mossy fibers and CA3 pyramidal cells, in which KARs are abundant and play a prominent physiological role. We observed a decrease of the immunostaining for GluK2 in the stratum lucidum in CA3, and of the amplitude and decay time of synaptic currents mediated by GluK2-containing KARs in an amyloid mouse model (APP/PS1) of AD. Interestingly, a similar phenotype was observed in CA3 pyramidal cells in male and female mice with a genetic deletion of either presenilin or APP/APLP2 as well as in organotypic cultures treated with γ-secretase inhibitors. Finally, the GluK2 protein interacts with full-length and C-terminal fragments of APP. Overall, our data suggest that APP stabilizes KARs at synapses, possibly through a transsynaptic mechanism, and this interaction is under the control the γ-secretase proteolytic activity of presenilin. SIGNIFICANCE STATEMENT Synaptic impairment correlates strongly with cognitive deficits in Alzheimer's disease (AD). In this context, many studies have addressed the dysregulation of AMPA and NMDA ionotropic glutamate receptors. Kainate receptors (KARs), which form the third family of iGluRs, represent an underestimated actor in the regulation of neuronal circuits and have not yet been examined in the context of AD. Here we provide evidence that synaptic KARs are markedly impaired in a mouse model of AD. Additional experiments indicate that the γ-secretase activity of presenilin acting on the amyloid precursor protein controls synaptic expression of KAR. This study clearly indicates that KARs should be taken into consideration whenever addressing synaptic dysfunction and related cognitive deficits in the context of AD., (Copyright © 2022 the authors.)

In addition to its role in Alzheimer's disease, amyloid precursor protein (APP) has physiological roles in synapse development and function. APP induces presynaptic differentiation when presented to axons, but the mechanism is unknown. Here we show that APP binds neurexin to mediate this synaptogenic activity. APP specifically binds β not α neurexins modulated by splice site 4. Binding to neurexin heparan sulfate glycan and LNS protein domains is required for high-affinity interaction and for full-length APP to recruit axonal neurexin. The synaptogenic activity of APP is abolished by triple knockdown of neurexins in hippocampal neurons pooled from male and female rats. Based on these and previous results, our model is that a dendritic-axonal trans dimer of full-length APP binds to axonal neurexin-β to promote synaptic differentiation and function. Furthermore, soluble sAPPs also bind neurexin-β and inhibit its interaction with neuroligin-1, raising the possibility that disruption of neurexin function by altered levels of full-length APP and its cleavage products may contribute to early synaptic deficits in Alzheimer's disease. SIGNIFICANCE STATEMENT The prevailing model for the basis of Alzheimer's disease is the amyloid cascade triggered by altered cleavage of amyloid precursor protein (APP). APP also has physiological roles at the synapse, but the molecular basis for its synaptic functions is not well understood. Here, we show that APP binds the presynaptic organizing protein neurexin-β and that neurexin is essential for the synaptogenic activity of APP. Furthermore, soluble APP forms generated by APP cleavage also bind neurexin-β and can block interaction with transmembrane synaptogenic ligands of neurexin. These findings reveal a role for neurexin-APP interaction in synapse development and raise the possibility that disruptions of neurexin function may contribute to synaptic and cognitive deficits in the critical early stage of Alzheimer's disease., (Copyright © 2022 the authors.)

Individuals who have Down syndrome (DS) frequently develop early onset Alzheimer's disease (AD), a neurodegenerative condition caused by the buildup of aggregated amyloid-β (Aβ) and tau proteins in the brain. Aβ is produced by amyloid precursor protein ( APP ), a gene located on chromosome 21. People who have DS have three copies of chromosome 21 and thus also an additional copy of APP ; this genetic change drives the early development of AD in these individuals. Here we use a combination of next-generation mouse models of DS (Tc1, Dp3Tyb, Dp(10)2Yey and Dp(17)3Yey) and a knockin mouse model of Aβ accumulation ( App NL-F ) to determine how chromosome 21 genes, other than APP , modulate APP/Aβ in the brain when in three copies. Using both male and female mice, we demonstrate that three copies of other chromosome 21 genes are sufficient to partially ameliorate Aβ accumulation in the brain. We go on to identify a subregion of chromosome 21 that contains the gene(s) causing this decrease in Aβ accumulation and investigate the role of two lead candidate genes, Dyrk1a and Bace2 Thus, an additional copy of chromosome 21 genes, other than APP , can modulate APP/Aβ in the brain under physiological conditions. This work provides critical mechanistic insight into the development of disease and an explanation for the typically later age of onset of dementia in people who have AD in DS, compared with those who have familial AD caused by triplication of APP SIGNIFICANCE STATEMENT Trisomy of chromosome 21 is a commonly occurring genetic risk factor for early-onset Alzheimer's disease (AD), which has been previously attributed to people with Down syndrome having three copies of the amyloid precursor protein ( APP ) gene, which is encoded on chromosome 21. However, we have shown that an extra copy of other chromosome 21 genes modifies AD-like phenotypes independently of APP copy number (Wiseman et al., 2018; Tosh et al., 2021). Here, we use a mapping approach to narrow down the genetic cause of the modulation of pathology, demonstrating that gene(s) on chromosome 21 decrease Aβ accumulation in the brain, independently of alterations to full-length APP or C-terminal fragment abundance and that just 38 genes are sufficient to cause this., (Copyright © 2022 the authors.)

Alzheimer's disease (AD) is characterized by progressive cognitive impairment associated with synaptic dysfunction and dendritic spine loss and the pathologic hallmarks of β-amyloid (Aβ) plaques and hyperphosphorylated tau tangles. 14-3-3 proteins are a highly conserved family of proteins whose functions include regulation of protein folding, neuronal architecture, and synaptic function. Additionally, 14-3-3s interact with both Aβ and tau, and reduced levels of 14-3-3s have been shown in the brains of AD patients and in AD mouse models. Here, we examine the neuroprotective potential of the 14-3-3θ isoform in AD models. We demonstrate that 14-3-3θ overexpression is protective and 14-3-3θ inhibition is detrimental against oligomeric Aβ-induced neuronal death in primary cortical cultures. Overexpression of 14-3-3θ using an adeno-associated viral (AAV) vector failed to improve performance on behavioral tests, improve Aβ pathology, or affect synaptic density in the J20 AD mouse model. Similarly, crossing a second AD mouse model, the App NL-G-F knock-in (APP KI) mouse, with 14-3-3θ transgenic mice failed to rescue behavioral deficits, reduce Aβ pathology, or impact synaptic density in the APP KI mouse model. 14-3-3θ is likely partially insolubilized in the APP models, as demonstrated by proteinase K digestion. These findings do not support increasing 14-3-3θ expression as a therapeutic approach for AD., (Copyright © 2022 Gannon et al.)

Alzheimer's disease (AD) is a debilitating dementia characterized by progressive memory loss and aggregation of amyloid-β (Aβ) protein into amyloid plaques in patient brains. Mutations in presenilin (PS) lead to abnormal generation of Aβ, which is the major cause of familial AD (FAD), and apolipoprotein E4 (ApoE4) is the major genetic risk factor for sporadic AD (SAD) onset. However, whether dysfunction of PS is involved in the pathogenesis of SAD is largely unknown. We found that ApoE secretion was completely abolished in PS-deficient cells and markedly decreased by inhibition of γ-secretase activity. Blockade of γ-secretase activity by a γ-secretase inhibitor, DAPT, decreased ApoE secretion, suggesting an important role of γ-secretase activity in ApoE secretion. Reduced ApoE secretion is also observed in nicastrin-deficient cells with reduced γ-secretase activity. PS deficiency enhanced nuclear translocation of ApoE and binding of ApoE to importin α4, a nuclear transport receptor. Moreover, the expression of PS mutants in PS-deficient cells suppressed the restoration effects on ApoE secretion compared with the expression of wild-type PS. Plasma ApoE levels were lower in FAD patients carrying PS1 mutations compared with normal control subjects. Our findings suggest a novel role of PS contributing to the pathogenesis of SAD by regulating ApoE secretion. SIGNIFICANCE STATEMENT Familial AD (FAD) typically results from mutations in the genes encoding amyloid precursor protein, presenilin 1 (PS1), or PS2. Many PS mutants have been found to exert impaired γ-secretase activity and increased amyloid-β 42 (Aβ42)/Aβ40 ratio, which induce early amyloid deposition and FAD. On the other hand, apolipoprotein E4 (ApoE4) is the major genetic risk factor for sporadic AD (SAD) and contributes to AD pathogenesis because it has reduced Aβ clearance capability compared with ApoE3 and ApoE2. FAD and SAD have long been considered to be caused by these two independent mechanisms; however, for the first time, we demonstrated that PS is essential for ApoE secretion and PS mutants affected ApoE secretion in vitro and in human samples, suggesting a novel mechanism by which PS is also involved in SAD pathogenesis., (Copyright © 2022 the authors.)

The central noradrenergic (NA) system is critical for the maintenance of attention, behavioral flexibility, spatial navigation, and learning and memory, those cognitive functions lost first in early Alzheimer's disease (AD). In fact, the locus coeruleus (LC), the sole source of norepinephrine (NE) for >90% of the brain, is the first site of pathologic tau accumulation in human AD with axon loss throughout forebrain, including hippocampus. The dentate gyrus is heavily innervated by LC-NA axons, where released NE acts on β-adrenergic receptors (ARs) at excitatory synapses from entorhinal cortex to facilitate long-term synaptic plasticity and memory formation. These synapses experience dysfunction in early AD before cognitive impairment. In the TgF344-AD rat model of AD, degeneration of LC-NA axons in hippocampus recapitulates human AD, providing a preclinical model to investigate synaptic and behavioral consequences. Using immunohistochemistry, Western blot analysis, and brain slice electrophysiology in 6- to 9-month-old wild-type and TgF344-AD rats, we discovered that the loss of LC-NA axons coincides with the heightened β-AR function at medial perforant path-dentate granule cell synapses that is responsible for the increase in LTP magnitude at these synapses. Furthermore, novel object recognition is facilitated in TgF344-AD rats that requires β-ARs, and pharmacological blockade of β-ARs unmasks a deficit in extinction learning only in TgF344-AD rats, indicating a greater reliance on β-ARs in both behaviors. Thus, a compensatory increase in β-AR function during prodromal AD in TgF344-AD rats heightens synaptic plasticity and preserves some forms of learning and memory. SIGNIFICANCE STATEMENT The locus coeruleus (LC), a brain region located in the brainstem which is responsible for attention and arousal, is damaged first by Alzheimer's disease (AD) pathology. The LC sends axons to hippocampus where released norepinephrine (NE) modulates synaptic function required for learning and memory. How degeneration of LC axons and loss of NE in hippocampus in early AD impacts synaptic function and learning and memory is not well understood despite the importance of LC in cognitive function. We used a transgenic AD rat model with LC axon degeneration mimicking human AD and found that heightened function of β-adrenergic receptors in the dentate gyrus increased synaptic plasticity and preserved learning and memory in early stages of the disease., (Copyright © 2021 the authors.)

Alzheimer's disease (AD) is an incurable neurodegenerative disorder and a major cause of dementia. Some of the hallmarks of AD include presence of amyloid plaques in brain parenchyma, calcium dysregulation within individual neurons, and neuroinflammation. A promising therapeutic would reverse or stymie these pathophysiologies in an animal model of AD. We tested the effect of NB-02, previously known as DA-9803, a novel multimodal therapeutic, on amyloid deposition, neuronal calcium regulation and neuroinflammation in 8- to 10-month-old APP/PS1 mice, an animal model of AD. In vivo multiphoton microscopy revealed that two-month-long administration of NB-02 halted amyloid plaque deposition and cleared amyloid in the cortex. Postmortem analysis verified NB-02-dependent decrease in plaque deposition in the cortex as well as hippocampus. Furthermore, drug treatment reversed neuronal calcium elevations, thus restoring neuronal function. Finally, NB-02 restored spine density and transformed the morphology of astrocytes as well as microglia to a more phagocytic state, affecting neuroinflammation. NB-02 was effective at reversing AD neuropathophysiology in an animal model. Therefore, in addition to serving as a promising preventative agent, NB-02 holds potential as a treatment for AD in the clinic., (Copyright © 2021 Lee et al.)

Diverse gene products contribute to the pathogenesis of Alzheimer's disease (AD). Experimental models have helped elucidate their mechanisms and impact on brain functions. Human amyloid precursor protein (hAPP) transgenic mice from line J20 (hAPP-J20 mice) are widely used to simulate key aspects of AD. However, they also carry an insertional mutation in noncoding sequence of one Zbtb20 allele, a gene involved in neural development. We demonstrate that heterozygous hAPP-J20 mice have reduced Zbtb20 expression in some AD-relevant brain regions, but not others, and that Zbtb20 levels are higher in hAPP-J20 mice than heterozygous Zbtb20 knock-out ( Zbtb20 +/- ) mice. Whereas hAPP-J20 mice have premature mortality, severe deficits in learning and memory, other behavioral alterations, and prominent nonconvulsive epileptiform activity, Zbtb20 +/- mice do not. Thus, the insertional mutation in hAPP-J20 mice does not ablate the affected Zbtb20 allele and is unlikely to account for the AD-like phenotype of this model., (Copyright © 2021 Gulbranson et al.)

Vascular dysfunction is a universal feature of aging and decreased cerebral blood flow has been identified as an early event in the pathogenesis of Alzheimer's disease (AD). Cerebrovascular dysfunction in AD includes deficits in neurovascular coupling (NVC), a mechanism that ensures rapid delivery of energy substrates to active neurons through the blood supply. The mechanisms underlying NVC impairment in AD, however, are not well understood. We have previously shown that mechanistic/mammalian target of rapamycin (mTOR) drives cerebrovascular dysfunction in models of AD by reducing the activity of endothelial nitric oxide synthase (eNOS), and that attenuation of mTOR activity with rapamycin is sufficient to restore eNOS-dependent cerebrovascular function. Here we show mTOR drives NVC impairments in an AD model through the inhibition of neuronal NOS (nNOS)- and non-NOS-dependent components of NVC, and that mTOR attenuation with rapamycin is sufficient to restore NVC and even enhance it above WT responses. Restoration of NVC and concomitant reduction of cortical amyloid-β levels effectively treated memory deficits in 12-month-old hAPP(J20) mice. These data indicate that mTOR is a critical driver of NVC dysfunction and underlies cognitive impairment in an AD model. Together with our previous findings, the present studies suggest that mTOR promotes cerebrovascular dysfunction in AD, which is associated with early disruption of nNOS activation, through its broad negative impact on nNOS as well as on non-NOS components of NVC. Our studies highlight the potential of mTOR attenuation as an efficacious treatment for AD and potentially other neurologic diseases of aging. SIGNIFICANCE STATEMENT Failure of the blood flow response to neuronal activation [neurovascular coupling (NVC)] in a model of AD precedes the onset of AD-like cognitive symptoms and is driven, to a large extent, by mammalian/mechanistic target of rapamycin (mTOR)-dependent inhibition of nitric oxide synthase activity. Our studies show that mTOR also drives AD-like failure of non-nitric oxide (NO)-mediated components of NVC. Thus, mTOR attenuation may serve to treat AD, where we find that neuronal NO synthase is profoundly reduced early in disease progression, and potentially other neurologic diseases of aging with cerebrovascular dysfunction as part of their etiology., (Copyright © 2021 the authors.)

Neuronal and network-level hyperexcitability is commonly associated with increased levels of amyloid-β (Aβ) and contribute to cognitive deficits associated with Alzheimer's disease (AD). However, the mechanistic complexity underlying the selective loss of basal forebrain cholinergic neurons (BFCNs), a well-recognized characteristic of AD, remains poorly understood. In this study, we tested the hypothesis that the oligomeric form of amyloid-β (oAβ 42 ), interacting with α7-containing nicotinic acetylcholine receptor (nAChR) subtypes, leads to subnucleus-specific alterations in BFCN excitability and impaired cognition. We used single-channel electrophysiology to show that oAβ 42 activates both homomeric α7- and heteromeric α7β2-nAChR subtypes while preferentially enhancing α7β2-nAChR open-dwell times. Organotypic slice cultures were prepared from male and female ChAT-EGFP mice, and current-clamp recordings obtained from BFCNs chronically exposed to pathophysiologically relevant level of oAβ 42 showed enhanced neuronal intrinsic excitability and action potential firing rates. These resulted from a reduction in action potential afterhyperpolarization and alterations in the maximal rates of voltage change during spike depolarization and repolarization. These effects were observed in BFCNs from the medial septum diagonal band and horizontal diagonal band, but not the nucleus basalis. Last, aged male and female APP/PS1 transgenic mice, genetically null for the β2 nAChR subunit gene, showed improved spatial reference memory compared with APP/PS1 aged-matched littermates. Combined, these data provide a molecular mechanism supporting a role for α7β2-nAChR in mediating the effects of oAβ 42 on excitability of specific populations of cholinergic neurons and provide a framework for understanding the role of α7β2-nAChR in oAβ 42 -induced cognitive decline., Competing Interests: The authors declare no competing financial interests., (Copyright © 2021 the authors.)

The amyloid-β (Aβ) peptide, a key pathogenic factor in Alzheimer's disease, attenuates the increase in cerebral blood flow (CBF) evoked by neural activity (functional hyperemia), a vital homeostatic response in which NMDA receptors (NMDARs) play a role through nitric oxide, and the CBF increase produced by endothelial factors. Tissue plasminogen activator (tPA), which is reduced in Alzheimer's disease and in mouse models of Aβ accumulation, is required for the full expression of the NMDAR-dependent component of functional hyperemia. Therefore, we investigated whether tPA is involved in the neurovascular dysfunction of Aβ. tPA activity was reduced, and the tPA inhibitor plasminogen inhibitor-1 (PAI-1) was increased in male mice expressing the Swedish mutation of the amyloid precursor protein (tg2576). Counteracting the tPA reduction with exogenous tPA or with pharmacological inhibition or genetic deletion of PAI-1 completely reversed the attenuation of the CBF increase evoked by whisker stimulation but did not ameliorate the response to the endothelium-dependent vasodilator acetylcholine. The tPA deficit attenuated functional hyperemia by suppressing NMDAR-dependent nitric oxide production during neural activity. Pharmacological inhibition of PAI-1 increased tPA activity, prevented neurovascular uncoupling, and ameliorated cognition in 11- to 12-month-old tg2576 mice, effects associated with a reduction of cerebral amyloid angiopathy but not amyloid plaques. The data unveil a selective role of the tPA in the suppression of functional hyperemia induced by Aβ and in the mechanisms of cerebral amyloid angiopathy, and support the possibility that modulation of the PAI-1-tPA pathway may be beneficial in diseases associated with amyloid accumulation. SIGNIFICANCE STATEMENT Amyloid-β (Aβ) peptides have profound neurovascular effects that may contribute to cognitive impairment in Alzheimer's disease. We found that Aβ attenuates the increases in blood flow evoked by neural activation through a reduction in tissue plasminogen activator (tPA) caused by upregulation of its endogenous inhibitor plasminogen inhibitor-1 (PAI-1). tPA deficiency prevents NMDA receptors from triggering nitric oxide production, thereby attenuating the flow increase evoked by neural activity. PAI-1 inhibition restores tPA activity, rescues neurovascular coupling, reduces amyloid deposition around blood vessels, and improves cognition in a mouse model of Aβ accumulation. The findings demonstrate a previously unappreciated role of tPA in Aβ-related neurovascular dysfunction and in vascular amyloid deposition. Restoration of tPA activity could be of therapeutic value in diseases associated with amyloid accumulation., (Copyright © 2020 the authors.)

The amyloid precursor protein (APP) has been extensively studied as the precursor of the β-amyloid (Aβ) peptide, the major component of the senile plaques found in the brain of Alzheimer's disease (AD) patients. However, the function of APP per se in neuronal physiology remains to be fully elucidated. APP is expressed at high levels in the brain. It resembles a cell adhesion molecule or a membrane receptor, suggesting that its function relies on cell-cell interaction and/or activation of intracellular signaling pathways. In this respect, the APP intracellular domain (AICD) was reported to act as a transcriptional regulator. Here, we used a transcriptome-based approach to identify the genes transcriptionally regulated by APP in the rodent embryonic cortex and on maturation of primary cortical neurons. Surprisingly, the overall transcriptional changes were subtle, but a more detailed analysis pointed to genes clustered in neuronal-activity dependent pathways. In particular, we observed a decreased transcription of neuronal PAS domain protein 4 (NPAS4) in APP-/- neurons. NPAS4 is an inducible transcription factor (ITF) regulated by neuronal depolarization. The downregulation of NPAS4 co-occurs with an increased production of the inhibitory neurotransmitter GABA and a reduced expression of the GABA A receptors α1. CRISPR-Cas-mediated silencing of NPAS4 in neurons led to similar observations. Patch-clamp investigation did not reveal any functional decrease of GABA A receptors activity, but long-term potentiation (LTP) measurement supported an increased GABA component in synaptic transmission of APP-/- mice. Together, NPAS4 appears to be a downstream target involved in APP-dependent regulation of inhibitory synaptic transmission., (Copyright © 2020 Opsomer et al.)

Alzheimer's disease (AD) patients often suffer from sleep disturbances. Alterations in sleep, especially rapid eye movement sleep (REMS), can precede the onset of dementia. To accurately characterize the sleep impairments accompanying AD and their underlying mechanisms using animal models, it is crucial to use models in which brain areas are affected in a manner similar to that observed in the actual patients. Here, we focused on App NL-G-F mice, in which expression levels and patterns of mutated amyloid precursor protein (APP) follow the endogenous patterns. We characterized the sleep architecture of male App NL-G-F homozygous and heterozygous mice at two ages (six and 12 months). At six months, homozygous mice exhibited reduced REMS, which was further reduced at 12 months together with a slight reduction in non-REMS (NREMS). By contrast, heterozygous mice exhibited an overall normal sleep architecture. Homozygous mice also exhibited decreased electroencephalogram γ to δ power ratio during REMS from six months, resembling the electroencephalogram slowing phenomenon observed in preclinical or early stages of AD. In addition, homozygous mice showed learning and memory impairments in the trace fear conditioning (FC) at both ages, and task performance strongly correlated with REMS amount at 12 months. Finally, histologic analyses revealed that amyloid-β accumulation in the pontine tegmental area and ventral medulla followed a course similar to that of the REMS reduction. These findings support the notion that changes in REMS are an early marker of AD and provide a starting point to address the mechanism of sleep deficits in AD and the effects on cognition., (Copyright © 2020 Maezono et al.)

TREM2 is an Alzheimer's disease (AD) risk gene expressed in microglia. To study the role of Trem2 in a mouse model of β-amyloidosis, we compared PS2APP transgenic mice versus PS2APP mice lacking Trem2 (PS2APP;Trem2 ko ) at ages ranging from 4 to 22 months. Microgliosis was impaired in PS2APP;Trem2 ko mice, with Trem2 -deficient microglia showing compromised expression of proliferation/Wnt-related genes and marked accumulation of ApoE. Plaque abundance was elevated in PS2APP;Trem2 ko females at 6-7 months; but by 12 or 19-22 months of age, it was notably diminished in female and male PS2APP;Trem2 ko mice, respectively. Across all ages, plaque morphology was more diffuse in PS2APP;Trem2 ko brains, and the Aβ42:Aβ40 ratio was elevated. The amount of soluble, fibrillar Aβ oligomers also increased in PS2APP;Trem2 ko hippocampi. Associated with these changes, axonal dystrophy was exacerbated from 6 to 7 months onward in PS2APP;Trem2 ko mice, notwithstanding the reduced plaque load at later ages. PS2APP;Trem2 ko mice also exhibited more dendritic spine loss around plaque and more neurofilament light chain in CSF. Thus, aggravated neuritic dystrophy is a more consistent outcome of Trem2 deficiency than amyloid plaque load, suggesting that the microglial packing of Aβ into dense plaque is an important neuroprotective activity. SIGNIFICANCE STATEMENT Genetic studies indicate that TREM2 gene mutations confer increased Alzheimer's disease (AD) risk. We studied the effects of Trem2 deletion in the PS2APP mouse AD model, in which overproduction of Aβ peptide leads to amyloid plaque formation and associated neuritic dystrophy. Interestingly, neuritic dystrophies were intensified in the brains of Trem2 -deficient mice, despite these mice displaying reduced plaque accumulation at later ages (12-22 months). Microglial clustering around plaques was impaired, plaques were more diffuse, and the Aβ42:Aβ40 ratio and amount of soluble, fibrillar Aβ oligomers were elevated in Trem2 -deficient brains. These results suggest that the Trem2-dependent compaction of Aβ into dense plaques is a protective microglial activity, limiting the exposure of neurons to toxic Aβ species., (Copyright © 2020 Meilandt et al.)

Impaired mitochondrial function and aberrant neuronal network activity are believed to be early events in the pathogenesis of Alzheimer's disease (AD), but how mitochondrial alterations contribute to aberrant activity in neuronal circuits is unknown. In this study, we examined the function of mitochondrial protein deacetylase sirtuin 3 (SIRT3) in the pathogenesis of AD. Compared with AppPs1 mice, Sirt3-haploinsufficient AppPs1 mice (Sirt3 +/- AppPs1) exhibit early epileptiform EEG activity and seizure. Both male and female Sirt3 +/- AppPs1 mice were observed to die prematurely before 5 months of age. When comparing male mice among different genotypes, Sirt3 haploinsufficiency renders GABAergic interneurons in the cerebral cortex vulnerable to degeneration and associated neuronal network hyperexcitability. Feeding Sirt3 +/- AppPs1 AD mice with a ketone ester-rich diet increases SIRT3 expression and prevents seizure-related death and the degeneration of GABAergic neurons, indicating that the aggravated GABAergic neuron loss and neuronal network hyperexcitability in Sirt3 +/- AppPs1 mice are caused by SIRT3 reduction and can be rescued by increase of SIRT3 expression. Consistent with a protective role in AD, SIRT3 levels are reduced in association with cerebral cortical Aβ pathology in AD patients. In summary, SIRT3 preserves GABAergic interneurons and protects cerebral circuits against hyperexcitability, and this neuroprotective mechanism can be bolstered by dietary ketone esters. SIGNIFICANCE STATEMENT GABAergic neurons provide the main inhibitory control of neuronal activity in the brain. By preserving mitochondrial function, SIRT3 protects parvalbumin and calretinin interneurons against Aβ-associated dysfunction and degeneration in AppPs1 Alzheimer's disease mice, thus restraining neuronal network hyperactivity. The neuronal network dysfunction that occurs in Alzheimer's disease can be partially reversed by physiological, dietary, and pharmacological interventions to increase SIRT3 expression and enhance the functionality of GABAergic interneurons., (Copyright © 2020 the authors.)

Type 2 diabetes mellitus (T2DM) is a risk factor for the development of late-onset Alzheimer's disease (AD). However, the mechanism underlying the development of late-onset AD is largely unknown. Here we show that levels of the endothelial-enriched protein caveolin-1 (Cav-1) are reduced in the brains of T2DM patients compared with healthy aging, and inversely correlated with levels of β-amyloid (Aβ). Depletion of Cav-1 is recapitulated in the brains of db/db ( Lepr db ) diabetic mice and corresponds with recognition memory deficits as well as the upregulation of amyloid precursor protein (APP), BACE-1, a trending increase in β-amyloid Aβ 42/40 ratio and hyperphosphorylated tau (p-tau) species. Importantly, we show that restoration of Cav-1 levels in the brains of male db/db mice using adenovirus overexpressing Cav-1 (AAV-Cav-1) rescues learning and memory deficits and reduces pathology (i.e., APP, BACE-1 and p-tau levels). Knocking down Cav-1 using shRNA in HEK cells expressing the familial AD-linked APPswe mutant variant upregulates APP, APP carboxyl terminal fragments, and Aβ levels. In turn, rescue of Cav-1 levels restores APP metabolism. Together, these results suggest that Cav-1 regulates APP metabolism, and that depletion of Cav-1 in T2DM promotes the amyloidogenic processing of APP and hyperphosphorylation of tau. This may suggest that depletion of Cav-1 in T2DM underlies, at least in part, the development of AD and imply that restoration of Cav-1 may be a therapeutic target for diabetic-associated sporadic AD. SIGNIFICANCE STATEMENT More than 95% of the Alzheimer's patients have the sporadic late-onset form (LOAD). The cause for late-onset Alzheimer's disease is unknown. Patients with Type 2 diabetes mellitus have considerably higher incidence of cognitive decline and AD compared with the general population, suggesting a common mechanism. Here we show that the expression of caveolin-1 (Cav-1) is reduced in the brain in Type 2 diabetes mellitus. In turn, reduced Cav-1 levels induce AD-associated neuropathology and learning and memory deficits. Restoration of Cav-1 levels rescues these deficits. This study unravels signals underlying LOAD and suggests that restoration of Cav-1 may be an effective therapeutic target., (Copyright © 2019 the authors.)

Lysosomal failure underlies pathogenesis of numerous congenital neurodegenerative disorders and is an early and progressive feature of Alzheimer's disease (AD) pathogenesis. Here, we report that lysosomal dysfunction in Down ayndrome (trisomy 21), a neurodevelopmental disorder and form of early onset AD, requires the extra gene copy of amyloid precursor protein (APP) and is specifically mediated by the β cleaved carboxy terminal fragment of APP (APP-βCTF, C99). In primary fibroblasts from individuals with DS, lysosomal degradation of autophagic and endocytic substrates is selectively impaired, causing them to accumulate in enlarged autolysosomes/lysosomes. Direct measurements of lysosomal pH uncovered a significant elevation (0.6 units) as a basis for slowed LC3 turnover and the inactivation of cathepsin D and other lysosomal hydrolases known to be unstable or less active when lysosomal pH is persistently elevated. Normalizing lysosome pH by delivering acidic nanoparticles to lysosomes ameliorated lysosomal deficits, whereas RNA sequencing analysis excluded a transcriptional contribution to hydrolase declines. Cortical neurons cultured from the Ts2 mouse model of DS exhibited lysosomal deficits similar to those in DS cells. Lowering APP expression with siRNA or BACE1 inhibition reversed cathepsin deficits in both fibroblasts and neurons. Deleting one Bace1 allele from adult Ts2 mice had similar rescue effects in vivo The modest elevation of endogenous APP-βCTF needed to disrupt lysosomal function in DS is relevant to sporadic AD where APP-βCTF, but not APP, is also elevated. Our results extend evidence that impaired lysosomal acidification drives progressive lysosomal failure in multiple forms of AD. SIGNIFICANCE STATEMENT Down syndrome (trisomy 21) (DS) is a neurodevelopmental disorder invariably leading to early-onset Alzheimer's disease (AD). We showed in cells from DS individuals and neurons of DS models that one extra copy of a normal amyloid precursor protein ( APP ) gene impairs lysosomal acidification, thereby depressing lysosomal hydrolytic activities and turnover of autophagic and endocytic substrates, processes vital to neuronal survival. These deficits, which were reversible by correcting lysosomal pH, are mediated by elevated levels of endogenous β-cleaved carboxy-terminal fragment of APP (APP-βCTF). Notably, similar endosomal-lysosomal pathobiology emerges early in sporadic AD, where neuronal APP-βCTF is also elevated, underscoring its importance as a therapeutic target and underscoring the functional and pathogenic interrelationships between the endosomal-lysosomal pathway and genes causing AD., (Copyright © 2019 the authors.)

Alzheimer's disease (AD) begins several decades before the onset of clinical symptoms, at a time when women may still undergo reproductive cycling. Whether ovarian functions alter substrates of AD pathogenesis is unknown. Here we show that ovarian cycle stages significantly modulate AD-related alterations in neural network patterns, cognitive impairments, and pathogenic protein production in the hAPP-J20 mouse model of AD. Female hAPP mice spent more time in estrogen-dominant cycle stages and these ovarian stages worsened AD-related network dysfunction and cognitive impairments. In contrast, progesterone-dominant stages and gonadectomy attenuated these AD-related deficits. Further studies revealed a direct role for estradiol in stimulating neural network excitability and susceptibility to seizures in hAPP mice and increasing amyloid beta levels. Understanding dynamic effects of the ovarian cycle on the female nervous system in disease, including AD, is of critical importance and may differ from effects on a healthy brain. The pattern of ovarian cycle effects on disease-related networks, cognition, and pathogenic protein expression may be relevant to young women at risk for AD.

Alzheimer's disease (AD) is a neurodegenerative disorder that has become a compelling global public health concern. Besides pathological hallmarks such as extracellular amyloid plaques, intracellular neurofibrillary tangles (NFTs), and loss of neurons and synapses, clinical reports have shown that epileptiform activity, even seizures, can occur early in the disease. Aberrant synaptic and network activities as well as epileptiform discharges have also been observed in various mouse models of AD. The new App NL-F mouse model is generated by a gene knock-in approach and there are limited studies on basic synaptic properties in App NL-F mice. Therefore, we applied quantitative methods to analyze spontaneous excitatory and inhibitory synaptic events in parietal cortex layer 2/3 pyramidal cells. First, by an objective amplitude distribution analysis, we found decreased amplitudes of spontaneous IPSCs (sIPSCs) in aged App NL-F mice caused by a reduction in the amplitudes of the large sIPSCs with fast rates of rise, consistent with deficits in the function of parvalbumin-expressing interneurons (PV INs). Second, we calculated the burstiness and memory in a series of successive synaptic events. Lastly, by using a novel approach to determine the excitation-to-inhibition (E/I) ratio, we found no changes in the App NL-F mice, indicating that homeostatic mechanisms may have maintained the overall balance of excitation and inhibition in spite of a mildly impaired PV IN function.

HIV-associated neurocognitive disorders (HANDs) share common symptoms with Alzheimer's disease (AD), which is characterized by amyloid-β (Aβ) plaques. Plaques are formed by aggregation of Aβ oligomers, which may be the toxic species in AD pathogenesis, and oligomers are generated by cleavage of amyloid precursor protein (APP) by β-site amyloid precursor protein cleaving enzyme 1 (BACE1). BACE1 inhibitors reverse neuronal loss and cognitive decline in animal models of AD. Although studies have also found evidence of altered APP processing in HIV + patients, it is unknown whether increased BACE1 expression or Aβ oligomer production is a common neuropathological feature of HAND. Moreover, it is unknown whether BACE1 or APP is involved in the excitotoxic, NMDAR-dependent component of HIV-associated neurotoxicity in vitro Herein, we hypothesize that HIV-associated neurotoxicity is mediated by NMDAR-dependent elevation of BACE1 and subsequent altered processing of APP. Supporting this, we observed elevated levels of BACE1 and Aβ oligomers in CNS of male and female HIV + patients. In a model of HIV-associated neurotoxicity in which rat neurons are treated with supernatants from HIV-infected human monocyte-derived macrophages, we observed NMDAR-dependent elevation of BACE1 protein. NMDA treatment also increased BACE1 and both pharmacological BACE1 inhibition and genetic loss of APP were partially neuroprotective. Moreover, in APP knock-out (APP -/- ) mouse neurons, NMDA-induced toxicity was BACE1 independent, indicating that cytotoxicity of BACE1 is dependent upon APP cleavage. Our findings suggest that increased BACE1 and the resultant Aβ oligomer production may contribute to HIV-associated neuropathogenesis and inhibition of BACE1 could have therapeutic potential in HANDs. SIGNIFICANCE STATEMENT HIV-associated neurocognitive disorders (HANDs) represent a range of cognitive impairments affecting ∼50% of HIV + individuals. The specific causes of HAND are unknown, but evidence suggests that HIV-infected macrophage infiltration into the brain may cause neuronal damage. Herein, we show that neurons treated with conditioned media from HIV-infected macrophages have increased expression of β-site amyloid precursor protein cleaving enzyme 1 (BACE1), a protein implicated in Alzheimer's disease pathogenesis. Moreover, inhibition of BACE1 prevented neuronal loss after conditioned media exposure, but had no effect on HIV-associated neurotoxicity in neurons lacking its cleavage target amyloid precursor protein. We also observed increased BACE1 expression in HIV + patient brain tissue, confirming the potential relevance of BACE1 as a therapeutic target in HANDs., (Copyright © 2018 the authors 0270-6474/18/384288-13$15.00/0.)

γ-Secretase is an intramembrane cleaving protease that is responsible for the generation of amyloid-β peptides, which are linked to the pathogenesis of Alzheimer disease. Recently, γ-secretase modulators (GSMs) have been shown to specifically decrease production of the aggregation-prone and toxic longer Aβ species, and concomitantly increase the levels of shorter Aβ. We previously found that phenylimidazole-type GSMs bind to presenilin 1 (PS1), the catalytic subunit of the γ-secretase, and allosterically modulate γ-secretase activity. However, the precise conformational alterations in PS1 remained unclear. Here we mapped the amino acid residues in PS1 that is crucial for the binding and pharmacological actions of E2012, a phenylimidazole-type GSM, using photoaffinity labeling and the substituted cysteine accessibility method. We also demonstrated that a piston-like vertical motion of transmembrane domain (TMD) 1 occurs during modulation of Aβ production. Taking these results together, we propose a model for the molecular mechanism of phenylimidazole-type GSMs, in which the trimming activity of γ-secretase is modulated by the position of the TMD1 of PS1 in the lipid bilayer. SIGNIFICANCE STATEMENT Reduction of the toxic longer amyloid-β peptide is one of the therapeutic approaches for Alzheimer disease. A subset of small compounds called γ-secretase modulators specifically decreases the longer amyloid-β production, although its mechanistic action remains unclear. Here we found that the modulator compound E2012 targets to the hydrophilic loop 1 of presenilin 1, which is a catalytic subunit of the γ-secretase. Moreover, E2012 triggers the piston movement of the transmembrane domain 1 of presenilin 1, which impacts on the γ-secretase activity. These results illuminate how γ-secretase modulators allosterically affect the proteolytic activity, and highlight the importance of the structural dynamics of presenilin 1 in the complexed process of the intramembrane cleavage., Competing Interests: Conflict-of-interest: M.Y. is a full-time employee of Eisai Company, Ltd. T.C. and T.T. declare no competing financial interests., (Copyright © 2017 the authors 0270-6474/17/3712272-09$15.00/0.)

The amyloid precursor protein (APP) is a receptor-like membrane protein. Although APP processing and β-amyloid production play a central role in Alzheimer's disease (AD) pathogenesis, the physiological function of APP remains elusive. Here, we identify APP as a novel receptor for Slit that mediates axon guidance and neural circuit formation. APP deficiency abolishes the Slit repulsive effect in a 3D olfactory explant culture, consistent with its callosal projection deficit in vivo and reminiscent of Slit loss. Inactivation of APP ortholog APL-1 in Caenorhabditis elegans results in pioneer axon mistargeting and genetic analysis places APL-1 in the SLT-1 (Slit)/SAX-3 (Robo) repulsive pathway. Slit binds to APP through the E1 domain, which triggers APP ectodomain shedding and recruitment of the intracellular FE65 and Pak1 complex and associated Rac1 GTPase activation. Our study establishes APP as a novel receptor for Slit ligand mediating axon guidance and neural circuit formation.

The amyloid precursor protein (APP), a key player in Alzheimer's disease, belongs to the family of synaptic adhesion molecules (SAMs) due to its impact on synapse formation and synaptic plasticity. These functions are mediated by both the secreted APP ectodomain that acts as a neurotrophic factor and full-length APP forming trans -cellular dimers. Two homologs of APP exist in mammals: the APP like proteins APLP1 and APLP2, exhibiting functions that partly overlap with those of APP. Here we tested whether APLP1 and APLP2 also show features of SAMs. We found that all three family members were upregulated during postnatal development coinciding with synaptogenesis. We observed presynaptic and postsynaptic localization of all APP family members and could show that heterologous expression of APLP1 or APLP2 in non-neuronal cells induces presynaptic differentiation in contacting axons of cocultured neurons, similar to APP and other SAMs. Moreover, APP/APLPs all bind to synaptic-signaling molecules, such as MINT/X11. Furthermore, we report that aged APLP1 knock-out mice show impaired basal transmission and a reduced mEPSC frequency, likely resulting from reduced spine density. This demonstrates an essential nonredundant function of APLP1 at the synapse. Compared to APP, APLP1 exhibits increased trans -cellular binding and elevated cell-surface levels due to reduced endocytosis. In conclusion, our results establish that APLPs show typical features of SAMs and indicate that increased surface expression, as observed for APLP1, is essential for proper synapse formation in vitro and synapse maintenance in vivo SIGNIFICANCE STATEMENT According to the amyloid-cascade hypothesis, Alzheimer's disease is caused by the accumulation of Aβ peptides derived from sequential cleavage of the amyloid precursor protein (APP) by β-site APP cleaving enzyme 1 (BACE1) and γ-secretase. Here we show that all mammalian APP family members (APP, APLP1, and APLP2) exhibit synaptogenic activity, involving trans -synaptic dimerization, similar to other synaptic cell adhesion molecules, such as Neuroligin/Neurexin. Importantly, our study revealed that the loss of APLP1, which is one of the major substrates of BACE1, causes reduced spine density in aged mice. Because some therapeutic interventions target APP processing (e.g., BACE inhibitors), those strategies may alter APP/APLP physiological function. This should be taken into account for the development of pharmaceutical treatments of Alzheimer's disease., (Copyright © 2017 the authors 0270-6474/17/375346-21$15.00/0.)

It is widely accepted that amyloid β (Aβ) generated from amyloid precursor protein (APP) oligomerizes and fibrillizes to form neuritic plaques in Alzheimer's disease (AD), yet little is known about the contribution of APP to intracellular signaling events preceding AD pathogenesis. The data presented here demonstrate that APP expression and neuronal exposure to oligomeric Aβ42 enhance Ras/ERK signaling cascade and glycogen synthase kinase 3 (GSK-3) activation. We find that RNA interference (RNAi)-directed knockdown of APP in B103 rat neuroblastoma cells expressing APP inhibits Ras-ERK signaling and GSK-3 activation, indicating that APP acts upstream of these signal transduction events. Both ERK and GSK-3 are known to induce hyperphosphorylation of tau and APP at Thr668, and our findings suggest that aberrant signaling by APP facilitates these events. Supporting this notion, analysis of human AD brain samples showed increased expression of Ras, activation of GSK-3, and phosphorylation of APP and tau, which correlated with Aβ levels in the AD brains. Furthermore, treatment of primary rat neurons with Aβ recapitulated these events and showed enhanced Ras-ERK signaling, GSK-3 activation, upregulation of cyclin D1, and phosphorylation of APP and tau. The finding that Aβ induces Thr668 phosphorylation on APP, which enhances APP proteolysis and Aβ generation, denotes a vicious feedforward mechanism by which APP and Aβ promote tau hyperphosphorylation and neurodegeneration in AD. Based on these results, we hypothesize that aberrant proliferative signaling by APP plays a fundamental role in AD neurodegeneration and that inhibition of this would impede cell cycle deregulation and neurodegeneration observed in AD.

Tau pathology was recently identified as a key driver of disease progression and an attractive therapeutic target in Alzheimer's disease (AD). Selenomethionine (Se-Met), a major bioactive form of selenium (Se) in organisms with significant antioxidant capacity, reduced the levels of total tau and hyperphosphorylated tau and ameliorated cognitive deficits in younger triple transgenic AD (3xTg-AD) mice. Whether Se-Met has a similar effect on tau pathology and the specific mechanism of action in older 3xTg-AD mice remains unknown. Autophagy is a major self-degradative process to maintain cellular homeostasis and function. Autophagic dysfunction has been implicated in the pathogenesis of multiple age-dependent diseases, including AD. Modulation of autophagy has been shown to retard the accumulation of misfolded and aggregated proteins and to delay the progression of AD. Here, we found that 3xTg-AD mice showed significant improvement in cognitive ability after a 3-month treatment with Se-Met beginning at 8 months of age. In addition to attenuating the hyperphosphorylation of tau by modulating the activity of Akt/glycogen synthase kinase-3β and protein phosphatase 2A, Se-Met-induced reduction of tau was also mediated by an autophagy-based pathway. Specifically, Se-Met improved the initiation of autophagy via the AMP-activated protein kinase-mTOR (mammalian target of rapamycin) signaling pathway and enhanced autophagic flux to promote the clearance of tau in 3xTg-AD mice and primary 3xTg neurons. Thus, our results demonstrate for the first time that Se-Met mitigates cognitive decline by targeting both the hyperphosphorylation of tau and the autophagic clearance of tau in AD mice. These data strongly support Se-Met as a potent nutraceutical for AD therapy. SIGNIFICANCE STATEMENT Selenium has been widely recognized as a vital trace element abundant in the brain with effects of antioxidant, anticancer, and anti-inflammation. In this study, we report that selenomethionine rescues spatial learning and memory impairments in aged 3xTg-AD mice via decreasing the level of tau protein and tau hyperphosphorylation. We find that selenomethionine promotes the initiation of autophagy via the AMPK-mTOR pathway and enhances autophagic flux, thereby facilitating tau clearance in vivo and in vitro We have now identified an additional, novel mechanism by which selenomethionine improves the cognitive function of AD mice. Specifically, our data suggest the effect of selenium/selenomethionine on an autophagic pathway in Alzheimer's disease., (Copyright © 2017 the authors 0270-6474/17/372449-14$15.00/0.)

Hyperinsulinemia is a risk factor for late-onset Alzheimer's disease (AD). In vitro experiments describe potential connections between insulin, insulin signaling, and amyloid-β (Aβ), but in vivo experiments are needed to validate these relationships under physiological conditions. First, we performed hyperinsulinemic-euglycemic clamps with concurrent hippocampal microdialysis in young, awake, behaving APP swe /PS1 dE9 transgenic mice. Both a postprandial and supraphysiological insulin clamp significantly increased interstitial fluid (ISF) and plasma Aβ compared with controls. We could detect no increase in brain, ISF, or CSF insulin or brain insulin signaling in response to peripheral hyperinsulinemia, despite detecting increased signaling in the muscle. Next, we delivered insulin directly into the hippocampus of young APP/PS1 mice via reverse microdialysis. Brain tissue insulin and insulin signaling was dose-dependently increased, but ISF Aβ was unchanged by central insulin administration. Finally, to determine whether peripheral and central high insulin has differential effects in the presence of significant amyloid pathology, we repeated these experiments in older APP/PS1 mice with significant amyloid plaque burden. Postprandial insulin clamps increased ISF and plasma Aβ, whereas direct delivery of insulin to the hippocampus significantly increased tissue insulin and insulin signaling, with no effect on Aβ in old mice. These results suggest that the brain is still responsive to insulin in the presence of amyloid pathology but increased insulin signaling does not acutely modulate Aβ in vivo before or after the onset of amyloid pathology. Peripheral hyperinsulinemia modestly increases ISF and plasma Aβ in young and old mice, independent of neuronal insulin signaling., Significance Statement: The transportation of insulin from blood to brain is a saturable process relevant to understanding the link between hyperinsulinemia and AD. In vitro experiments have found direct connections between high insulin and extracellular Aβ, but these mechanisms presume that peripheral high insulin elevates brain insulin significantly. We found that physiological hyperinsulinemia in awake, behaving mice does not increase CNS insulin to an appreciable level yet modestly increases extracellular Aβ. We also found that the brain of aged APP/PS1 mice was not insulin resistant, contrary to the current state of the literature. These results further elucidate the relationship between insulin, the brain, and AD and its conflicting roles as both a risk factor and potential treatment., (Copyright © 2016 the authors 0270-6474/16/3611704-12$15.00/0.)

Unlabelled: Intraneuronal calcium stimulates the calpain-dependent conversion of p35 to p25, a CDK5 activator. It is widely believed that amyloid β peptide (Aβ) induces this conversion that, in turn, has an essential role in Alzheimer's disease pathogenesis. However, in vivo studies on p25 generation used transgenic mice overexpressing mutant amyloid precursor protein (APP) and presenilin (PS). Here, using single App knock-in mice, we show that p25 generation is an artifact caused by membrane protein overexpression. We show that massive Aβ42 accumulation without overexpression of APP or presenilin does not produce p25, whereas p25 generation occurred with APP/PS overexpression and in postmortem mouse brain. We further support this finding using mice deficient for calpastatin, the sole calpain-specific inhibitor protein. Thus, the intracerebral environment of the APP/PS mouse brain and postmortem brain is an unphysiological state., Significance Statement: We recently estimated using single App knock-in mice that accumulate amyloid β peptide without transgene overexpression that 60% of the phenotypes observed in Alzheimer's model mice overexpressing mutant amyloid precursor protein (APP) or APP and presenilin are artifacts (Saito et al., 2014). The current study further supports this estimate by invalidating key results from papers that were published in Cell These findings suggest that more than 3000 publications based on APP and APP/PS overexpression must be reevaluated., (Copyright © 2016 Saito et al.)

Unlabelled: Vascular cognitive impairment and dementia (VCID) is the second most common form of dementia behind Alzheimer's disease (AD). It is estimated that 40% of AD patients also have some form of VCID. One promising therapeutic for AD is anti-Aβ immunotherapy, which uses antibodies against Aβ to clear it from the brain. While successful in clearing Aβ and improving cognition in mice, anti-Aβ immunotherapy failed to reach primary cognitive outcomes in several different clinical trials. We hypothesized that one potential reason the anti-Aβ immunotherapy clinical trials were unsuccessful was due to this high percentage of VCID comorbidity in the AD population. We used our unique model of VCID-amyloid comorbidity to test this hypothesis. We placed 9-month-old wild-type and APP/PS1 mice on either a control diet or a diet that induces hyperhomocysteinemia (HHcy). After being placed on the diet for 3 months, the mice then received intraperotineal injections of either IgG2a control or 3D6 for another 3 months. While we found that treatment of our comorbidity model with 3D6 resulted in decreased total Aβ levels, there was no cognitive benefit of the anti-Aβ immunotherapy in our AD/VCID mice. Further, microhemorrhages were increased by 3D6 in the APP/PS1/control but further increased in an additive fashion when 3D6 was administered to the APP/PS1/HHcy mice. This suggests that the use of anti-Aβ immunotherapy in patients with both AD and VCID would be ineffective on cognitive outcomes., Significance Statement: Despite significant mouse model data demonstrating both pathological and cognitive efficacy of anti-Aβ immunotherapy for the treatment of Alzheimer's disease, clinical trial outcomes have been underwhelming, failing to meet any primary endpoints. We show here that vascular cognitive impairment and dementia (VCID) comorbidity eliminates cognitive efficacy of anti-Aβ immunotherapy, despite amyloid clearance. Further, cerebrovascular adverse events of the anti-Aβ immunotherapy are significantly exacerbated by the VCID comorbidity. These data suggest that VCID comorbidity with Alzheimer's disease may mute the response to anti-Aβ immunotherapy., (Copyright © 2016 the authors 0270-6474/16/369896-12$15.00/0.)

Unlabelled: Despite the demonstration that amyloid-β (Aβ) can trigger increased tau phosphorylation and neurofibrillary tangle (NFT) formation in vivo, the molecular link associating Aβ and tau pathologies remains ill defined. Here, we observed that exposure of cultured primary neurons to Aβ trimers isolated from brain tissue of subjects with Alzheimer's disease led to a specific conformational change of tau detected by the antibody Alz50. A similar association was supported by postmortem human brain analyses. To study the role of Aβ trimers in vivo, we created a novel bigenic Tg-Aβ+Tau mouse line by crossing Tg2576 (Tg-Aβ) and rTg4510 (Tg-Tau) mice. Before neurodegeneration and amyloidosis, apparent Aβ trimers were increased by ∼2-fold in 3-month-old Tg-Aβ and Tg-Aβ+Tau mice compared with younger mice, whereas soluble monomeric Aβ levels were unchanged. Under these conditions, the expression of soluble Alz50-tau conformers rose by ∼2.2-fold in the forebrains of Tg-Aβ+Tau mice compared with nontransgenic littermates. In parallel, APP accumulated intracellularly, suggestive of a putative dysfunction of anterograde axonal transport. We found that the protein abundance of the kinesin-1 light chain (KLC1) was reduced selectively in vivo and in vitro when soluble Aβ trimers/Alz50-tau were present. Importantly, the reduction in KLC1 was prevented by the intraneuronal delivery of Alz50 antibodies. Collectively, our findings reveal that specific soluble conformers of Aβ and tau cooperatively disrupt axonal transport independently from plaques and tangles. Finally, these results suggest that not all endogenous Aβ oligomers trigger the same deleterious changes and that the role of each assembly should be considered separately., Significance Statement: The mechanistic link between amyloid-β (Aβ) and tau, the two major proteins composing the neuropathological lesions detected in brain tissue of Alzheimer's disease subjects, remains unclear. Here, we report that the trimeric Aβ species induce a pathological modification of tau in cultured neurons and in bigenic mice expressing Aβ and human tau. This linkage was also observed in postmortem brain tissue from subjects with mild cognitive impairment, when Aβ trimers are abundant. Further, this modification of tau was associated with the intracellular accumulation of the precursor protein of Aβ, APP, as a result of the selective decrease in kinesin light chain 1 expression. Our findings suggest that Aβ trimers might cause axonal transport deficits in AD., (Copyright © 2016 the authors 0270-6474/16/369647-12$15.00/0.)

Unlabelled: Microglial dysfunction is increasingly recognized as a key contributor to the pathogenesis of Alzheimer's disease (AD). Environmental enrichment (EE) is well documented to enhance neuronal form and function, but almost nothing is known about whether and how it alters the brain's innate immune system. Here we found that prolonged exposure of naive wild-type mice to EE significantly altered microglial density and branching complexity in the dentate gyrus of hippocampus. In wild-type mice injected intraventricularly with soluble Aβ oligomers (oAβ) from hAPP-expressing cultured cells, EE prevented several morphological features of microglial inflammation and consistently prevented oAβ-mediated mRNA changes in multiple inflammatory genes both in vivo and in primary microglia cultured from the mice. Microdialysis in behaving mice confirmed that EE normalized increases in the extracellular levels of the key cytokines (CCL3, CCL4, TNFα) identified by the mRNA analysis. Moreover, EE prevented the changes in microglial gene expression caused by ventricular injection of oAβ extracted directly from AD cerebral cortex. We conclude that EE potently alters the form and function of microglia in a way that prevents their inflammatory response to human oAβ, suggesting that prolonged environmental enrichment could protect against AD by modulating the brain's innate immune system., Significance Statement: Environmental enrichment (EE) is a potential therapy to delay Alzheimer's disease (AD). Microglial inflammation is associated with the progression of AD, but the influence of EE on microglial inflammation is unclear. Here we systematically applied in vivo methods to show that EE alters microglia in the dentate gyrus under physiological conditions and robustly prevents microglial inflammation induced by human Aβ oligomers, as shown by neutralized microglial inflammatory morphology, mRNA changes, and brain interstitial fluid cytokine levels. Our findings suggest that EE alters the innate immune system and could serve as a therapeutic approach to AD and provide new targets for drug discovery. Further, we propose that the therapeutic benefits of EE could extend to other neurodegenerative diseases involving microglial inflammation., (Copyright © 2016 the authors 0270-6474/16/369041-16$15.00/0.)

Unlabelled: Amyloid precursor protein (APP) was originally identified as the source of β-amyloid peptides that accumulate in Alzheimer's disease (AD), but it also has been implicated in the control of multiple aspects of neuronal motility. APP belongs to an evolutionarily conserved family of transmembrane proteins that can interact with a variety of adapter and signaling molecules. Recently, we showed that both APP and its insect ortholog [APPL (APP-Like)] directly bind the heterotrimeric G-protein Goα, supporting the model that APP can function as an unconventional Goα-coupled receptor. We also adapted a well characterized assay of neuronal migration in the hawkmoth, Manduca sexta, to show that APPL-Goα signaling restricts ectopic growth within the developing nervous system, analogous to the role postulated for APP family proteins in controlling migration within the mammalian cortex. Using this assay, we have now identified Manduca Contactin (MsContactin) as an endogenous ligand for APPL, consistent with previous work showing that Contactins interact with APP family proteins in other systems. Using antisense-based knockdown protocols and fusion proteins targeting both proteins, we have shown that MsContactin is selectively expressed by glial cells that ensheath the migratory neurons (expressing APPL), and that MsContactin-APPL interactions normally prevent inappropriate migration and outgrowth. These results provide new evidence that Contactins can function as authentic ligands for APP family proteins that regulate APP-dependent responses in the developing nervous system. They also support the model that misregulated Contactin-APP interactions might provoke aberrant activation of Goα and its effectors, thereby contributing to the neurodegenerative sequelae that typify AD., Significance Statement: Members of the amyloid precursor protein (APP) family participate in many aspects of neuronal development, but the ligands that normally activate APP signaling have remained controversial. This research provides new evidence that members of the Contactin family function as authentic ligands for APP and its orthologs, and that this evolutionarily conserved class of membrane-attached proteins regulates key aspects of APP-dependent migration and outgrowth in the embryonic nervous system. By defining the normal role of Contactin-APP signaling during development, these studies also provide the framework for investigating how the misregulation of Contactin-APP interactions might contribute to neuronal dysfunction in the context of both normal aging and neurodegenerative conditions, including Alzheimer's disease., (Copyright © 2016 the authors 0270-6474/16/368757-19$15.00/0.)

Unlabelled: Recent evidence implicates exosomes in the aggregation of Aβ and spreading of tau in Alzheimer's disease. In neural cells, exosome formation can be blocked by inhibition or silencing of neutral sphingomyelinase-2 (nSMase2). We generated genetically nSMase2-deficient 5XFAD mice (fro;5XFAD) to assess AD-related pathology in a mouse model with consistently reduced ceramide generation. We conducted in vitro assays to assess Aβ42 aggregation and glial clearance with and without exosomes isolated by ultracentrifugation and determined exosome-induced amyloid aggregation by particle counting. We analyzed brain exosome content, amyloid plaque formation, neuronal degeneration, sphingolipid, Aβ42 and phospho-tau levels, and memory-related behaviors in 5XFAD versus fro;5XFAD mice using contextual and cued fear conditioning. Astrocyte-derived exosomes accelerated aggregation of Aβ42 and blocked glial clearance of Aβ42 in vitro Aβ42 aggregates were colocalized with extracellular ceramide in vitro using a bifunctional ceramide analog preloaded into exosomes and in vivo using anticeramide IgG, implicating ceramide-enriched exosomes in plaque formation. Compared with 5XFAD mice, the fro;5XFAD mice had reduced brain exosomes, ceramide levels, serum anticeramide IgG, glial activation, total Aβ42 and plaque burden, tau phosphorylation, and improved cognition in a fear-conditioned learning task. Ceramide-enriched exosomes appear to exacerbate AD-related brain pathology by promoting the aggregation of Aβ. Reduction of exosome secretion by nSMase2 loss of function improves pathology and cognition in the 5XFAD mouse model., Significance Statement: We present for the first time evidence, using Alzheimer's disease (AD) model mice deficient in neural exosome secretion due to lack of neutral sphingomyelinase-2 function, that ceramide-enriched exosomes exacerbate AD-related pathologies and cognitive deficits. Our results provide rationale to pursue a means of inhibiting exosome secretion as a potential therapy for individuals at risk for developing AD., (Copyright © 2016 the authors 0270-6474/16/368653-15$15.00/0.)

Unlabelled: Acute cerebral ischemia and chronic neurovascular diseases share various common mechanisms with neurodegenerative diseases, such as disturbed cellular calcium and energy homeostasis and accumulation of toxic metabolites. A link between these conditions may be constituted by amyloid precursor protein (APP), which plays a pivotal role in the pathogenesis of Alzheimer's disease, but has also been associated with the response to acute hypoxia and regulation of calcium homeostasis. We therefore studied hypoxia-induced loss of function and recovery upon reoxygenation in hippocampal slices of mice lacking APP (APP(-/-)) or selectively expressing its soluble extracellular domain (APPsα-KI). Transient hypoxia disrupted electrical activity at the network and cellular level. In mice lacking APP, these impairments were significantly more severe, showing increased rise of intracellular calcium, faster loss of function, and higher incidence of spreading depression. Likewise, functional recovery upon reoxygenation was much slower and less complete than in controls. Most of these deficits were rescued by selective expression of the soluble extracellular fragment APPsα, or by pharmacological block of L-type calcium channels. We conclude that APP supports neuronal resistance toward acute hypoxia. This effect is mediated by the secreted APPsα-domain and involves L-type calcium channels., Significance Statement: Amyloid precursor protein (APP) is involved in the pathophysiology of Alzheimer's disease, but its normal function in the brain remains elusive. Here, we describe a neuroprotective role of the protein in acute hypoxia. Functional recovery of mouse hippocampal networks after transient reduction of oxygen supply was strongly impaired in animals lacking APP. Most protective effects are mediated by the soluble extracellular fragment APPsα and involve L-type calcium channels. Thus, APP contributes to calcium homeostasis in situations of metabolic stress. This finding may shed light on the physiological function of APP and may be important for understanding mechanisms of neurodegenerative diseases., (Copyright © 2016 the authors 0270-6474/16/368356-16$15.00/0.)

Unlabelled: Prior work suggests that amyloid precursor protein (APP) can function as a proinflammatory receptor on immune cells, such as monocytes and microglia. Therefore, we hypothesized that APP serves this function in microglia during Alzheimer's disease. Although fibrillar amyloid β (Aβ)-stimulated cytokine secretion from both wild-type and APP knock-out (mAPP(-/-)) microglial cultures, oligomeric Aβ was unable to stimulate increased secretion from mAPP(-/-) cells. This was consistent with an ability of oligomeric Aβ to bind APP. Similarly, intracerebroventricular infusions of oligomeric Aβ produced less microgliosis in mAPP(-/-) mice compared with wild-type mice. The mAPP(-/-) mice crossed to an APP/PS1 transgenic mouse line demonstrated reduced microgliosis and cytokine levels and improved memory compared with wild-type mice despite robust fibrillar Aβ plaque deposition. These data define a novel function for microglial APP in regulating their ability to acquire a proinflammatory phenotype during disease., Significance Statement: A hallmark of Alzheimer's disease (AD) brains is the accumulation of amyloid β (Aβ) peptide within plaques robustly invested with reactive microglia. This supports the notion that Aβ stimulation of microglial activation is one source of brain inflammatory changes during disease. Aβ is a cleavage product of the ubiquitously expressed amyloid precursor protein (APP) and is able to self-associate into a wide variety of differently sized and structurally distinct multimers. In this study, we demonstrate both in vitro and in vivo that nonfibrillar, oligomeric forms of Aβ are able to interact with the parent APP protein to stimulate microglial activation. This provides a mechanism by which metabolism of APP results in possible autocrine or paracrine Aβ production to drive the microgliosis associated with AD brains., (Copyright © 2016 the authors 0270-6474/16/368471-16$15.00/0.)

In Alzheimer's disease (AD), the accumulation and deposition of amyloid-β (Aβ) peptides in the brain is a central event. Aβ is cleaved from amyloid precursor protein (APP) by β-secretase and γ-secretase mainly in neurons. Although mutations inAPP,PS1, orPS2cause early-onset familial AD,ABCA7encoding ATP-binding cassette transporter A7 is one of the susceptibility genes for late-onset AD (LOAD), in which itsloss-of-functionvariants increase the disease risk. ABCA7 is homologous to a major lipid transporter ABCA1 and is highly expressed in neurons and microglia in the brain. Here, we show that ABCA7 deficiency altered brain lipid profile and impaired memory in ABCA7 knock-out (Abca7(-/-)) mice. When bred to amyloid model APP/PS1 mice, plaque burden was exacerbated by ABCA7 deficit.In vivomicrodialysis studies indicated that the clearance rate of Aβ was unaltered. Interestingly, ABCA7 deletion facilitated the processing of APP to Aβ by increasing the levels of β-site APP cleaving enzyme 1 (BACE1) and sterol regulatory element-binding protein 2 (SREBP2) in primary neurons and mouse brains. Knock-down of ABCA7 expression in neurons caused endoplasmic reticulum stress highlighted by increased level of protein kinase R-like endoplasmic reticulum kinase (PERK) and increased phosphorylation of eukaryotic initiation factor 2α (eIF2α). In the brains of APP/PS1;Abca7(-/-)mice, the level of phosphorylated extracellular regulated kinase (ERK) was also significantly elevated. Together, our results reveal novel pathways underlying the association of ABCA7 dysfunction and LOAD pathogenesis., Significance Statement: Gene variants inABCA7encoding ATP-binding cassette transporter A7 are associated with the increased risk for late-onset Alzheimer's disease (AD). Importantly, we found the altered brain lipid profile and impaired memory in ABCA7 knock-out mice. The accumulation of amyloid-β (Aβ) peptides cleaved from amyloid precursor protein (APP) in the brain is a key event in AD pathogenesis and we also found that ABCA7 deficit exacerbated brain Aβ deposition in amyloid AD model APP/PS1 mice. Mechanistically, we found that ABCA7 deletion facilitated the processing of APP and Aβ production by increasing the levels of β-secretase 1 (BACE1) in primary neurons and mouse brains without affecting the Aβ clearance rate in APP/PS1 mice. Our study demonstrates a novel mechanism underlying how dysfunctions of ABCA7 contribute to the risk for AD., (Copyright © 2016 the authors 0270-6474/16/363849-12$15.00/0.)

Aerobic glycolysis and lactate production in the brain plays a key role in memory, yet the role of this metabolism in the cognitive decline associated with Alzheimer's disease (AD) remains poorly understood. Here we examined the relationship between cerebral lactate levels and memory performance in an APP/PS1 mouse model of AD, which progressively accumulates amyloid-β. In vivo (1)H-magnetic resonance spectroscopy revealed an age-dependent decline in lactate levels within the frontal cortex of control mice, whereas lactate levels remained unaltered in APP/PS1 mice from 3 to 12 months of age. Analysis of hippocampal interstitial fluid by in vivo microdialysis revealed a significant elevation in lactate levels in APP/PS1 mice relative to control mice at 12 months of age. An age-dependent decline in the levels of key aerobic glycolysis enzymes and a concomitant increase in lactate transporter expression was detected in control mice. Increased expression of lactate-producing enzymes correlated with improved memory in control mice. Interestingly, in APP/PS1 mice the opposite effect was detected. In these mice, increased expression of lactate producing enzymes correlated with poorer memory performance. Immunofluorescent staining revealed localization of the aerobic glycolysis enzymes pyruvate dehydrogenase kinase and lactate dehydrogenase A within cortical and hippocampal neurons in control mice, as well as within astrocytes surrounding amyloid plaques in APP/PS1 mice. These observations collectively indicate that production of lactate, via aerobic glycolysis, is beneficial for memory function during normal aging. However, elevated lactate levels in APP/PS1 mice indicate perturbed lactate processing, a factor that may contribute to cognitive decline in AD., Significance Statement: Lactate has recently emerged as a key metabolite necessary for memory consolidation. Lactate is the end product of aerobic glycolysis, a unique form of metabolism that occurs within certain regions of the brain. Here we detected an age-dependent decline in the expression of aerobic glycolysis enzymes and a concomitant decrease in lactate levels within the frontal cortex of wild-type mice. Improved memory performance in wild-type mice correlated with elevated expression of aerobic glycolysis enzymes. Surprisingly, lactate levels remained elevated with age and increased aerobic glycolysis enzyme expression correlated with poorer memory performance in APP/PS1 mice. These findings suggest that while lactate production is beneficial for memory in the healthy aging brain, it might be detrimental in an Alzheimer's disease context., (Copyright © 2016 the authors 0270-6474/16/361871-08$15.00/0.)

The accumulation of β-amyloid (Aβ) peptides, a pathological hallmark of Alzheimer's disease (AD), has been associated with functional alterations, often in an episodic memory system with a particular emphasis on medial temporal lobe function. The topography of Aβ deposition, however, largely overlaps with frontoparietal control (FPC) regions implicated in cognitive control that has been shown to be impaired in early mild AD. To understand the neural mechanism underlying early changes in cognitive control with AD, we examined the impact of Aβ deposition on task-evoked FPC activation using functional magnetic resonance imaging (fMRI) in humans. Forty-three young and 62 cognitively normal older adults underwent an fMRI session during an executive contextual task in which task difficulty varied: single (either letter case or vowel/consonant judgment task) vs dual (switching between letter case and vowel/consonant decisions) task. Older subjects additionally completed (18)F-florbetaben positron emission tomography scans and were classified as either amyloid positive (Aβ+) or negative (Aβ-). Consistent with previous reports, age-related increases in brain activity were found in FPC regions commonly identified across groups. For both task conditions, Aβ-related increases in brain activity were found compared with baseline activity. For higher cognitive control load, however, Aβ+ elderly showed reduced task-switching activation in the right inferior frontal cortex. Our findings suggest that with Aβ deposition, brain activation in the cognitive control region reaches a maximum with lower control demand and decreases with higher control demand, which may underlie early impairment in cognitive control with AD progression., Significance Statement: The accumulation of β-amyloid (Aβ) peptides, a pathological hallmark of Alzheimer's disease, spatially overlaps with frontoparietal control (FPC) regions implicated in cognitive control, but the impact of Aβ deposition on FPC regions is largely unknown. Using functional magnetic resonance imaging with a task-switching task, we found Aβ-related increases in FPC regions compared with baseline activity. For higher cognitive control load, however, Aβ-related hypoactivity was found in the right inferior frontal cortex, a region highly implicated in cognitive control. The findings suggest that with Aβ deposition, task-related brain activity may reach a plateau early and undergo downstream pathways of neural dysfunction, which may relate to the early impairment of cognitive control seen in the progression of Aβ pathology., (Copyright © 2016 the authors 0270-6474/16/361962-09$15.00/0.)