Amyloid Precursor Protein Knock-in Research Articles

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

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

Disrupted mitochondrial response to nutrients is a presymptomatic event in the cortex of the APPSAA knock-in mouse model of Alzheimer's disease.

Reduced brain energy metabolism, mammalian target of rapamycin (mTOR) dysregulation, and extracellular amyloid beta (Aβ) oligomer (xcAβO) buildup are some well-known Alzheimer's disease (AD) features; how they promote neurodegeneration is poorly understood. We previously reported that xcAβOs inhibit nutrient-induced mitochondrial activity (NiMA) in cultured neurons. We now report NiMA disruption in vivo. Brain energy metabolism and oxygen consumption were recorded in heterozygous amyloid precursor protein knock-in (APPSAA) mice using two-photon fluorescence lifetime imaging and multiparametric photoacoustic microscopy. NiMA is inhibited in APPSAA mice before other defects are detected in these Aβ-producing animals that do not overexpress APP or contain foreign DNA inserts into genomic DNA. Glycogen synthase kinase 3 (GSK3β) signals through mTORC1 to regulate NiMA independently of mitochondrial biogenesis. Inhibition of GSK3β with TWS119 stimulates NiMA in cultured human neurons, and mitochondrial activity and oxygen consumption in APPSAA mice. NiMA disruption in vivo occurs before plaques, neuroinflammation, and cognitive decline in APPSAA mice, and may represent an early stage in human AD. Amyloid beta blocks communication between lysosomes and mitochondria in vivo. Nutrient-induced mitochondrial activity (NiMA) is disrupted long before the appearance of Alzheimer's disease (AD) histopathology in heterozygous amyloid precursor protein knock-in (APPSAA/+) mice. NiMA is disrupted long before learning and memory deficits in APPSAA/+ mice. Pharmacological interventions can rescue AD-related NiMA disruption in vivo.

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

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

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

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

Molecular Mechanisms of Amyloid-β Self-Assembly Seeded by In Vivo-Derived Fibrils and Inhibitory Effects of the BRICHOS Chaperone.

Self-replication of amyloid-β-peptide (Aβ) fibril formation is a hallmark in Alzheimer's disease (AD). Detailed insights have been obtained in Aβ self-assembly in vitro, yet whether similar mechanisms are relevant in vivo has remained elusive. Here, we investigated the ability of in vivo-derived Aβ fibrils from two different amyloid precursor protein knock-in AD mouse models to seed Aβ42 aggregation, where we quantified the microscopic rate constants. We found that the nucleation mechanism of in vivo-derived fibril-seeded Aβ42 aggregation can be described with the same kinetic model as that in vitro. Further, we identified the inhibitory mechanism of the anti-amyloid BRICHOS chaperone on seeded Aβ42 fibrillization, revealing a suppression of secondary nucleation and fibril elongation, which is strikingly similar as observed in vitro. These findings hence provide a molecular understanding of the Aβ42 nucleation process triggered by in vivo-derived Aβ42 propagons, providing a framework for the search for new AD therapeutics.

Cellular cholesterol loss by DHCR24 knockdown leads to Aβ production by changing APP intracellular localization

For the past 20years, the majority of cell culture studies reported that increasing cholesterol level increases amyloid-β (Aβ) production. Conversely, other studies and genetic evidences support that cellular cholesterol loss leads to Aβ generation. As a highly controversial issue in Alzheimer's disease pathogenesis, the apparent contradiction prompted us to again explore the role of cellular cholesterol in Aβ production. Here, we adopted new neuronal and astrocytic cell models induced by 3β-hydroxysterol-Δ24 reductase (DHCR24), which obviously differ from the widely used cell models with overexpressing amyloid precursor protein (APP) in the majority of previous studies. In neuronal and astrocytic cell model, we found that deficiency of cellular cholesterol by DHCR24 knockdown obviously increased intracellular and extracellular Aβ generation. Importantly, in cell models with overexpressing APP, we found that APP overexpression could disrupt cellular cholesterol homeostasis and affect function of cells, coupled with the increase of APP β-cleavage product, 99-residue transmembrane C-terminal domain. Therefore, we suppose the results derived from the APP knockin models will need to be re-evaluated. One rational explanation for the discrepancy between our outcomes and the previous studies could be attributed to the two different cell models. Mechanistically, we showed that cellular cholesterol loss obviously altered APP intracellular localization by affecting cholesterol-related trafficking protein of APP. Therefore, our outcomes strongly support cellular cholesterol loss by DHCR24 knockdown leads to Aβ production.

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

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

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

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

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

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

Impaired sharp-wave ripple coordination between the medial entorhinal cortex and hippocampal CA1 of knock-in model of Alzheimer's disease.

Clinical evidence suggests that the entorhinal cortex is a primary brain area triggering memory impairments in Alzheimer’s disease (AD), but the underlying brain circuit mechanisms remain largely unclear. In healthy brains, sharp-wave ripples (SWRs) in the hippocampus and entorhinal cortex play a critical role in memory consolidation. We tested SWRs in the MEC layers 2/3 of awake amyloid precursor protein knock-in (APP-KI) mice, recorded simultaneously with SWRs in the hippocampal CA1. We found that MEC→CA1 coordination of SWRs, found previously in healthy brains, was disrupted in APP-KI mice even at a young age before the emergence of spatial memory impairments. Intriguingly, long-duration SWRs critical for memory consolidation were mildly diminished in CA1, although SWR density and amplitude remained intact. Our results point to SWR incoordination in the entorhinal-hippocampal circuit as an early network symptom that precedes memory impairment in AD.

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

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

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

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

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

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

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

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

Probiotic Bifidobacterium breve decreases Aβ production via the upregulation of ADAM10 level and attenuates microglia activation in an APP knock‐in mouse model of Alzheimer’s disease

Probiotics supplementation reinstates microbiome diversity and improves brain function, including memory and learning abilities, although the molecular mechanisms have not been fully elucidated. In the current study, we investigated the effects of orally supplemented Bifidobacterium breve MCC1274 (B. breve MCC1274, synonym B. breve A1) on memory impairment and Alzheimer's disease (AD) pathogenesis in amyloid precursor protein knock-in (APP-KI) mice, (AppNL-G-F ).Three-month-old AppNL-G-F mice were orally supplemented with B. breve MCC1274 for four months. Novel object recognition test was used to evaluate memory-ameliorating effect of B. breve MCC1274. Amyloid plaques, Aβ levels, amyloid precursor protein (APP) and its processing enzymes, its metabolic products, and glial activity were assessed by immunohistochemistry, Aβ ELISA, Western blotting, and immunofluorescence staining. The mRNA expression levels of neuroinflammatory cytokines determined by quantitative RT-PCR analysis.Oral supplementation of B. breve MCC1274 rescued cognitive deficits in AppNL-G-F mice and down-regulated hippocampal Aβ40 and Aβ42 levels via the enhancement of non-amyloidogenic processing of APP, resulting from the enhancement of a-disintegrin and metalloproteinase 10 (ADAM10) protein level. Moreover, oral supplementation of B. breve MCC1274 activated the ERK/HIF-1α signaling pathway, which is responsible for the enhancement of ADAM10 level. In addition, oral supplementation ofB. breve MCC1274 attenuated microglial activation, which in turn led to down-regulation of the neuroinflammatory cytokines such as interleukin-6 (IL-6) and IL-1β mRNA expression levels in the brain. Moreover, B. breve MCC1274 supplementation increased postsynaptic protein levels in the hippocampus.Our study sheds light on the mechanism by which oral supplementation of B. breve MCC1274 reduced Aβ production and deposition through the enhancement of ERK-HIF-1α-ADAM10 signaling pathway, consequently rescuing cognitive deficits. Therefore, our findings suggest that oral supplementation of B. breve MCC1274 could be used as a potential therapy for preventing AD progression.

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

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

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

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

Mitochondria Damage as Early Indicator in an APP NL‐G‐F Mouse Model of Alzheimer's Disease

Introduction Traditional transgenic mouse models of Alzheimer's disease (AD) overexpress mutant amyloid precursor protein (APP) in order to increase pathological amyloid b peptide. However, this results in artificial phenotypes due to the non-clinically relevant overexpression of APP peptide species. To overcome these limitations, mice with a single humanized APP knock-in (KI) carrying Swedish (K670N/M671L), Arctic (E693G) and Beyreuther/Iberian (I716F) mutations (APPNL-G-F) have been developed to better reflect the human disease condition. Several groups have characterized the amyloidogenic, neuroinflammatory, and behavioral phenotypes of this mouse. However, to date no group has investigated and characterized the metabolic and mitochondrial phenotype of this mouse at different stages of development. Methods Mitochondria play a key role in both disease onset and progression, therefore detailed mechanistic characterization of mitochondrial and associated-metabolic dysfunction in this novel AD model is essential. Here, we evaluated mitochondria function, dynamics (fission/fusion) and morphology in APPNL-G-F mice using flowcytometry, Immunoblotting and high-resolution mitochondrial respirometry. Results Mitochondria dysfunction was detected as early as 3-4 months (m) of age in the hippocampus of AppNL-G-F mice, with further pathological progression detected at 6-8 m. Hippocampal mitochondria from 6-8 m AppNL-G-F miceexhibited higher membrane potential, decreased ATP production and increased reactive oxygen species (ROS) production. Morphologically, these mitochondria were larger in volume accompanied by lower dynamics (i.e., altered fission/fusion) at 6-8 m. Interestingly, plasma isolated from these 6-8 m AppNL-G-F mice resulted in a mitochondrial dysfunction in Hela cells and human differentiated neurons derived from induced pluripotent stem cells in vitro. Conclusion Our data revealed early mitochondrial dysfunction and associated metabolic changes in the hippocampus and circulatory plasma of AppNL-G-F mice.Further research may help to develop early AD detection based on mitochondrial/metabolic dysfunction.

Integrated analysis of behavioral, epigenetic, and gut microbiome analyses in AppNL-G-F, AppNL-F, and wild type mice

Epigenetic mechanisms occurring in the brain as well as alterations in the gut microbiome composition might contribute to Alzheimer’s disease (AD). Human amyloid precursor protein knock-in (KI) mice contain the Swedish and Iberian mutations (AppNL-F) or those two and also the Arctic mutation (AppNL-G-F). In this study, we assessed whether behavioral and cognitive performance in 6-month-old AppNL-F, AppNL-G-F, and C57BL/6J wild-type (WT) mice was associated with the gut microbiome, and whether the genotype modulates this association. The genotype effects observed in behavioral tests were test-dependent. The biodiversity and composition of the gut microbiome linked to various aspects of mouse behavioral and cognitive performance but differences in genotype modulated these relationships. These genotype-dependent associations include members of the Lachnospiraceae and Ruminococcaceae families. In a subset of female mice, we assessed DNA methylation in the hippocampus and investigated whether alterations in hippocampal DNA methylation were associated with the gut microbiome. Among other differentially methylated regions, we identified a 1 Kb region that overlapped ing 3′UTR of the Tomm40 gene and the promoter region of the Apoe gene that and was significantly more methylated in the hippocampus of AppNL-G-F than WT mice. The integrated gut microbiome hippocampal DNA methylation analysis revealed a positive relationship between amplicon sequence variants (ASVs) within the Lachnospiraceae family and methylation at the Apoe gene. Hence, these microbes may elicit an impact on AD-relevant behavioral and cognitive performance via epigenetic changes in AD-susceptibility genes in neural tissue or that such changes in the epigenome can elicit alterations in intestinal physiology that affect the growth of these taxa in the gut microbiome.

Comparison of memory, affective behavior, and neuropathology in APPNLGF knock-in mice to 5xFAD and APP/PS1 mice

Transgenic mouse models of Aβ amyloidosis generated by knock-in of a humanized Aβ sequence can offer some advantages over the transgenic models that overexpress amyloid precursor protein (APP). However, systematic comparison of memory, behavioral, and neuropathological phenotypes between these models has not been well documented. In this study, we compared memory and affective behavior in APPNLGF mice, an APP knock-in model, to two widely used mouse models of Alzheimer's disease, 5xFAD and APP/PS1 mice, at 10 months of age. We found that, despite similar deficits in working memory, object recognition, and social recognition memory, APPNLGF and 5xFAD mice but not APP/PS1 mice show compelling anxiety- and depressive-like behavior, and exhibited a marked impairment of social interaction. We quantified corticolimbic Aβ plaques, which were lowest in APPNLGF, intermediate in APP/PS1, and highest in 5xFAD mice. Interestingly, analysis of plaque size revealed that plaques were largest in APP/PS1 mice, intermediate in 5xFAD mice, and smallest in APPNLGF mice. Finally, we observed a significantly higher percentage of the area occupied by plaques in both 5xFAD and APP/PS1 relative to APPNLGF mice. Overall, our findings suggest that the severity of Aβ neuropathology is not directly correlated with memory and affective behavior impairments between these three transgenic mouse models. Additionally, APPNLGF may represent a valid mouse model for studying AD comorbid with anxiety and depression.