Female 3xTg-AD Mice Research Articles

Age- and Sex-Associated Wnt Signaling Dysregulation is Exacerbated from the Early Stages of Neuropathology in an Alzheimer’s Disease Model

Emerging studies suggest that Wnt signaling is dysregulated in the brains of AD patients, suggesting that this pathway may also contribute to disease progression. However, it remains to be determined whether alterations in the Wnt pathway are the cause or consequence of this disease and which elements of Wnt signaling mainly contribute to the appearance of AD histopathological markers early in disease compared to what occurs during normal aging. The present study aimed to describe the status of several canonical Wnt pathway components and the expression of the AD marker p-tau in the hippocampi of female and male 3xTg-AD mice during disease progression compared to those during normal aging. We analyzed the levels of the canonical Wnt components Wnt7a, Dkk-1, LRP6 and GSK3β as well as the levels of p-tau and BDNF at 3, 6, 9–12 and 18 months of age. We found a gradual increase in Dkk-1 levels during aging prior to Wnt7a and LRP5/6 depletion, which was strongly exacerbated in 3xTg-AD mice even at young ages and correlated with GSK3β activation and p-tau-S202/Thr205 expression. Dkk-1 upregulation, as well as the level of p-tau, was significantly greater in females than in males. Our results suggest that Dkk-1 upregulation is involved in the expression of several features of AD at early stages, which supports the possibility of positively modulating the canonical Wnt pathway as a therapeutic tool to delay this disease at early stages.

Glomerular Hypertrophy and Splenic Red Pulp Degeneration Concurrent with Oxidative Stress in 3xTg-AD Mice Model for Alzheimer's Disease and Its Exacerbation with Sex and Social Isolation.

The continuously expanding field of Alzheimer's disease (AD) research is now beginning to defocus the brain to take a more systemic approach to the disease, as alterations in the peripheral organs could be related to disease progression. One emerging hypothesis is organ involvement in the process of Aβ clearance. In the present work, we aimed to examine the status and involvement of the kidney as a key organ for waste elimination and the spleen, which is in charge of filtering the blood and producing lymphocytes, and their influence on AD. The results showed morphological and structural changes due to acute amyloidosis in the kidney (glomeruli area) and spleen (red pulp area and red/white pulp ratio) together with reduced antioxidant defense activity (GPx) in 16-month-old male and female 3xTg-AD mice when compared to their age- and sex-matched non-transgenic (NTg) counterparts. All these alterations correlated with the anxious-like behavioral phenotype of this mouse model. In addition, forced isolation, a cause of psychological stress, had a negative effect by intensifying genotype differences and causing differences to appear in NTg animals. This study further supports the relevance of a more integrative view of the complex interplay between systems in aging, especially at advanced stages of Alzheimer's disease.

Thermoneutral temperature exposure enhances slow-wave sleep with a correlated improvement in amyloid pathology in a triple-transgenic mouse model of Alzheimer's disease.

Accumulation of amyloid-β (Aβ) plays an important role in Alzheimer's disease (AD) pathology. There is growing evidence that disordered sleep may accelerate AD pathology by impeding the physiological clearance of Aβ from the brain that occurs in normal sleep. Therapeutic strategies for improving sleep quality may therefore help slow disease progression. It is well documented that the composition and dynamics of sleep are sensitive to ambient temperature. We therefore compared Aβ pathology and sleep metrics derived from polysomnography in 12-month-old female 3xTg-AD mice (n = 8) exposed to thermoneutral temperatures during the light period over 4 weeks to those of age- and sex-matched controls (n = 8) that remained at normal housing temperature (22°C) during the same period. The treated group experienced greater proportions of slow wave sleep (SWS)-i.e. epochs of elevated 0.5-2 Hz EEG slow wave activity during non-rapid eye movement (NREM) sleep-compared to controls. Assays performed on mouse brain tissue harvested at the end of the experiment showed that exposure to thermoneutral temperatures significantly reduced levels of DEA-soluble (but not RIPA- or formic acid-soluble) Aβ40 and Aβ42 in the hippocampus, though not in the cortex. With both groups pooled together and without regard to treatment condition, NREM sleep continuity and any measure of SWS within NREM at the end of the treatment period were inversely correlated with DEA-soluble Aβ40 and Aβ42 levels, again in the hippocampus but not in the cortex. These findings suggest that experimental manipulation of SWS could offer useful clues into the mechanisms and treatment of AD.

Modulation of hippocampal protein expression by a brain penetrant biologic TNF-α inhibitor in the 3xTg Alzheimer’s disease mice

BackgroundBiologic TNF-α inhibitors (bTNFIs) can block cerebral TNF-α in Alzheimer’s disease (AD) if these macromolecules can cross the blood–brain barrier (BBB). Thus, a model bTNFI, the extracellular domain of type II TNF-α receptor (TNFR), which can bind to and sequester TNF-α, was fused with a mouse transferrin receptor antibody (TfRMAb) to enable brain delivery via BBB TfR-mediated transcytosis. Previously, we found TfRMAb-TNFR to be protective in a mouse model of amyloidosis (APP/PS1) and tauopathy (PS19), and herein we investigated its effects in mice that combine both amyloidosis and tauopathy (3xTg-AD).MethodsEight-month-old female 3xTg-AD mice were injected intraperitoneally with saline (n = 11) or TfRMAb-TNFR (3 mg/kg; n = 11) three days per week for 12 weeks. Age-matched wild-type (WT) mice (n = 9) were treated similarly with saline. Brains were processed for immunostaining and high-resolution multiplex NanoString GeoMx spatial proteomics.ResultsWe observed regional differences in proteins relevant to Aβ, tau, and neuroinflammation in the hippocampus of 3xTg-AD mice compared with WT mice. From 64 target proteins studied using spatial proteomics, a comparison of the Aβ-plaque bearing vs. plaque-free regions in the 3xTg-AD mice yielded 39 differentially expressed proteins (DEP) largely related to neuroinflammation (39% of DEP) and Aβ and tau pathology combined (31% of DEP). Hippocampal spatial proteomics revealed that the majority of the proteins modulated by TfRMAb-TNFR in the 3xTg-AD mice were relevant to microglial function (⁓ 33%). TfRMAb-TNFR significantly reduced mature Aβ plaques and increased Aβ-associated microglia around larger Aβ deposits in the 3xTg-AD mice. Further, TfRMAb-TNFR increased mature Aβ plaque-associated microglial TREM2 in 3xTg-AD mice.ConclusionOverall, despite the low visual Aβ load in the 11-month-old female 3xTg-AD mice, our results highlight region-specific AD-relevant DEP in the hippocampus of these mice. Chronic TfRMAb-TNFR dosing modulated several DEP involved in AD pathology and showed a largely microglia-centric mechanism of action in the 3xTg-AD mice.

Food Finding Test without Deprivation: A Sensorial Paradigm Sensitive to Sex, Genotype, and Isolation Shows Signatures of Derangements in Old Mice with Alzheimer's Disease Pathology and Normal Aging.

The Food Finding Test (FFT) olfactory paradigm without overnight food deprivation examined olfaction in aged (16-months-old) animals. Ethograms of three goal-directed behaviors towards hidden food (sniffing, finding and eating) elicited in male and female 3xTg-AD mice for Alzheimer's disease (AD) and their age-matched C57BL/6 wild-type counterparts with normal aging were meticulously analyzed with the support of video recordings. The new FFT protocol elicited longer ethograms than previously reported with the standard deprivation protocol. However, it was sensitive when identifying genotype- and sex-dependent olfactory signatures for the temporal patterns of slow sniffing, finding, and eating in AD and males, but it had a striking consistency in females. The impact of forced social isolation was studied and it was found to exert sex-dependent modifications of the ethogram, mostly in males. Still, in both sexes, a functional derangement was detected since the internal correlations among the behaviors decreased or were lost under isolated conditions. In conclusion, the new paradigm without overnight deprivation was sensitive to sex (males), genotype (AD), and social context (isolation-dependent changes) in its ethogram and functional correlation. At the translational level, it is a warning about the impact of isolation in the advanced stages of the disease, paying notable attention to the male sex.

Signature of paraoxonases in the altered redox homeostasis in Alzheimer's disease

Paraoxonase (PON) enzymes (PON1, PON2 and PON3) exert antioxidant properties through arylesterase, lactonase and paraoxonase activities. Increasing findings suggested their potential involvement, particularly PON1 and PON2, in Alzheimer's disease (AD), a neurodegenerative pathology characterized by early oxidative stress. Specifically, decreased serum PON1-arylesterase and lactonase activities seem to be associated with an increased brain oxidative damage in early AD, leading to hypothesize that PON activity alterations might be an early event in AD. To address this hypothesis, the levels of 4-hydroxynonenal (4-HNE; i.e. a marker of oxidative stress damage) along with the protein expression and enzymatic activity of PON1 and PON2 have been investigated in the brain and serum of young [Postnatal day (PD)8–10, 20–25 and 60–65] asymptomatic 3xTg-AD female mice, one of the most used transgenic models of AD.At PD 8–10, there were no differences in hippocampus and prefrontal cortex (PFC) 4-HNE expression levels between 3xTg-AD mice compared to controls (Non-Tg mice). On the other hand, significant increased levels of 4-HNE were detected in PD 20–30 3xTg-AD mice hippocampus, while a significant reduction was observed in 3xTg-AD group at PD 60–65. In the PFC, 4-HNE levels were significantly reduced in 3xTg-AD mice brain at PD 20–30, while no differences in 4-HNE levels were detected at PD 60–65.No significant differences in arylesterase and lactonase activities were observed in the plasma of 3xTg-AD and Non-Tg mice at the different considered ages. Compared to Non-Tg mice, a reduction of brain arylesterase activity was found in 3xTg-AD female at PD 20–30 and PD 60–65, but it was significant only in the younger group. Finally, a similar trend was observed also for PON1 and PON2 protein levels, with both significantly, and solely, decreased in 3xTg-AD mice brain at PD 20–30.Overall, these findings suggest that the altered oxidative stress homeostasis in the 3xTg-AD female mice may be related to an early reduction in activity and expression of PONs enzymes most likely via a reduced brain arylesterases activity.

Serial reversal learning in an olfactory discrimination task in 3xTg-AD mice.

Male and female 3xTg-AD mice between 5 and 24 mo of age and their B6129F2/J wild-type controls were tested on a series of 18 olfactory discrimination and reversal tasks in an operant olfactometer. All mice learned the odor discriminations and reversals to a criterion of 85% correct, but the 3xTg-AD mice made fewer errors than the B6129F2/J mice in the odor discriminations and in the first six reversal learning tasks. Many mice showed evidence of near errorless learning, and on the reversal tasks the 3xTg-AD mice showed more instances of near errorless learning than the B6129F2/J mice. There was no evidence of an age effect on odor discrimination, but there was a decrease in errorless reversal learning in aged B6129F2/J mice. In long-term memory tests, there was an increase in the number of errors made but no genotype difference. The high level of performance indicates that the mice were able to develop a "learning to learn" strategy. The finding that the 3xTg-AD mice outperformed their littermate controls provides an example of paradoxical functional facilitation in these mice.

Estrogen receptor β exerts neuroprotective effects by fine-tuning mitochondrial homeostasis through NRF1/PGC-1α

BackgroundEstrogen deficiency causes mitochondrial defects that precede pathological changes related to Alzheimer's disease (AD) in the mouse model of postmenopause. The aim of this study was to investigate in such a mouse model whether and how estrogen receptor β (ERβ) was involved in prevention of mitochondrial damage and protection of neurons in the hippocampus. MethodsA mouse model of postmenopausal AD was created by ovariectomizing female 3xTg-AD mice, some of which were subcutaneously injected for six weeks with the non-steroidal ERβ agonist diarylpropionitrile. ERβ expression in female C57BL/6J mice was knocked down using shRNA interference. The different groups of animals were compared in terms of cognitive function using the Y-maze test, new object recognition test, and Morris water maze test, expression of numerous proteins related to mitochondrial biogenesis, mitophagy, apoptosis, and mitochondrial membrane potential, as well as deposition of amyloid β and neurofibrillary tangles. To complement these in vivo studies, we probed the effects of diarylpropionitrile on ERβ expression, apoptosis, and mitochondrial homeostasis in primary rat hippocampal neurons treated with amyloid β. ResultsERβ knockdown in C57BL/6J mice produced cognitive impairment, reduced mitochondrial biogenesis by downregulating PGC-1α, NRF1, mtTFA, and TOM20, and decreased mitophagy by downregulating Pink1, Parkin, and LC3B while upregulating PARIS and p62. ERβ knockdown promoted neuronal apoptosis by upregulating Cleaved-Caspase 9, Cleaved-Caspase 3, and Bax, while downregulating Bcl2 in hippocampus. Diarylpropionitrile mitigated cognitive decline in ovariectomized 3xTg-AD mice, which was associated with downregulation of BACE1, reduction of Aβ deposition, neurofibrillary tangles, and tau hyperphosphorylation, and upregulation of ERβ, increases in mitochondrial biogenesis and mitophagy, and decreases in apoptosis. The effects of diarylpropionitrile in mice were recapitulated in Aβ-injured primary rat hippocampal neurons. ConclusionsERβ activation can support learning and memory and alleviate AD symptoms in the postmenopausal AD model, which may involve regulation of neuronal mitochondrial biogenesis and mitophagy via NRF1/PGC-1α. This study supports further research on ERβ as a therapeutic target for postmenopausal women with AD.

Survival Bias, Non-Lineal Behavioral and Cortico-Limbic Neuropathological Signatures in 3xTg-AD Mice for Alzheimer's Disease from Premorbid to Advanced Stages and Compared to Normal Aging.

Pre-clinical research in aging is hampered by the scarcity of studies modeling its heterogeneity and complexity forged by pathophysiological conditions throughout the life cycle and under the sex perspective. In the case of Alzheimer's disease, the leading cause of dementia in older adults, we recently described in female wildtype and APP23 mice a survival bias and non-linear chronology of behavioral signatures from middle age to long life. Here, we present a comprehensive and multidimensional (physical, cognitive, and neuropsychiatric-like symptoms) screening and underlying neuropathological signatures in male and female 3xTg-AD mice at 2, 4, 6, 12, and 16 months of age and compared to their non-transgenic counterparts with gold-standard C57BL/6J background. Most variables studied detected age-related differences, whereas the genotype factor was specific to horizontal and vertical activities, thigmotaxis, coping with stress strategies, working memory, and frailty index. A sex effect was predominantly observed in classical emotional variables and physical status. Sixteen-month-old mice exhibited non-linear age- and genotype-dependent behavioral signatures, with higher heterogeneity in females, and worsened in naturalistically isolated males, suggesting distinct compensatory mechanisms and survival bias. The underlying temporal and spatial progression of Aβ and tau pathologies pointed to a relevant cortico-limbic substrate roadmap: premorbid intracellular Aβ immunoreactivity and pSer202/pThr205 tau phosphorylation in the amygdala and ventral hippocampus, and the entorhinal cortex and ventral hippocampus as the areas most affected by Aβ plaques. Therefore, depicting phenotypic signatures and neuropathological correlates can be critical to unveiling preventive/therapeutic research and intervention windows and studying adaptative behaviors and maladaptive responses relevant to psychopathology.

Hepatic Oxi-Inflammation and Neophobia as Potential Liver-Brain Axis Targets for Alzheimer's Disease and Aging, with Strong Sensitivity to Sex, Isolation, and Obesity.

Research on Alzheimer's disease (AD) has classically focused on alterations that occur in the brain and their intra- and extracellular neuropathological hallmarks. However, the oxi-inflammation hypothesis of aging may also play a role in neuroimmunoendocrine dysregulation and the disease's pathophysiology, where the liver emerges as a target organ due to its implication in regulating metabolism and supporting the immune system. In the present work, we demonstrate organ (hepatomegaly), tissue (histopathological amyloidosis), and cellular oxidative stress (decreased glutathione peroxidase and increased glutathione reductase enzymatic activities) and inflammation (increased IL-6 and TNF𝛼) as hallmarks of hepatic dysfunction in 16-month-old male and female 3xTg-AD mice at advanced stages of the disease, and as compared to age- and sex-matched non-transgenic (NTg) counterparts. Moreover, liver-brain axis alterations were found through behavioral (increased neophobia) and HPA axis correlations that were enhanced under forced isolation. In all cases, sex (male) and isolation (naturalistic and forced) were determinants of worse hepatomegaly, oxidative stress, and inflammation progression. In addition, obesity in old male NTg mice was translated into a worse steatosis grade. Further research is underway determine whether these alterations could correlate with a worse disease prognosis and to establish potential integrative system targets for AD research.

Postsynaptic Protein Shank3a Deficiency Synergizes with Alzheimer's Disease Neuropathology to Impair Cognitive Performance in the 3xTg-AD Murine Model.

Synaptic loss is intrinsically linked to Alzheimer's disease (AD) neuropathology and symptoms, but its direct impact on clinical symptoms remains elusive. The postsynaptic protein Shank3 (SH3 and multiple ankyrin repeat domains) is of particular interest, as the loss of a single allele of the SHANK3 gene is sufficient to cause profound cognitive symptoms in children. We thus sought to determine whether a SHANK3 deficiency could contribute to the emergence or worsening of AD symptoms and neuropathology. We first found a 30%-50% postmortem loss of SHANK3a associated with cognitive decline in the parietal cortex of individuals with AD. To further probe the role of SHANK3 in AD, we crossed male and female 3xTg-AD mice modelling Aβ and tau pathologies with Shank3a-deficient mice (Shank3Δex4-9). We observed synergistic deleterious effects of Shank3a deficiency and AD neuropathology on object recognition memory at 9, 12, and 18 months of age and on anxious behavior at 9 and 12 months of age in hemizygous Shank3Δex4-9-3xTg-AD mice. In addition to the expected 50% loss of Shank3a, levels of other synaptic proteins, such as PSD-95, drebrin, and homer1, remained unchanged in the parietotemporal cortex of hemizygous Shank3Δex4-9 animals. However, Shank3a deficiency increased the levels of soluble Aβ42 and human tau at 18 months of age compared with 3xTg-AD mice with normal Shank3 expression. The results of this study in human brain samples and in transgenic mice are consistent with the hypothesis that Shank3 deficiency makes a key contribution to cognitive impairment in AD.SIGNIFICANCE STATEMENT Although the loss of several synaptic proteins has been described in Alzheimer's disease (AD), it remains unclear whether their reduction contributes to clinical symptoms. The results of this study in human samples show lower levels of SHANK3a in AD brain, correlating with cognitive decline. Data gathered in a novel transgenic mouse suggest that Shank3a deficiency synergizes with AD neuropathology to induce cognitive impairment, consistent with a causal role in AD. Therefore, treatment aiming at preserving Shank3 in the aging brain may be beneficial to prevent AD.

An 8-cage imaging system for automated analyses of mouse behavior

The analysis of mouse behavior is used in biomedical research to study brain function in health and disease. Well-established rapid assays allow for high-throughput analyses of behavior but have several drawbacks, including measurements of daytime behaviors in nocturnal animals, effects of animal handling, and the lack of an acclimation period in the testing apparatus. We developed a novel 8-cage imaging system, with animated visual stimuli, for automated analyses of mouse behavior in 22-h overnight recordings. Software for image analysis was developed in two open-source programs, ImageJ and DeepLabCut. The imaging system was tested using 4–5 month-old female wild-type mice and 3xTg-AD mice, a widely-used model to study Alzheimer’s disease (AD). The overnight recordings provided measurements of multiple behaviors including acclimation to the novel cage environment, day and nighttime activity, stretch-attend postures, location in various cage areas, and habituation to animated visual stimuli. The behavioral profiles were different in wild-type and 3xTg-AD mice. AD-model mice displayed reduced acclimation to the novel cage environment, were hyperactive during the first hour of darkness, and spent less time at home in comparison to wild-type mice. We propose that the imaging system may be used to study various neurological and neurodegenerative disorders, including Alzheimer’s disease.

Sex differences in hippocampal β-amyloid accumulation in the triple-transgenic mouse model of Alzheimer's disease and the potential role of local estrogens.

Epidemiological studies show that women have a higher prevalence of Alzheimer's disease (AD) than men. Peripheral estrogen reduction during aging in women is proposed to play a key role in this sex-associated prevalence, however, the underlying mechanism remains elusive. We previously found that transcription factor early growth response-1 (EGR1) significantly regulates cholinergic function. EGR1 stimulates acetylcholinesterase (AChE) gene expression and is involved in AD pathogenesis. We aimed to investigate whether the triple-transgenic AD (3xTg-AD) mice harboring PS1 M146V , APP Swe , and Tau P301L show sex differences in β-amyloid (Aβ) and hyperphosphorylated tau (p-Tau), the two primary AD hallmarks, and how local 17β-estradiol (E2) may regulate the expression of EGR1 and AChE. We first sacrificed male and female 3xTg-AD mice at 3-4, 7-8, and 11-12 months and measured the levels of Aβ, p-Tau, EGR1, and AChE in the hippocampal complex. Second, we infected SH-SY5Y cells with lentivirus containing the amyloid precursor protein construct C99, cultured with or without E2 administration we measured the levels of extracellular Aβ and intracellular EGR1 and AChE. Female 3xTg-AD mice had higher levels of Aβ compared to males, while no p-Tau was found in either group. In SH-SY5Y cells infected with lentivirus containing the amyloid precursor protein construct C99, we observed significantly increased extracellular Aβ and decreased expression of intracellular EGR1 and AChE. By adding E2 to the culture medium, extracellular Aβ(l-42) was significantly decreased while intracellular EGR1 and AChE expression were elevated. This data shows that the 3xTg-AD mouse model can be useful for studying the human sex differences of AD, but only in regards to Ap. Furthermore, in vitro data shows local E2 may be protective for EGR1 and cholinergic functions in AD while suppressing soluble Aβ(1-42) levels. Altogether, this study provides further in vivo and in vitro data supporting the human epidemiological data indicating a higher prevalence of AD in women is related to changes in brain estrogen levels.

Predicting Neurodegenerative Disease Using Prepathology Gut Microbiota Composition: a Longitudinal Study in Mice Modeling Alzheimer's Disease Pathologies.

The gut microbiota-brain axis is suspected to contribute to the development of Alzheimer's disease (AD), a neurodegenerative disease characterized by amyloid-β plaque deposition, neurofibrillary tangles, and neuroinflammation. To evaluate the role of the gut microbiota-brain axis in AD, we characterized the gut microbiota of female 3xTg-AD mice modeling amyloidosis and tauopathy and wild-type (WT) genetic controls. Fecal samples were collected fortnightly from 4 to 52 weeks, and the V4 region of the 16S rRNA gene was amplified and sequenced on an Illumina MiSeq. RNA was extracted from the colon and hippocampus, converted to cDNA, and used to measure immune gene expression using reverse transcriptase quantitative PCR (RT-qPCR). Diversity metrics were calculated using QIIME2, and a random forest classifier was applied to predict bacterial features that are important in predicting mouse genotype. Gene expression of glial fibrillary acidic protein (GFAP; indicating astrocytosis) was elevated in the colon at 24 weeks. Markers of Th1 inflammation (il6) and microgliosis (mrc1) were elevated in the hippocampus. Gut microbiota were compositionally distinct early in life between 3xTg-AD mice and WT mice (permutational multivariate analysis of variance [PERMANOVA], 8 weeks, P = 0.001, 24 weeks, P = 0.039, and 52 weeks, P = 0.058). Mouse genotypes were correctly predicted 90 to 100% of the time using fecal microbiome composition. Finally, we show that the relative abundance of Bacteroides species increased over time in 3xTg-AD mice. Taken together, we demonstrate that changes in bacterial gut microbiota composition at prepathology time points are predictive of the development of AD pathologies. IMPORTANCE Recent studies have demonstrated alterations in the gut microbiota composition in mice modeling Alzheimer's disease (AD) pathologies; however, these studies have only included up to 4 time points. Our study is the first of its kind to characterize the gut microbiota of a transgenic AD mouse model, fortnightly, from 4 weeks of age to 52 weeks of age, to quantify the temporal dynamics in the microbial composition that correlate with the development of disease pathologies and host immune gene expression. In this study, we observed temporal changes in the relative abundances of specific microbial taxa, including the genus Bacteroides, that may play a central role in disease progression and the severity of pathologies. The ability to use features of the microbiota to discriminate between mice modeling AD and wild-type mice at prepathology time points indicates a potential role of the gut microbiota as a risk or protective factor in AD.

KIR channel regulation of electrical conduction along cerebrovascular endothelium: Enhanced modulation during Alzheimer's disease.

Endothelial cell (EC) coupling occurs through gap junctions and underlies cerebral blood flow regulation governed by inward-rectifying (KIR ) channels. This study addressed effects of KIR channel activity on EC coupling before and during Alzheimer's disease (AD). Intact EC tubes (width: ~90-100 μm; length: ~0.5 mm) were freshly isolated from posterior cerebral arteries of young Pre-AD (1-3 months) and aged AD (13-18 months) male and female 3xTg-AD mice. Dual intracellular microelectrodes applied simultaneous current injections (±0.5-3 nA) and membrane potential (Vm ) recordings in ECs at distance ~400 μm. Elevated extracellular potassium ([K+ ]E ; 8-15 mmol L-1 ; reference, 5 mmol L-1 ) activated KIR channels. Conducted Vm (∆Vm ) responses ranged from ~-30 to 30 mV in response to -3 to +3 nA (linear regression, R2 ≥0.99) while lacking rectification for charge polarity or axial direction of spread. Conduction slope decreased ~10-20% during 15 mmol L-1 [K+ ]E in Pre-AD males and AD females. 15 mmol L-1 [K+ ]E decreased conduction at lower thresholds in AD animals (~±20 mV) versus Pre-AD (~±25 mV). AD increased (~10-15%) conducted hyperpolarization during 8-12 mmol L-1 [K+ ]E . Brain endothelial KIR channel activity modulates bidirectional spread of vasoreactive signals with enhanced regulation of EC coupling during AD pathology.

Sex- and Neuropsychiatric-Dependent Circadian Alterations in Daily Voluntary Physical Activity Engagement and Patterns in Aged 3xTg-AD Mice.

Alzheimer's disease (AD) patients suffer from circadian rhythm alterations affecting their daily physical activity patterns with less willingness to perform a voluntary exercise. In preclinical studies, there is no clarity on whether animal models of AD can replicate these impairments. Here, we provide a proof of concept of the performance and behavioral effects of four weeks of voluntary wheel running (VWR) in a group of 14-month-old male and female 3xTg-AD mice at advanced stages of AD and the daily variance (behavioral circadian rhythmicity) of VWR associated with sex and their neuropsychiatric-like phenotype. Higher levels of horizontal exploration in the open field (OF) test were found in mice submitted to exercise. A linear mixed effect model showed significant sex-dependent differences in the VWR activity performed on the first night of follow-up, with high-NIBI males running less than high-NIBI females. Thus, an influence of NPS-like symptoms on the circadian patterns of VWR may account for such differences. In addition, males remained more active than females during diurnal periods. We hypothesize that this increment in energy expenditure during resting periods may be related to hyperactive behavior, similar to that observed in humans' exacerbated agitation or sundowning behavior. These findings support the usage of the 3xTg-AD mouse as a reliable model for studying circadian rhythm alterations in AD and, at the translational level, the importance of tailored and individualized physical activity programs in clinical settings.

Sex-dependent worsening of NMDA-induced responses, anxiety, hypercortisolemia, and organometry of early peripheral immunoendocrine impairment in adult 3xTg-AD mice and their long-lasting ontogenic modulation by neonatal handling

The neuroimmunomodulation hypothesis for Alzheimer’s disease (AD) postulates that alterations in the innate immune system triggered by damage signals result in adverse effects on neuronal functions. The peripheral immune system and neuroimmunoendocrine communication are also impaired. Here we provide further evidence using a longitudinal design that also studied the long-lasting effects of an early life sensorial intervention (neonatal handling, from postnatal day 1–21) in 6-month-old (early stages of the disease) male and female 3xTg-AD mice compared to age- and sex-matched non-transgenic (NTg) mice with normal aging. The behavioral patterns elicited by the direct exposure to an open field, and the motor depression response evoked by NMDA (25 mg/kg, i.p) were found correlated to the organometry of peripheral immune-endocrine organs (thymus involution, splenomegaly, and adrenal glands’ hypertrophy) and increased corticosterone levels, suggesting their potential value for diagnostic and biomonitoring.The NMDA-induced immediate and depressant motor activity and endocrine (corticosterone) responses were sensitive to sex and AD-genotype, suggesting worse endogenous susceptibility/neuroprotective response to glutamatergic excitotoxicity in males and in the AD-genotype. 3xTg-AD females showed a reduced immediate response, whereas the NTg showed higher responsiveness to subsequent NMDA-induced depressant effect than their male counterparts. The long-lasting ontogenic modulation by handling was shown as a potentiation of NMDA-depressant effect in NTg males and females, while sex × treatment effects were found in 3xTg-AD mice. Finally, NMDA-induced corticosterone showed sex, genotype and interaction effects with sexual dimorphism enhanced in the AD-genotype, suggesting different endogenous vulnerability/neuroprotective capacities and modulation of the neuroimmunoendocrine system.

Selective orexin 1 receptor antagonist SB-334867 aggravated cognitive dysfunction in 3xTg-AD mice

Cognitive dysfunction is the main clinical manifestation of Alzheimer’s disease (AD). Previous research found that elevated orexin level in the cerebrospinal fluid was closely related to the course of AD, and orexin-A treatment could increase amyloid β protein (Aβ) deposition and aggravate spatial memory impairment in APP/PS1 mice. Furthermore, recent research found that dual orexin receptor (OXR) antagonist might affect Aβ level and cognitive dysfunction in AD, but the effects of OX1R or OX2R alone is unreported until now. Considering that OX1R is highly expressed in the hippocampus and plays important roles in learning and memory, the effects of OX1R in AD cognitive dysfunction and its possible mechanism should be investigated. In the present study, selective OX1R antagonist SB-334867 was used to block OX1R. Then, different behavioral tests were performed to observe the effects of OX1R blockade on cognitive function of 3xTg-AD mice exhibited both Aβ and tau pathology, in vivo electrophysiological recording and western blot were used to investigate the potential mechanism. The results showed that chronic OX1R blockade aggravated the impairments of short-term working memory, long-term spatial memory and synaptic plasticity in 9-month-old female 3xTg-AD mice, increased levels of soluble Aβ oligomers and p-tau, and decreased PSD-95 expression in the hippocampus of 3xTg-AD mice. These results indicate that the detrimental effects of SB-334867 on cognitive behaviors in 3xTg-AD mice are closely related to the decrease of PSD-95 and depression of in vivo long-term potentiation (LTP) caused by increased Aβ oligomers and p-tau.

Ovariectomy-induced hormone deprivation aggravates Aβ1-42 deposition in the basolateral amygdala and cholinergic fiber loss in the cortex but not cognitive behavioral symptoms in a triple transgenic mouse model of Alzheimer's disease.

Alzheimer’s disease is the most common type of dementia, being highly prevalent in elderly women. The advanced progression may be due to decreased hormone synthesis during post-menopause as estradiol and progesterone both have neuroprotective potentials. We aimed to confirm that female hormone depletion aggravates the progression of dementia in a triple transgenic mouse model of Alzheimer’s disease (3xTg-AD). As pathological hallmarks are known to appear in 6-month-old animals, we expected to see disease-like changes in the 4-month-old 3xTg-AD mice only after hormone depletion. Three-month-old female 3xTg-AD mice were compared with their age-matched controls. As a menopause model, ovaries were removed (OVX or Sham surgery). After 1-month recovery, the body composition of the animals was measured by an MRI scan. The cognitive and anxiety parameters were evaluated by different behavioral tests, modeling different aspects (Y-maze, Morris water maze, open-field, social discrimination, elevated plus maze, light–dark box, fox odor, operant conditioning, and conditioned fear test). At the end of the experiment, uterus was collected, amyloid-β accumulation, and the cholinergic system in the brain was examined by immunohistochemistry. The uterus weight decreased, and the body weight increased significantly in the OVX animals. The MRI data showed that the body weight change can be due to fat accumulation. Moreover, OVX increased anxiety in control, but decreased in 3xTg-AD animals, the later genotype being more anxious by default based on the anxiety z-score. In general, 3xTg-AD mice moved less. In relation to cognition, neither the 3xTg-AD genotype nor OVX surgery impaired learning and memory in general. Despite no progression of dementia-like behavior after OVX, at the histological level, OVX aggravated the amyloid-β plaque deposition in the basolateral amygdala and induced early cholinergic neuronal fiber loss in the somatosensory cortex of the transgenic animals. We confirmed that OVX induced menopausal symptoms. Removal of the sexual steroids aggravated the appearance of AD-related alterations in the brain without significantly affecting the behavior. Thus, the OVX in young, 3-month-old 3xTg-AD mice might be a suitable model for testing the effect of new treatment options on structural changes; however, to reveal any beneficial effect on behavior, a later time point might be needed.

White matter pathology in alzheimer’s transgenic mice with chronic exposure to low-level ambient fine particulate matter

BackgroundAir pollution, especially fine particulate matter (PM), can cause brain damage, cognitive decline, and an increased risk of neurodegenerative disease, especially alzheimer’s disease (AD). Typical pathological findings of amyloid and tau protein accumulation have been detected in the brain after exposure in animal studies. However, these observations were based on high levels of PM exposure, which were far from the WHO guidelines and those present in our environment. In addition, white matter involvement by air pollution has been less reported. Thus, this experiment was designed to simulate the true human world and to discuss the possible white matter pathology caused by air pollution.Results6 month-old female 3xTg-AD mice were divided into exposure and control groups and housed in the Taipei Air Pollutant Exposure System (TAPES) for 5 months. The mice were subjected to the Morris water maze test after exposure and were then sacrificed with brain dissection for further analyses. The mean mass concentration of PM2.5 during the exposure period was 13.85 μg/m3. After exposure, there was no difference in spatial learning function between the two groups, but there was significant decay of memory in the exposure group. Significantly decreased total brain volume and more neuronal death in the cerebral and entorhinal cortex and demyelination of the corpus callosum were noted by histopathological staining after exposure. However, there was no difference in the accumulation of amyloid or tau on immunohistochemistry staining. For the protein analysis, amyloid was detected at significantly higher levels in the cerebral cortex, with lower expression of myelin basic protein in the white matter. A diffuse tensor image study also revealed insults in multiple white matter tracts, including the optic tract.ConclusionsIn conclusion, this pilot study showed that even chronic exposure to low PM2.5 concentrations still caused brain damage, such as gross brain atrophy, cortical neuron damage, and multiple white matter tract damage. Typical amyloid cascade pathology did not appear prominently in the vulnerable brain region after exposure. These findings imply that multiple pathogenic pathways induce brain injury by air pollution, and the optic nerve may be another direct invasion route in addition to olfactory nerve.