Male 5xFAD Mice Research Articles

Restoring capillary-to-arteriole signaling in Alzheimer's Disease

There is growing evidence for vascular contributions to cognitive impairments in Alzheimer’s Disease (AD). Functional hyperemia, which refers to local increases in blood flow in response to local increases in brain activity, is impaired early in patients and animal models of AD. The mechanisms underlying functional hyperemia are referred to as neurovascular coupling (NVC). A key player in NVC is the inward rectifying K+ channel (Kir2.1) in capillary endothelial cells (cECs). Activation of Kir2.1 channels initiates a retrograde hyperpolarizing signal which causes upstream arterioles to dilate and increase local blood flow. Studies have shown that Kir2.1 channel function is impaired in the 5xFAD mouse model for AD, and administration of Kir2.1 cofactor, phosphatidylinositol 4,5-bisphosphate (PIP2), was suffcient to restore channel function and functional hyperemia deficits. The objective of this study is to characterize the effect of AD on NVC. Since AD is characterized by the deposition of amyloid beta (Aβ), we hypothesize that Aβ is impairing Kir2.1 function in cECs at an early stage of the disease. Using 6-month-old female and male 5xFAD mice, we performed our ex vivo capillary-parenchymal arteriole (CaPA) technique to investigate NVC responses by directly stimulating capillaries and recording upstream arteriolar dilation in real time. We further tested the direct effect of Aβ oligomers (200 nM) and reactive oxygen species (ROS) scavengers (superoxide dismutase 120 U/mL + catalase 1000 U/mL), to probe their effect on the NVC response. Finally, to test the ability of PIP2 to rescue the NVC response, systemic administration via intraperitoneal injection was performed one hour before CaPA experiment. Consistent with a NVC impairment in AD, capillary stimulation with 10 mM K+ failed to evoke upstream arteriolar dilation in 5xFAD mice at an early stage, and this impairment was recapitulated in wild type mice after treatment with Aβ. Importantly, Aβ only caused a modest arteriole constriction (20.11% ± 5.24 %) but did not impair its ability to dilate in response to direct stimulation with 10 mM K+, suggesting an effect on cECs. ROS scavenging during Aβ treatment prevented NVC impairment as 10 mM K+ capillary stimulation induced an upstream arteriolar dilation of 59% of max dilation. Finally, systemic administration of PIP2 also rescued the NVC response to the levels observed in control conditions. Taken together, our results suggest that Aβ is causing impairment in NVC via increasing ROS production, specifically at the capillary endothelial level. This increase in ROS is likely leading to PIP2 degradation and causing Kir2.1 channel dysfunction. Administration of PIP2 was suffcient to rescue the NVC response, suggesting a possible strategy for rescuing functional hyperemia in AD. This study was supported by a research grant from the Center for Women’s Health Research located at the University of Colorado Anschutz Medical Campus; 2 research grants from the University of Pennsylvania Orphan Disease Center in partnership with the cureCADASIL (2019 and 2022), the National Institute of Neurological Disorders and Stroke RF1/R01NS129022, and the National Heart, Lung, and Blood Institute R01HL136636 to FD. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

147 Medial Septal Nucleus Theta Stimulation Improves Spatial Working Memory and Increases Hippocampal Neurovascular Activity in a Mouse Model of Alzheimers Disease (5xFAD)

INTRODUCTION: Alzheimer’s disease (AD) remains among the most prolific neurodegenerative disorders. The pathogenesis is the result, in part, of aberrant cerebral circuitry; in particular, the mesolimbic-septohippocampal circuit and its associated medial septal nucleus (MSN). Interestingly, the administration of theta stimulation via deep brain stimulation (DBS) to the MSN has been demonstrated to increase hippocampal theta oscillations, which are critical to the role of the hippocampus in learning and memory. Recently, functional ultrasound imaging (fUSI) has emerged as a method of evaluating large-scale neural activity with both high spatiotemporal resolution (∼100 μm3) and sensitivity to slow blood flow (∼1 mm/s). fUSI allows for the in-vivo analyses of neurovascular changes secondary to our theta-burst neuromodulatory intervention. METHODS: 12 male 5xFAD mice were partitioned into two groups, one received theta-burst stimulation targeting the MSN (n = 4) and the other acted as controls (n = 8). Following stimulation, spatial working memory was assessed via the Barnes maze task. An additional 44 C57BL/6 mice were partitioned into three groups: one receiving theta (7.7 Hz) stimulation to the MSN (n = 16), one receiving gamma stimulation to the MSN (100 Hz, n = 14), and the remaining group acting as the control (n = 14). RESULTS: The MSN theta stimulation group of 5xFAD mice demonstrated decreased latency compared to controls (p = 0.003). CBF volumes in the hippocampus were relatively increased in the theta-burst group when compared to both control and gamma stimulation groups. CONCLUSIONS: Our results demonstrate significant improvement in cognitive performance secondary to MSN theta-burst stimulation. Additionally, these changes were mirrored by CBF volume increases in the hippocampus in a frequency-dependent manner. These findings provide evidence for the therapeutic role of theta MSN stimulation in the treatment of AD and furthers fUSI’s role in evaluating in-vivo CBF.

Sex-specific changes in protein expression of membrane transporters in the brain cortex of 5xFAD mouse model of Alzheimer's disease.

Membrane transporters playing an important role in the passage of drugs, metabolites and nutrients across the membranes of the brain cells have been shown to be involved in pathogenesis of Alzheimer's disease (AD). However, little is known about sex-specific changes in transporter protein expression at the brain in AD. Here, we investigated sex-specific alterations in protein expression of three ATP-binding cassette (ABC) and five solute carriers (SLC) transporters in the prefrontal cortex of a commonly used model of familial AD (FAD), 5xFAD mice. Sensitive liquid chromatography tandem mass spectrometry-based quantitative targeted absolute proteomic analysis was applied for absolute quantification of transporter protein expression. We compared the changes in transporter protein expressions in 7-month-old male and female 5xFAD mice versus sex-matched wild-type mice. The study revealed a significant sex-specific increase in protein expression of ABCC1 (p = 0.007) only in male 5xFAD mice as compared to sex-matched wild-type animals. In addition, the increased protein expression of glucose transporter 1 (p = 0.01), 4F2 cell-surface antigen heavy chain (p = 0.01) and long-chain fatty acid transport protein 1 (p = 0.02) were found only in female 5xFAD mice as compared to sex-matched wild-type animals. Finally, protein expression of alanine/serine/cysteine/threonine transporter 1 was upregulated in both male (p = 0.02) and female (p = 0.002) 5xFAD mice. The study provides important information about sex-specific changes in brain cortical transporter expression in 5xFAD mice, which will facilitate drug development of therapeutic strategies for AD targeting these transporters and drug delivery research.

Proteomic Signaling of Dual-Specificity Phosphatase 4 (DUSP4) in Alzheimer's Disease.

DUSP4 is a member of the DUSP (dual-specificity phosphatase) subfamily that is selective to the mitogen-activated protein kinases (MAPK) and has been implicated in a range of biological processes and functions in Alzheimer's disease (AD). In this study, we utilized the stereotactic delivery of adeno-associated virus (AAV)-DUSP4 to overexpress DUSP4 in the dorsal hippocampus of 5xFAD and wildtype (WT) mice, then used mass spectrometry (MS)-based proteomics along with the label-free quantification to profile the proteome and phosphoproteome in the hippocampus. We identified protein expression and phosphorylation patterns modulated in 5xFAD mice and examined the sex-specific impact of DUSP4 overexpression on the 5xFAD proteome/phosphoproteome. In 5xFAD mice, a substantial number of proteins were up- or down-regulated in both male and female mice in comparison to age and sex-matched WT mice, many of which are involved in AD-related biological processes, such as activated immune response or suppressed synaptic activities. Many proteins in pathways, such as immune response were found to be suppressed in response to DUSP4 overexpression in male 5xFAD mice. In contrast, such a shift was absent in female mice. For the phosphoproteome, we detected an array of phosphorylation sites regulated in 5xFAD compared to WT and modulated via DUSP4 overexpression in each sex. Interestingly, 5xFAD- and DUSP4-associated phosphorylation changes occurred in opposite directions. Strikingly, both the 5xFAD- and DUSP4-associated phosphorylation changes were found to be mostly in neurons and play key roles in neuronal processes and synaptic functions. Site-centric pathway analysis revealed that both the 5xFAD- and DUSP4-associated phosphorylation sites were enriched for a number of kinase sets in females but only a limited number of sets of kinases in male mice. Taken together, our results suggest that male and female 5xFAD mice responded to DUSP4 overexpression via shared and sex-specific molecular mechanisms, which might underly similar reductions in amyloid pathology in both sexes while learning deficits were reduced in only females with DUSP4 overexpression. Finally, we validated our findings with the sex-specific AD-associated proteomes in human cohorts and further developed DUSP4-centric proteomic network models and signaling maps for each sex.

Dual-specificity protein phosphatase 6 (DUSP6) overexpression reduces amyloid load and improves memory deficits in male 5xFAD mice.

Dual specificity protein phosphatase 6 (DUSP6) was recently identified as a key hub gene in a causal VGF gene network that regulates late-onset Alzheimer's disease (AD). Importantly, decreased DUSP6 levels are correlated with an increased clinical dementia rating (CDR) in human subjects, and DUSP6 levels are additionally decreased in the 5xFAD amyloidopathy mouse model. To investigate the role of DUSP6 in AD, we stereotactically injected AAV5-DUSP6 or AAV5-GFP (control) into the dorsal hippocampus (dHc) of both female and male 5xFAD or wild type mice, to induce overexpression of DUSP6 or GFP. Barnes maze testing indicated that DUSP6 overexpression in the dHc of 5xFAD mice improved memory deficits and was associated with reduced amyloid plaque load, Aß1-40 and Aß1-42 levels, and amyloid precursor protein processing enzyme BACE1, in male but not in female mice. Microglial activation, which was increased in 5xFAD mice, was significantly reduced by dHc DUSP6 overexpression in both males and females, as was the number of "microglial clusters," which correlated with reduced amyloid plaque size. Transcriptomic profiling of female 5xFAD hippocampus revealed upregulation of inflammatory and extracellular signal-regulated kinase pathways, while dHc DUSP6 overexpression in female 5xFAD mice downregulated a subset of genes in these pathways. Gene ontology analysis of DEGs (p < 0.05) identified a greater number of synaptic pathways that were regulated by DUSP6 overexpression in male compared to female 5xFAD. In summary, DUSP6 overexpression in dHc reduced amyloid deposition and memory deficits in male but not female 5xFAD mice, whereas reduced neuroinflammation and microglial activation were observed in both males and females, suggesting that DUSP6-induced reduction of microglial activation did not contribute to sex-dependent improvement in memory deficits. The sex-dependent regulation of synaptic pathways by DUSP6 overexpression, however, correlated with the improvement of spatial memory deficits in male but not female 5xFAD.

DRhQ: a novel therapeutic that improves cognition and mitochondrial function and reduces neuroinflammation in beta‐amyloid overexpressing Mice

AbstractBackgroundNeuroinflammation and mitochondrial dysfunction are central to the progression of Alzheimer’s disease (AD), provoking neuronal loss and cognitive decline. Identifying strategies for mitigating these responses could therefore be of significant therapeutic relevance. Evidence suggests that the inflammatory axis mediated by macrophage migration inhibitory factory (MIF) binding to its receptor, CD74, plays an important role in many central nervous system (CNS) disorders like AD. Our group has developed DRhQ, a novel CD74 binding construct that competitively inhibits MIF binding, blocks T‐cell and macrophage activation and migration into the CNS, enhances anti‐inflammatory microglia cell numbers and reduces pro‐inflammatory gene expression. DRhQ treatment resulted in reduced neuroinflammation and disease severity in a mouse model of multiple sclerosis. Because neuroinflammation is a prominent in the AD brain as well we hypothesized that DRhQ could be a similarly effective neuroprotective agent in the context of AD. We evaluated this hypothesis using the 5xFAD mouse model of β‐amyloid (Aβ) accumulation.MethodSix month old male and female 5xFAD mice and their wild‐type littermates will be treated with 0 or 100 ug of DRhQ subcutaneously for five days followed by a 3 day booster series weekly for the next 3 weeks. During the 4th week of treatment mice were evaluated cognitively using the novel object recognition test and brain tissue was harvested to evaluate mitochondrial function, Aβ pathology as well as markers of microglial activation and neuroinflammation.ResultDRhQ improved cognition and reduced neuroinflammatory markers in both male and female 5xFAD mice. DRhQ also significantly attenuated deficits in cortical mitochondrial function in female 5xFAD mice and elicited a similar, though non‐significant, effect in male 5xFAD mice. Aβ plaque burden was likewise reduced in the hippocampus and cortex of female 5xFAD mice but not significantly reduced in male 5xFAD animals.ConclusionThese results suggest that DRhQ attenuates Aβ pathology, mitochondrial dysfunction and neuroinflammation and improves cognitive function in 5xFAD mice. Future experiments will explore dose response effects and investigate the apparent sex differences in the magnitude of effect of DRhQ on specific endpoints in these mice as well as evaluate the effects of DRhQ in models of tauopathy.

The olfactory working memory capacity paradigm: A more sensitive and robust method of assessing cognitive function in male 5XFAD mice.

The olfactory working memory capacity (OWMC) paradigm is able to detect cognitive deficits in 5XFAD mice (an animal model of Alzheimer's disease [TG]) as early as 3 months of age, while other behavioral paradigms detect cognitive deficits only at 4-5 months of age. Therefore, we aimed to demonstrate that the OWMC paradigm is more sensitive and consistent in the early detection of declines in cognitive function than other commonly used behavioral paradigms. The prefrontal cortex (PFC), retrosplenial cortex (RSC), subiculum (SUB), and amygdala (AMY) of 5XFAD mice were harvested and subjected to immunostaining to detect the expression of β-amyloid (Aβ). Additionally, we compared the performance of 3-month-old male 5XFAD mice on common behavioral paradigms for assessing cognitive function (i.e., the open field [OF] test, novel object recognition [NOR] test, novel object location [NOL] test, Y-maze, and Morris water maze [MWM]) with that on the OWMC task. In the testing phase of the OWMC task, we varied the delay periods to evaluate the working memory capacity (WMC) of wild-type (WT) mice. Significant amyloid plaque deposition was observed in the PFC, RSC, SUB, and AMY of 3-month-old male 5XFAD mice. However, aside from the OWMC task, the other behavioral tests failed to detect cognitive deficits in 5XFAD mice. Additionally, to demonstrate the efficacy of the OWMC task in assessing WMC, we varied the retention delay periods; we found that the WMC of WT mice decreased with longer delay periods. The OWMC task is a sensitive and robust behavioral assay for detecting changes in cognitive function.

Upregulation of sFRP1 Is More Profound in Female than Male 5xFAD Mice and Positively Associated with Amyloid Pathology.

The prevalence of Alzheimer's disease is greater in women, but the underlying mechanisms remain to be elucidated. We herein demonstrated that α-secretase ADAM10 was downregulated and ADAM10 inhibitor sFRP1 was upregulated in 5xFAD mice. While there were no sex effects on ADAM10 protein and sFRP1 mRNA levels, female 5xFAD and age-matched non-transgenic mice exhibited higher levels of sFRP1 protein than corresponding male mice. Importantly, female 5xFAD mice accumulated more Aβ than males, and sFRP1 protein levels were positively associated with Aβ42 levels in 5xFAD mice. Our study suggests that sFRP1 is associated with amyloid pathology in a sex-dependent manner.

Effects of Striatal Amyloidosis on the Dopaminergic System and Behavior: A Comparative Study in Male and Female 5XFAD Mice.

Nearly two-thirds of patients diagnosed with Alzheimer's disease (AD) are female. In addition, female patients with AD have more significant cognitive impairment than males at the same disease stage. This disparity suggests there are sex differences in AD progression. While females appear to be more affected by AD, most published behavioral studies utilize male mice. In humans, there is an association between antecedent attention-deficit/hyperactivity disorder and increased risk of dementia. Functional connectivity studies indicate that dysfunctional cortico-striatal networks contribute to hyperactivity in attention deficit hyperactivity disorder. Higher plaque density in the striatum accurately predicts the presence of clinical AD pathology. In addition, there is a link between AD-related memory dysfunction and dysfunctional dopamine signaling. With the need to consider sex as a biological variable, we investigated the influence of sex on striatal plaque burden, dopaminergic signaling, and behavior in prodromal 5XFAD mice. Six-month-old male and female 5XFAD and C57BL/6J mice were evaluated for striatal amyloid plaque burden, locomotive behavior, and changes in dopaminergic machinery in the striatum. 5XFAD female mice had a higher striatal amyloid plaque burden than male 5XFAD mice. 5XFAD females, but not males, were hyperactive. Hyperactivity in female 5XFAD mice was associated with increased striatal plaque burden and changes in dopamine signaling in the dorsal striatum. Our results indicate that the progression of amyloidosis involves the striatum in females to a greater extent than in males. These studies have significant implications for using male-only cohorts in the study of AD progression.

GABA-Positive Astrogliosis in Sleep-Promoting Areas Associated with Sleep Disturbance in 5XFAD Mice.

Sleep disturbances, a debilitating symptom of Alzheimer's disease (AD), are associated with neuropathological changes. However, the relationship between these disturbances and regional neuron and astrocyte pathologies remains unclear. This study examined whether sleep disturbances in AD result from pathological changes in sleep-promoting brain areas. Male 5XFAD mice underwent electroencephalography (EEG) recordings at 3, 6, and 10 months, followed by an immunohistochemical analysis of three brain regions associated with sleep promotion. The findings showed that 5XFAD mice demonstrated reduced duration and bout counts of nonrapid eye movement (NREM) sleep by 6 months and reduced duration and bout counts of rapid eye movement (REM) sleep by 10 months. Additionally, peak theta EEG power frequency during REM sleep decreased by 10 months. Sleep disturbances correlated with the total number of GFAP-positive astrocytes and the ratio of GFAP- and GABA-positive astrocytes across all three sleep-associated regions corresponding to their roles in sleep promotion. The presence of GABRD in sleep-promoting neurons indicated their susceptibility to inhibition by extrasynaptic GABA. This study reveals that neurotoxic reactive astrogliosis in NREM and REM sleep-promoting areas is linked to sleep disturbances in 5XFAD mice, which suggests a potential target for the treatment of sleep disorders in AD.

Search strategy analysis reveals sex‐specific deficits in spatial reference memory in 5xFAD mice

AbstractBackgroundSpatial disorientation is one of the earliest symptoms in Alzheimer’s disease. Search strategy classification systems to study navigational deficits in Alzheimer disease have been developed but aren’t widely available. Hence, we studied the navigational strategies in the 5xFAD murine model and evaluated differences between sexes.MethodWe studied spatial reference memory in 3‐month‐, 7‐month‐ and 12‐month‐old female and male 5xFAD mice and their aged‐matched controls in the Morris water maze. Furthermore, a detailed analysis of the swimming strategies using “Pathfinder” was performed. Navigational strategies were classified in spatial (“direct path”, “directed search”, “focal search”, “indirect search”) and non‐spatial categories (“chaining”, “scanning”, “random search”, thigmotaxis”).ResultEscape latency was prolonged in 5xFAD mice compared to WT, only at 7 and 12 months, with greater magnitude in females than males. Regarding search strategies during acquisition training, the most common spatial strategy was “indirect search” whereas “random search” was the most frequent non‐spatial strategy. At 3 months, no clear difference was seen between 5xFAD and WT animals. However, at 7 months, 5xFAD mice used more frequently non‐spatial strategies. Although transgenic mice progressively shifted to more spatial strategies as they learned (mainly “indirect search”), WT animals applied more frequently spatial strategies with higher cognitive value such as “direct path”, “directed search” and “focal search”. In comparison, 12 month‐old 5xFAD mice used more non‐spatial strategies, mainly “random search”, than the WT and their transition to “indirect search” occurred more rarely than in younger mice. Overall, all animals increased the use of non‐spatial strategies as they aged but this increment was greater and earlier in 5xFAD females than in 5xFAD males. Lastly, in the probe trial, 5xFAD female mice at 3 and 12 months applied “random search” more often, while the WT chose “indirect search” with higher frequency. No difference was seen in males during probe trial.ConclusionConventional behavior analysis of escape latencies revealed spatial reference memory deficits in 7m and 12m 5XFAD mice. Deficits in spatial memory were more pronounced in female 5XFAD mice than in male mice. 5XFAD mice, regardless of sex, used a qualitatively and quantitatively different search strategy pattern than WT animals.

Search Strategy Analysis of 5xFAD Alzheimer Mice in the Morris Water Maze Reveals Sex- and Age-Specific Spatial Navigation Deficits

Spatial disorientation and navigational impairments are not only some of the first memory deficits in Alzheimer's disease, but are also very disease-specific. In rodents, the Morris Water Maze is used to investigate spatial navigation and memory. Here, we examined the spatial memory in the commonly used 5xFAD Alzheimer mouse model in a sex- and age-dependent manner. Our findings show first spatial learning deficits in 7-month-old female 5xFAD and 12-month-old male 5xFAD mice, respectively. While the assessment of spatial working memory using escape latencies provides a global picture of memory performance, it does not explain how an animal solves a spatial task. Therefore, a detailed analysis of swimming strategies was performed to better understand the behavioral differences between 5xFAD and WT mice. 5xFAD mice used a qualitatively and quantitatively different search strategy pattern than wildtype animals that used more non-spatial strategies and showed allocentric-specific memory deficits. Furthermore, a detailed analysis of swimming strategies revealed allocentric memory deficits in the probe trial in female 3-month-old and male 7-month-old 5xFAD animals before the onset of severe reference memory deficits. Overall, we could demonstrate that spatial navigation deficits in 5xFAD mice are age- and sex-dependent, with female mice being more severely affected. In addition, the implementation of a search strategy classification system allowed an earlier detection of behavioral differences and therefore could be a powerful tool for preclinical drug testing in the 5xFAD model.

The Helico Maze Detects Early Impairment of Reference Memory at Three Months of Age in the 5XFAD Mouse Model of Alzheimer's Disease.

The 5XFAD model of Alzheimer's disease (AD) bearing five familial mutations of Alzheimer's disease on human APP and PSEN1 transgenes shows deposits of amyloid-β peptide (Aβ) as early as 2 months, while deficits in long-term memory can be detected at 4 months using the highly sensitive olfactory-dependent tests that we previously reported. Given that detecting early dysfunctions in AD prior to overt pathology is of major interest in the field, we sought to detect memory deficits at earlier stages of the disease in 3-month-old male 5XFAD mice. To this end, we used the Helico Maze, a behavioral task that was recently developed and patented. This device allows deeper analysis of learning and subcategories of hippocampal-dependent long-term memory using olfactory cues. Eight male 5XFAD and 6 male wild-type (WT: C57Bl6 background) mice of 3 months of age were tested in the Helico Maze. The results demonstrated, for the first time, a starting deficit of pure reference long-term memory. Interestingly, memory impairment was clearly correlated with Aβ deposits in the hippocampus. While we also found significant differences in astrogliosis between 5XFAD and WT mice, this was not correlated with memory abilities. Our results underline the efficiency of this new olfactory-dependent behavioral task, which is easy to use, with a small cohort of mice. Using the Helico Maze may open new avenues to validate the efficacy of treatments that target early events related to the amyloid-dependent pathway of the disease and AD progression.

Loss of body weight in old 5xFAD mice and the alteration of gut microbiota composition.

The cause of age-related body weight loss in Alzheimer's disease (AD) is unclear. We compared the differences in food intake, malabsorption, locomotor activity, and gut microbiota composition between 5xFAD mice, a useful model of AD, and wild-type (WT) mice to investigate the mechanisms underlying lower body weight in 5xFAD mice. Fifteen-month-old male 5xFAD mice and age-matched WT mice were divided into four groups: a control diet (CD) or a high-fat diet (HFD). After feeding CD or HFD for eight to nine weeks, 5xFAD mice had a significantly lower body weight than WT mice regardless of diet (p<0.05). Additionally, the 5xFAD mice did not show a reduction in food intake compared to the WT mice regardless of diet. To evaluate malabsorption, we performed a fecal fat test. There was no obvious fecal fat in both the 5xFAD mice and WT mice. However, 5xFAD mice showed greater locomotor activity than WT mice in the Y-maze test. The comprehensive analysis of gut microbiota composition showed that 15-month-old 5xFAD mice had more Proteobacteria population and fewer Actinobacteria and Bifidobacteriales populations than WT mice. To investigate the effects of fructooligosaccharides (FOS), we administrated FOS to 15-month-old 5xFAD mice. FOS administration decreased Proteobacteria and increased Actinobacteria population, although that did not change Bifidobacteriales population. Moreover, cognitive impairment and body weight of 5xFAD mice were not changed by FOS administration. In conclusion, loss of body weight in 15-month-old 5xFAD mice might be partially derived from excess energy output by hyperactivity. Moreover, 15-month-old 5xFAD mice might have unique alteration of gut microbiota composition and the potential resistance to FOS.

Transcorneal electrical stimulation enhances cognitive functions in aged and 5XFAD mouse models.

Dementia is a major burden on global health for which there are no effective treatments. The use of noninvasive visual stimulation to ameliorate cognitive deficits is a novel concept that may be applicable for treating dementia. In this study, we investigated the effects of transcorneal electrical stimulation (TES) on memory enhancement using two mouse models, in aged mice and in the 5XFAD model of Alzheimer's disease. After 3 weeks of TES treatment, mice were subjected to Y-maze and Morris water maze tests to assess hippocampal-dependent learning and memory. Immunostaining of the hippocampus of 5XFAD mice was also performed to examine the effects of TES on amyloid plaque pathology. The results showed that TES improved the performance of both aged and 5XFAD mice in memory tests. TES also reduced hippocampal plaque deposition in male, but not female, 5XFAD mice. Moreover, TES significantly reversed the downregulated level of postsynaptic protein 95 in the hippocampus of male 5XFAD mice, suggesting the effects of TES involve a postsynaptic mechanism. Overall, these findings support further investigation of TES as a potential treatment for cognitive dysfunction and mechanistic studies of TES effects in other dementia models.

Systemic inflammation elicits distinct brain immune signaling dynamics in female and male mice with AD pathology

Women are disproportionately affected by Alzheimer's disease (AD), even after adjusting for age and socioeconomic status. Sex-based biological differences in immune response, including hormonal and X-linked immune genes, may contribute to differences in disease progression. However, we have limited understanding of the role of sex in modulating immune response and how these differences are affected by AD pathology. Here, we hypothesized that female and male mice would exhibit distinctive neural immune signaling in response to challenge with the pro-inflammatory stimulus lipopolysaccharides (LPS), and that these differences would be exacerbated in model of AD pathology compared to wild-type controls.Female and male six-month-old 5xFAD amyloid beta (Aβ) and wild type (WT) littermate mice were interperitoneally injected with LPS or saline vehicle once daily for 0,1, or 4 days (N=4). Luminex multiplexed ELISAs was used to quantify inflammatory cytokines, MAPK intracellular signaling phospho-proteins in cortical tissues.In the saline group, we observed no differences between females and males in either 5xFAD or WT mice. Additionally, in wild type mice, we observed no significant differences between females and males after either one or four daily injections of LPS. In contrast, in 5xFAD mice injected with LPS, we found significant differences between females and males after either one or four once daily injections. In particular, female 5xFAD mice after a single injection exhibited significantly increased profile (p=0.014, two tailed t-test) of phospho-proteins and cytokines, including MEK, cJUN, IL-13, IL-17, IL-10, and IL-1α compared to males. After four once-daily LPS injections, female 5xFAD mice exhibited a significantly different profile (p=0.016, two tailed t-test) consisting of increased phosphorylation of ATF2, Mek, cJUN, and HSP-27, together with decreased expression of multiple proinflammatory cytokines including IL-9, VEGF, MIP-1β compared to males.Our data reveal that although sex is not a significant determinant of neural immune response in WT mice, it significantly affects neural immune response to LPS challenge in 5xFAD mice. These data suggest distinctive immune processes in female vs male 5xFAD mice that are not present in healthy controls. Future work will consider the effects of Aβ pathology on these results.

CERTL reduces C16 ceramide, amyloid-β levels and inflammation in a model of Alzheimer's disease.

Dysregulation of ceramide and sphingomyelin levels have been suggested to contribute to the pathogenesis of Alzheimer's disease (AD). Ceramide transfer proteins (CERTs) are ceramide carriers which are crucial for ceramide and sphingomyelin balance in cells. Extracellular forms of CERTs co-localize with amyloid-β (Aβ) plaques in AD brains. To date, the significance of these observations for the pathophysiology of AD remains uncertain. A plasmid expressing CERTL , the long isoform of CERTs, was used to study the interaction of CERTL with amyloid precursor protein (APP) by co-immunoprecipitation and immunofluorescence in HEK cells. The recombinant CERTL protein was employed to study interaction of CERTL with amyloid-β (Aβ), Aβ aggregation process in presence of CERTL , and the resulting changes in Aβ toxicity in neuroblastoma cells. CERTL was overexpressed in neurons by adeno associated virus (AAV) in a mouse model of familial AD (5xFAD). Ten weeks after transduction, animals were challenged with behavior tests for memory, anxiety and locomotion. At week twelve, brains were investigated for sphingolipid levels by mass spectrometry, plaques and neuroinflammation by immunohistochemistry, gene expression and/or immunoassay. Here, we report that CERTL , binds to APP, modifies Aβ aggregation and reduces Aβ neurotoxicity in vitro. Furthermore, we show that intracortical injection of AAV, mediating the expression of CERTL , decreases levels of ceramide d18:1/16:0 and increases sphingomyelin levels in the brain of male 5xFAD mice. CERTL in vivo over-expression has a mild effect on animal locomotion, decreases Aβ formation and modulates microglia by decreasing their pro-inflammatory phenotype. Our results demonstrate a crucial role of CERTL in regulating ceramide levels in the brain, in amyloid plaque formation and neuroinflammation, thereby opening research avenues for therapeutic targets of AD and other neurodegenerative diseases.

Quantitative Brain Positron Emission Tomography in Female 5XFAD Alzheimer Mice: Pathological Features and Sex-Specific Alterations.

Successful back-translating clinical biomarkers and molecular imaging methods of Alzheimer's disease (AD), including positron emission tomography (PET), are very valuable for the evaluation of new therapeutic strategies and increase the quality of preclinical studies. 18F-Fluorodeoxyglucose (FDG)–PET and 18F-Florbetaben–PET are clinically established biomarkers capturing two key pathological features of AD. However, the suitability of 18F-FDG– and amyloid–PET in the widely used 5XFAD mouse model of AD is still unclear. Furthermore, only data on male 5XFAD mice have been published so far, whereas studies in female mice and possible sex differences in 18F-FDG and 18F-Florbetaben uptake are missing. The aim of this study was to evaluate the suitability of 18F-FDG– and 18F-Florbetaben–PET in 7-month-old female 5XFAD and to assess possible sex differences between male and female 5XFAD mice. We could demonstrate that female 5XFAD mice showed a significant reduction in brain glucose metabolism and increased cerebral amyloid deposition compared with wild type animals, in accordance with the pathology seen in AD patients. Furthermore, we showed for the first time that the hypometabolism in 5XFAD mice is gender-dependent and more pronounced in female mice. Therefore, these results support the feasibility of small animal PET imaging with 18F-FDG- and 18F-Florbetaben in 5XFAD mice in both, male and female animals. Moreover, our findings highlight the need to account for sex differences in studies working with 5XFAD mice.

CERTL reduces C16 ceramide, amyloid-\u03b2 levels, and inflammation in a model of Alzheimer\u2019s disease

BackgroundDysregulation of ceramide and sphingomyelin levels have been suggested to contribute to the pathogenesis of Alzheimer’s disease (AD). Ceramide transfer proteins (CERTs) are ceramide carriers which are crucial for ceramide and sphingomyelin balance in cells. Extracellular forms of CERTs co-localize with amyloid-β (Aβ) plaques in AD brains. To date, the significance of these observations for the pathophysiology of AD remains uncertain.MethodsA plasmid expressing CERTL, the long isoform of CERTs, was used to study the interaction of CERTL with amyloid precursor protein (APP) by co-immunoprecipitation and immunofluorescence in HEK cells. The recombinant CERTL protein was employed to study interaction of CERTL with amyloid-β (Aβ), Aβ aggregation process in presence of CERTL, and the resulting changes in Aβ toxicity in neuroblastoma cells. CERTL was overexpressed in neurons by adeno-associated virus (AAV) in a mouse model of familial AD (5xFAD). Ten weeks after transduction, animals were challenged with behavior tests for memory, anxiety, and locomotion. At week 12, brains were investigated for sphingolipid levels by mass spectrometry, plaques, and neuroinflammation by immunohistochemistry, gene expression, and/or immunoassay.ResultsHere, we report that CERTL binds to APP, modifies Aβ aggregation, and reduces Aβ neurotoxicity in vitro. Furthermore, we show that intracortical injection of AAV, mediating the expression of CERTL, decreases levels of ceramide d18:1/16:0 and increases sphingomyelin levels in the brain of male 5xFAD mice. CERTL in vivo over-expression has a mild effect on animal locomotion, decreases Aβ formation, and modulates microglia by decreasing their pro-inflammatory phenotype.ConclusionOur results demonstrate a crucial role of CERTL in regulating ceramide levels in the brain, in amyloid plaque formation and neuroinflammation, thereby opening research avenues for therapeutic targets of AD and other neurodegenerative diseases.

Crocetin promotes clearance of amyloid-β by inducing autophagy via the STK11/LKB1-mediated AMPK pathway

ABSTRACT Alzheimer disease (AD) is usually accompanied by two prominent pathological features, cerebral accumulation of amyloid-β (Aβ) plaques and presence of MAPT/tau neurofibrillary tangles. Dysregulated clearance of Aβ largely contributes to its accumulation and plaque formation in the brain. Macroautophagy/autophagy is a lysosomal degradative process, which plays an important role in the clearance of Aβ. Failure of autophagic clearance of Aβ is currently acknowledged as a contributing factor to increased accumulation of Aβ in AD brains. In this study, we have identified crocetin, a pharmacologically active constituent from the flower stigmas of Crocus sativus, as a potential inducer of autophagy in AD. In the cellular model, crocetin induced autophagy in N9 microglial and primary neuron cells through STK11/LKB1 (serine/threonine kinase 11)-mediated AMP-activated protein kinase (AMPK) pathway activation. Autophagy induction by crocetin significantly increased Aβ clearance in N9 cells. Moreover, crocetin crossed the blood-brain barrier and induced autophagy in the brains’ hippocampi of wild-type male C57BL/6 mice. Further studies in transgenic male 5XFAD mice, as a model of AD, revealed that one-month treatment with crocetin significantly reduced Aβ levels and neuroinflammation in the mice brains and improved memory function by inducing autophagy that was mediated by AMPK pathway activation. Our findings support further development of crocetin as a pharmacological inducer of autophagy to prevent, slow down progression, and/or treat AD. Abbreviations: Aβ: amyloid-β; ABCB1/P-gp/P-glycoprotein: ATP-binding cassette, subfamily B (MDR/TAP), member 1; AD: Alzheimer disease; AMPK/PRKAA: AMP-activated protein kinase; APP: amyloid beta (A4) precursor protein; ATG: autophagy related; BBB: blood-brain barrier; BECN1: beclin 1, autophagy related; CAMKK2/CaMKKβ: calcium/calmodulin-dependent protein kinase kinase 2, beta; CSE: Crocus sativus extract; CTSB: cathepsin B; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; GFAP: glial fibrillary acidic protein; GSK3B/GSK3β: glycogen synthase kinase 3 beta; Kp: brain partition coefficient; LRP1: low density lipoprotein receptor-related protein 1; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MAP2: microtubule-associated protein 2; MAPK/ERK: mitogen-activated protein kinase; MAPT/tau: microtubule-associated protein tau; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MTOR: mechanistic target of rapamycin kinase; MWM: Morris water maze; NFKB/NF-κB: nuclear factor of kappa light polypeptide gene enhancer in B cells; NMDA: N-methyl-d-aspartic acid; RPTOR: regulatory associated protein of MTOR; RPS6KB1/p70S6K: ribosomal protein S6 kinase 1; SQSTM1: sequestosome 1; SRB: sulforhodamine B; STK11/LKB1: serine/threonine kinase 11; TFEB: transcription factor EB; TSC2: TSC complex subunit 2; ULK1: unc-51 like kinase 1.