Female Transgenic Mice Research Articles

MPO/HOCl Facilitates Apoptosis and Ferroptosis in the SOD1G93A Motor Neuron of Amyotrophic Lateral Sclerosis.

Background Oxidative stress and reactive oxygen species (ROS) are important in the pathogenesis of amyotrophic lateral sclerosis (ALS). Hypochlorous acid (HOCl) is a powerful oxidant of the reactive oxygen species (ROS) family. HOCl's role in the progress of ALS remains unclear due to the lack of an effective HOCl detection method. Cumulative evidence supports oxidative damage incurred by mutant hSOD1 contributing to motor neuron death; however, whether HOCl as well as its catalytic enzyme myeloperoxidase (MPO) function in the cell death of SOD1G93A ALS remains elusive. Methods The hSOD1WT and hSOD1G93A NSC-34 cell and SOD1G93A ALS mouse models were employed. With a novel fluorescent HOCl probe, HKOCl-3, we detected the expressions of HOCl and its catalytic enzyme, MPO, in the above models in vitro and in vivo. The regulation of MPO/HOCl by hSOD1G93A mutation and cell deaths by MPO/HOCl were also assayed, including apoptosis, ferroptosis, and autophagy. Results Our results showed that hSOD1G93A mutation promoted the activation of the MPO/HOCl pathway in SOD1G93A ALS cell models. The activation of MPO/HOCl pathways facilitated apoptosis and ferroptosis through increasing the Bax/Bcl-2 ratio and expression of caspase-3 or inhibiting the expressions of GPX4 and NQO1 and thus leading to irreversible lipid peroxidation. Overexpressed FSP1, a glutathione-independent suppressor, could ameliorate ferroptosis. In vivo, we demonstrated that the activation of the MPO/HOCl pathway occurred differently in motor neurons of the motor cortices, brain stems, and spinal cords in male and female SOD1G93A transgenic mice. In addition, inhibiting MPO improved the motor performance of SOD1G93A transgenic mice, as demonstrated by the rotarod test. Conclusions We concluded that aggregation of mutant hSOD1 proteins contributed to activation of the MPO/HOCl pathway, triggering apoptosis and ferroptosis in motor neuronal deaths and exerting impaired motor performance.

Assessing Sex-Specific Circadian, Metabolic, and Cognitive Phenotypes in the AβPP/PS1 and APPNL-F/NL-F Models of Alzheimer's Disease.

Circadian disruption has long been recognized as a symptom of Alzheimer's disease (AD); however, emerging data suggests that circadian dysfunction occurs early on in disease development, potentially preceding any noticeable cognitive deficits. This study compares the onset of AD in male and female wild type (C57BL6/J), transgenic (AβPP/PS1), and knock-in (APPNL-F/NL-F) AD mouse models from the period of plaque initiation (6 months) through 12 months. Rhythmic daily activity patterns, glucose sensitivity, cognitive function (Morris water maze, MWM), and AD pathology (plaques formation) were assessed. A comparison was made across sexes. Sex-dependent hyperactivity in AβPP/PS1 mice was observed. In comparison to C57BL/6J animals, 6-month-old male AβPP/PS1 demonstrated nighttime hyperactivity, as did 12-month-old females. Female AβPP/PS1 animals performed significantly worse on a MWM task than AβPP/PS1 males at 12 months and trended toward increased plaque pathology. APPNL-F/NL-F 12-month-old males performed significantly worse on the MWM task compared to 12-month-old females. Significantly greater plaque pathology occurred in AβPP/PS1 animals as compared to APPNL-F/NL-F animals. Female AβPP/PS1 animals performed significantly worse than APPNL-F/NL-F animals in spatial learning and memory tasks, though this was reversed in males. Taken together, this study provides novel insights into baseline sex differences, as well as characterizes baseline diurnal activity variations, in the AβPP/PS1 and APPNL-F/NL-F AD mouse models.

Exercise training worsens cardiac performance in males but does not change ejection fraction and improves hypertrophy in females in a mouse model of metabolic syndrome

BackgroundMetabolic syndrome (MetS) refers to a cluster of co-existing cardio-metabolic risk factors, including visceral obesity, dyslipidemia, hyperglycemia with insulin resistance, and hypertension. As there is a close link between MetS and cardiovascular diseases, we aimed to investigate the sex-based differences in MetS-associated heart failure (HF) and cardiovascular response to regular exercise training (ET).MethodsHigh-fat diet-fed male and female APOB-100 transgenic (HFD/APOB-100, 3 months) mice were used as MetS models, and age- and sex-matched C57BL/6 wild-type mice on standard diet served as healthy controls (SD/WT). Both the SD/WT and HFD/APOB-100 mice were divided into sedentary and ET groups, the latter running on a treadmill (0.9 km/h) for 45 min 5 times per week for 7 months. At month 9, transthoracic echocardiography was performed to monitor cardiac function and morphology. At the termination of the experiment at month 10, blood was collected for serum low-density lipoprotein (LDL)- and high-density lipoprotein (HDL)-cholesterol measurements and homeostatic assessment model for insulin resistance (HOMA-IR) calculation. Cardiomyocyte hypertrophy and fibrosis were assessed by histology. Left ventricular expressions of selected genes associated with metabolism, inflammation, and stress response were investigated by qPCR.ResultsBoth HFD/APOB-100 males and females developed obesity and hypercholesterolemia; however, only males showed insulin resistance. ET did not change these metabolic parameters. HFD/APOB-100 males showed echocardiographic signs of mild HF with dilated ventricles and thinner walls, whereas females presented the beginning of left ventricular hypertrophy. In response to ET, SD/WT males developed increased left ventricular volumes, whereas females responded with physiologic hypertrophy. Exercise-trained HFD/APOB-100 males presented worsening HF with reduced ejection fraction; however, ET did not change the ejection fraction and reversed the echocardiographic signs of left ventricular hypertrophy in HFD/APOB-100 females. The left ventricular expression of the leptin receptor was higher in females than males in the SD/WT groups. Left ventricular expression levels of stress response-related genes were higher in the exercise-trained HFD/APOB-100 males and exercise-trained SD/WT females than exercise-trained SD/WT males.ConclusionsHFD/APOB-100 mice showed sex-specific cardiovascular responses to MetS and ET; however, left ventricular gene expressions were similar between the groups except for leptin receptor and several stress response-related genes.

Disrupting the Balance of Protein Quality Control Protein UBQLN2 Accelerates Tau Proteinopathy.

Tau protein accumulation drives toxicity in several neurodegenerative disorders. To better understand the pathways regulating tau homeostasis in disease, we investigated the role of ubiquilins (UBQLNs)-a class of proteins linked to ubiquitin-mediated protein quality control (PQC) and various neurodegenerative diseases-in regulating tau. Cell-based assays identified UBQLN2 as the primary brain-expressed UBQLN to regulate tau. UBQLN2 efficiently lowered wild-type tau levels regardless of aggregation, suggesting that UBQLN2 interacts with and regulates tau protein under normal conditions or early in disease. Moreover, UBQLN2 itself proved to be prone to accumulation as insoluble protein in male and female tau transgenic mice and the human tauopathy progressive supranuclear palsy. Genetic manipulation of UBQLN2 in a tauopathy mouse model demonstrated that a physiological UBQLN2 balance is required for tau homeostasis. UBQLN2 overexpression exacerbated phosphorylated tau pathology and toxicity in mice expressing P301S mutant tau, whereas P301S mice lacking UBQLN2 showed significantly reduced phosphorylated tau. Further studies support the view that an imbalance of UBQLN2 perturbs ubiquitin-dependent PQC and autophagy. We conclude that changes in UBQLN2 levels, whether because of pathogenic mutations or secondary to disease states, such as tauopathy, contribute to proteostatic imbalances that exacerbate neurodegeneration.SIGNIFICANCE STATEMENT We defined a role for the protein quality control protein Ubiquilin-2 (UBQLN2), in age-related neurodegenerative tauopathies. This group of disorders is characterized by the accumulation of tau protein aggregates, which differ when UBQLN2 levels are altered. Given the lack of effective disease-modifying therapies for tauopathies and the function of UBQLN2 in handling various disease-linked proteins, we explored the role of UBQLN2 in regulating tau. We found that UBQLN2 reduced tau levels in cell models but behaved differently in mouse brain, where it accelerated mutant tau pathology and tau-mediated toxicity. A better understanding of the diverse functions of regulatory proteins like UBQLN2 can elucidate some of the causative factors in neurodegenerative disease and outline new routes to therapeutic intervention.

Neurogranin as an important regulator in swimming training to improve the spatial memory dysfunction of mice with chronic cerebral hypoperfusion

BackgroundVascular cognitive impairment caused by chronic cerebral hypoperfusion (CCH) has become a hot issue worldwide. Aerobic exercise positively contributes to the preservation or restoration of cognitive abilities; however, the specific mechanism has remained inconclusive. And recent studies found that neurogranin (Ng) is a potential biomarker for cognitive impairment. This study aims to investigate the underlying role of Ng in swimming training to improve cognitive impairment. MethodsTo test this hypothesis, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system was utilized to construct a strain of Ng conditional knockout (Ng cKO) mice, and bilateral common carotid artery stenosis (BCAS) surgery was performed to prepare the model. In Experiment 1, 2-month-old male and female transgenic mice were divided into a control group (wild-type littermate, n = 9) and a Ng cKO group (n = 9). Then, 2-month-old male and female C57BL/6 mice were divided into a sham group (C57BL/6, n = 12) and a BCAS group (n = 12). In Experiment 2, 2-month-old male and female mice were divided into a sham group (wild-type littermate, n = 12), BCAS group (n = 12), swim group (n = 12), BCAS + Ng cKO group (n = 12), and swim + Ng cKO group (n = 12). Then, 7 days after BCAS, mice were given swimming training for 5 weeks (1 week for adaptation and 4 weeks for training, 5 days a week, 60 min a day). After intervention, laser speckle was used to detect cerebral blood perfusion in the mice, and the T maze and Morris water maze were adopted to test their spatial memory. Furthermore, electrophysiology and Western blotting were conducted to record long-term potential and observe the expressions of Ca2+ pathway-related proteins, respectively. Immunohistochemistry was applied to analyze the expression of relevant markers in neuronal damage, inflammation, and white matter injury. ResultsThe figures showed that spatial memory impairment was detected in Ng cKO mice, and a sharp decline of cerebral blood flow and an impairment of progressive spatial memory were observed in BCAS mice. Regular swimming training improved the spatial memory impairment of BCAS mice. This was achieved by preventing long-term potential damage and reversing the decline of Ca2+ signal transduction pathway-related proteins. At the same time, the results suggested that swimming also led to improvements in neuronal death, inflammation, and white matter injury induced by CCH. Further study adopted the use of Ng cKO transgenic mice, and the results indicated that the positive effects of swimming training on cognitive impairments, synaptic plasticity, and related pathological changes caused by CCH could be abolished by the knockout of Ng. ConclusionSwimming training can mediate the expression of Ng to enhance hippocampal synaptic plasticity and improve related pathological changes induced by CCH, thereby ameliorating the spatial memory impairment of vascular cognitive impairment.

Role of ADCC, CDC, and CDCC in Vaccine-Mediated Protection against Her2 Mammary Carcinogenesis.

Amplification or mutation of the Her2 oncoantigen in human mammary glands leads to the development of an aggressive breast carcinoma. Several features of this breast carcinoma are reproduced in mammary carcinomas that spontaneously arise in female transgenic mice bearing the activated rat Her2 oncogene under transcriptional control of the mouse mammary tumor virus promoter-BALB-neuT (neuT) mice. We previously demonstrated that carcinoma progression in neuT mice can be prevented by DNA vaccination with RHuT, a plasmid coding for a chimeric rat/human Her2 protein. RHuT vaccination exerts an antitumor effect, mostly mediated by the induction of a strong anti-rat Her2 antibody response. IgG induced by RHuT vaccine mainly acts by blocking Her2 signaling, thus impairing cell cycle progression and inducing apoptosis of cancer cells, but other indirect effector mechanisms could be involved in the antibody-mediated protection. The recruitment of cells with perforin-dependent cytotoxic activity, able to perform antibody-dependent cellular cytotoxicity, has already been investigated. Less is known about the role of the complement system in sustaining antitumor response through complement-dependent cytotoxicity and cellular cytotoxicity in vaccinated mice. This work highlights that the weight of such mechanisms in RHuT-induced cancer protection is different in transplantable versus autochthonous Her2+ tumor models. These results may shed new light on the effector mechanisms involved in antibody-dependent anti-cancer responses, which might be exploited to ameliorate the therapy of Her2+ breast cancer.

Development of ovarian tumour causes significant loss of muscle and adipose tissue: a novel mouse model for cancer cachexia study.

BackgroundCancer‐associated cachexia (CAC) is a complex syndrome of progressive muscle wasting and adipose loss with metabolic dysfunction, severely increasing the morbidity and mortality risk in cancer patients. However, there are limited studies focused on the underlying mechanisms of the progression of CAC due to the complexity of this syndrome and the lack of preclinical models that mimics its stagewise progression.MethodsWe characterized the initiation and progression of CAC in transgenic female mice with ovarian tumours. We measured proposed CAC biomarkers (activin A, GDF15, IL‐6, IL‐1β, and TNF‐α) in sera (n = 6) of this mouse model. The changes of activin A and GDF15 (n = 6) were correlated with the decline of bodyweight over time. Morphometry and signalling markers of muscle atrophy (n ≥ 6) and adipose tissue wasting (n ≥ 7) were assessed during CAC progression.ResultsCancer‐associated cachexia symptoms of the transgenic mice model used in this study mimic the progression of CAC seen in humans, including drastic body weight loss, skeletal muscle atrophy, and adipose tissue wasting. Serum levels of two cachexia biomarkers, activin A and GDF15, increased significantly during cachexia progression (76‐folds and 10‐folds, respectively). Overactivation of proteolytic activity was detected in skeletal muscle through up‐regulating muscle‐specific E3 ligases Atrogin‐1 and Murf‐1 (16‐folds and 14‐folds, respectively) with decreasing cross‐sectional area of muscle fibres (P < 0.001). Muscle wasting mechanisms related with p‐p38 MAPK, FOXO3, and p‐AMPKα were highly activated in concurrence with an elevation in serum activin A. Dramatic fat loss was also observed in this mouse model with decreased fat mass (n ≥ 6) and white adipocytes sizes (n = 6) (P < 0.0001). The adipose tissue wasting was based on thermogenesis, supported by the up‐regulation of uncoupling protein 1 (UCP1). Fibrosis in adipose tissue was also observed in concurrence with adipose tissue loss (n ≥ 13) (p < 0.0001).ConclusionsOur novel preclinical CAC mouse model mimics human CAC phenotypes and serum biomarkers. The mouse model in this study showed proteolysis in muscle atrophy, browning in adipose tissue wasting, elevation of serum activin A and GDF15, and atrophy of pancreas and liver. This mouse line would be the best preclinical model to aid in clarifying molecular mediators of CAC and dissecting metabolic dysfunction and tissue atrophy during the progression of CAC.

Impaired Oligodendrocyte Maturation Is an Early Feature in SCA3 Disease Pathogenesis.

Spinocerebellar ataxia Type 3 (SCA3), the most common dominantly inherited ataxia, is a polyglutamine neurodegenerative disease for which there is no disease-modifying therapy. The polyglutamine-encoding CAG repeat expansion in the ATXN3 gene results in expression of a mutant form of the ATXN3 protein, a deubiquitinase that causes selective neurodegeneration despite being widely expressed. The mechanisms driving neurodegeneration in SCA3 are unclear. Research to date, however, has focused almost exclusively on neurons. Here, using equal male and female age-matched transgenic mice expressing full-length human mutant ATXN3, we identified early and robust transcriptional changes in selectively vulnerable brain regions that implicate oligodendrocytes in disease pathogenesis. We mapped transcriptional changes across early, mid, and late stages of disease in two selectively vulnerable brain regions: the cerebellum and brainstem. The most significant disease-associated module through weighted gene coexpression network analysis revealed dysfunction in SCA3 oligodendrocyte maturation. These results reflect a toxic gain-of-function mechanism, as ATXN3 KO mice do not exhibit any impairments in oligodendrocyte maturation. Genetic crosses to reporter mice revealed a marked reduction in mature oligodendrocytes in SCA3-disease vulnerable brain regions, and ultrastructural microscopy confirmed abnormalities in axonal myelination. Further study of isolated oligodendrocyte precursor cells from SCA3 mice established that this impairment in oligodendrocyte maturation is a cell-autonomous process. We conclude that SCA3 is not simply a disease of neurons, and the search for therapeutic strategies and disease biomarkers will need to account for non-neuronal involvement in SCA3 pathogenesis.SIGNIFICANCE STATEMENT Despite advances in spinocerebellar ataxia Type 3 (SCA3) disease understanding, much remains unknown about how the disease gene causes brain dysfunction ultimately leading to cell death. We completed a longitudinal transcriptomic analysis of vulnerable brain regions in SCA3 mice to define the earliest and most robust changes across disease progression. Through gene network analyses followed up with biochemical and histologic studies in SCA3 mice, we provide evidence for severe dysfunction in oligodendrocyte maturation early in SCA3 pathogenesis. Our results advance understanding of SCA3 disease mechanisms, identify additional routes for therapeutic intervention, and may provide broader insight into polyglutamine diseases beyond SCA3.

Transgenic overexpression of CTRP3 does not prevent alcohol induced hepatic steatosis in female mice.

Alcoholic liver disease (ALD) is one of the leading causes of morbidity and mortality from hepatic complications. C1q/TNF-related protein 3 (CTRP3) is an adiponectin paralog and, in male mice, increased levels of circulating CTRP3 prevents ALD. Therefore, the purpose of this study was to replicate the observed hepatoprotective effect of elevated circulating CTRP3 levels in female mice. Twelve-week-old female wildtype and CTRP3 overexpressing transgenic mice were fed the Lieber-DeCarli alcohol-containing liquid diet (5% vol/vol) for 6 weeks. Unlike the previous study with male mice, CTRP3 overexpression provided no attenuation to alcohol-induced hepatic lipid accumulation, cytokine production, or overall mortality. In conclusion, there appears to be a clear sex-specific effect of CTRP3 in response to alcohol consumption that needs to be explored further.

Impact of APOE Genotype on Diet-induced Mitochondrial Adaptations in Mouse Skeletal Muscle

Lifestyle interventions based on behaviour change principles may provide a useful mechanism in reducing dementia risk amongst older adults, however intervention acceptability remains relatively unexplored. We assessed the feasibility and acceptability of BRAIN BOOTCAMP, an Australian initiative aiming to improve dementia literary and reduce dementia risk by delivering a brain health box addressing multiple lifestyle factors through education, physical prompts and an individualised brain health profile. Semi-structured phone interviews were conducted with participants (N=94) at completion of the program (3-months) using a theoretical sampling approach to select a range of participants with varying brain health scores, age, gender, education and locality. Interview topics included participants’ overall experience and suggestions for program improvement. Interviews were transcribed and analysed using thematic analysis. Participants were mostly female (79%), with a mean age of 72.6 years (SD=5.4), from an English-speaking background (89.4%) and resided in metropolitan areas (76.6%). Participants positively perceived the program, resulting in high usability and acceptability. Valuable aspects included building dementia awareness in an innovative way, and having re-assessments which identified areas for personal improvement. Participants further discussed how the program prompted lifestyle change, including setting goals (e.g., physical activity) and facilitated a general awareness of their brain health. Suggested improvements included shorter surveys, regular check-ins, and specific tailoring of the program to be more inclusive for older adults with varying levels of health. Our study demonstrated that a simple, innovative program could be a promising medium for delivering comprehensive educational resources and induce lifestyle change for older adults.

Impact of APOE Genotype on Diet-induced Mitochondrial Adaptations in Mouse Skeletal Muscle

The forecasted adoption of autonomous vehicles (AVs) will lead to major changes to the job of truck driving. These changes may be particularly challenging for drivers, as the population of truck drivers skews much older than that of other occupations. In this study we sought to understand truck drivers’ attitudes towards AVs and the longevity of their job. We conducted focus groups with truck drivers, their supervisors, and upper-level managers of trucking companies. We relate supervisors’ and managers’ experiences working with drivers through the rollout of new technologies to further understand drivers’ initial reactions to automation and how their attitudes may develop. Based on qualitative open coding our analysis uncovered two overarching themes. The first theme is the unknown. With AVs, companies expect that experience will be less important, so they can hire younger workers. In response, drivers have expressed fear of being displaced and anxiety over the uncertainty of not knowing how their jobs will be affected. The second theme is adaptability, and desire to adapt. Older drivers have expressed resistance to adapting to AVs and to their job changing. Concerningly however, managers envision the need for a driving workforce that has experience working with technology and is adaptable. Our study identifies key challenges concerning older workers’ reactions and career decisions in response to automation. Accounting for driver reactions to AVs is necessary not only to build theory and understanding on worker reactions to automation, but also for workforce planning and to support employees, particularly older workers.

CHOP is not a main contributor to tau‐mediated toxicity

AbstractBackgroundIn Alzheimer's disease (AD), the accumulation of the microtubule associated protein tau is linked with neuronal loss, but the major cell death pathway remains to be identified. Pathogenic tau promotes endoplasmic reticulum (ER) stress and subsequent unfolded protein response (UPR) activation. However, the contribution of ER stress to tau‐mediated toxicity in neurons is unknown. A previous study demonstrated that mice lacking CHOP/Ddit3, an important regulator of ER stress‐induced cell death, were protected from ER stress induced neuronal loss. We hypothesized that silencing CHOP would prevent tau toxicity through the ER stress pathway and reveal the contribution of tau toxicity that is mediated through this pathway.MethodTo investigate this, we generated a mouse‐targeting CHOP/Ddit3 shRNA AAV9 that also expresses GFP by a separate promoter. We injected shCHOP AAV9 or a shScrambled control AAV9 in the brains of 8‐month‐old male and female rTg4510 tau transgenic mice and harvested them at 12‐months of age, when ER stress is activated, and neuronal loss is occurring in these mice. We performed behavioral studies using the Y‐Maze, Open field, and 2‐day Radial‐Arm water maze task with a reversal paradigm. Unbiased stereology was used to determine neuronal health and effect of CHOP shRNA on CHOP levels was evaluated by immunofluorescence.ResultAAV9 CHOP shRNA did not alter spatial learning and memory in tau transgenic mice. Evaluation of the tissue revealed a modest, but non‐significant increase in neuronal loss from CHOP knock down, while the DG shows a non‐significant decrease in neuronal loss. Tissue volume was unchanged in both regions. Very surprising to us, and opposite of what was expected, we found that in these regions of interest the CHOP levels increased in the animal expressing the CHOP.ConclusionOverall, this suggests that shCHOP AAV9 does not alter tau‐modified behaviors such as learning and memory particularly at this timepoint. Our results also suggest that there may be a compensatory increase in CHOP signaling in neighboring cells, suggesting that the activation of the UPR in the brains of these mice may be a protective mechanism.

Reduction in Aha1 chaperone preserves hippocampal neurons and prevents tau‐related pathology in rTg4510 tau transgenic mice

Several molecular chaperones have been identified as important modulators of tau protein in aging and neurodegenerative illnesses like Alzheimer's diseases. Tau-related diseases, collectively known as tauopathies, are characterized by abnormal deposition of insoluble tau protein leading to neuronal degeneration and the formation of neurofibrillary tangles in the brain. Although the exact mechanism is largely unknown, discrete molecular chaperones are known to regulate tau stability. Our laboratory demonstrated the activator of Hsp90 ATPase Activity (Aha1) potently promotes tau fibrilization and its expression positively correlates with features of tau pathology in brains from tau overexpressing mice and individuals with Alzheimer's disease. In this study, we investigate whether reduction of Aha1 levels in tau transgenic mice is sufficient to slow the progression of tau pathology in brains of rTg4510 tau transgenic mice.To test this, we designed three AAV9 plasmids targeting Aha1 and a scramble shRNA was used as control. Aha1 knockdown was confirmed in N2A cells using Western blot analysis. Following this, 4-month-old rTg4510 male and female tau transgenic mice were injected into the hippocampus with Aha1-shRNA or scramble-shRNA (control). Two months were allowed for viral expression followed by the collection of brains for protein and tissue analysis.n N2A cells, one out of three Aha1-targeting shRNAs effectively reduced Aha1 expression after 72 hours of viral transfection. In animals, Aha1 knockdown decreased pS396/404 and AT8 phosphorylated tau as well Gallyas-silver positive tau tangles in the hippocampus. Total or oligomeric tau were not affected in mice injected with AAV9-shAha1 when compared to AAV9-scramble animals. Using unbiased stereology, we found that hippocampal CA1 neurons were preserved after reducing Aha1.Our findings demonstrate that Aha1 knockdown reduces tau phosphorylated species, prevents tau tangle formation, and rescued CA1 hippocampal neurons in rTg4510 mice. Overall, these in vivo observations suggest that Aha1 knockdown could prevent development of pathological features like tangle formation in tauopathies. Although further examinations in human brain are needed, Aha1 inhibition could be a potential therapeutic target to slow or prevent tau pathologies.

Contribution of ER stress to tau-mediated toxicity.

There are many ways cells in the brain can die. In Alzheimer's disease (AD), the accumulation of tau is linked with cell death, but the major cell death pathway remains to be identified. Prior work has shown that pathogenic tau promotes endoplasmic reticulum (ER) stress and subsequent unfolded protein response (UPR) activation. However, the contribution of ER stress-induced cell death to tau-mediated neurotoxicity is unknown. Interestingly, mice lacking CHOP/Ddit3, an important regulator of ER stress-induced cell death, have been shown to be protected from ER stress induced neuronal loss. We hypothesized that silencing CHOP would prevent tau toxicity through the ER stress pathway and reveal the contribution of tau toxicity that is mediated through this pathway. To investigate this, we generated a mouse-targeting CHOP/Ddit3 shRNA AAV9 that also expresses GFP by a separate promoter. We injected shCHOP AAV9 or a shScrambled control AAV9 in the brains of 8-month-old male and female rTg4510 tau transgenic mice and harvested them at 12-months of age, when ER stress is activated, and neuronal loss is occurring in these mice. We performed behavioral studies using the Y-Maze, Open field, and 2-day Radial-Arm water maze task with a reversal paradigm. Unbiased stereology was used to determine neuronal health and effect of CHOP shRNA on CHOP levels was evaluated by immunofluorescence. AAV9 CHOP shRNA did not alter learning and memory in tau transgenic mice. Evaluation of the tissue revealed a modest, but non-significant increase in neuronal loss from CHOP knock down, while the DG shows a non-significant decrease in neuronal loss. Tissue volume was unchanged in both regions. Very surprising to us, and opposite of what was expected, we found that in these regions of interest the CHOP levels increased in the animal expressing the CHOP. Overall, this suggests that shCHOP AAV9 does not alter tau-modified behaviors such as learning and memory particularly at this timepoint. Our results also suggest that there may be a compensatory increase in CHOP signaling in neighboring cells, suggesting that the activation of the UPR in the brains of these mice may be a protective mechanism.

A time-restricted feeding intervention reduces alterations in circadian behaviors and pathology in a mouse model of Alzheimer's disease.

Circadian rhythm disruptions impact nearly all Alzheimer's disease (AD) patients, with daytime sleepiness, sundowning, sleep fragmentation, and night-time insomnia being common disturbances. These early and progressive declines support the role of circadian dysfunction as a driver of AD pathology, hence the therapeutic potential of circadian-modulating interventions needs to be investigated. One of the most powerful regulators of the circadian system is the daily feed/fast cycle. We have recently shown that early, presymptomatic time-restricted feeding (TRF) can ameliorate a range of symptoms related to neurodegenerative conditions, including improving sleep/wake timing and circadian rhythmicity. At 12 months of age, the APP23 mouse model of AD (expressing the human APP gene containing the Swedish mutation) exhibits disrupted behavior resembling sundowning. We hypothesized that imposing early-age TRF in APP23 mice would improve circadian rhythmicity and deter the disruptions observed to emerge and progress with advancing neuropathology. Adult male and female APP23 transgenic (AD) and littermate non-transgenic (control) mice were housed individually and subjected to ab libitum (ALF) feeding or a TRF protocol consisting of 6-hours feeding in the middle of the active phase followed by 18-hours fasting for 3 months. Behavioral sleep (48 hours) and ongoing cage locomotor activity were recorded from an early age. Our analysis shows that AD mice exhibit disruptions in total sleep (P < 0.001) and the emergence of excessive wakefulness (P < 0.001). AD mice also show significant active-phase agitation (P < 0.05) accompanied by excessive sleep-phase fragmentation (P < 0.05). TRF induced nutritional ketosis (β-Hydroxybutyrate > 0.5 mM) accompanied by reduced blood glucose when compared to ALF mice, although mice consumed equivalent volumes of food in both experimental groups. Importantly, TRF improved total and sleep-phase sleep in AD mice (P < 0.05) and significantly reduced agitation and fragmentation (P < 0.05). Early application of TRF can forestall circadian disruptions in AD mice. TRF has been reported to modulate expression of clock and clock-controlled genes, transcriptional regulators, metabolism related genes, BDNF, and other neurotrophic factors. Our results suggest that these changes may also impact sleep, circadian behaviors, and agitation associated with AD.

Evidence that the gut microbiota regulates progression of neurodegeneration in a mouse model of tauopathy, in a sex‐ and ApoE isoform‐dependent manner

Alzheimer's disease (AD) is a fatal progressive neurodegenerative disease. Mounting evidence supports that an unbalanced gut microbiota (GM) is linked to amyloid-β deposition potentially by disrupting neuroinflammation and metabolic homeostasis. However, the contribution of the GM to tau-mediated neurodegeneration is poorly characterized. Furthermore, recent studies have reported that the configuration of the GM is also affected by ApoE isoforms, which is the strongest genetic risk factor for late-onset AD. Here, we explore the hypothesis that the GM regulates tau-mediated neurodegeneration in a sex- and ApoE isoform- dependent manner, using a mouse model of tauopathy.Male and female P301S tau transgenic mice were crossed with human ApoE knock-in mice (ApoE3 or ApoE4) or ApoE knockout mice (EKO) to generate P301S::ApoE3/E3, ApoE4/E4, or P301S/EKO mice, termed TE3F, TE4F, and TEKO, respectively. The GM in each group was perturbed by gastric gavage of a combination of antibiotics (ABX) from post-natal day 16-22; controls were gavaged with water (H2O). Mice were housed in a specific pathogen-free facility and fed a standard mouse chow diet ad libitum until they were euthanized at 9 months of age. In addition, a separate group of male and female TE4F mice were housed under germ-free conditions. Collected brains were sectioned, stained with 0.1% Sudan black and used to measure volumes of regions of interest.In males, TE3F and TE4F mice treated with H2O showed significant hippocampal atrophy compared to P301S mice lacking a functional ApoE gene (TEKO). However, TE3F mice treated with a short-term ABX showed significantly milder hippocampal atrophy compared to the water-treated group (p < 0.001, t-test). ABX treatment of TE4F animals was also associated with milder atrophy, although the effect did not reach statistical significance (p = 0.09, t-test). There were no effects in TEKO mice treated with ABX. Remarkably, these phenotypic effects of ABX treatment were not observed in females. Moreover, germ-free TE4F mice showed a marked decrease in brain atrophy compared to conventionally-raised animals; this was true for both sexes.Our results indicate that tau-mediated neurodegeneration occurs in a ApoE- dependent manner and is influenced by the gut microbiota and gender.

Binge Ethanol Exposure in Mice Represses Expression of Genes Involved in Osteoblast Function and Induces Expression of Genes Involved in Osteoclast Differentiation Independently of Endogenous Catalase.

Excessive ethanol consumption is a risk factor for osteopenia. Since a previous study showed that transgenic female mice with overexpression of catalase are partially protected from ethanol-mediated trabecular bone loss, we investigated the role of endogenous catalase in skeletal ethanol toxicity comparing catalase knockout to wild-type mice. We hypothesized that catalase depletion would exacerbate ethanol effects. The mice were tested in a newly designed binge ethanol model, in which 12-week-old mice were exposed to 4 consecutive days of gavage with ethanol at 3, 3, 4, and 4.5 g ethanol/kg body weight. Binge ethanol decreased the concentration of serum osteocalcin, a marker of bone formation. The catalase genotype did not affect the osteocalcin levels. RNA sequencing of femoral shaft RNA from males was conducted. Ethanol exposure led to significant downregulation of genes expressed in cells of the osteoblastic lineage with a role in osteoblastic function and collagen synthesis, including the genes encoding major structural bone proteins. Binge ethanol further induced a smaller set of genes with a role in osteoclastic differentiation. Catalase depletion affected genes with expression in erythroblasts and erythrocytes. There was no clear interaction between binge ethanol and the catalase genotype. In an independent experiment, we confirmed that the binge ethanol effects on gene expression were reproducible and occurred throughout the skeleton in males. In conclusion, the binge ethanol exposure, independently of endogenous catalase, reduces expression of genes involved in osteoblastic function and induces expression of genes involved in osteoclast differentiation throughout the skeleton in males.

Brain-Restricted Inhibition of IL-6 Trans-Signaling Mildly Affects Metabolic Consequences of Maternal Obesity in Male Offspring.

Maternal obesity greatly affects next generations, elevating obesity risk in the offspring through perinatal programming and flawed maternal and newborn nutrition. The exact underlying mechanisms are poorly understood. Interleukin-6 (IL-6) mediates its effects through a membrane-bound receptor or by trans-signaling (tS), which can be inhibited by the soluble form of the co-receptor gp130 (sgp130). As IL-6 tS mediates western-style diet (WSD) effects via chronic low-grade inflammation (LGI) and LGI is an important mediator in brain–adipose tissue communication, this study aims at determining the effects of maternal obesity in a transgenic mouse model of brain-restricted IL-6tS inhibition (GFAPsgp130) on offspring’s short- and long-term body composition and epigonadal white adipose tissue (egWAT) metabolism. Female wild type (WT) or transgenic mice were fed either standard diet (SD) or WSD pregestationally, during gestation, and lactation. Male offspring received SD from postnatal day (P)21 to P56 and were metabolically challenged with WSD from P56 to P120. At P21, offspring from WT and transgenic dams that were fed WSD displayed increased body weight and egWAT mass, while glucose tolerance testing showed the strongest impairment in GFAPsgp130WSD offspring. Simultaneously, egWAT proteome reveals a characteristic egWAT expression pattern in offspring as a result of maternal conditions. IL-6tS inhibition in transgenic mice was in tendency associated with lower body weight in dams on SD and their respective offspring but blunted by the WSD. In conclusion, maternal nutrition affects offspring’s body weight and egWAT metabolism predominantly independent of IL-6tS inhibition, emphasizing the importance of maternal and newborn nutrition for long-term offspring health.

Second-Generation Probiotic Lactobacillus Reuteri Producing IL-22 (LR-IL-22) Protects the Intestine to Facilitate Whole Abdomen Irradiation (WAI) in Ovarian Cancer

We delivered intraoral (gavage) of LR-IL-22 (Zhang, et al., In Vivo, 34: 139-150, 2020) prior to whole abdominal irradiation (WAI) of C57BL/6J mice and quantitated survival, intestinal barrier integrity, and levels of irradiation-induced inflammatory cytokines. We measured the effect on fractionated WAI of LR-IL-22 in transgenic C57BL/6J-MUC1 (human-MUC1) female mice with widespread intra-abdominal cisplatin-resistant ovarian cancer.C57BL/6J mice were irradiated to 19.75 Gy WAI, then evaluated for the primary endpoint of improved 30-day survival. Fluorescent beads (F13083 FluoSpheres™ Polystyrene Microspheres, 1.0 μm, red fluorescent (580/605), for tracer studies) were administered intraorally three hours prior to sacrifice at days 2 or 5 after WAI, and blood was assayed for beads. Luminex assay of plasma and intestinal cells were also assayed at days 0, 1, 2, 3, and 5 after WAI for radiation-induced inflammatory cytokines. Murine ovarian cancer 2F8cis cells were injected intraperitoneally into female transgenic C57BL/6J-MUC1 mice (1 × 106 cells per mouse). Seventy-two hours later, mice were irradiated to 16 Gy WAI and followed for tumor growth. In a separate experiment, female mice harboring 2F8cis tumors were irradiated to 6 Gy WAI daily for 4 successive days. Overall survival was compared between groups: irradiation only, LR-IL-22 gavage three hours prior to each fraction, and/or weekly anti-PD-L1 antibody treatment.Mice receiving LR-IL-22 (109 bacteria in 100 µL of saline) 24-hours and 3-hours prior to a single fraction of 19.75 Gy WAI, and 24-hours following irradiation, showed improved survival and barrier integrity compared to those receiving control LR or IL-22 protein (P = 0.0167 or 0.0276, n = 15). LR-IL-22 gavage maintained intestinal barrier integrity and decreased levels of intestinal pro-inflammatory cytokines, including KC (CXCL1) (P = 0.002), and IFN- γ (P = 0.0024), as well as profibrotic plasma Eotaxin (CCL11) (P = 0.0088). In MUC1 transgenic female mice with tumors, nonirradiated mice had over 100 disseminated tumor nodules throughout the abdomen at day 10, while control WAI mice or those treated with intraoral LR-IL-22 had less than 10 nodules at day 10 (P = 0.0471); furthermore, LR-IL-22 treated mice showed reduced intestinal edema. C57BL/6J-MUC1 female mice with intra-abdominal 2F8cis tumor showed significantly improved 30-day survival by adding LR-IL-22 and anti-PD-L1 to 6 Gy × 4 WAI (P = 0.0004).Intraoral LR-IL-22 prior to WAI protects the intestine from radiation-induced toxicity and facilitates fractionated therapeutic WAI to improve survival in cis-platinum resistant ovarian cancer.D.F. Hamade: None. M. Epperly: None. R. Fisher: None. W. Hou: None. J.S. Greenberger: None.

Abstract P375: Gender-based Cardio-protective Functional Dimorphism Of Bone Marrow Endothelial Progenitor Cells And Their Exosomes: Estrogen-independent Epigenetic Mechanisms

Introduction: Estrogen or estrogen receptor-dependent mechanisms in enhancing the cardioprotective efficacy of bone marrow endothelial progenitor cells (BM-EPC) is well-established in preclinical studies. However, the efficacy of estrogen does not reflect in the data from randomized cardiovascular clinical trials, suggesting an estrogen-independent role of female BM-EPC in eliciting enhanced cardiac protection compared to males. Hypothesis: Epigenetic mechanisms may contribute to the sex-specific dimorphism of Sca-1 + /CD31 + BM-EPC in regulating cell-homing, pro-angiogenic and anti-inflammatory functions in the ischemic myocardium leading to enhanced reparative function of female progenitor cells. Methods &amp; Results: Transplantation of GFP-BM-mononuclear cells from male and female GFP transgenic mice into the BM of lethally irradiated recipient male C57BL/6 mice resulted in the enhanced mobilization of female Sca-1 + CD31 + /GFP + BM-EPC into circulation post-MI. A higher number of female BM-EPC homed to the ischemic myocardium and significantly improved LV functions and capillary density post-MI compared to male BM-EPC. Female BM-EPC showed increased expression of bFGF, VEGFR2, SDF-1α, and IL-10 genes, thereby efficiently promoted endothelial tube formation in vitro compared to male BM-EPC. Transplantation of female BM-EPC and their exosomes into post-MI male mice improved LV cardiac function, reduced scar size, and improved capillary density compared to male BM-EPC and exosomes. Male BM-EPC showed an increased expression of G9a/Ehmt2, an H3K9me3 methyltransferase, and Dnmt3a DNA methyltransferase compared to female BM-EPC. In contrast, Kdm6b/JMJD3, H3K27me3 demethylase was highly expressed in female BM-EPC compared to males. Treatment of BM-EPC of both sexes with 17-β-estradiol did not alter the expression of Kdm6b/JMJD3. Male BM-EPC highly expressed repressive gene marks, H3K9me3, and H3K27me3 compared to females. Compared to the male, BM-EPC from female and ovariectomized (OXV) female mice showed equally high expression of angiogenic genes ANGPT-1, MDK, PLAU, Tie-2, and VEGFR2 and lower levels of inflammatory cytokines, TNFα, IFNγ, IL-1β, and CCL3. Conditioned medium from female and OVX BM-EPC equally promoted enhanced migration and tube formation of HUVEC in vitro, compared to male BM-EPC. Conclusions: An estrogen-independent epigenetic mechanism likely governs the enhanced cardiac reparative properties of female BM-EPC.