Changes In Tau Phosphorylation Research Articles

Temperature-induced Artifacts in Tau Phosphorylation: Implications for Reliable Alzheimer's Disease Research.

In preclinical research on Alzheimer's disease and related tauopathies, tau phosphorylation analysis is routinely employed in both cellular and animal models. However, recognizing the sensitivity of tau phosphorylation to various extrinsic factors, notably temperature, is vital for experimental accuracy. Hypothermia can trigger tau hyperphosphorylation, while hyperthermia leads to its dephosphorylation. Nevertheless, the rapidity of tau phosphorylation in response to unintentional temperature variations remains unknown. In cell cultures, the most significant temperature change occurs when the cells are removed from the incubator before harvesting, and in animal models, during anesthesia prior to euthanasia. In this study, we investigate the kinetics of tau phosphorylation in N2a and SH-SY5Y neuronal cell lines, as well as in mice exposed to anesthesia. We observed changes in tau phosphorylation within the few seconds upon transferring cell cultures from their 37°C incubator to room temperature conditions. However, cells placed directly on ice post-incubation exhibited negligible phosphorylation changes. In vivo, isoflurane anesthesia rapidly resulted in tau hyperphosphorylation within the few seconds needed to lose the pedal withdrawal reflex in mice. These findings emphasize the critical importance of preventing temperature variation in researches focused on tau. To ensure accurate results, we recommend avoiding anesthesia before euthanasia and promptly placing cells on ice after removal from the incubator. By controlling temperature fluctuations, the reliability and validity of tau phosphorylation studies can be significantly enhanced.

APOEε4 potentiates the effects of Aβ pathology on the deposition of neurofibrillary tangles via tau phosphorylation

AbstractBackgroundThe mechanisms by which the apolipoprotein E ε4 (APOEε4) allele influences Alzheimer’s disease (AD) pathophysiological progression are poorly understood. Here, we tested the association of APOEε4 carriership and amyloid‐β (Aβ) burden with longitudinal tau pathology progression.MethodWe studied 104 individuals across the aging and AD clinical spectrum from the McGill TRIAD cohort. Study participants underwent clinical assessments, APOE genotyping, magnetic resonance imaging, positron emission tomography (PET) for Aβ ([18F]AZD4694) and tau ([18F]MK6240) at baseline, as well as an additional follow‐up tau‐PET scan (mean follow‐up, 2.4 years). We further assessed longitudinal changes in tau phosphorylation (plasma phosphorylated tau at threonine 217 [p‐tau217+]), brain atrophy (gray matter density), and clinical function (clinical dementia rating scale sum of boxes).ResultWe found that APOEε4 carriership potentiates Aβ effects on longitudinal tau tangle accumulation over two years (Figure 1). Interestingly, the APOEε4‐potentiated Aβ effects on tangles were mediated by longitudinal plasma p‐tau217+ increase (Figure 2). In addition, this longitudinal tau accumulation as measured by PET was accompanied by brain atrophy and clinical decline during the follow‐up period (Figure 3).ConclusionOur results support a model in which the APOEε4 allele plays a key role in Aβ downstream effects on the aggregation of phosphorylated tau in the form of neurofibrillary tangles in the living human brain, which is a key factor in the development of dementia. These observations have important implications for the design of future trials by suggesting that the combination of therapies targeting both ApoeE4 and Aβ pathology might have the potential to synergistically halt tau progression in AD.

APOEε4 potentiates the effects of Aβ pathology on the deposition of neurofibrillary tangles via tau phosphorylation

AbstractBackgroundThe mechanisms by which the apolipoprotein E e4 (APOEe4) allele influences Alzheimer’s disease (AD) pathophysiological progression are poorly understood. Here, we tested the association of APOEe4 carriership and amyloid‐ß (Aß) burden with longitudinal tau pathology progression.MethodWe studied 104 individuals across the aging and AD clinical spectrum from the McGill TRIAD cohort. Study participants underwent clinical assessments, APOE genotyping, magnetic resonance imaging, positron emission tomography (PET) for Aß ([18F]AZD4694) and tau ([18F]MK6240) at baseline, as well as an additional follow‐up tau‐PET scan (mean follow‐up, 2.4 years). We further assessed longitudinal changes in tau phosphorylation (plasma phosphorylated tau at threonine 217 [p‐tau217+]), brain atrophy (gray matter density), and clinical function (clinical dementia rating scale sum of boxes).ResultWe found that APOEe4 carriership potentiates Aß effects on longitudinal tau tangle accumulation over two years (Figure 1). Interestingly, the APOEe4‐potentiated Aß effects on tangles were mediated by longitudinal plasma p‐tau217+ increase (Figure 2). In addition, this longitudinal tau accumulation as measured by PET was accompanied by brain atrophy and clinical decline during the follow‐up period (Figure 3).ConclusionOur results support a model in which the APOEe4 allele plays a key role in Aß downstream effects on the aggregation of phosphorylated tau in the form of neurofibrillary tangles in the living human brain, which is a key factor in the development of dementia. These observations have important implications for the design of future trials by suggesting that the combination of therapies targeting both ApoeE4 and Aß pathology might have the potential to synergistically halt tau progression in AD.

Repopulated microglia induce expression of Cxcl13 with differential changes in Tau phosphorylation but do not impact amyloid pathology

BackgroundAdult microglia rely on self-renewal through division to repopulate and sustain their numbers. However, with aging, microglia display morphological and transcriptional changes that reflect a heightened state of neuroinflammation. This state threatens aging neurons and other cells and can influence the progression of Alzheimer’s disease (AD). In this study, we sought to determine whether renewing microglia through a forced partial depletion/repopulation method could attenuate AD pathology in the 3xTg and APP/PS1 mouse models.MethodsWe pharmacologically depleted the microglia of two cohorts of 21- to 22-month-old 3xTg mice and one cohort of 14-month-old APP/PS1 mice using PLX5622 formulated in chow for 2 weeks. Following depletion, we returned the mice to standard chow diet for 1 month to allow microglial repopulation. We assessed the effect of depletion and repopulation on AD pathology, microglial gene expression, and surface levels of homeostatic markers on microglia using immunohistochemistry, single-cell RNAseq and flow cytometry.ResultsAlthough we did not identify a significant impact of microglial repopulation on amyloid pathology in either of the AD models, we observed differential changes in phosphorylated-Tau epitopes after repopulation in the 3xTg mice. We provide evidence that repopulated microglia in the hippocampal formation exhibited changes in the levels of homeostatic microglial markers. Lastly, we identified novel subpopulations of microglia by performing single-cell RNAseq analysis on CD45int/+ cells from hippocampi of control and repopulated 3xTg mice. In particular, one subpopulation induced after repopulation is characterized by heightened expression of Cxcl13.ConclusionOverall, we found that depleting and repopulating microglia causes overexpression of microglial Cxcl13 with disparate effects on Tau and amyloid pathologies.

Temporal and Region‐Specific Tau Protein Hyper‐Phosphorylation in the Brain Coincides with the Development of Impaired Baroreflex Control of Arterial Pressure and Spatial Learning in Male Obese Zucker Rats

Obesity and other traits of metabolic syndrome (MetS) are recognized risk factors for the development of neurodegenerative diseases such as Alzheimer’s Disease (AD) and related dementias. Emerging evidence indicates pathophysiologies common to MetS and AD, such as obesity, hypertension, insulin resistance, and impaired glycemic control, occur with development of tauopathies and cognitive decline. Male obese Zucker rats (OZRs), a hyperphagic genetic model of MetS, develop these deleterious traits with predictable ages of onset and time courses. Juvenile OZRs display prominently elevated triglycerides and insulin compared to age‐matched lean Zucker rats (LZRs). By 12‐14 weeks of age although fasting glucose is still comparable in OZRs and LZRs, with access to food the OZRs exhibit chronic, marked hyperglycemia. At this age male OZRs also become hypertensive with impaired short‐term control of arterial pressure by baroreflexes, which are improved by treatments that normalize blood glucose. We hypothesized that young adult male OZRs would develop age‐dependent expression of markers of neurodegeneration in medulla and hippocampus coincident with deficits in functions related to these brain regions. Cohorts of age‐matched male OZRs and LZRs underwent cognitive testing using the Morris water maze (MWM). At 18‐19 weeks, the escape latencies and path lengths were comparable in male OZRs and LZRs (n=12/group). In contrast, at 28 weeks of age, OZRs (n=16) exhibited a greater than 2‐fold higher escape latency and significantly longer path lengths (p<0.05) in the acquisition and retention phases of the MWM compared to age‐matched LZRs (n=15), suggesting the emergence of impaired spatial learning ability in the OZRs. The 18‐19‐week‐old male OZRs exhibited tau hyper‐phosphorylation (S396 and S199,and S202) in the medulla, but the expressions of these markers in the hippocampus were comparable in OZRs and LZRs (n=6/group) at this age. In contrast, 27‐28‐week‐old OZRs (n=6) exhibited tau hyperphosphorylation at S396in hippocampus as well as medulla compared to LZRs (n=6). Our data suggest that changes in tau phosphorylation in male OZRs occur in a temporal and spatial manner coincident with changes in brain function in these regions. In male OZRs, earlier manifestation in the medulla occurred with impaired short‐term regulation of arterial pressure by the medulla. Later manifestation of tau hyperphosphorylation in hippocampus coincided with deficits in spatial learning. Understanding mechanisms by which these histological, physiological, and behavioral changes occur in male OZRs may provide important insights into connections between the development of MetS and AD‐related dementias in humans to yield improved early‐stage interventions.

Simple, Single-Shot Phosphoproteomic Analysis of Heat-Stable Tau Identifies Age-Related Changes in pS235- and pS396-Tau Levels in Non-human Primates.

Age is the most significant risk factor for Alzheimer’s disease (AD), and understanding its role in specific aspects of AD pathology will be critical for therapeutic development. Neurofibrillary tangles composed of hyperphosphorylated tau are a quintessential hallmark of AD. To study age-related changes in tau phosphorylation, we developed a simple, antibody-free approach for single shot analysis of tau phosphorylation across the entire protein by liquid-chromatography tandem mass spectrometry. This methodology is species independent; thus, while initially developed in a rodent model, we utilized this technique to analyze 36 phosphorylation sites on rhesus monkey tau from the prefrontal cortex (PFC), a region vulnerable to AD-linked degeneration. Data are available via ProteomeXchange with identifier PXD027971. We identified novel, age-related changes in tau phosphorylation in the rhesus monkey PFC and analyzed patterns of phosphorylation change across domains of the protein. We confirmed a significant increase and positive correlation with age of phosphorylated serine 235 tau and phosphorylated serine 396 tau levels in an expanded cohort of 14 monkeys. Histology showed robust labeling for tau phosphorylated at these sites in vulnerable layer III pyramidal cells in the PFC. The results presented in this study suggest an important role of the natural aging process in tau phosphorylation in rhesus monkey.

Evaluating Effects of Glatiramer Acetate Treatment on Amyloid Deposition and Tau Phosphorylation in the 3xTg Mouse Model of Alzheimer's Disease.

Neuroinflammation driven by the accumulation of amyloid β (Aβ) can lead to neurofibrillary tangle formation in Alzheimer’s Disease (AD). To test the hypothesis that an anti-inflammatory immunomodulatory agent might have beneficial effects on amyloid and tau pathology, as well as microglial phenotype, we evaluated glatiramer acetate (GA), a multiple sclerosis drug thought to bias type 2 helper T (Th2) cell responses and alternatively activate myeloid cells. We administered weekly subcutaneous injections of GA or PBS to 15-month-old 3xTg AD mice, which develop both amyloid and tau pathology, for a period of 8 weeks. We found that subcutaneous administration of GA improved behavioral performance in novel object recognition and decreased Aβ plaque in the 3xTg AD mice. Changes in tau phosphorylation were mixed with specific changes in phosphoepitopes seen in immunohistochemistry but not observed in western blot. In addition, we found that there was a trend toward increased microglia complexity in 3xTg mice treated with GA, suggesting a shift toward homeostasis. These findings correlated with subtle changes in the microglial transcriptome, in which the most striking difference was the upregulation of Dcstamp. Lastly, we found no evidence of changes in proportions of major helper T cell (Th) subtypes in the periphery. Overall, our study provides further evidence for the benefits of immunomodulatory therapies that alter the adaptive immune system with the goal of modifying microglia responses for the treatment of Alzheimer’s Disease.

Effects of concentrated ambient ultrafine particulate matter on hallmarks of Alzheimer’s disease in the 3xTgAD mouse model

BackgroundExposure to air pollution has been identified as a possible environmental contributor to Alzheimer’s Disease (AD) risk. As the number of people with AD worldwide continues to rise, it becomes vital to understand the nature of this potential gene-environment interaction. This study assessed the effects of short-term exposures to concentrated ambient ultrafine particulates (UFP, <100 nm) on measurements of amyloid-β, tau, and microglial morphology. MethodsTwo cohorts of aged (12.5–14 months) 3xTgAD and NTg mice were exposed to concentrated ambient UFP or filtered air for 2 weeks (4-h/day, 4 days/week). Bronchoalveolar lavage fluid and brain tissue were collected twenty-four hours following the last exposure to evaluate lung inflammation, tau pathology, amyloid-β pathology, and glial cell morphology. ResultsNo exposure- or genotype-related changes were found with any of the measures of lung inflammation or in the hippocampal staining density of astrocyte marker glial fibrillary acidic protein. The microglia marker, ionized calcium binding adaptor molecule 1, and amyloid-β marker, 6E10, exhibited significant genotype by exposure interactions such that levels were lower in the UFP-exposed as compared to filtered air-exposed 3xTgAD mice. When microglia morphology was assessed by Sholl analysis, microglia from both NTg mouse groups were ramified. The 3xTgAD air-exposed mice had the most ameboid microglia, while the 3xTgAD UFP-exposed mice had microglia that were comparatively more ramified. The 3xTgAD air-exposed mice had more plaques per region of interest as measured by Congo red staining as well as more plaque-associated microglia than the 3xTgAD UFP-exposed mice. The number of non-plaque-associated microglia was not affected by genotype or exposure. Levels of soluble and insoluble human amyloid-β42 protein were measured in both 3xTgAD groups and no exposure effect was found. In contrast, UFP-exposure led to significant elevations in phosphorylated tau in 3xTgAD mice as compared to those that were exposed to air, as measured by pT205 staining. ConclusionsExposure to environmentally relevant levels of ultrafine particulates led to changes in tau phosphorylation and microglial morphology in the absence of overt lung inflammation. Such changes highlight the need to develop greater mechanistic understanding of the link between air pollution exposure and Alzheimer’s disease.

Risk of neurodegenerative disease due to tau phosphorylation changes and arsenic exposure via drinking water.

It is estimated that around 140 million people are drinking highly contaminated water with arsenic (As) as a natural earth's crust component. On the other hand, the prevalence of neurodegenerative disorders, especially Alzheimer's disease, is constantly increasing. The aim of the present study was to investigate the correlation between oral arsenic trioxide exposure and its impact on tau protein phosphorylation at Ser262. Fifty-four male mice were randomly divided into three groups and were freely accessed to food and contaminated water of 1 and 10ppm arsenic trioxide for 3months, except for control subjects. At the end of each month, As concentration and tau phosphorylation were checked with graphite furnace atomic absorption spectrometer and western blot analysis, respectively. Surprisingly, it was observed that the amount of measured brain arsenic in 10ppm-exposed subjects was significantly increased after 3months (P-value ˂ 0.0001). The significant changes in tau phosphorylation were not seen in the 1ppm-exposed subjects, and it was observed that Ser262 phosphorylation significantly increased after 2 and 3months in the 10ppm group (P-value < 0.05). Our results demonstrated that arsenic accumulated in the brain time-dependently and increased Ser262 tau phosphorylation, which is very important in several tauopathies. In conclusion, it could be inferred that environmental arsenic exposure even at very low concentrations could be considered as a reason for increasing the risk of developing neurodegenerative disease.

Haploinsufficiency of tau decreases survival of the mouse model of Niemann-Pick disease type C1 but does not alter tau phosphorylation.

Niemann-Pick C1 (NPC1) mouse models show neurofibrillary tangles as do human patients. A previous study in NPC1/tau double-null mutant mice showed that tau knockout nulls and heterozygotes unexpectedly had decreased survival when compared with NPC1 single mutants (Pacheco et al., Hum Molec Genetics 18:956-965, 2009). This was done in a null model of NPC1 (Npc1-/-). We have extended these results to a hypomorphic model (Npc1nmf164) and additionally studied tau phosphorylation, which has not been previously done in a tau heterozygote. As before, NPC1/tau double-mutant mice had shortened survival when compared with the NPC1 single mutant. Tau dosage was not affected by the Npc1 mutation. The increased phosphorylation of tau-ser396 previously noted in NPC1 mouse models was also present, but unaffected by the tau knockout, indicating that changes in tau phosphorylation are not the cause of decreased survival in NPC1/tau double mutants. Thus, the reason for shortened survival of NPC1 mouse models with concomitant tau haploinsufficiency is uncertain.

Evaluating the Effect of Interleukin-4 in the 3xTg Mouse Model of Alzheimer's Disease.

Chronic neuroinflammation has long been hypothesized to be involved in Alzheimer’s Disease (AD) progression. Previous research suggests that both anti-inflammatory and inflammatory microglia ameliorate amyloid pathology, but the latter worsen tau pathology. In this study, we sought to determine whether induction of arginase-1 positive microglia with the anti-inflammatory cytokine IL-4 modulates pathology in the 3xTg mouse model of AD. Our findings indicate that a single intracranial IL-4 injection positively modulated performance of 3xTg AD mice in a Novel Object Recognition task, and locally increased the levels of arginase-1 positive myeloid cells when assessed one-week post injection. Furthermore, immunohistochemical analysis revealed decreased tau phosphorylation in IL-4 injected animals; however, we were not able to detect significant changes in tau phosphorylation utilizing Western blot. Lastly, IL-4 injection did not appear to cause significant changes in amyloid β load. In conclusion, acute intracranial IL-4 led to some positive benefits in the 3xTg mouse model of AD. Although more work remains, these results support therapeutic strategies aimed at modifying microglial activation states in neurodegenerative diseases.

Research Progress on Alzheimer's Disease and Resveratrol.

Alzheimer's disease (AD), a common irreversible neurodegenerative disease characterized by amyloid-β plaques, neurofibrillary tangles, and changes in tau phosphorylation, is accompanied by memory loss and symptoms of cognitive dysfunction. Increases in disease incidence due to the ageing of the population have placed a great burden on society. To date, the mechanism of AD and the identities of adequate drugs for AD prevention and treatment have eluded the medical community. It has been confirmed that phytochemicals have certain neuroprotective effects against AD. For example, some progress has been made in research on the use of resveratrol, a natural polyphenolic phytochemical, for the prevention and treatment of AD in recent years. Elucidation of the pathogenesis of AD will create a solid foundation for drug treatment. In addition, research on resveratrol, including its mechanism of action, the roles of signalling pathways and its therapeutic targets, will provide new ideas for AD treatment, which is of great significance. In this review, we discuss the possible relationships between AD and the following factors: synapses, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs), silent information regulator 1 (SIRT1), and estrogens. We also discuss the findings of previous studies regarding these relationships in the context of AD treatment and further summarize research progress related to resveratrol treatment.

Increasing Tau 4R Tau Levels Exacerbates Hippocampal Tau Hyperphosphorylation in the hTau Model of Tauopathy but Also Tau Dephosphorylation Following Acute Systemic Inflammation.

Inflammation is considered a mechanistic driver of Alzheimer's disease, thought to increase tau phosphorylation, the first step to the formation of neurofibrillary tangles (NFTs). To further understand how inflammation impacts the development of tau pathology, we used (hTau) mice, which express all six, non-mutated, human tau isoforms, but with an altered ratio of tau isoforms favoring 3R tau due to the concomitant loss of murine tau (mTau) that is predominantly 4R. Such an imbalance pattern has been related to susceptibility to NFTs formation, but whether or not this also affects susceptibility to systemic inflammation and related changes in tau phosphorylation is not known. To reduce the predominance of 3R tau by increasing 4R tau availability, we bred hTau mice on a heterozygous mTau background and compared the impact of systemic inflammation induced by lipopolysaccharide (LPS) in hTau mice hetero- or homozygous mTau knockout. Three-month-old male wild-type (Wt), mTau+/−, mTau−/−, hTau/mTau+/−, and hTau/mTau−/− mice were administered 100, 250, or 330 μg/kg of LPS or its vehicle phosphate buffer saline (PBS) [intravenously (i.v.), n = 8–9/group]. Sickness behavior, reflected by behavioral suppression in the spontaneous alternation task, hippocampal tau phosphorylation, measured by western immunoblotting, and circulating cytokine levels were quantified 4 h after LPS administration. The persistence of the LPS effects (250 μg/kg) on these measures, and food burrowing behavior, was assessed at 24 h post-inoculation in Wt, mTau+/−, and hTau/mTau+/− mice (n = 9–10/group). In the absence of immune stimulation, increasing 4R tau levels in hTau/mTau+/− exacerbated pS202 and pS396/404 tau phosphorylation, without altering total tau levels or worsening early behavioral perturbations characteristic of hTau/mTau−/− mice. We also show for the first time that modulating 4R tau levels in hTau mice affects the response to systemic inflammation. Behavior was suppressed in all genotypes 4 h following LPS administration, but hTau/mTau+/− exhibited more severe sickness behavior at the 100 μg/kg dose and a milder behavioral and cytokine response than hTau/mTau−/− mice at the 330 μg/kg dose. All LPS doses decreased tau phosphorylation at both epitopes in hTau/mTau+/− mice, but pS202 levels were selectively reduced at the 100 μg/kg dose in hTau/mTau−/− mice. Behavioral suppression and decreased tau phosphorylation persisted at 24 h following LPS administration in hTau/mTau+/− mice.

Kainic acid hyperphosphorylates tau via inflammasome activation in MAPT transgenic mice.

The excitotoxicity induced by kainic acid (KA) is thought to contribute to the development of Alzheimer’s disease (AD); however, the mechanisms underlying this excitotoxicity remain unknown. In the current study, we investigated the dynamic changes in tau phosphorylation and their associations with the excitotoxicity induced by intraperitoneal injection of KA in the mouse brain. We found that KA-induced excitotoxicity led to sustained hyperphosphorylation of tau in MAPT transgenic (Tg) mice. By using cultured microglia and mouse brains, we showed that KA treatment specifically induced endoplasmic reticulum (ER) stress, which was characterized by activation of the major biomarkers of ER, such as ATF6, GRP78, and IRE1, and resulted in stimulation of inflammasomes. KA receptors (KARs), such as Girk1, were determined to be involved in this KA-induced ER stress. ER stress was also shown to activate inflammasomes by stimulating the expression of the two major components of inflammasomes, nucleotide binding oligomerization domain (NOD)-like receptor (NLR) protein 3 (NLRP3) and nuclear factor (NF)-κB, and eventually causing the production of interleukin-1β (IL-1β). Inhibition of NLRP3 or NF-κB by Bay11-7082 resulted in reduction of KA-induced IL-1β production. Our results also revealed the positive effects of IL-1β on tau phosphorylation, which was blocked by Bay11-7082. Notably, the results indicate that Bay11-7082 acts against KA-induced neuronal degeneration, tau phosphorylation, and memory defects via inflammasomes, which further highlight the protective role of Bay11-7082 in KA-induced neuronal defects.

Circadian and sleep/wake-dependent variations in tau phosphorylation are driven by temperature.

Aggregates of hyperphosphorylated tau protein are a hallmark of Alzheimer's disease (AD) and other tauopathies. Sleep disturbances are common in AD patients, and insufficient sleep may be a risk factor for AD. Recent evidence suggests that tau phosphorylation is dysregulated by sleep disturbances in mice. However, the physiological regulation of tau phosphorylation during the sleep-wake cycle is currently unknown. We thus aimed to determine whether tau phosphorylation is regulated by circadian rhythms, inherently linked to the sleep-wake cycle. To answer these questions, we analyzed by Western blotting tau protein and associated kinases and phosphatases in the brains of awake, sleeping, and sleep-deprived B6 mice. We also recorded their temperature. We found that tau phosphorylation undergoes sleep-driven circadian variations as it is hyperphosphorylated during sleep but not during acute sleep deprivation. Moreover, we demonstrate that the mechanism behind these changes involves temperature, as tau phosphorylation was inversely correlated with circadian- and sleep deprivation-induced variations in body temperature, and prevented by housing the animals at a warmer temperature. Notably, similar changes in tau phosphorylation were reproduced in neuronal cells exposed to temperatures recorded during the sleep-wake cycle. Our results also suggest that inhibition of protein phosphatase 2A (PP2A) may explain the hyperphosphorylation of tau during sleep-induced hypothermia. Taken together, our results demonstrate that tau phosphorylation follows a circadian rhythm driven mostly by body temperature and sleep, and provide the physiological basis for further understanding how sleep deregulation can affect tau and ultimately AD pathology.

3528 Common Mechanisms Underlying Epilepsy and Tauopathy

OBJECTIVES/SPECIFIC AIMS: Neurologic disorders are among the most significant health challenges facing society today. Although different neurologic disorders are often thought to be distinct from one another, evidence suggests similar processes may contribute to pathology in different diseases. Previous studies suggest that common disease mechanisms contribute to the development of epilepsy and tauopathy. The purpose of this study is to better characterize this relationship and explore potential therapeutic avenues to slow disease progress. METHODS/STUDY POPULATION: This study uses the pilocarpine-induced status epilepticus model of temporal lobe epilepsy to explore the effect of severe seizures on tau pathology. Brains were collected from mice at 6 or 24 hours after induced status epilepticus. Homogenates were analyzed via Western blot to look for changes in tau phosphorylation or activity of two major regulators of tau phosphorylation, GSK3β and PP2A. These data show that changes in tau phosphorylation dynamics occur at a much earlier time point after status epilepticus than has previously been described. RESULTS/ANTICIPATED RESULTS: GSK3β activity increased within 6 hours and remained elevated by 24 hours. PP2A activity initially decreased but returned to normal by 24 hours. These data show that changes in tau phosphorylation dynamics occur at a much earlier time point after status epilepticus than has previously been described. DISCUSSION/SIGNIFICANCE OF IMPACT: The current project supports previous observations that seizures promote tau phosphorylation in vivo, but suggests that changes begin much earlier than previously thought. Further work is needed to understand how post-seizure changes in tau phosphorylation develop over longer periods of time. Additionally, future work will characterize the effect of tauopathy on electrical activity in vivo and in vivo.

The cysteine-rich whey protein supplement, Immunocal®, preserves brain glutathione and improves cognitive, motor, and histopathological indices of traumatic brain injury in a mouse model of controlled cortical impact

Traumatic brain injury (TBI) is a major public health problem estimated to affect nearly 1.7 million people in the United States annually. Due to the often debilitating effects of TBI, novel preventative agents are highly desirable for at risk populations. Here, we tested a whey protein supplement, Immunocal®, for its potential to enhance resilience to TBI. Immunocal® is a non-denatured whey protein preparation which has been shown to act as a cysteine delivery system to increase levels of the essential antioxidant glutathione (GSH). Twice daily oral supplementation of CD1 mice with Immunocal® for 28 days prior to receiving a moderate TBI prevented an ~ 25% reduction in brain GSH/GSSG observed in untreated TBI mice. Immunocal® had no significant effect on the primary mechanical injury induced by TBI, as assessed by MRI, changes in Tau phosphorylation, and righting reflex time or apnea. However, pre-injury supplementation with Immunocal® resulted in statistically significant improvements in motor function (beam walk and rotarod) and cognitive function (Barnes maze). We also observed a significant preservation of corpus callosum width (axonal myelination), a significant decrease in degenerating neurons, a reduction in Iba1 (microglial marker), decreased lipid peroxidation, and preservation of brain-derived neurotrophic factor (BDNF) in the brains of Immunocal®-pretreated mice compared to untreated TBI mice. Taken together, these data indicate that pre-injury supplementation with Immunocal® significantly enhances the resilience to TBI induced by a moderate closed head injury in mice. We conclude that Immunocal® may hold significant promise as a preventative agent for TBI, particularly in certain high risk populations such as athletes and military personnel.

NAD-biosynthetic enzyme NMNAT1 reduces early behavioral impairment in the htau mouse model of tauopathy

NAD metabolism and the NAD biosynthetic enzymes nicotinamide nucleotide adenylyltransferases (NMNATs) are thought to play a key neuroprotective role in tauopathies, including Alzheimer’s disease. Here, we investigated whether modulating the expression of the NMNAT nuclear isoform NMNAT1, which is important for neuronal maintenance, influences the development of behavioral and neuropathological abnormalities in htau mice, which express non-mutant human tau isoforms and represent a model of tauopathy relevant to Alzheimer’s disease. Prior to the development of cognitive symptoms, htau mice exhibit tau hyperphosphorylation associated with a selective deficit in food burrowing, a behavior reminiscent to activities of daily living which are impaired early in Alzheimer’s disease. We crossed htau mice with Nmnat1 transgenic and knockout mice and tested the resulting offspring until the age of 6 months. We show that overexpression of NMNAT1 ameliorates the early deficit in food burrowing characteristic of htau mice. At 6 months of age, htau mice did not show neurodegenerative changes in both the cortex and hippocampus, and these were not induced by downregulating NMNAT1 levels. Modulating NMNAT1 levels produced a corresponding effect on NMNAT enzymatic activity but did not alter NAD levels in htau mice. Although changes in local NAD levels and subsequent modulation of NAD-dependent enzymes cannot be ruled out, this suggests that the effects seen on behavior may be due to changes in tau phosphorylation. Our results suggest that increasing NMNAT1 levels can slow the progression of symptoms and neuropathological features of tauopathy, but the underlying mechanisms remain to be established.

Differential effects of voluntary treadmill exercise and caloric restriction on tau pathogenesis in a mouse model of Alzheimer's disease-like tau pathology fed with Western diet

BackgroundTau is a microtubule-associated protein that becomes pathological when it undergoes hyperphosphorylation and aggregation as seen in Alzheimer's disease (AD). AD is mostly sporadic, with environmental, biological and/or genetic risks factors, interacting together to promote the disease. In the past decade, reports have suggested that obesity in midlife could be one of these risk factors. On the other hand, caloric restriction and physical exercise have been reported to reduce the incidence and outcome of obesity as well as AD. MethodsWe evaluated the impact of voluntary physical exercise and caloric restriction on tau pathology during 2months in hTau mice under high caloric diet in order to evaluate if these strategies could prevent AD-like pathology in obese conditions. ResultsWe found no effects of obesity induced by Western diet on both Tau phosphorylation and aggregation compared to controls. However, exercise reduced tau phosphorylation while caloric restriction exacerbated its aggregation in the brains of obese hTau mice. We then examined the mechanisms underlying changes in tau phosphorylation and aggregation by exploring major tau kinases and phosphatases and key proteins involved in autophagy. However, there were no significant effects of voluntary exercise and caloric restriction on these proteins in hTau mice that could explain our results. ConclusionIn this study, we report differential effects of voluntary treadmill exercise and caloric restriction on tau pathogenesis in our obese mice, namely beneficial effect of exercise on tau phosphorylation and deleterious effect of caloric restriction on tau aggregation. Our results suggest that lifestyle strategies used to reduce metabolic disorders and AD must be selected and studied carefully to avoid exacerbation of pathologies.

Tau accumulation in the retina promotes early neuronal dysfunction and precedes brain pathology in a mouse model of Alzheimer\u2019s disease

BackgroundTau is an axon-enriched protein that binds to and stabilizes microtubules, and hence plays a crucial role in neuronal function. In Alzheimer’s disease (AD), pathological tau accumulation correlates with cognitive decline. Substantial visual deficits are found in individuals affected by AD including a preferential loss of retinal ganglion cells (RGCs), the neurons that convey visual information from the retina to the brain. At present, however, the mechanisms that underlie vision changes in these patients are poorly understood. Here, we asked whether tau plays a role in early retinal pathology and neuronal dysfunction in AD.MethodsAlterations in tau protein and gene expression, phosphorylation, and localization were investigated by western blots, qPCR, and immunohistochemistry in the retina and visual pathways of triple transgenic mice (3xTg) harboring mutations in the genes encoding presenilin 1 (PS1M146 V), amyloid precursor protein (APPSwe), and tau (MAPTP301L). Anterograde axonal transport was assessed by intraocular injection of the cholera toxin beta subunit followed by quantification of tracer accumulation in the contralateral superior colliculus. RGC survival was analyzed on whole-mounted retinas using cell-specific markers. Reduction of tau expression was achieved following intravitreal injection of targeted siRNA.ResultsOur data demonstrate an age-related increase in endogenous retinal tau characterized by epitope-specific hypo- and hyper-phosphorylation in 3xTg mice. Retinal tau accumulation was observed as early as three months of age, prior to the reported onset of behavioral deficits, and preceded tau aggregation in the brain. Intriguingly, tau build up occurred in RGC soma and dendrites, while tau in RGC axons in the optic nerve was depleted. Tau phosphorylation changes and missorting correlated with substantial defects in anterograde axonal transport that preceded RGC death. Importantly, targeted siRNA-mediated knockdown of endogenous tau improved anterograde transport along RGC axons.ConclusionsOur study reveals profound tau pathology in the visual system leading to early retinal neuron damage in a mouse model of AD. Importantly, we show that tau accumulation promotes anterograde axonal transport impairment in vivo, and identify this response as an early feature of neuronal dysfunction that precedes cell death in the AD retina. These findings provide the first proof-of-concept that a global strategy to reduce tau accumulation is beneficial to improve axonal transport and mitigate functional deficits in AD and tauopathies.