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

Accumulation of amyloid-ß (Aß) plays an important role in Alzheimer's disease (AD) pathology. There is growing evidence that disordered sleep may accelerate AD pathology by impeding the physiological clearance of Aß from the brain that occurs in normal sleep. Therapeutic strategies for improving sleep quality may therefore help slow disease progression. It is well documented that the composition and dynamics of sleep are sensitive to ambient temperature. We therefore compared Aß pathology and sleep metrics derived from polysomnography in 12-month-old female 3xTg-AD mice (n = 8) exposed to thermoneutral temperatures during the light period over 4 weeks to those of age- and sex-matched controls (n = 8) that remained at normal housing temperature (22°C) during the same period. The treated group experienced greater proportions of slow wave sleep (SWS)-i.e. epochs of elevated 0.5-2 Hz EEG slow wave activity during non-rapid eye movement (NREM) sleep-compared to controls. Assays performed on mouse brain tissue harvested at the end of the experiment showed that exposure to thermoneutral temperatures significantly reduced levels of DEA-soluble (but not RIPA- or formic acid-soluble) Aß40 and Aß42 in the hippocampus, though not in the cortex. With both groups pooled together and without regard to treatment condition, NREM sleep continuity and any measure of SWS within NREM at the end of the treatment period were inversely correlated with DEA-soluble Aß40 and Aß42 levels, again in the hippocampus but not in the cortex. These findings suggest that experimental manipulation of SWS could offer useful clues into the mechanisms and treatment of AD.

Sex and Sleep Disruption as Contributing Factors in Alzheimer's Disease.

Alzheimer's disease (AD) affects more women than men, with women throughout the menopausal transition potentially being the most under researched and at-risk group. Sleep disruptions, which are an established risk factor for AD, increase in prevalence with normal aging and are exacerbated in women during menopause. Sex differences showing more disrupted sleep patterns and increased AD pathology in women and female animal models have been established in literature, with much emphasis placed on loss of circulating gonadal hormones with age. Interestingly, increases in gonadotropins such as follicle stimulating hormone are emerging to be a major contributor to AD pathogenesis and may also play a role in sleep disruption, perhaps in combination with other lesser studied hormones. Several sleep influencing regions of the brain appear to be affected early in AD progression and some may exhibit sexual dimorphisms that may contribute to increased sleep disruptions in women with age. Additionally, some of the most common sleep disorders, as well as multiple health conditions that impair sleep quality, are more prevalent and more severe in women. These conditions are often comorbid with AD and have bi-directional relationships that contribute synergistically to cognitive decline and neuropathology. The association during aging of increased sleep disruption and sleep disorders, dramatic hormonal changes during and after menopause, and increased AD pathology may be interacting and contributing factors that lead to the increased number of women living with AD.

The amyloid-ß peptide: Guilty as charged?

Recent years have seen both considerable progress and controversy in the Alzheimer's disease (AD) field. After decades of slow to negligible movement towards the development of disease modifying therapies, promising outcomes in recent clinical trials with several monoclonal antibodies targeting various forms of the amyloid-ß (Aß) peptide have at last opened a possible way forward. In fact, at this point multiple anti-Aß therapeutics are close to receiving (or have already received) regulatory approval. Although these outcomes are not without some degree of divisiveness, the fact remains that targeting amyloid for removal has finally shown at least modest efficacy in slowing the otherwise relentless progression of the disease. Although the validation of the long standing amyloid cascade hypothesis would seem to be at hand, what remains is the puzzling issue of why - if Aß indeed causes AD - does removing it from the brain not stop the disease entirely.

The db mutation improves memory in younger mice in a model of Alzheimer's disease.

Alzheimer's disease (AD) is the most common age-related neurodegenerative disease, while obesity is a major global public health problem associated with the metabolic disorder type 2 diabetes mellitus (T2DM). Chronic obesity and T2DM have been identified as invariant risk factors for dementia and late-onset AD, while their impacts on the occurrence and development of AD remain unclear. As shown in our previous study, the diabetic mutation (db, Leprdb/db) induces mixed or vascular dementia in mature to middle-aged APPΔNL/ΔNL x PS1P264L/P264L knock-in mice (db/AD). In the present study, the impacts of the db mutation on young AD mice at 10 weeks of age were evaluated. The db mutation not only conferred young AD mice with severe obesity, impaired glucose regulation and activated mammalian target of rapamycin (mTOR) signaling pathway in the mouse cortex, but lead to a surprising improvement in memory. At this young age, mice also had decreased cerebral Aß content, which we have not observed at older ages. This was unlikely to be related to altered Aß synthesis, as both ß- and γ-secretase were unchanged. The db mutation also reduced the cortical IL-1ß mRNA level and IBA1 protein level in young AD mice, with no significant effect on the activation of microglia and astrocytes. We conclude that the db mutation could transitorily improve the memory of young AD mice, a finding that may be partially explained by the relatively improved glucose homeostasis in the brains of db/AD mice compared to their counterpart AD mice, suggesting that glucose regulation could be a strategy for prevention and treatment of neurodegenerative diseases like AD.

Ketogenic diet enhances neurovascular function with altered gut microbiome in young healthy mice.

Neurovascular integrity, including cerebral blood flow (CBF) and blood-brain barrier (BBB) function, plays a major role in determining cognitive capability. Recent studies suggest that neurovascular integrity could be regulated by the gut microbiome. The purpose of the study was to identify if ketogenic diet (KD) intervention would alter gut microbiome and enhance neurovascular functions, and thus reduce risk for neurodegeneration in young healthy mice (12-14 weeks old). Here we show that with 16 weeks of KD, mice had significant increases in CBF and P-glycoprotein transports on BBB to facilitate clearance of amyloid-beta, a hallmark of Alzheimer's disease (AD). These neurovascular enhancements were associated with reduced mechanistic target of rapamycin (mTOR) and increased endothelial nitric oxide synthase (eNOS) protein expressions. KD also increased the relative abundance of putatively beneficial gut microbiota (Akkermansia muciniphila and Lactobacillus), and reduced that of putatively pro-inflammatory taxa (Desulfovibrio and Turicibacter). We also observed that KD reduced blood glucose levels and body weight, and increased blood ketone levels, which might be associated with gut microbiome alteration. Our findings suggest that KD intervention started in the early stage may enhance brain vascular function, increase beneficial gut microbiota, improve metabolic profile, and reduce risk for AD.

Calcineurin/NFAT Signaling in Activated Astrocytes Drives Network Hyperexcitability in Aß-Bearing Mice.

Hyperexcitable neuronal networks are mechanistically linked to the pathologic and clinical features of Alzheimer's disease (AD). Astrocytes are a primary defense against hyperexcitability, but their functional phenotype during AD is poorly understood. Here, we found that activated astrocytes in the 5xFAD mouse model were strongly associated with proteolysis of the protein phosphatase calcineurin (CN) and the elevated expression of the CN-dependent transcription factor nuclear factor of activated T cells 4 (NFAT4). Intrahippocampal injections of adeno-associated virus vectors containing the astrocyte-specific promoter Gfa2 and the NFAT inhibitory peptide VIVIT reduced signs of glutamate-mediated hyperexcitability in 5xFAD mice, measured in vivo with microelectrode arrays and ex vivo brain slices, using whole-cell voltage clamp. VIVIT treatment in 5xFAD mice led to increased expression of the astrocytic glutamate transporter GLT-1 and to attenuated changes in dendrite morphology, synaptic strength, and NMDAR-dependent responses. The results reveal astrocytic CN/NFAT4 as a key pathologic mechanism for driving glutamate dysregulation and neuronal hyperactivity during AD.SIGNIFICANCE STATEMENT Neuronal hyperexcitability and excitotoxicity are increasingly recognized as important mechanisms for neurodegeneration and dementia associated with Alzheimer's disease (AD). Astrocytes are profoundly activated during AD and may lose their capacity to regulate excitotoxic glutamate levels. Here, we show that a highly active calcineurin (CN) phosphatase fragment and its substrate transcription factor, nuclear factor of activated T cells (NFAT4), appear in astrocytes in direct proportion to the extent of astrocyte activation. The blockade of astrocytic CN/NFAT signaling in a common mouse model of AD, using adeno-associated virus vectors normalized glutamate signaling dynamics, increased astrocytic glutamate transporter levels and alleviated multiple signs of neuronal hyperexcitability. The results suggest that astrocyte activation drives hyperexcitability during AD through a mechanism involving aberrant CN/NFAT signaling and impaired glutamate transport.

Down syndrome: age-dependence of PiB binding in postmortem frontal cortex across the lifespan.

Beta-amyloid (Aß) deposition in brain accumulates as a function of age in people with Down syndrome (DS) with subsequent development into Alzheimer disease neuropathology, typically by 40 years of age. In vivo imaging using the Pittsburgh compound B (PiB) ligand has facilitated studies linking Aß, cognition, and dementia in DS. However, there are no studies of PiB binding across the lifespan in DS. The current study describes in vitro 3H-PiB binding in the frontal cortex of autopsy cases with DS compared to non-DS controls. Tissue from 64 cases included controls (n = 25) and DS (n = 39). In DS, 3H-PiB binding was significantly associated with age. After age 40 years in DS, 3H-PiB binding rose dramatically along with increasing individual variability. 3H-PiB binding correlated with the amount of Aß42. Using fixed frontal tissue and fluorescent 6-CN-PiB, neuritic and cored plaques along with extensive cerebral amyloid angiopathy showed 6-CN-PiB binding. These results suggest that cortical PiB binding as shown by positron emission tomography imaging reflects plaques and cerebral amyloid angiopathy in DS brain.

Aß vaccination in combination with behavioral enrichment in aged beagles: effects on cognition, Aß, and microhemorrhages.

Beta-amyloid (Aß) immunotherapy is a promising intervention to slow Alzheimer's disease. Aging dogs naturally accumulate Aß and show cognitive decline. An active vaccine against fibrillar Aß 1-42 (VAC) in aged beagles resulted in maintenance but not improvement of cognition along with reduced brain Aß. Behavioral enrichment (ENR) led to cognitive benefits but no reduction in Aß. We hypothesized cognitive outcomes could be improved by combining VAC with ENR in aged dogs. Aged dogs (11-12 years) were placed into 4 groups: (1) control/control (C/C); (2) control/VAC (C/V); (3) ENR/control (E/C); and (4) ENR/VAC (E/V) and treated for 20 months. VAC decreased brain Aß, pyroglutamate Aß, increased cerebrospinal fluid Aß 42 and brain-derived neurotrophic factor RNA levels but also increased microhemorrhages. ENR reduced brain Aß and prevented microhemorrhages. The combination treatment resulted in a significant maintenance of learning over time, reduced Aß, and increased brain-derived neurotrophic factor mRNA despite increased microhemorrhages; however, there were no benefits to memory. These results suggest that the combination of immunotherapy with behavioral enrichment leads to cognitive maintenance associated with reduced neuropathology that may benefit people with Alzheimer's disease.

A Novel Small Molecule Modulator of Amyloid Pathology.

Because traditional approaches to drug development for Alzheimer's disease are becoming increasingly expensive and in many cases disappointingly unsuccessful, alternative approaches are required to shift the paradigm. Following leads from investigations of dihydropyridine calcium channel blockers, we observed unique properties from a class of functionalized naphthyridines and sought to develop these as novel therapeutics that minimize amyloid pathology without the adverse effects associated with current therapeutics. Our data show methyl 2,4-dimethyl-5-oxo-5,6-dihydrobenzo[c][2,7]naphthyridine-1-carboxylate (BNC-1) significantly decreases amyloid burden in a well-established mouse model of amyloid pathology through a unique mechanism mediated by Elk-1, a transcriptional repressor of presenilin-1. Additionally, BNC-1 treatment leads to increased levels of synaptophysin and synapsin, markers of synaptic integrity, but does not adversely impact presenilin-2 or processing of Notch-1, thus avoiding negative off target effects associated with pan-gamma secretase inhibition. Overall, our data show BNC-1 significantly decreases amyloid burden and improves markers of synaptic integrity in a well-established mouse model of amyloid deposition by promoting phosphorylation and activation of Elk-1, a transcriptional repressor of presenilin-1 but not presenilin-2. These data suggest BNC-1 might be a novel, disease-modifying therapeutic that will alter the pathogenesis of Alzheimer's disease.

Vascular cognitive impairment: Modeling a critical neurologic disease in vitro and in vivo.

BACKGROUND: Vascular contributions to cognitive impairment and dementia (VCID) is a complex form of dementia, combining aspects of vascular disease and other forms of dementia, such as Alzheimer's disease. VCID encompasses a wide spectrum of cerebrovascular-driven cognitive impairment, from mild cognitive impairment to fully developed dementia. This disease state is further complicated by metabolic disorders, such as type 2 diabetes and hypertension, and lifestyle factors, like obesity and high fat diets. SCOPE OF REVIEW: This manuscript is meant to both define VCID and review the in vitro and in vivo models of the disease state. This includes in vitro models of the neurovascular unit, models of chronic cerebral hypoperfusion, animals with NOTCH3 mutations as a model of small vessel disease, large animals with cerebral amyloid angiopathy (CAA), and animal models of mixed dementia. MAJOR CONCLUSIONS: Synthetic microvessels are a promising technique to study the neurovascular unit and canines, despite the cost, are an excellent model to study CAA. While there are several good models of individual aspects of VCID, the heterogeneity of the disease states prevents them from being a model of all aspects of the disease. Therefore, VCID needs to be further defined into disease states that exist within this umbrella term. This includes specific guidelines for stroke counts and stroke locations and further categorization of overlapping cerebrovascular and AD pathologies that contribute to dementia. This will allow for better models and a more thorough understanding of how vascular disease contributes to dementia. GENERAL SIGNIFICANCE: VCID is the second most common form of dementia and is expected to increase in coming years. The heterogeneity of VCID makes it difficult to study, but without better definitions and models, VCID presents a major public health problem for our aging population. This article is part of a Special Issue entitled: Vascular Contributions to Cognitive Impairment and Dementia, edited by M. Paul Murphy, Roderick A. Corriveau and Donna M. Wilcock.

Down syndrome individuals with Alzheimer's disease have a distinct neuroinflammatory phenotype compared to sporadic Alzheimer's disease.

Down syndrome (DS) is the most common genetic cause of intellectual disability and is primarily caused by the triplication of chromosome 21. The overexpression of amyloid precursor protein gene may be sufficient to drive Alzheimer's disease (AD) neuropathology that is observed in virtually all individuals with DS by the age of 40 years. There is relatively little information about inflammation in the DS brain and how the genetics of DS may alter inflammatory responses and modify the course of AD pathogenesis in this disorder. Using the macrophage classification system of M1, M2a, M2b, and M2c inflammatory phenotypes, we have shown that the early stages of AD are associated with a bias toward an M1 or M2a phenotype. In later stages of AD, markers of M1, M2a and M2c are elevated. We now report the inflammatory phenotype in a DS autopsy series to compare this with the progression in sporadic AD. Tissue from young DS cases (under 40 years of age, pre-AD) show a bias toward M1 and M2b states with little M2a or M2c observed. Older DS cases (over 40 with AD pathology) show a distinct bias toward an M2b phenotype. Importantly, this is distinct from sporadic AD where the M2b phenotype has been rarely, if ever observed in postmortem studies. Stimulated by immune complex activation of microglial cells and toll-like receptor activation, the M2b phenotype represents a unique neuroinflammatory state in diseased brain and may have significant implications for therapeutic intervention for persons with DS.

Bridging integrator 1 (BIN1) protein expression increases in the Alzheimer's disease brain and correlates with neurofibrillary tangle pathology.

Recent genome wide association studies have implicated bridging integrator 1 (BIN1) as a late-onset Alzheimer's disease (AD) susceptibility gene. There are at least 15 different known isoforms of BIN1, with many being expressed in the brain including the longest isoform (iso1), which is brain-specific and localizes to axon initial segments and nodes of Ranvier. It is currently unknown what role BIN1 plays in AD. We analyzed BIN1 protein expression from a large number (n = 71) of AD cases and controls from five different brain regions (hippocampus, inferior parietal cortex, inferior temporal cortex, frontal cortex (BA9), and superior and middle temporal gyri). We found that the amount of the largest isoform of BIN1 was significantly reduced in the AD brain compared to age-matched controls, and smaller BIN1 isoforms were significantly increased. Further, BIN1 was significantly correlated with the amount of neurofibrillary tangle (NFT) pathology but not with either diffuse or neuritic plaques, or with the amount of amyloid-ß peptide. BIN1 is known to be abnormally expressed in another human disease, myotonic dystrophy, which also features prominent NFT pathology. These data suggest that BIN1 is likely involved in AD as a modulator of NFT pathology, and that this role may extend to other human diseases that feature tau pathology.

Synaptophysin and synaptojanin-1 in Down syndrome are differentially affected by Alzheimer's disease.

Adults with Down syndrome (DS) develop Alzheimer's disease (AD) neuropathology by 40 years of age. Synaptophysin (SYN) consistently declines with age and is further reduced with sporadic AD. Thus, we hypothesized that SYN would be reduced in DS with AD. The gene for synaptojanin-1 (SYNJ1), involved in synaptic vesicle recycling, is on chromosome 21. We measured SYN and SYNJ1 in an autopsy series of 39 cases with DS and 28 without DS, along with 7 sporadic AD cases. SYN was significantly lower in DSAD compared with DS alone and similar to sporadic AD. Reduced SYN is associated with AD neuropathology and with Aß levels in DS, as is seen in sporadic AD. SYNJ1 was significantly higher in DS and correlated with several measures of Aß. SYNJ1 was higher in DSAD and significantly higher than SYNJ1 in sporadic AD. Although significantly higher in DS, SYNJ1 is further increased with AD neuropathology suggesting interesting differences in a synapse-associated protein that is overexpressed in trisomy 21.

Antisense oligonucleotide against GSK-3ß in brain of SAMP8 mice improves learning and memory and decreases oxidative stress: Involvement of transcription factor Nrf2 and implications for Alzheimer disease.

Glycogen synthase kinase (GSK)-3ß is a multifunctional protein that has been implicated in the pathological characteristics of Alzheimer's disease (AD), including the heightened levels of neurofibrillary tangles, amyloid-beta (Aß), and neurodegeneration. In this study we used 12-month-old SAMP8 mice, an AD model, to examine the effects GSK-3ß may cause regarding the cognitive impairment and oxidative stress associated with AD. To suppress the level of GSK-3ß, SAMP8 mice were treated with an antisense oligonucleotide (GAO) directed at this kinase. We measured a decreased level of GSK-3ß in the cortex of the mice, indicating the success of the antisense treatment. Learning and memory assessments of the SAMP8 mice were tested post-antisense treatment using an aversive T-maze and object recognition test, both of which observably improved. In cortex samples of the SAMP8 mice, decreased levels of protein carbonyl and protein-bound HNE were measured, indicating decreased oxidative stress. Nuclear factor erythroid-2-related factor 2 (Nrf2) is a transcription factor known to increase the level of many antioxidants, including glutathione-S transferase (GST), and is negatively regulated by the activity of GSK-3ß. Our results indicated the increased nuclear localization of Nrf2 and level of GST, suggesting the increased activity of the transcription factor as a result of GSK-3ß suppression, consistent with the decreased oxidative stress observed. Consistent with the improved learning and memory, and consistent with GSK-3b being a tau kinase, we observed decreased tau phosphorylation in brain of GAO-treated SAMP8 mice compared to that of RAO-treated SAMP8 mice. Lastly, we examined the ability of GAO to cross the blood-brain barrier and determined it to be possible. The results presented in this study demonstrate that reducing GSK-3 with a phosphorothionated antisense against GSK-3 improves learning and memory, reduces oxidative stress, possibly coincident with increased levels of the antioxidant transcriptional activity of Nrf2, and decreases tau phosphorylation. Our study supports the notion of GAO as a possible treatment for AD.

Oxidative modification of lipoic acid by HNE in Alzheimer disease brain.

Alzheimer disease (AD) is an age-related neurodegenerative disease characterized by the presence of three pathological hallmarks: synapse loss, extracellular senile plaques (SP) and intracellular neurofibrillary tangles (NFTs). The major component of SP is amyloid ß-peptide (Aß), which has been shown to induce oxidative stress. The AD brain shows increased levels of lipid peroxidation products, including 4-hydroxy-2-nonenal (HNE). HNE can react covalently with Cys, His, or Lys residues on proteins, altering structure and function of the latter. In the present study we measured the levels of the HNE-modified lipoic acid in brain of subjects with AD and age-matched controls. Lipoic acid is a key co-factor for a number of proteins including pyruvate dehydrogenase and α-ketoglutarate dehydrogenase, key complexes for cellular energetics. We observed a significant decrease in the levels of HNE-lipoic acid in the AD brain compared to that of age-matched controls. To investigate this phenomenon further, the levels and activity of lipoamide dehydrogenase (LADH) were measured in AD and control brains. Additionally, LADH activities were measured after in-vitro HNE-treatment to mice brains. Both LADH levels and activities were found to be significantly reduced in AD brain compared to age-matched control. HNE-treatment also reduced the LADH activity in mice brain. These data are consistent with a two-hit hypothesis of AD: oxidative stress leads to lipid peroxidation that, in turn, causes oxidative dysfunction of key energy-related complexes in mitochondria, triggering neurodegeneration. This study is consonant with the notion that lipoic acid supplementation could be a potential treatment for the observed loss of cellular energetics in AD and potentiate the antioxidant defense system to prevent or delay the oxidative stress in and progression of this devastating dementing disorder.

Transgenic models of Alzheimer's disease: better utilization of existing models through viral transgenesis.

Animal models have been used for decades in the Alzheimer's disease (AD) research field and have been crucial for the advancement of our understanding of the disease. Most models are based on familial AD mutations of genes involved in the amyloidogenic process, such as the amyloid precursor protein (APP) and presenilin 1 (PS1). Some models also incorporate mutations in tau (MAPT) known to cause frontotemporal dementia, a neurodegenerative disease that shares some elements of neuropathology with AD. While these models are complex, they fail to display pathology that perfectly recapitulates that of the human disease. Unfortunately, this level of pre-existing complexity creates a barrier to the further modification and improvement of these models. However, as the efficacy and safety of viral vectors improves, their use as an alternative to germline genetic modification is becoming a widely used research tool. In this review we discuss how this approach can be used to better utilize common mouse models in AD research. This article is part of a Special Issue entitled: Animal Models of Disease.

Impairment of proteostasis network in Down syndrome prior to the development of Alzheimer's disease neuropathology: redox proteomics analysis of human brain.

DS is the most frequent genetic cause of intellectual disability characterized by the anomalous presence of three copies of chromosome 21. One of the peculiar features of DS is the onset of Alzheimer's disease neuropathology after the age of 40years characterized by deposition of senile plaques and neurofibrillary tangles. Growing studies demonstrated that increased oxidative damage, accumulation of unfolded/damaged protein aggregates and dysfunction of intracellular degradative system are key players in neurodegenerative processes. In this study, redox proteomics approach was used to analyze the frontal cortex from DS subjects under the age of 40 compared with age-matched controls, and proteins found to be increasingly carbonylated were identified. Interestingly, our results showed that oxidative damage targets specifically different components of the intracellular quality control system such as GRP78, UCH-L1, V0-ATPase, cathepsin D and GFAP that couples with decreased activity of the proteasome and autophagosome formation observed. We also reported a slight but consistent increase of Aß 1-42 SDS- and PBS-soluble form and tau phosphorylation in DS versus CTR. We suggest that disturbance in the proteostasis network could contribute to the accumulation of protein aggregates, such as amyloid deposits and NFTs, which occur very early in DS. It is likely that a sub-optimal functioning of degradative systems occur in DS neurons, which in turn provide the basis for further accumulation of toxic protein aggregates. The results of this study suggest that oxidation of protein members of the proteostatis network is an early event in DS and might contribute to neurodegenerative phenomena.