Investigating the plasma-liver-brain axis of omega-3 fatty acid metabolism in mouse knock-in for the human apolipoprotein E epsilon 4 allele.

The metabolism of docosahexaenoic acid (DHA), an omega-3 fatty acid, is different in carriers of APOE4, the main genetic risk factor for late-onset Alzheimer's disease. The brain relies on the plasma DHA pool for its need, but the plasma-liver-brain axis in relation to cognition remains obscure. We hypothesized that this relationship is compromised in APOE4 mice considering the differences in fatty acid metabolism between APOE3 and APOE4 mice. Male and female APOE3 and APOE4 mice were fed either a diet enriched with DHA (0.7 g DHA/100 g diet) or a control diet for 8 months. There was a significant genotype × diet interaction for DHA concentration in the liver and adipose tissue. In the cortex, a genotype effect was found where APOE4 mice had a higher concentration of DHA than APOE3 mice fed the control diet. There was a significant genotype × diet interaction for the liver and hippocampal arachidonic acid (AA). APOE4 mice had 20-30% lower plasma DHA and AA concentrations than APOE3 mice, independent of diet. Plasma and liver DHA levels were significantly correlated in APOE3 and APOE4 mice. In APOE4 mice, there was a significant correlation between plasma, adipose tissues, cortex DHA and the Barnes maze and/or with a better recognition index. Moreover, higher AA levels in the liver and the hippocampus of APOE4 mice were correlated with lower cognitive performance. Our results suggest that there is a plasma-liver-brain axis of DHA that is modified in APOE4 mice. Moreover, our data support that APOE4 mice rely more on plasma DHA than APOE3 mice, especially in cognitive performance. Any disturbance in plasma DHA metabolism might have a greater impact on cognition in APOE4 carriers.

Astrocytes amplify neurovascular coupling to sustained activation of neocortex in awake mice.

Functional hyperemia occurs when enhanced neuronal activity signals to increase local cerebral blood flow (CBF) to satisfy regional energy demand. Ca2+ elevation in astrocytes can drive arteriole dilation to increase CBF, yet affirmative evidence for the necessity of astrocytes in functional hyperemia in vivo is lacking. In awake mice, we discovered that functional hyperemia is bimodal with a distinct early and late component whereby arteriole dilation progresses as sensory stimulation is sustained. Clamping astrocyte Ca2+ signaling in vivo by expressing a plasma membrane Ca2+ ATPase (CalEx) reduces sustained but not brief sensory-evoked arteriole dilation. Elevating astrocyte free Ca2+ using chemogenetics selectively augments sustained hyperemia. Antagonizing NMDA-receptors or epoxyeicosatrienoic acid production reduces only the late component of functional hyperemia, leaving brief increases in CBF to sensory stimulation intact. We propose that a fundamental role of astrocyte Ca2+ is to amplify functional hyperemia when neuronal activation is prolonged.

A diet rich in docosahexaenoic acid enhances reactive astrogliosis and ramified microglia morphology in apolipoprotein E epsilon 4-targeted replacement mice.

Docosahexaenoic acid (DHA) consumption reduces spatial memory impairment in mice carrying the human apolipoprotein E ε4 (APOE4) allele. The current study evaluated whether astrocyte and microglia morphology contribute to the mechanism of this result. APOE3 and APOE4 mice were fed either a DHA-enriched diet or a control diet from 4 to 12 months of age. Coronal brain sections were immunostained for GFAP, Iba1, and NeuN. Astrocytes from APOE4 mice exhibited signs of reactive astrogliosis compared to APOE3 mice. Consumption of DHA exacerbated reactive astrocyte morphology in APOE4 carriers. Microglia from APOE4-control mice exhibited characteristics of amoeboid morphology and other characteristics of ramified morphology (more processes, greater process complexity, and greater distance between neighboring microglia). DHA enhanced ramified microglia morphology in APOE4 mice. In addition, APOE4 mice fed the DHA diet had lower hippocampal concentrations of interleukin-7, lipopolysaccharide-induced CXC chemokine and monocyte chemoattractant protein 1, and higher concentration of interferon-gamma compared to APOE4-control mice. Our results indicate that a diet rich in DHA enhances reactive astrogliosis and ramified microglia morphology in APOE4 mice.

Metabolic determinants of Alzheimer's disease: A focus on thermoregulation.

Alzheimer's disease (AD) is a complex age-related neurodegenerative disease, associated with central and peripheral metabolic anomalies, such as impaired glucose utilization and insulin resistance. These observations led to a considerable interest not only in lifestyle-related interventions, but also in repurposing insulin and other anti-diabetic drugs to prevent or treat dementia. Body temperature is the oldest known metabolic readout and mechanisms underlying its maintenance fail in the elderly, when the incidence of AD rises. This raises the possibility that an age-associated thermoregulatory deficit contributes to energy failure underlying AD pathogenesis. Brown adipose tissue (BAT) plays a central role in thermogenesis and maintenance of body temperature. In recent years, the modulation of BAT activity has been increasingly demonstrated to regulate energy expenditure, insulin sensitivity and glucose utilization, which could also provide benefits for AD. Here, we review the evidence linking thermoregulation, BAT and insulin-related metabolic defects with AD, and we propose mechanisms through which correcting thermoregulatory impairments could slow the progression and delay the onset of AD.

Astrocytes regulate ultra-slow arteriole oscillations via stretch-mediated TRPV4-COX-1 feedback.

Very-low-frequency oscillations in microvascular diameter cause fluctuations in oxygen delivery that are important for fueling the brain and for functional imaging. However, little is known about how the brain regulates ongoing oscillations in cerebral blood flow. In mouse and rat cortical brain slice arterioles, we find that selectively enhancing tone is sufficient to recruit a TRPV4-mediated Ca2+ elevation in adjacent astrocyte endfeet. This endfoot Ca2+ signal triggers COX-1-mediated "feedback vasodilators" that limit the extent of evoked vasoconstriction, as well as constrain fictive vasomotion in slices. Astrocyte-Ptgs1 knockdown in vivo increases the power of arteriole oscillations across a broad range of very low frequencies (0.01-0.3 Hz), including ultra-slow vasomotion (∼0.1 Hz). Conversely, clamping astrocyte Ca2+in vivo reduces the power of vasomotion. These data demonstrate bidirectional communication between arterioles and astrocyte endfeet to regulate oscillatory microvasculature activity.

High-Fat Diet Modulates Hepatic Amyloid ß and Cerebrosterol Metabolism in the Triple Transgenic Mouse Model of Alzheimer's Disease.

Obesity and diabetes are strongly associated not only with fatty liver but also cognitive dysfunction. Moreover, their presence, particularly in midlife, is recognized as a risk factor for Alzheimer's disease (AD). AD, the most common cause of dementia, is increasingly considered as a metabolic disease, although underlying pathogenic mechanisms remain unclear. The liver plays a major role in maintaining glucose and lipid homeostasis, as well as in clearing the AD neuropathogenic factor amyloid-ß (Aß) and in metabolizing cerebrosterol, a cerebral-derived oxysterol proposed as an AD biomarker. We hypothesized that liver impairment induced by obesity contributes to AD pathogenesis. We show that the AD triple transgenic mouse model (3xTg-AD) fed a chow diet presents a hepatic phenotype similar to nontransgenic controls (NTg) at 15 months of age. A high-fat diet (HFD), started at the age of 6 months and continued for 9 months, until sacrifice, induced hepatic steatosis in NTg, but not in 3xTg-AD mice, whereas HFD did not induce changes in hepatic fatty acid oxidation, de novo lipogenesis, and gluconeogenesis. HFD-induced obesity was associated with a reduction of insulin-degrading enzyme, one of the main hepatic enzymes responsible for Aß clearance. The hepatic rate of cerebrosterol glucuronidation was lower in obese 3xTg-AD than in nonobese controls (P < 0.05) and higher compared with obese NTg (P < 0.05), although circulating levels remained unchanged. Conclusion: Modulation of hepatic lipids, Aß, and cerebrosterol metabolism in obese 3xTg-AD mice differs from control mice. This study sheds light on the liver-brain axis, showing that the chronic presence of NAFLD and changes in liver function affect peripheral AD features and should be considered during development of biomarkers or AD therapeutic targets.

Behavioral Deficits in Mice with Postnatal Disruption of Ndel1 in Forebrain Excitatory Neurons: Implications for Epilepsy and Neuropsychiatric Disorders.

Dysfunction of nuclear distribution element-like 1 (Ndel1) is associated with schizophrenia, a neuropsychiatric disorder characterized by cognitive impairment and with seizures as comorbidity. The levels of Ndel1 are also altered in human and models with epilepsy, a chronic condition whose hallmark feature is the occurrence of spontaneous recurrent seizures and is typically associated with comorbid conditions including learning and memory deficits, anxiety, and depression. In this study, we analyzed the behaviors of mice postnatally deficient for Ndel1 in forebrain excitatory neurons (Ndel1 CKO) that exhibit spatial learning and memory deficits, seizures, and shortened lifespan. Ndel1 CKO mice underperformed in species-specific tasks, that is, the nest building, open field, Y maze, forced swim, and dry cylinder tasks. We surveyed the expression and/or activity of a dozen molecules related to Ndel1 functions and found changes that may contribute to the abnormal behaviors. Finally, we tested the impact of Reelin glycoprotein that shows protective effects in the hippocampus of Ndel1 CKO, on the performance of the mutant animals in the nest building task. Our study highlights the importance of Ndel1 in the manifestation of species-specific animal behaviors that may be relevant to our understanding of the clinical conditions shared between neuropsychiatric disorders and epilepsy.

Reelin Improves Cognition and Extends the Lifespan of Mutant Ndel1 Mice with Postnatal CA1 Hippocampus Deterioration.

The glycoprotein Reelin maintains neuronal positioning and regulates neuronal plasticity in the adult brain. Reelin deficiency has been associated with neurological diseases. We recently showed that Reelin is depleted in mice with a targeted disruption of the Ndel1 gene in forebrain postnatal excitatory neurons (Ndel1 conditional knockout (CKO)). Ndel1 CKO mice exhibit fragmented microtubules in CA1 pyramidal neurons, profound deterioration of the CA1 hippocampus and a shortened lifespan (~10 weeks). Here we report that Ndel1 CKO mice (of both sexes) experience spatial learning and memory deficits that are associated with deregulation of neuronal cell adhesion, plasticity and neurotransmission genes, as assessed by genome-wide transcriptome analysis of the hippocampus. Importantly, a single injection of Reelin protein in the hippocampus of Ndel1 CKO mice improves spatial learning and memory function and this is correlated with reduced intrinsic hyperexcitability of CA1 pyramidal neurons, and normalized gene deregulation in the hippocampus. Strikingly, when treated with Reelin, Ndel1 CKO animals that die from an epileptic phenotype, live twice as long as nontreated, or vehicle-treated CKO animals. Thus, Reelin confers striking beneficial effects in the CA1 hippocampus, and at both behavioral and organismal levels.

Dietary intake of branched-chain amino acids in a mouse model of Alzheimer's disease: Effects on survival, behavior, and neuropathology.

INTRODUCTION: High levels of plasmatic branched-chain amino acids (BCAA), commonly used as dietary supplements, are linked to metabolic risk factors for Alzheimer's disease (AD). BCAA directly influence amino acid transport to the brain and, therefore, neurotransmitter levels. We thus investigated the impact of BCAA on AD neuropathology in a mouse model. METHODS: 3xTg-AD mice were fed either a control diet or a high-fat diet from 6 to 18 months of age. For the last 2 months, dietary BCAA content was adjusted to high (+50%), normal (+0%), or low (-50%). RESULTS: Mice fed a BCAA-supplemented high-fat diet displayed higher tau neuropathology and only four out of 13 survived. Mice on the low-BCAA diet showed higher threonine and tryptophan cortical levels while performing better on the novel object recognition task. DISCUSSION: These preclinical data underscore a potential risk of combining high-fat and high BCAA consumption, and possible benefits from BCAA restriction in AD.

In vitro models and systems for evaluating the dynamics of drug delivery to the healthy and diseased brain.

The blood-brain barrier (BBB) plays a crucial role in maintaining brain homeostasis and transport of drugs to the brain. The conventional animal and Transwell BBB models along with emerging microfluidic-based BBB-on-chip systems have provided fundamental functionalities of the BBB and facilitated the testing of drug delivery to the brain tissue. However, developing biomimetic and predictive BBB models capable of reasonably mimicking essential characteristics of the BBB functions is still a challenge. In addition, detailed analysis of the dynamics of drug delivery to the healthy or diseased brain requires not only biomimetic BBB tissue models but also new systems capable of monitoring the BBB microenvironment and dynamics of barrier function and delivery mechanisms. This review provides a comprehensive overview of recent advances in microengineering of BBB models with different functional complexity and mimicking capability of healthy and diseased states. It also discusses new technologies that can make the next generation of biomimetic human BBBs containing integrated biosensors for real-time monitoring the tissue microenvironment and barrier function and correlating it with the dynamics of drug delivery. Such integrated system addresses important brain drug delivery questions related to the treatment of brain diseases. We further discuss how the combination of in vitro BBB systems, computational models and nanotechnology supports for characterization of the dynamics of drug delivery to the brain.

Docosahexaenoic acid prevents cognitive deficits in human apolipoprotein E epsilon 4-targeted replacement mice.

At a population level, dietary consumption of fish rich in docosahexaenoic acid (DHA) is associated with prevention of cognitive decline but this association is not clear in carriers of the apolipoprotein E epsilon 4 allele (E4). Plasma and liver DHA concentrations show significant alterations in E4 carriers, in part corrected by DHA supplementation. However, whether DHA sufficiency in E4 carriers has consequences on cognition is unknown. Mice expressing human E4 or apolipoprotein E epsilon 3 allele (E3) were fed either a control diet or a diet containing DHA for 8 months and cognitive performance was tested using the object recognition test and the Barnes maze test. In E4 mice fed the control diet, impaired memory was detected and arachidonic acid concentrations were elevated in the hippocampus compared to E3 mice fed the control diet. DHA consumption prevented memory decline and restored arachidonic acid concentrations in the hippocampus of E4 mice. Our results suggest that long-term high-dose DHA intake may prevent cognitive decline in E4 carriers.

Association of Neuropathological Markers in the Parietal Cortex With Antemortem Cognitive Function in Persons With Mild Cognitive Impairment and Alzheimer Disease.

The associations between cognitive function and neuropathological markers in patients with mild cognitive impairment (MCI) and Alzheimer disease (AD) remain only partly defined. We investigated relationships between antemortem global cognitive scores and ß-amyloid (Aß), tau, TDP-43, synaptic proteins and other key AD neuropathological markers assessed by biochemical approaches in postmortem anterior parietal cortex samples from 36 subjects (12 MCI, 12 AD and 12 not cognitively impaired) from the Religious Orders Study. Overall, the strongest negative correlation coefficients associated with global cognitive scores were obtained for insoluble phosphorylated tau (r2 = -0.484), insoluble Aß42 (r2 = -0.389) and neurofibrillary tangle counts (r2 = -0.494) (all p < 0.001). Robust inverse associations with cognition scores were also established for TDP-43-positive cytoplasmic inclusions (r2 = -0.476), total insoluble tau (r2 = -0.385) and Aß plaque counts (r2 = -0.426). Sarkosyl (SK)- or formic acid (FA)-extracted tau showed similar interrelations. On the other hand, synaptophysin (r2 = +0.335), pS403/404 TDP-43 (r2 = +0.265) and septin-3 (r2 = +0.257) proteins positively correlated with cognitive scores. This study suggests that tau and Aß42 in their insoluble aggregated forms, synaptic proteins and TDP-43 are the markers in the parietal cortex that are most strongly associated with cognitive function. This further substantiates the relevance of investigating these markers to understand the pathogenesis of AD and develop therapeutic tools.

Altered cerebral insulin response in transgenic mice expressing the epsilon-4 allele of the human apolipoprotein E gene.

Apolipoprotein E epsilon-4 (APOEε4 or APOE4), an allelic variation of the APOE gene, not only increases the risk of developing the late-onset form of Alzheimer's disease (AD), but also influences the outcome of treatment. Indeed, data from clinical studies show that the beneficial effect of insulin on cognition is blunted in APOE4 carriers. To investigate how APOE impacts insulin response, we assessed the effects of an acute insulin injection in APOE3- and APOE4-targeted replacement mice that respectively express the human APOE3 or APOE4 isoform instead of the endogenous murine ApoE protein. We evaluated cognition, insulin signaling and proteins implicated in Aß transport and tau phosphorylation in the cortex and brain capillaries. We found that a single acute insulin injection increased Akt pSer473 in APOE4 compared to APOE3 mice (+113% versus +78.5%), indicating that APOE4 carriage potentiates activation of insulin upstream signaling pathway in the brain. Insulin also led to decreased concentrations of the receptor for advanced glycation endproducts (RAGE) in brain capillaries in both groups of mice. Moreover, higher phosphorylation of tau at Ser202, one of the key markers of AD neuropathology, was observed in insulin-injected APOE4 mice (+44%), consistent with findings in human APOE4 carriers (+400% compared to non-carriers). Therefore, our data suggest that APOE4 carriage leads to an increased insulin-induced activation of cerebral Akt pathway, associated with higher AD-like tau neuropathology. Our results provide evidence of altered insulin signaling in APOE4 carriers as well as a possible mechanism to explain the absence of cognitive benefit from insulin therapy in these individuals.

Old age potentiates cold-induced tau phosphorylation: linking thermoregulatory deficit with Alzheimer's disease.

Thermoregulatory deficits coincide with a rise in the incidence of Alzheimer's disease (AD) in old age. Lower body temperature increases tau phosphorylation, a neuropathological hallmark of AD. To determine whether old age potentiates cold-induced tau phosphorylation, we compared the effects of cold exposure (4 °C, 24 hours) in 6- and 18-month-old mice. Cold-induced changes in body temperature, brown adipose tissue activity, and phosphorylation of tau at Ser202 were not different between 6- and 18-month-old mice. However, following cold exposure, only old mice displayed a significant rise in soluble tau pThr181 and pThr231, which was correlated with body temperature. Inactivation of glycogen synthase kinase 3ß was more prominent in young mice, suggesting a protective mechanism against cold-induced tau phosphorylation. These results suggest that old age confers higher susceptibility to tau hyperphosphorylation following a change in body temperature, thereby contributing to an enhanced risk of developing AD.

eEF2K inhibition blocks Aß42 neurotoxicity by promoting an NRF2 antioxidant response.

Soluble oligomers of amyloid-ß (Aß) impair synaptic plasticity, perturb neuronal energy homeostasis, and are implicated in Alzheimer's disease (AD) pathogenesis. Therefore, significant efforts in AD drug discovery research aim to prevent the formation of Aß oligomers or block their neurotoxicity. The eukaryotic elongation factor-2 kinase (eEF2K) plays a critical role in synaptic plasticity, and couples neurotransmission to local dendritic mRNA translation. Recent evidence indicates that Aß oligomers activate neuronal eEF2K, suggesting a potential link to Aß induced synaptic dysfunction. However, a detailed understanding of the role of eEF2K in AD pathogenesis, and therapeutic potential of eEF2K inhibition in AD, remain to be determined. Here, we show that eEF2K activity is increased in postmortem AD patient cortex and hippocampus, and in the hippocampus of aged transgenic AD mice. Furthermore, eEF2K inhibition using pharmacological or genetic approaches prevented the toxic effects of Aß42 oligomers on neuronal viability and dendrite formation in vitro. We also report that eEF2K inhibition promotes the nuclear factor erythroid 2-related factor (NRF2) antioxidant response in neuronal cells, which was crucial for the beneficial effects of eEF2K inhibition in neurons exposed to Aß42 oligomers. Accordingly, NRF2 knockdown or overexpression of the NRF2 inhibitor, Kelch-Like ECH-Associated Protein-1 (Keap1), significantly attenuated the neuroprotection associated with eEF2K inhibition. Finally, genetic deletion of the eEF2K ortholog efk-1 reduced oxidative stress, and improved chemotaxis and serotonin sensitivity in C. elegans expressing human Aß42 in neurons. Taken together, these findings highlight the potential utility of eEF2K inhibition to reduce Aß-mediated oxidative stress in AD.

Impaired thermoregulation and beneficial effects of thermoneutrality in the 3×Tg-AD model of Alzheimer's disease.

The sharp rise in the incidence of Alzheimer's disease (AD) at an old age coincides with a reduction in energy metabolism and core body temperature. We found that the triple-transgenic mouse model of AD (3×Tg-AD) spontaneously develops a lower basal body temperature and is more vulnerable to a cold environment compared with age-matched controls. This was despite higher nonshivering thermogenic activity, as evidenced by brown adipose tissue norepinephrine content and uncoupling protein 1 expression. A 24-hour exposure to cold (4 °C) aggravated key neuropathologic markers of AD such as: tau phosphorylation, soluble amyloid beta concentrations, and synaptic protein loss in the cortex of 3×Tg-AD mice. Strikingly, raising the body temperature of aged 3×Tg-AD mice via exposure to a thermoneutral environment improved memory function and reduced amyloid and synaptic pathologies within a week. Our results suggest the presence of a vicious cycle between impaired thermoregulation and AD-like neuropathology, and it is proposed that correcting thermoregulatory deficits might be therapeutic in AD.

Can insulin signaling pathways be targeted to transport Aß out of the brain?

Although the causal role of Amyloid-ß (Aß) in Alzheimer's disease (AD) is unclear, it is still reasonable to expect that lowering concentrations of Aß in the brain may decrease the risk of developing the neurocognitive symptoms of the disease. Brain capillary endothelial cells forming the blood-brain barrier (BBB) express transporters regulating the efflux of Aß out of the cerebral tissue. Age-related BBB dysfunctions, that have been identified in AD patients, might impair Aß clearance from the brain. Thus, targeting BBB outward transport systems has been suggested as a way to stimulate the clearance of Aß from the brain. Recent data indicate that the increase in soluble brain Aß and behavioral impairments in 3×Tg-AD mice generated by months of intake of a high-fat diet can be acutely reversed by the administration of a single dose of insulin. A concomitant increase in plasma Aß suggests that clearance from the brain through the BBB is a likely mechanism for this rapid effect of insulin. Here, we review how BBB insulin response pathways could be stimulated to decrease brain Aß concentrations and improve cognitive performance, at least on the short term.

Age-dependent impairment of glucose tolerance in the 3xTg-AD mouse model of Alzheimer's disease.

Alzheimer's disease (AD) has been associated with type II diabetes (T2D) and obesity in several epidemiologic studies. To determine whether AD neuropathology can cause peripheral metabolic impairments, we investigated metabolic parameters in the triple-transgenic (3xTg)-AD mouse model of AD, compared with those in nontransgenic (non-Tg) controls, at 6, 8, and 14 mo of age. We found a more pronounced cortical Aß accumulation (2- and 3.5-fold increase in Aß42 in the soluble and insoluble protein fractions, respectively) in female 3xTg-AD mice than in the males. Furthermore, female 3xTg-AD mice displayed a significant deterioration in glucose tolerance (AUC, +118% vs. non-Tg mice at 14 mo). Fasting plasma insulin levels rose 2.5-fold from 6 to 14 mo of age in female 3xTg-AD mice. Glucose intolerance and cortical amyloid pathology worsened with age, and both were more pronounced in the females. Pancreatic amyloidopathy was revealed and could underlie the observed deficit in glycemic response in 3xTg-AD mice. The present results suggest that AD-like neuropathology extends to the pancreas in the 3xTg-AD mouse, leading to glucose intolerance and contributing to a pathologic self-amplifying loop between AD and T2D.

The benefit of docosahexaenoic acid for the adult brain in aging and dementia.

A brief overview of the evidence for omega-3 fatty acids and, in particular, of docosahexaenoic acid (DHA), involvement in cognition and in dementia is given. Two studies are presented in this regard in which the key intervention is a DHA supplement. The fist, the MIDAS Study demonstrated that DHA can be of benefit for episodic memory in healthy adults with a mild memory complaint. The second, the ADCS AD trial found no benefit of DHA in the primary outcomes but found an intriguing benefit for cognitive score in ApoE4 negative allele patients. This leads to a consideration of the mechanisms of action and role of ApoE and its modulation by DHA. Given the fundamental role of ApoE in cellular lipid transport and metabolism in the brain and periphery, it is no surprise that ApoE affects n-3 PUFA brain function as well. It remains to be seen to what extent ApoE4 deleterious effect in AD is associated with n-3 PUFA-related cellular mechanisms in the brain and, more specifically, whether ApoE4 directly impairs the transport of DHA into the brain, as has been suggested.