Regional differences in Alzheimer's disease pathology confound behavioural rescue after amyloid-ß attenuation.

Failure of Alzheimer's disease clinical trials to improve or stabilize cognition has led to the need for a better understanding of the driving forces behind cognitive decline in the presence of active disease processes. To dissect contributions of individual pathologies to cognitive function, we used the TgF344-AD rat model, which recapitulates the salient hallmarks of Alzheimer's disease pathology observed in patient populations (amyloid, tau inclusions, frank neuronal loss, and cognitive deficits). scyllo-Inositol treatment attenuated amyloid-ß peptide in disease-bearing TgF344-AD rats, which rescued pattern separation in the novel object recognition task and executive function in the reversal learning phase of the Barnes maze. Interestingly, neither activities of daily living in the burrowing task nor spatial memory in the Barnes maze were rescued by attenuating amyloid-ß peptide. To understand the pathological correlates leading to behavioural rescue, we examined the neuropathology and in vivo electrophysiological signature of the hippocampus. Amyloid-ß peptide attenuation reduced hippocampal tau pathology and rescued adult hippocampal neurogenesis and neuronal function, via improvements in cross-frequency coupling between theta and gamma bands. To investigate mechanisms underlying the persistence of spatial memory deficits, we next examined neuropathology in the entorhinal cortex, a region whose input to the hippocampus is required for spatial memory. Reduction of amyloid-ß peptide in the entorhinal cortex had no effect on entorhinal tau pathology or entorhinal-hippocampal neuronal network dysfunction, as measured by an impairment in hippocampal response to entorhinal stimulation. Thus, rescue or not of cognitive function is dependent on regional differences of amyloid-ß, tau and neuronal network dysfunction, demonstrating the importance of staging disease in patients prior to enrolment in clinical trials. These results further emphasize the need for combination therapeutic approaches across disease progression.

In vivo neurovascular response to focused photoactivation of Channelrhodopsin-2.

The rapid growth in the use of optogenetics for neuroscience applications is largely driven by two important advantages: highly specific cellular targeting through genetic manipulations; and precise temporal control of neuronal activation via temporal modulation of the optical stimulation. The difference between the most commonly used stimulation modalities, namely diffused (i.e. synchronous) and focused (i.e. asynchronous) stimulation has not been described. Furthermore, full realization of optogenetics' potential is hindered by our incomplete understanding of the cellular and network level response to photoactivation. Here we address these gaps by examining the neuronal and cerebrovascular responses to focused and diffuse photostimulation of channelrhodopsin in the Thy1-ChR2 mouse. We presented the responses of photoactivation via 470-nm fiber optic illumination (diffuse) alongside 458-nm raster-scan (focused) stimulation of the barrel field. Local field potentials (LFP) assessment of intracerebral electrophysiology and two-photon fluorescence microscopy measurements of red blood cell (RBC) speed (vRBC) in cortical penetrating vessels revealed ∼40% larger LFP responses (p = 0.05) and twice as large cerebrovascular responses (p = 0.002) under focused vs. diffuse photostimulation (focused: 1.64 ±â€¯0.84 mV LFP amplitude and 75 ±â€¯48% increase in vRBC; diffuse: 1.14 ±â€¯0.75 mV LFP amplitude and 35 ±â€¯23% increase in vRBC). Compared to diffuse photostimulation, focused photostimulation resulted in a ∼65% increase in the yield of cerebrovascular responses (73 ±â€¯10% for focused and 42 ±â€¯29% for diffuse photostimulation) and a doubling of the signal-to-noise ratio of the cerebrovascular response (20.9 ±â€¯14.7 for focused and 10.4 ±â€¯1.4 for diffuse photostimulation). These data reveal important advantages of focused optogenetic photoactivation, which can be easily integrated into single- or two-photon fluorescence microscopy platforms, as a means of assessing neuronal excitability and cerebrovascular reactivity, thus paving the way for broader application of optogenetics in preclinical models of CNS diseases.

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.

Imaging the Effects of ß-Hydroxybutyrate on Peri-Infarct Neurovascular Function and Metabolism.

Background and Purpose- Recent evidence suggests great potential of metabolically targeted interventions for treating neurological disorders. We investigated the use of the endogenous ketone body ß-hydroxybutyrate (BHB) as an alternate metabolic substrate for the brain in the acute phase of ischemia because postischemic hyperglycemia and brain glucose metabolism elevation compromise functional recovery. Methods- We delivered BHB (or vehicle) 1 hour after ischemic insult induced by cortical microinjection of endothelin-1 in sensorimotor cortex of rats. Two days after ischemic insult, the rats underwent multimodal characterization of the BHB effects. We examined glucose uptake on 2-Deoxy-d-glucose chemical exchange saturation transfer magnetic resonance imaging, cerebral hemodynamics on continuous arterial spin labeling magnetic resonance imaging, resting-state field potentials by intracerebral multielectrode arrays, Neurological Deficit Score, reactive oxygen species production, and astrogliosis and neuronal death. Results- When compared with vehicle-administered animals, BHB-treated cohort showed decreased peri-infarct neuronal glucose uptake which was associated with reduced oxidative stress, diminished astrogliosis and neuronal death. Functional examination revealed ameliorated neuronal functioning, normalized perilesional resting perfusion, and ameliorated cerebrovascular reactivity to hypercapnia, suggesting improved functioning. Cellular and functional recovery of the neurogliovascular unit in the BHB-treated animals was associated with improved performance on the withdrawal test. Conclusions- We characterize the effects of the ketone body BHB administration at cellular and system levels after focal cortical stroke. The results demonstrate that BHB curbs the peri-infarct glucose-metabolism driven production of reactive oxygen species and astrogliosis, culminating in improved neurogliovascular and functional recovery.

Early-stage attenuation of phase-amplitude coupling in the hippocampus and medial prefrontal cortex in a transgenic rat model of Alzheimer's disease.

Alzheimer's disease (AD) is pathologically characterized by amyloid-ß peptide (Aß) accumulation, neurofibrillary tangle formation, and neurodegeneration. Preclinical studies on neuronal impairments associated with progressive amyloidosis have demonstrated some Aß-dependent neuronal dysfunction including modulation of gamma-aminobutyric acid-ergic signaling. The present work focuses on the early stage of disease progression and uses TgF344-AD rats that recapitulate a broad repertoire of AD-like pathologies to investigate the neuronal network functioning using simultaneous intracranial recordings from the hippocampus (HPC) and the medial prefrontal cortex (mPFC), followed by pathological analyses of gamma-aminobutyric acid (GABAA ) receptor subunits α1, α5, and δ, and glutamic acid decarboxylases (GAD65 and GAD67). Concomitant to amyloid deposition and tau hyperphosphorylation, low-gamma band power was strongly attenuated in the HPC and mPFC of TgF344-AD rats in comparison to those in non-transgenic littermates. In addition, the phase-amplitude coupling of the neuronal networks in both areas was impaired, evidenced by decreased modulation of theta band phase on gamma band amplitude in TgF344-AD animals. Finally, the gamma coherence between HPC and mPFC was attenuated as well. These results demonstrate significant neuronal network dysfunction at an early stage of AD-like pathology. This network dysfunction precedes the onset of cognitive deficits and is likely driven by Aß and tau pathologies. This article is part of the Special Issue "Vascular Dementia".

Leptin regulates amyloid ß production via the γ-secretase complex.

Alzheimer's disease (AD) is the most common age-related neurodegenerative disease, affecting an estimated 5.3million people in the United States. While many factors likely contribute to AD progression, it is widely accepted that AD is driven by the accumulation of ß-amyloid (Aß), a small, fibrillogenic peptide generated by the sequential proteolysis of the amyloid precursor protein by the ß- and γ-secretases. Though the underlying causes of Aß accumulation in sporadic AD are myriad, it is clear that lifestyle and overall health play a significant role. The adipocyte-derived hormone leptin has varied systemic affects, including neuropeptide release and neuroprotection. A recent study by Lieb et al. (2009) showed that individuals with low plasma leptin levels are at greater risk of developing AD, through unknown mechanisms. In this report, we show that plasma leptin is a strong negative predictor of Aß levels in the mouse brain, supporting a protective role for the hormone in AD onset. We also show that the inhibition of Aß accumulation is due to the downregulation of transcription of the γ-secretase components. On the other hand, ß-secretase expression is either unchanged (BACE1) or increased (BACE2). Finally, we show that only presenilin 1 (PS1) is negatively correlated with plasma leptin at the protein level (p<0.0001). These data are intriguing and may highlight a role for leptin in regulating the onset of amyloid pathology and AD.

Prolonged diet induced obesity has minimal effects towards brain pathology in mouse model of cerebral amyloid angiopathy: implications for studying obesity-brain interactions in mice.

Cerebral amyloid angiopathy (CAA) occurs in nearly every individual with Alzheimer's disease (AD) and Down's syndrome, and is the second largest cause of intracerebral hemorrhage. Mouse models of CAA have demonstrated evidence for increased gliosis contributing to CAA pathology. Nearly two thirds of Americans are overweight or obese, with little known about the effects of obesity on the brain, although increasingly the vasculature appears to be a principle target of obesity effects on the brain. In the current study we describe for the first time whether diet induced obesity (DIO) modulates glial reactivity, amyloid levels, and inflammatory signaling in a mouse model of CAA. In these studies we identify surprisingly that DIO does not significantly increase Aß levels, astrocyte (GFAP) or microglial (IBA-1) gliosis in the CAA mice. However, within the hippocampal gyri a localized increase in reactive microglia were increased in the CA1 and stratum oriens relative to CAA mice on a control diet. DIO was observed to selectively increase IL-6 in CAA mice, with IL-1ß and TNF-α not increased in CAA mice in response to DIO. Taken together, these data show that prolonged DIO has only modest effects towards Aß in a mouse model of CAA, but appears to elevate some localized microglial reactivity within the hippocampal gyri and selective markers of inflammatory signaling. These data are consistent with the majority of the existing literature in other models of Aß pathology, which surprisingly show a mixed profile of DIO effects towards pathological processes in mouse models of neurodegenerative disease. The importance for considering the potential impact of ceiling effects in pathology within mouse models of Aß pathogenesis, and the current experimental limitations for DIO in mice to fully replicate metabolic dysfunction present in human obesity, are discussed. This article is part of a Special Issue entitled: Animal Models of Disease.

Nucleic acid oxidation: an early feature of Alzheimer's disease.

Studies of oxidative damage during the progression of Alzheimer's disease (AD) suggest its central role in disease pathogenesis. To investigate levels of nucleic acid oxidation in both early and late stages of AD, levels of multiple base adducts were quantified in nuclear and mitochondrial DNA from the superior and middle temporal gyri (SMTG), inferior parietal lobule (IPL), and cerebellum (CER) of age-matched normal control subjects, subjects with mild cognitive impairment, preclinical AD, late-stage AD, and non-AD neurological disorders (diseased control; DC) using gas chromatography/mass spectrometry. Median levels of multiple DNA adducts in nuclear and mitochondrial DNA were significantly (p ≤ 0.05) elevated in the SMTG, IPL, and CER in multiple stages of AD and in DC subjects. Elevated levels of fapyguanine and fapyadenine in mitochondrial DNA suggest a hypoxic environment early in the progression of AD and in DC subjects. Overall, these data suggest that oxidative damage is an early event not only in the pathogenesis of AD but is also present in neurodegenerative diseases in general. Levels of oxidized nucleic acids in nDNA and mtDNA were found to be significantly elevated in mild cognitive impairment (MCI), preclinical Alzheimer's disease (PCAD), late-stage AD (LAD), and a pooled diseased control group (DC) of frontotemporal dementia (FTD) and dementia with Lewy bodies (DLB) subjects compared to normal control (NC) subjects. Nucleic acid oxidation peaked early in disease progression and remained elevated. The study suggests nucleic acid oxidation is a general event in neurodegeneration.

Targeting astrocytes ameliorates neurologic changes in a mouse model of Alzheimer's disease.

Astrocytes are the most abundant cell type in the brain and play a critical role in maintaining healthy nervous tissue. In Alzheimer's disease (AD) and most other neurodegenerative disorders, many astrocytes convert to a chronically "activated" phenotype characterized by morphologic and biochemical changes that appear to compromise protective properties and/or promote harmful neuroinflammatory processes. Activated astrocytes emerge early in the course of AD and become increasingly prominent as clinical and pathological symptoms progress, but few studies have tested the potential of astrocyte-targeted therapeutics in an intact animal model of AD. Here, we used adeno-associated virus (AAV) vectors containing the astrocyte-specific Gfa2 promoter to target hippocampal astrocytes in APP/PS1 mice. AAV-Gfa2 vectors drove the expression of VIVIT, a peptide that interferes with the immune/inflammatory calcineurin/NFAT (nuclear factor of activated T-cells) signaling pathway, shown by our laboratory and others to orchestrate biochemical cascades leading to astrocyte activation. After several months of treatment with Gfa2-VIVIT, APP/PS1 mice exhibited improved cognitive and synaptic function, reduced glial activation, and lower amyloid levels. The results confirm a deleterious role for activated astrocytes in AD and lay the groundwork for exploration of other novel astrocyte-based therapies.

Association between frontal cortex oxidative damage and beta-amyloid as a function of age in Down syndrome.

Down syndrome (DS) is the most common genetic cause of intellectual disability in children, and the number of adults with DS reaching old age is increasing. By the age of 40 years, virtually all people with DS have sufficient neuropathology for a postmortem diagnosis of Alzheimer disease (AD). Trisomy 21 in DS leads to an overexpression of many proteins, of which at least two are involved in oxidative stress and AD: superoxide dismutase 1 (SOD1) and amyloid precursor protein (APP). In this study, we tested the hypothesis that DS brains with neuropathological hallmarks of AD have more oxidative and nitrosative stress than those with DS but without significant AD pathology, as compared with similarly aged-matched non-DS controls. The frontal cortex was examined in 70 autopsy cases (n=29 control and n=41 DS). By ELISA, we quantified soluble and insoluble Aß40 and Aß42, as well as oligomers. Oxidative and nitrosative stress levels (protein carbonyls, 4-hydroxy-2-trans-nonenal (HNE)-bound proteins, and 3-nitrotyrosine) were measured by slot-blot. We found that soluble and insoluble amyloid beta peptide (Aß) and oligomers increase as a function of age in DS frontal cortex. Of the oxidative stress markers, HNE-bound proteins were increased overall in DS. Protein carbonyls were correlated with Aß40 levels. These results suggest that oxidative damage, but not nitrosative stress, may contribute to the onset and progression of AD pathogenesis in DS. Conceivably, treatment with antioxidants may provide a point of intervention to slow pathological alterations in DS.

BACE2 expression increases in human neurodegenerative disease.

ß-Secretase, the rate-limiting enzymatic activity in the production of the amyloid-ß (Aß) peptide, is a major target of Alzheimer's disease (AD) therapeutics. There are two forms of the enzyme: ß-site Aß precursor protein cleaving enzyme (BACE) 1 and BACE2. Although BACE1 increases in late-stage AD, little is known about BACE2. We conducted a detailed examination of BACE2 in patients with preclinical to late-stage AD, including amnestic mild cognitive impairment, and age-matched controls, cases of frontotemporal dementia, and Down's syndrome. BACE2 protein and enzymatic activity increased as early as preclinical AD and were found in neurons and astrocytes. Although the levels of total BACE2 mRNA were unchanged, the mRNA for BACE2 splice form C (missing exon 7) increased in parallel with BACE2 protein and activity. BACE1 and BACE2 were strongly correlated with each other at all levels, suggesting that their regulatory mechanisms may be largely shared. BACE2 was also elevated in frontotemporal dementia but not in Down's syndrome, even in patients with substantial Aß deposition. Thus, expression of both forms of ß-secretase are linked and may play a combined role in human neurologic disease. A better understanding of the normal functions of BACE1 and BACE2, and how these change in different disease states, is essential for the future development of AD therapeutics.

Pittsburgh compound B and the postmortem diagnosis of Alzheimer disease.

OBJECTIVE: Deposition of the amyloid-ß (Aß) peptide in neuritic plaques is a requirement for the diagnosis of Alzheimer disease (AD). Although the continued development of in vivo imaging agents such as Pittsburgh compound B (PiB) is promising, the diagnosis of AD is still challenging. This can be partially attributed to our lack of a detailed understanding of the interrelationship between the various pools and species of Aß and other common indices of AD pathology. We hypothesized that recent advances in our ability to accurately measure Aß postmortem (for example, using PiB), could form the basis of a simple means to deliver an accurate AD diagnosis. METHODS: We conducted a comprehensive analysis of the amount of Aß40 and Aß42 in increasingly insoluble fractions, oligomeric Aß, and fibrillar Aß (as defined by PiB binding), as well as plaques (diffuse and neuritic), and neurofibrillary tangles in autopsy specimens from age-matched, cognitively normal controls (n = 23) and AD (n = 22) cases, across multiple brain regions. RESULTS: Both PiB binding and the amount of sodium dodecyl sulfate (SDS)-soluble Aß were able to predict disease status; however, SDS-soluble Aß was a better measure. Oligomeric Aß was not a predictor of disease status. PiB binding was strongly related to plaque count, although diffuse plaques were a stronger correlate than neuritic plaques. INTERPRETATION: Although postmortem PiB binding was somewhat useful in distinguishing AD from control cases, SDS-soluble Aß measured by standard immunoassay was substantially better. These findings have important implications for the development of imaging-based biomarkers of AD.

Prostacyclin Promotes Degenerative Pathology in a Model of Alzheimer's Disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is the most common form of dementia in aged populations. A substantial amount of data demonstrates that chronic neuroinflammation can accelerate neurodegenerative pathologies. In AD, chronic neuroinflammation results in the upregulation of cyclooxygenase and increased production of prostaglandin H2, a precursor for many vasoactive prostanoids. While it is well-established that many prostaglandins can modulate the progression of neurodegenerative disorders, the role of prostacyclin (PGI2) in the brain is poorly understood. We have conducted studies to assess the effect of elevated prostacyclin biosynthesis in a mouse model of AD. Upregulated prostacyclin expression significantly worsened multiple measures associated with amyloid-ß (Aß) disease pathologies. Mice overexpressing both Aß and PGI2 exhibited impaired learning and memory and increased anxiety-like behavior compared with non-transgenic and PGI2 control mice. PGI2 overexpression accelerated the development of Aß accumulation in the brain and selectively increased the production of soluble Aß42. PGI2 damaged the microvasculature through alterations in vascular length and branching; Aß expression exacerbated these effects. Our findings demonstrate that chronic prostacyclin expression plays a novel and unexpected role that hastens the development of the AD phenotype.

Cognitive impairment in humanized APP×PS1 mice is linked to Aß(1-42) and NOX activation.

Cognitive impairment in Alzheimer's disease (AD) is strongly associated with both extensive deposition of amyloid ß peptides and oxidative stress, but the exact role of these indices in the development of dementia is not clear. This study was designed to determine the relationship between cognitive impairment, activation of the free radical producing enzyme NADPH oxidase (NOX), and progressive changes in Aß deposition and solubility in humanized APP×PS1 knock-in mice of increasing age. Data show that cognitive performance and expression of key synaptic proteins were progressively decreased in aging APP×PS1 mice. Likewise, NOX activity and expression of the specific NOX subunit NOX4 were significantly increased in APP×PS1 mice in an age-dependent manner, and NOX activity and cognitive impairment shared a significant linear relationship. Data further show that age-dependent increases in Aß(1-42) had a significant linear relationship with both NOX activity and cognitive performance in APP×PS1 knock-in mice. Collectively, these data show that NOX expression and activity are significantly upregulated with age in this humanized model of Aß pathogenesis, and suggest that NOX-associated redox pathways are intimately linked to both the loss of cognitive function and the deposition of Aß(1-42).

Covert strokes prior to Alzheimer's disease onset accelerate peri-lesional pathology but not cognitive deficits in an inducible APP mouse model.

It is estimated that up to 1 in 3 healthy middle-aged adults will have had a covert stroke during their lifetime. Furthermore, post-stroke, survivors are more than twice as likely to develop dementia. In the present study, we aimed to model the impact of focal subclinical ischemia prior to the onset of AD pathogenesis in a preclinical model. We utilized endothelin-1 to induce ischemia in an iducible transgenic mouse model of Alzheimer's disease, APPsi:tTA, allowing for temporal control of APP gene expression. We induced the focal subclinical ischemic events in the absence of APP expression, thus prior to AD onset. T2 structural magnetic resonance imaging confirmed the volume and location of focal subclinical ischemic lesions to the medial prefrontal cortex. Following recovery from surgery and 7 weeks of APP expression, we found that two subclinical ischemic lesions resulted in a significant localized increase in amyloid load and in microglial activation proximal to the lesion. However, no differences were found in astrogliosis. A battery of behaviour tests was conducted, in which no significant differences were detected in activities of daily living and cognitive function between stroked and sham cohorts. Overall, our results demonstrated that APP expression was the sole driving force behind behavioural deficits. In conclusion, our results suggest that a history of two subclinical strokes prior to AD onset does not worsen early disease trajectory in a mouse model.

Frequency selective neuronal modulation triggers spreading depolarizations in the rat endothelin-1 model of stroke.

Ischemia is one of the most common causes of acquired brain injury. Central to its noxious sequelae are spreading depolarizations (SDs), waves of persistent depolarizations which start at the location of the flow obstruction and expand outwards leading to excitotoxic damage. The majority of acute stage of stroke studies to date have focused on the phenomenology of SDs and their association with brain damage. In the current work, we investigated the role of peri-injection zone pyramidal neurons in triggering SDs by optogenetic stimulation in an endothelin-1 rat model of focal ischemia. Our concurrent two photon fluorescence microscopy data and local field potential recordings indicated that a ≥ 60% drop in cortical arteriolar red blood cell velocity was associated with SDs at the ET-1 injection site. SDs were also observed in the peri-injection zone, which subsequently exhibited elevated neuronal activity in the low-frequency bands. Critically, SDs were triggered by low- but not high-frequency optogenetic stimulation of peri-injection zone pyramidal neurons. Our findings depict a complex etiology of SDs post focal ischemia and reveal that effects of neuronal modulation exhibit spectral and spatial selectivity.

Cognitive decline in Alzheimer's disease is associated with selective changes in calcineurin/NFAT signaling.

Upon activation by calcineurin, the nuclear factor of activated T-cells (NFAT) translocates to the nucleus and guides the transcription of numerous molecules involved in inflammation and Ca(2+) dysregulation, both of which are prominent features of Alzheimer's disease (AD). However, NFAT signaling in AD remains relatively uninvestigated. Using isolated cytosolic and nuclear fractions prepared from rapid-autopsy postmortem human brain tissue, we show that NFATs 1 and 3 shifted to nuclear compartments in the hippocampus at different stages of neuropathology and cognitive decline, whereas NFAT2 remained unchanged. NFAT1 exhibited greater association with isolated nuclear fractions in subjects with mild cognitive impairment (MCI), whereas NFAT3 showed a strong nuclear bias in subjects with severe dementia and AD. Similar to NFAT1, calcineurin-Aalpha also exhibited a nuclear bias in the early stages of cognitive decline. But, unlike NFAT1 and similar to NFAT3, the nuclear bias for calcineurin became more pronounced as cognition worsened. Changes in calcineurin/NFAT3 were directly correlated to soluble amyloid-beta (Abeta((1-42))) levels in postmortem hippocampus, and oligomeric Abeta, in particular, robustly stimulated NFAT activation in primary rat astrocyte cultures. Oligomeric Abeta also caused a significant reduction in excitatory amino acid transporter 2 (EAAT2) protein levels in astrocyte cultures, which was blocked by NFAT inhibition. Moreover, inhibition of astrocytic NFAT activity in mixed cultures ameliorated Abeta-dependent elevations in glutamate and neuronal death. The results suggest that NFAT signaling is selectively altered in AD and may play an important role in driving Abeta-mediated neurodegeneration.

BACE1 and BACE2 enzymatic activities in Alzheimer's disease.

beta-Secretase is the rate limiting enzymatic activity in the production of the amyloid-beta peptide (Abeta) and is thought to be involved in Alzheimer's disease (AD) pathogenesis. Although BACE1 (beta-site APP Cleaving Enzyme 1, EC 3.4.23.46) has received significant attention, the related BACE2 (EC 3.4.23.45) has not. Though BACE2 is also expressed in the brain, its potential role in AD has not been resolved. In this study, we compared the activities of both BACE1 and BACE2, which were isolated from the same samples of frontal cortex from both AD-affected individuals and age-matched controls. BACE1 activity showed a significant positive correlation with the amount of extractable Abeta, and BACE1 protein and activity were significantly increased in AD cases. Unexpectedly, there were substantial total amounts of BACE2 protein and enzymatic activity in the human brain. BACE2 activity did not change significantly in the AD brain, and was not related to Abeta concentration. These data indicate that BACE1 likely accounts for most of the Abeta produced in the human brain, and that BACE2 activity is not a likely contributor. However, as both forms of BACE compete for the same substrate pool, even small changes in BACE2 activity could have consequences for human disease.

Preclinical Alzheimer disease: brain oxidative stress, Abeta peptide and proteomics.

Alzheimer disease (AD) is a neurodegenerative disorder characterized clinically by progressive memory loss and subsequent dementia and neuropathologically by senile plaques, neurofibrillary tangles, and synapse loss. Interestingly, a small percentage of individuals with normal antemortem psychometric scores meet the neuropathological criteria for AD (termed 'preclinical' AD (PCAD)). In this study, inferior parietal lobule (IPL) from PCAD and control subjects was compared for oxidative stress markers by immunochemistry, amyloid beta peptide by ELISA, and identification of protein expression differences by proteomics. We observed a significant increase in highly insoluble monomeric Abeta42, but no significant differences in oligomeric Abeta nor in oxidative stress measurements between controls and PCAD subjects. Expression proteomics identified proteins whose trends in PCAD are indicative of cellular protection, possibly correlating with previous studies showing no cell loss in PCAD. Our analyses may reveal processes involved in a period of protection from neurodegeneration that mimic the clinical phenotype of PCAD.

Effects of nonsteroidal anti-inflammatory drugs on amyloid-beta pathology in mouse skeletal muscle.

Sporadic inclusion body myositis (sIBM) is a common age-related inflammatory myopathy characterized by the presence of intracellular inclusions that contain the amyloid-beta (Abeta) peptide, a derivative of the amyloid precursor protein (APP). Abeta is believed to cause Alzheimer's disease (AD), suggesting that a link may exist between the two diseases. If AD and sIBM are linked, then treatments that lower Abeta in brain may prove useful for sIBM. To test this hypothesis, transgenic mice that overexpress APP in skeletal muscle were treated for 6 months with a variety of nonsteroidal anti-inflammatory drugs (NSAIDs; naproxen, ibuprofen, carprofen or R-flurbiprofen), a subset of which reduce Abeta in brain and cultured cells. Only ibuprofen lowered Abeta in muscle, and this was not accompanied by corresponding improvements in phenotype. These results indicate that the effects of NSAIDs in the brain may be different from other tissues and that Abeta alone cannot account for skeletal muscle dysfunction in these mice.