Bacterial sepsis increases hippocampal fibrillar amyloid plaque load and neuroinflammation in a mouse model of Alzheimer's disease.

BACKGROUND: Sepsis, a leading cause for intensive care unit admissions, causes both an acute encephalopathy and chronic brain dysfunction in survivors. A history of sepsis is also a risk factor for future development of dementia symptoms. Similar neuropathologic changes are associated with the cognitive decline of sepsis and Alzheimer's disease (AD), including neuroinflammation, neuronal death, and synaptic loss. Amyloid plaque pathology is the earliest pathological hallmark of AD, appearing 10 to 20 years prior to cognitive decline, and is present in 30% of people over 65. As sepsis is also more common in older adults, we hypothesized that sepsis might exacerbate amyloid plaque deposition and plaque-related injury, promoting the progression of AD-related pathology. METHODS: We evaluated whether the brain's response to sepsis modulates AD-related neurodegenerative changes by driving amyloid deposition and neuroinflammation in mice. We induced polymicrobial sepsis by cecal ligation and puncture (CLP) in APP/PS1-21 mice, a model of AD-related ß-amyloidosis. We performed CLP or sham surgery at plaque onset (2 months of age) and examined pathology 2 months after CLP in surviving mice. RESULTS: Sepsis significantly increased fibrillar amyloid plaque formation in the hippocampus of APP/PS1-21 mice. Sepsis enhanced plaque-related astrocyte activation and complement C4b gene expression in the brain, both of which may play a role in modulating amyloid formation. CLP also caused large scale changes in the gut microbiome of APP/PS1 mice, which have been associated with a pro-amyloidogenic and neuroinflammatory state. CONCLUSIONS: Our results suggest that experimental sepsis can exacerbate amyloid plaque deposition and plaque-related inflammation, providing a potential mechanism for increased dementia in older sepsis survivors.

Low-density lipoprotein receptor represents an apolipoprotein E-independent pathway of Aß uptake and degradation by astrocytes.

Accumulation of the amyloid ß (Aß) peptide within the brain is hypothesized to be one of the main causes underlying the pathogenic events that occur in Alzheimer disease (AD). Consequently, identifying pathways by which Aß is cleared from the brain is crucial for better understanding of the disease pathogenesis and developing novel therapeutics. Cellular uptake and degradation by glial cells is one means by which Aß may be cleared from the brain. In the current study, we demonstrate that modulating levels of the low-density lipoprotein receptor (LDLR), a cell surface receptor that regulates the amount of apolipoprotein E (apoE) in the brain, altered both the uptake and degradation of Aß by astrocytes. Deletion of LDLR caused a decrease in Aß uptake, whereas increasing LDLR levels significantly enhanced both the uptake and clearance of Aß. Increasing LDLR levels also enhanced the cellular degradation of Aß and facilitated the vesicular transport of Aß to lysosomes. Despite the fact that LDLR regulated the uptake of apoE by astrocytes, we found that the effect of LDLR on Aß uptake and clearance occurred in the absence of apoE. Finally, we provide evidence that Aß can directly bind to LDLR, suggesting that an interaction between LDLR and Aß could be responsible for LDLR-mediated Aß uptake. Therefore, these results identify LDLR as a receptor that mediates Aß uptake and clearance by astrocytes, and provide evidence that increasing glial LDLR levels may promote Aß degradation within the brain.

Targeting BACE1-mediated production of amyloid beta improves hippocampal synaptic function in an experimental model of ischemic stroke.

Post-stroke cognitive impairment and dementia (PSCID) affects many survivors of large vessel cerebral ischemia. The molecular pathways underlying PSCID are poorly defined but may overlap with neurodegenerative pathophysiology. Specifically, synaptic dysfunction after stroke may be directly mediated by alterations in the levels of amyloid beta (Aß), the peptide that accumulates in the brains of Alzheimer's disease (AD) patients. In this study, we use the transient middle cerebral artery occlusion (MCAo) model in young adult mice to evaluate if a large vessel stroke increases brain soluble Aß levels. We show that soluble Aß40 and Aß42 levels are increased in the ipsilateral hippocampus in MCAo mice 7 days after the injury. We also analyze the level and activity of ß-site amyloid precursor protein cleaving enzyme 1 (BACE1), an enzyme that generates Aß in the brain, and observe that BACE1 activity is increased in the ipsilateral hippocampus of the MCAo mice. Finally, we highlight that treatment of MCAo mice with a BACE1 inhibitor during the recovery period rescues stroke-induced deficits in hippocampal synaptic plasticity. These findings support a molecular pathway linking ischemia to alterations in BACE1-mediated production of Aß, and encourage future studies that evaluate whether targeting BACE1 activity improves the cognitive deficits seen with PSCID.

Haploinsufficiency of human APOE reduces amyloid deposition in a mouse model of amyloid-ß amyloidosis.

The ε4 allele of the apolipoprotein E (APOE) gene is the strongest genetic risk factor for Alzheimer's disease (AD). Evidence suggests that the effect of apoE isoforms on amyloid-ß (Aß) accumulation in the brain plays a critical role in AD pathogenesis. Like in humans, apoE4 expression in animal models that develop Aß amyloidosis results in greater Aß and amyloid deposition than with apoE3 expression. However, whether decreasing levels of apoE3 or apoE4 would promote or attenuate Aß-related pathology has not been directly addressed. To determine the effect of decreasing human apoE levels on Aß accumulation in vivo, we generated human APOE isoform haploinsufficient mouse models by crossing APPPS1-21 mice with APOE isoform knock-in mice. By genetically manipulating APOE gene dosage, we demonstrate that decreasing human apoE levels, regardless of isoform status, results in significantly decreased amyloid plaque deposition and microglial activation. These differences in amyloid load between apoE3- and apoE4-expressing mice were not due to apoE4 protein being present at lower levels than apoE3. These data suggest that current therapeutic strategies to increase apoE levels without altering its lipidation state may actually worsen Aß amyloidosis, while increasing apoE degradation or inhibiting its synthesis may be a more effective treatment approach.

Anti-apoE immunotherapy inhibits amyloid accumulation in a transgenic mouse model of Aß amyloidosis.

The apolipoprotein E (APOE) ε4 allele is the strongest genetic risk factor for Alzheimer's disease (AD). The influence of apoE on amyloid ß (Aß) accumulation may be the major mechanism by which apoE affects AD. ApoE interacts with Aß and facilitates Aß fibrillogenesis in vitro. In addition, apoE is one of the protein components in plaques. We hypothesized that certain anti-apoE antibodies, similar to certain anti-Aß antibodies, may have antiamyloidogenic effects by binding to apoE in the plaques and activating microglia-mediated amyloid clearance. To test this hypothesis, we developed several monoclonal anti-apoE antibodies. Among them, we administered HJ6.3 antibody intraperitoneally to 4-mo-old male APPswe/PS1ΔE9 mice weekly for 14 wk. HJ6.3 dramatically decreased amyloid deposition by 60-80% and significantly reduced insoluble Aß40 and Aß42 levels. Short-term treatment with HJ6.3 resulted in strong changes in microglial responses around Aß plaques. Collectively, these results suggest that anti-apoE immunization may represent a novel AD therapeutic strategy and that other proteins involved in Aß binding and aggregation might also be a target for immunotherapy. Our data also have important broader implications for other amyloidosis. Immunotherapy to proteins tightly associated with misfolded proteins might open up a new treatment option for many protein misfolding diseases.

Measurement of apolipoprotein E and amyloid ß clearance rates in the mouse brain using bolus stable isotope labeling.

BACKGROUND: Abnormal proteostasis due to alterations in protein turnover has been postulated to play a central role in several neurodegenerative diseases. Therefore, the development of techniques to quantify protein turnover in the brain is critical for understanding the pathogenic mechanisms of these diseases. We have developed a bolus stable isotope-labeling kinetics (SILK) technique coupled with multiple reaction monitoring mass spectrometry to measure the clearance of proteins in the mouse brain. RESULTS: Cohorts of mice were pulse labeled with 13C6-leucine and the brains were isolated after pre-determined time points. The extent of label incorporation was measured over time using mass spectrometry to measure the ratio of labeled to unlabeled apolipoprotein E (apoE) and amyloid ß (Aß). The fractional clearance rate (FCR) was then calculated by analyzing the time course of disappearance for the labeled protein species. To validate the technique, apoE clearance was measured in mice that overexpress the low-density lipoprotein receptor (LDLR). The FCR in these mice was 2.7-fold faster than wild-type mice. To demonstrate the potential of this technique for understanding the pathogenesis of neurodegenerative disease, we applied our SILK technique to determine the effect of ATP binding cassette A1 (ABCA1) on both apoE and Aß clearance. ABCA1 had previously been shown to regulate both the amount of apoE in the brain, along with the extent of Aß deposition, and represents a potential molecular target for lowering brain amyloid levels in Alzheimer's disease patients. The FCR of apoE was increased by 1.9- and 1.5-fold in mice that either lacked or overexpressed ABCA1, respectively. However, ABCA1 had no effect on the FCR of Aß, suggesting that ABCA1 does not regulate Aß metabolism in the brain. CONCLUSIONS: Our SILK strategy represents a straightforward, cost-effective, and efficient method to measure the clearance of proteins in the mouse brain. We expect that this technique will be applicable to the study of protein dynamics in the pathogenesis of several neurodegenerative diseases, and could aid in the evaluation of novel therapeutic agents.

In vivo human apolipoprotein E isoform fractional turnover rates in the CNS.

Apolipoprotein E (ApoE) is the strongest genetic risk factor for Alzheimer's disease and has been implicated in the risk for other neurological disorders. The three common ApoE isoforms (ApoE2, E3, and E4) each differ by a single amino acid, with ApoE4 increasing and ApoE2 decreasing the risk of Alzheimer's disease (AD). Both the isoform and amount of ApoE in the brain modulate AD pathology by altering the extent of amyloid beta (Aß) peptide deposition. Therefore, quantifying ApoE isoform production and clearance rates may advance our understanding of the role of ApoE in health and disease. To measure the kinetics of ApoE in the central nervous system (CNS), we applied in vivo stable isotope labeling to quantify the fractional turnover rates of ApoE isoforms in 18 cognitively-normal adults and in ApoE3 and ApoE4 targeted-replacement mice. No isoform-specific differences in CNS ApoE3 and ApoE4 turnover rates were observed when measured in human CSF or mouse brain. However, CNS and peripheral ApoE isoform turnover rates differed substantially, which is consistent with previous reports and suggests that the pathways responsible for ApoE metabolism are different in the CNS and the periphery. We also demonstrate a slower turnover rate for CSF ApoE than that for amyloid beta, another molecule critically important in AD pathogenesis.

The role of apolipoprotein E in Alzheimer's disease.

The epsilon4 allele of apolipoprotein E (APOE) is the major genetic risk factor for Alzheimer's disease (AD). Although there have been numerous studies attempting to elucidate the underlying mechanism for this increased risk, how apoE4 influences AD onset and progression has yet to be proven. However, prevailing evidence suggests that the differential effects of apoE isoforms on Abeta aggregation and clearance play the major role in AD pathogenesis. Other potential mechanisms, such as the differential modulation of neurotoxicity and tau phosphorylation by apoE isoforms as well as its role in synaptic plasticity and neuroinflammation, have not been ruled out. Inconsistent results among studies have made it difficult to define whether the APOE epsilon4 allele represents a gain of toxic function, a loss of neuroprotective function, or both. Therapeutic strategies based on apoE propose to reduce the toxic effects of apoE4 or to restore the physiological, protective functions of apoE.

Overexpression of low-density lipoprotein receptor in the brain markedly inhibits amyloid deposition and increases extracellular A beta clearance.

Apolipoprotein E (APOE) is the strongest genetic risk factor for Alzheimer's disease (AD). Previous studies suggest that the effect of apoE on amyloid-beta (A beta) accumulation plays a major role in AD pathogenesis. Therefore, understanding proteins that control apoE metabolism may provide new targets for regulating A beta levels. LDLR, a member of the LDL receptor family, binds to apoE, yet its potential role in AD pathogenesis remains unclear. We hypothesized that LDLR overexpression in the brain would decrease apoE levels, enhance A beta clearance, and decrease A beta deposition. To test our hypothesis, we created several transgenic mice that overexpress LDLR in the brain and found that apoE levels in these mice decreased by 50%-90%. Furthermore, LDLR overexpression dramatically reduced A beta aggregation and enhanced A beta clearance from the brain extracellular space. Plaque-associated neuroinflammatory responses were attenuated in LDLR transgenic mice. These findings suggest that increasing LDLR levels may represent a novel AD treatment strategy.

The low density lipoprotein receptor-related protein 1 mediates uptake of amyloid beta peptides in an in vitro model of the blood-brain barrier cells.

The metabolism of amyloid beta peptide (A beta) in the brain is crucial to the pathogenesis of Alzheimer disease. A body of evidence suggests that A beta is actively transported from brain parenchyma to blood across the blood-brain barrier (BBB), although the precise mechanism remains unclear. To unravel the cellular and molecular mechanism of A beta transport across the BBB, we established a new in vitro model of the initial internalization step of A beta transport using TR-BBB cells, a conditionally immortalized endothelial cell line from rat brain. We show that TR-BBB cells rapidly internalize A beta through a receptor-mediated mechanism. We also provide evidence that A beta internalization is mediated by LRP1 (low density lipoprotein receptor-related protein 1), since administration of LRP1 antagonist, receptor-associated protein, neutralizing antibody, or small interference RNAs all reduced A beta uptake. Despite the requirement of LRP1-dependent internalization, A beta does not directly bind to LRP1 in an in vitro binding assay. Unlike TR-BBB cells, mouse embryonic fibroblasts endogenously expressing functional LRP1 and exhibiting the authentic LRP1-mediated endocytosis (e.g. of tissue plasminogen activator) did not show rapid A beta uptake. Based on these data, we propose that the rapid LRP1-dependent internalization of A beta occurs under the BBB-specific cellular context and that TR-BBB is a useful tool for analyzing the molecular mechanism of the rapid transport of A beta across BBB.

Bi-compartmental communication contributes to the opposite proliferative behavior of Notch1-deficient hair follicle and epidermal keratinocytes.

Notch1-deficient epidermal keratinocytes become progressively hyperplastic and eventually produce tumors. By contrast, Notch1-deficient hair matrix keratinocytes have lower mitotic rates, resulting in smaller follicles with fewer cells. In addition, the ratio of melanocytes to keratinocytes is greatly reduced in hair follicles. Investigation into the underlying mechanism for these phenotypes revealed significant changes in the Kit, Tgfbeta and insulin-like growth factor (IGF) signaling pathways, which have not been previously shown to be downstream of Notch signaling. The level of Kitl (Scf) mRNA produced by Notch1-deficient follicular keratinocytes was reduced when compared with wild type, resulting in a decline in melanocyte population. Tgfbeta ligands were elevated in Notch1-deficient keratinocytes, which correlated with elevated expression of several targets, including the diffusible IGF antagonist Igfbp3 in the dermal papilla. Diffusible stromal targets remained elevated in the absence of epithelial Tgfbeta receptors, consistent with paracrine Tgfbeta signaling. Overexpression of Igf1 in the keratinocyte reversed the phenotype, as expected if Notch1 loss altered the IGF/insulin-like growth factor binding protein (IGFBP) balance. Conversely, epidermal keratinocytes contained less stromal Igfbp4 and might thus be primed to experience an increase in IGF signaling as animals age. These results suggest that Notch1 participates in a bi-compartmental signaling network that controls homeostasis, follicular proliferation rates and melanocyte population within the skin.