Mary Beth Finn

Synaptic Activity Regulates Interstitial Fluid Amyloid-β Levels In Vivo

Summary Aggregation of the amyloid-β (Aβ) peptide in the extracellular space of the brain is central to Alzheimers disease pathogenesis. Aβ aggregation is concentration dependent and brain region specific. Utilizing in vivo microdialysis concurrently with field potential recordings, we demonstrate that Aβ levels in the brain interstitial fluid are dynamically and directly influenced by synaptic activity on a timescale of minutes to hours. Using an acute brain slice model, we show that the rapid effects of synaptic activity on Aβ levels are primarily related to synaptic vesicle exocytosis. These results suggest that synaptic activity may modulate a neurodegenerative disease process, in this case by influencing Aβ metabolism and ultimately region-specific Aβ deposition. The findings also have important implications for treatment development.

P-glycoprotein deficiency at the blood-brain barrier increases amyloid-β deposition in an Alzheimer disease mouse model

Accumulation of amyloid-beta (Abeta) within extracellular spaces of the brain is a hallmark of Alzheimer disease (AD). In sporadic, late-onset AD, there is little evidence for increased Abeta production, suggesting that decreased elimination from the brain may contribute to elevated levels of Abeta and plaque formation. Efflux transport of Abeta across the blood-brain barrier (BBB) contributes to Abeta removal from the brain. P-glycoprotein (Pgp) is highly expressed on the luminal surface of brain capillary endothelial cells and contributes to the BBB. In Pgp-null mice, we show that [I]Abeta40 and [I]Abeta42 microinjected into the CNS clear at half the rate that they do in WT mice. When amyloid precursor protein-transgenic (APP-transgenic) mice were administered a Pgp inhibitor, Abeta levels within the brain interstitial fluid significantly increased within hours of treatment. Furthermore, APP-transgenic, Pgp-null mice had increased levels of brain Abeta and enhanced Abeta deposition compared with APP-transgenic, Pgp WT mice. These data establish a direct link between Pgp and Abeta metabolism in vivo and suggest that Pgp activity at the BBB could affect risk for developing AD as well as provide a novel diagnostic and therapeutic target.

ApoE isoform-specific disruption of amyloid β peptide clearance from mouse brain

Neurotoxic amyloid beta peptide (Abeta) accumulates in the brains of individuals with Alzheimer disease (AD). The APOE4 allele is a major risk factor for sporadic AD and has been associated with increased brain parenchymal and vascular amyloid burden. How apoE isoforms influence Abeta accumulation in the brain has, however, remained unclear. Here, we have shown that apoE disrupts Abeta clearance across the mouse blood-brain barrier (BBB) in an isoform-specific manner (specifically, apoE4 had a greater disruptive effect than either apoE3 or apoE2). Abeta binding to apoE4 redirected the rapid clearance of free Abeta40/42 from the LDL receptor-related protein 1 (LRP1) to the VLDL receptor (VLDLR), which internalized apoE4 and Abeta-apoE4 complexes at the BBB more slowly than LRP1. In contrast, apoE2 and apoE3 as well as Abeta-apoE2 and Abeta-apoE3 complexes were cleared at the BBB via both VLDLR and LRP1 at a substantially faster rate than Abeta-apoE4 complexes. Astrocyte-secreted lipo-apoE2, lipo-apoE3, and lipo-apoE4 as well as their complexes with Abeta were cleared at the BBB by mechanisms similar to those of their respective lipid-poor isoforms but at 2- to 3-fold slower rates. Thus, apoE isoforms differentially regulate Abeta clearance from the brain, and this might contribute to the effects of APOE genotype on the disease process in both individuals with AD and animal models of AD.

Pomegranate juice decreases amyloid load and improves behavior in a mouse model of Alzheimer's disease

Although there are no proven ways to delay onset or slow progression of Alzheimers disease (AD), studies suggest that diet can affect risk. Pomegranates contain very high levels of antioxidant polyphenolic substances as compared to other fruits and vegetables. Polyphenols have been shown to be neuroprotective in different model systems. We asked whether dietary supplementation with pomegranate juice (PJ) would influence behavior and AD-like pathology in a transgenic mouse model. Transgenic mice (APP(sw)/Tg2576) received either PJ or sugar water control from 6 to 12.5 months of age. PJ-treated mice learned water maze tasks more quickly and swam faster than controls. Mice treated with PJ had significantly less (approximately 50%) accumulation of soluble Abeta42 and amyloid deposition in the hippocampus as compared to control mice. These results suggest that further studies to validate and determine the mechanism of these effects, as well as whether substances in PJ may be useful in AD, should be considered.

In Vivo Microdialysis Reveals Age-Dependent Decrease of Brain Interstitial Fluid Tau Levels in P301S Human Tau Transgenic Mice

Although tau is a cytoplasmic protein, it is also found in brain extracellular fluids, e.g., CSF. Recent findings suggest that aggregated tau can be transferred between cells and extracellular tau aggregates might mediate spread of tau pathology. Despite these data, details of whether tau is normally released into the brain interstitial fluid (ISF), its concentration in ISF in relation to CSF, and whether ISF tau is influenced by its aggregation are unknown. To address these issues, we developed a microdialysis technique to analyze monomeric ISF tau levels within the hippocampus of awake, freely moving mice. We detected tau in ISF of wild-type mice, suggesting that tau is released in the absence of neurodegeneration. ISF tau was significantly higher than CSF tau and their concentrations were not significantly correlated. Using P301S human tau transgenic mice (P301S tg mice), we found that ISF tau is fivefold higher than endogenous murine tau, consistent with its elevated levels of expression. However, following the onset of tau aggregation, monomeric ISF tau decreased markedly. Biochemical analysis demonstrated that soluble tau in brain homogenates decreased along with the deposition of insoluble tau. Tau fibrils injected into the hippocampus decreased ISF tau, suggesting that extracellular tau is in equilibrium with extracellular or intracellular tau aggregates. This technique should facilitate further studies of tau secretion, spread of tau pathology, the effects of different disease states on ISF tau, and the efficacy of experimental treatments.

Rapid Microglial Response Around Amyloid Pathology after Systemic Anti-Aβ Antibody Administration in PDAPP Mice

Aggregation of amyloid-β (Aβ) peptide in the brain in the form of neuritic plaques and cerebral amyloid angiopathy (CAA) is a key feature of Alzheimers disease (AD). Microglial cells surround aggregated Aβ and are believed to play a role in AD pathogenesis. A therapy for AD that has entered clinical trials is the administration of anti-Aβ antibodies. One mechanism by which certain anti-Aβ antibodies have been proposed to exert their effects is via antibody-mediated microglial activation. Whether, when, or to what extent microglial activation occurs after systemic administration of anti-Aβ antibodies has not been fully assessed. We administered an anti-Aβ antibody (m3D6) that binds aggregated Aβ to PDAPP mice, an AD mouse model that was bred to contain fluorescent microglia. Three days after systemic administration of m3D6, there was a marked increase in both the number of microglial cells and processes per cell visualized in vivo by multiphoton microscopy. These changes required the Fc domain of m3D6 and were not observed with an antibody specific to soluble Aβ. These findings demonstrate that some effects of antibodies that recognize aggregated Aβ are rapid, involve microglia, and provide insight into the mechanism of action of a specific passive immunotherapy for AD.

Haploinsufficiency of Human APOE Reduces Amyloid Deposition in a Mouse Model of Amyloid-β Amyloidosis

The ε4 allele of the apolipoprotein E (APOE) gene is the strongest genetic risk factor for Alzheimers disease (AD). Evidence suggests that the effect of apoE isoforms on amyloid-β (Aβ) accumulation in the brain plays a critical role in AD pathogenesis. Like in humans, apoE4 expression in animal models that develop Aβ amyloidosis results in greater Aβ and amyloid deposition than with apoE3 expression. However, whether decreasing levels of apoE3 or apoE4 would promote or attenuate Aβ-related pathology has not been directly addressed. To determine the effect of decreasing human apoE levels on Aβ 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 Aβ amyloidosis, while increasing apoE degradation or inhibiting its synthesis may be a more effective treatment approach.

Apolipoprotein E, Especially Apolipoprotein E4, Increases the Oligomerization of Amyloid β Peptide

Alzheimers disease (AD) is the most common progressive neurodegenerative disorder causing dementia. Massive deposition of amyloid β peptide (Aβ) as senile plaques in the brain is the pathological hallmark of AD, but oligomeric, soluble forms of Aβ have been implicated as the synaptotoxic component. The apolipoprotein E ε 4 (apoE ε4) allele is known to be a genetic risk factor for developing AD. However, it is still unknown how apoE impacts the process of Aβ oligomerization. Here, we found that the level of Aβ oligomers in APOE ε4/ε4 AD patient brains is 2.7 times higher than those in APOE ε3/ε3 AD patient brains, matched for total plaque burden, suggesting that apoE4 impacts the metabolism of Aβ oligomers. To test this hypothesis, we examined the effect of apoE on Aβ oligomer formation. Using both synthetic Aβ and a split-luciferase method for monitoring Aβ oligomers, we observed that apoE increased the level of Aβ oligomers in an isoform-dependent manner (E2 < E3 < E4). This effect appears to be dependent on the ApoE C-terminal domain. Moreover, these results were confirmed using endogenous apoE isolated from the TBS-soluble fraction of human brain, which increased the formation of Aβ oligomers. Together, these data show that lipidated apoE, especially apoE4, increases Aβ oligomers in the brain. Higher levels of Aβ oligomers in the brains of APOE ε4/ε4 carriers compared with APOE ε3/ε3 carriers may increase the loss of dendritic spines and accelerate memory impairments, leading to earlier cognitive decline in AD.

Altered microglial response to Aβ plaques in APPPS1-21 mice heterozygous for TREM2.

BackgroundRecent genome-wide association studies linked variants in TREM2 to a strong increase in the odds of developing Alzheimer’s disease. The mechanism by which TREM2 influences the susceptibility to Alzheimer’s disease is currently unknown. TREM2 is expressed by microglia and is thought to regulate phagocytic and inflammatory microglial responses to brain pathology. Given that a single allele of variant TREM2, likely resulting in a loss of function, conferred an increased risk of developing Alzheimer’s disease, we tested whether loss of one functional trem2 allele would affect Aβ plaque deposition or the microglial response to Aβ pathology in APPPS1-21 mice.ResultsThere was no significant difference in Aβ deposition in 3-month old or 7-month old APPPS1-21 mice expressing one or two copies of trem2. However, 3-month old mice with one copy of trem2 exhibited a marked decrease in the number and size of plaque-associated microglia. While there were no statistically significant differences in cytokine levels or markers of microglial activation in 3- or 7-month old animals, there were trends towards decreased expression of NOS2, C1qa, and IL1a in 3-month old TREM2+/− vs. TREM2+/+ mice.ConclusionsLoss of a single copy of trem2 had no effect on Aβ pathology, but altered the morphological phenotype of plaque-associated microglia. These data suggest that TREM2 is important for the microglial response to Aβ deposition but that a 50% decrease inTREM2 expression does not affect Aβ plaque burden.

ApoE4 markedly exacerbates tau-mediated neurodegeneration in a mouse model of tauopathy

APOE4 is the strongest genetic risk factor for late-onset Alzheimer disease. ApoE4 increases brain amyloid-β pathology relative to other ApoE isoforms. However, whether APOE independently influences tau pathology, the other major proteinopathy of Alzheimer disease and other tauopathies, or tau-mediated neurodegeneration, is not clear. By generating P301S tau transgenic mice on either a human ApoE knock-in (KI) or ApoE knockout (KO) background, here we show that P301S/E4 mice have significantly higher tau levels in the brain and a greater extent of somatodendritic tau redistribution by three months of age compared with P301S/E2, P301S/E3, and P301S/EKO mice. By nine months of age, P301S mice with different ApoE genotypes display distinct phosphorylated tau protein (p-tau) staining patterns. P301S/E4 mice develop markedly more brain atrophy and neuroinflammation than P301S/E2 and P301S/E3 mice, whereas P301S/EKO mice are largely protected from these changes. In vitro, E4-expressing microglia exhibit higher innate immune reactivity after lipopolysaccharide treatment. Co-culturing P301S tau-expressing neurons with E4-expressing mixed glia results in a significantly higher level of tumour-necrosis factor-α (TNF-α) secretion and markedly reduced neuronal viability compared with neuron/E2 and neuron/E3 co-cultures. Neurons co-cultured with EKO glia showed the greatest viability with the lowest level of secreted TNF-α. Treatment of P301S neurons with recombinant ApoE (E2, E3, E4) also leads to some neuronal damage and death compared with the absence of ApoE, with ApoE4 exacerbating the effect. In individuals with a sporadic primary tauopathy, the presence of an ε4 allele is associated with more severe regional neurodegeneration. In individuals who are positive for amyloid-β pathology with symptomatic Alzheimer disease who usually have tau pathology, ε4-carriers demonstrate greater rates of disease progression. Our results demonstrate that ApoE affects tau pathogenesis, neuroinflammation, and tau-mediated neurodegeneration independently of amyloid-β pathology. ApoE4 exerts a ‘toxic’ gain of function whereas the absence of ApoE is protective.