Neuronal enhancers are hotspots for DNA single-strand break repair.

Defects in DNA repair frequently lead to neurodevelopmental and neurodegenerative diseases, underscoring the particular importance of DNA repair in long-lived post-mitotic neurons1,2. The cellular genome is subjected to a constant barrage of endogenous DNA damage, but surprisingly little is known about the identity of the lesion(s) that accumulate in neurons and whether they accrue throughout the genome or at specific loci. Here we show that post-mitotic neurons accumulate unexpectedly high levels of DNA single-strand breaks (SSBs) at specific sites within the genome. Genome-wide mapping reveals that SSBs are located within enhancers at or near CpG dinucleotides and sites of DNA demethylation. These SSBs are repaired by PARP1 and XRCC1-dependent mechanisms. Notably, deficiencies in XRCC1-dependent short-patch repair increase DNA repair synthesis at neuronal enhancers, whereas defects in long-patch repair reduce synthesis. The high levels of SSB repair in neuronal enhancers are therefore likely to be sustained by both short-patch and long-patch processes. These data provide the first evidence of site- and cell-type-specific SSB repair, revealing unexpected levels of localized and continuous DNA breakage in neurons. In addition, they suggest an explanation for the neurodegenerative phenotypes that occur in patients with defective SSB repair.

Apolipoprotein E4 impairs spontaneous blood brain barrier repair following traumatic brain injury.

BACKGROUND: Traumatic Brain Injury (TBI) is a major cause of disability and mortality, to which there is currently no comprehensive treatment. Blood Brain Barrier (BBB) dysfunction is well documented in human TBI patients, yet the molecular mechanisms that underlie this neurovascular unit (NVU) pathology remains unclear. The apolipoprotein-E (apoE) protein has been implicated in controlling BBB integrity in an isoform dependent manner, via suppression of Cyclophilin A (CypA)-Matrix metallopeptidase-9 (MMP-9) signaling cascades, however the contribution of this pathway in TBI-induced BBB permeability is not fully investigated. METHODS: We exposed C57Bl/6 mice to controlled cortical impact and assessed NVU and BBB permeability responses up to 21 days post-injury. We pharmacologically probed the role of the CypA-MMP-9 pathway in BBB permeability after TBI using Cyclosporin A (CsA, 20 mg/kg). Finally, as the apoE4 protein is known to be functionally deficient compared to the apoE3 protein, we used humanized APOE mice as a clinically relevant model to study the role of apoE on BBB injury and repair after TBI. RESULTS: In C57Bl/6 mice there was an inverse relationship between soluble apoE and BBB permeability, such that damaged BBB stabilizes as apoE levels increase in the days following TBI. TBI mice displayed acute pericyte loss, increased MMP-9 production and activity, and reduced tight-junction expression. Treatment with the CypA antagonist CsA in C57Bl/6 mice attenuates MMP-9 responses and enhances BBB repair after injury, demonstrating that MMP-9 plays an important role in the timing of spontaneous BBB repair after TBI. We also show that apoe mRNA is present in both astrocytes and pericytes after TBI. We report that APOE3 and APOE4 mice have similar acute BBB responses to TBI, but APOE3 mice display faster spontaneous BBB repair than APOE4 mice. Isolated microvessel analysis reveals delayed pericyte repopulation, augmented and sustained MMP-9 expression at the NVU, and impaired stabilization of Zonula Occludens-1, Occludin and Claudin-5 expression at tight junctions in APOE4 mice after TBI compared to APOE3 mice. CONCLUSIONS: These data confirm apoE as an important modulator of spontaneous BBB stabilization following TBI, and highlights the APOE4 allele as a risk factor for poor outcome after TBI.

Reduced cortical excitatory synapse number in APOE4 mice is associated with increased calcineurin activity.

Synaptic loss is a symptom of Alzheimer's disease (AD) that is associated with the onset of cognitive decline and the loss of executive function. The strongest genetic risk factor for AD is the APOE4 allele, which results in both a greater risk of developing AD as well as an earlier age of onset of AD. Dendritic spines, the anatomical substrate of the excitatory synapse, are reduced in the cortex of humanized APOE4 mice but the reason for this synaptic decline is unknown. Calcineurin, a calcium/calmodulin dependent phosphatase, is a mediator of dendritic spine retraction. We used humanized APOE mice to examine how APOE genotype altered calcineurin activity and found that APOE4 mice have 35% higher cortical calcineurin activity compared with APOE3 mice. This occurred in the absence of any increase in calcineurin protein levels or mRNA expression. The elevation in calcineurin was associated with 10% fewer dendritic spine number in layer II/III of the cortex. Treatment with the calcineurin inhibitor FK506 reduced calcineurin activity by 64% and resulted in normalization of dendritic spine numbers in APOE4 mice. In conclusion, we found that the APOE4 gene in mice was associated with elevated calcineurin activity and fewer dendritic spine numbers compared with APOE3 mice. Importantly, calcineurin in APOE4 remained sensitive to pharmacological inhibition and spine density can be rescued by treatment with FK506.

Dendritic Spine Loss and Chronic White Matter Inflammation in a Mouse Model of Highly Repetitive Head Trauma.

Mild traumatic brain injury (mTBI) is an emerging risk for chronic behavioral, cognitive, and neurodegenerative conditions. Athletes absorb several hundred mTBIs each year; however, rodent models of repeat mTBI (rmTBI) are often limited to impacts in the single digits. Herein, we describe the effects of 30 rmTBIs, examining structural and pathological changes in mice up to 365 days after injury. We found that single mTBI causes a brief loss of consciousness and a transient reduction in dendritic spines, reflecting a loss of excitatory synapses. Single mTBI does not cause axonal injury, neuroinflammation, or cell death in the gray or white matter. Thirty rmTBIs with a 1-day interval between each mTBI do not cause dendritic spine loss; however, when the interinjury interval is increased to 7 days, dendritic spine loss is reinstated. Thirty rmTBIs cause white matter pathology characterized by positive silver and Fluoro-Jade B staining, and microglial proliferation and activation. This pathology continues to develop through 60 days, and is still apparent at 365 days, after injury. However, rmTBIs did not increase ß-amyloid levels or tau phosphorylation in the 3xTg-AD mouse model of Alzheimer disease. Our data reveal that single mTBI causes a transient loss of synapses, but that rmTBIs habituate to repetitive injury within a short time period. rmTBI causes the development of progressive white matter pathology that continues for months after the final impact.

Experimental traumatic brain injury induces rapid aggregation and oligomerization of amyloid-beta in an Alzheimer's disease mouse model.

Soluble amyloid-beta (Aß) oligomers are hypothesized to be the pathogenic species in Alzheimer's disease (AD), and increased levels of oligomers in the brain subsequent to traumatic brain injury (TBI) may exacerbate secondary injury pathways and underlie increased risk of developing AD in later life. To determine whether TBI causes Aß aggregation and oligomerization in the brain, we exposed triple transgenic AD model mice to controlled cortical impact injury and measured levels of soluble, insoluble, and oligomeric Aß by enzyme-linked immunosorbent assay (ELISA) at 1, 3, and 7 days postinjury. TBI rapidly increased levels of both soluble and insoluble Aß40 and Aß42 in the injured cortex at 1 day postinjury. We confirmed previous findings that identified damaged axons as a major site of Aß accumulation using both immunohistochemistry and biochemistry. We also report that soluble Aß oligomers were significantly increased in the injured cortex, as demonstrated by both ELISA and Western blot. Interestingly, the mouse brain is able to rapidly clear trauma-induced Aß, with both soluble and insoluble Aß species returning to sham levels by 7 days postinjury. In conclusion, we demonstrate that TBI causes acute accumulation and aggregation of Aß in the brain, including the formation of low- and high-molecular-weight Aß oligomers. The formation and aggregation of Aß into toxic species acutely after injury may play a role in secondary injury cascades after trauma and, chronically, may contribute to increased risk of developing AD in later life.

Human immunodeficiency virus type 1 alters brain-derived neurotrophic factor processing in neurons.

The molecular mechanisms leading to synaptic simplification and neuronal apoptosis in human immunodeficiency virus type 1 (HIV-1)-positive subjects are unknown. The HIV protein gp120 reduced the length of neuronal processes similarly to the proneurotrophin pro-brain-derived neurotrophic factor (proBDNF). Intriguingly, the effects of both proBDNF and gp120 were blocked by inhibitors of the p75 neurotrophin receptor, suggesting that proBDNF and gp120 share a similar mechanism of neurotoxicity. Therefore, we tested the hypothesis that gp120 affects the release of proBDNF. Using rat primary neurons, we observed that gp120 promotes a time-dependent intracellular and extracellular accumulation of proBDNF concomitantly with a decrease in mature BDNF. A similar imbalance in the ratio proBDNF/mature BDNF was confirmed in postmortem brains of HIV-positive subjects cognitively impaired and motor impaired. Therefore, it is conceivable to formulate the hypothesis that HIV neurotoxicity includes a gp120-mediated alteration of BDNF processing. To determine the cellular mechanism whereby gp120 produces an accumulation of proBDNF, we examined the levels of intracellular and extracellular enzymes that proteolytically cleave proBDNF furin and tissue plasminogen, respectively. In rat neurons exposed to gp120, intracellular furin levels decreased before cell death, whereas tissue plasminogen changed only during apoptosis. Our data suggest that HIV, through gp120, reduces proBDNF processing by affecting furin levels, and therefore causes an altered balance between antiapoptotic and proapoptotic neurotrophins. Our studies identify a new mechanism that may explain how HIV promotes neuronal injury.

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 effect of injury severity on behavior: a phenotypic study of cognitive and emotional deficits after mild, moderate, and severe controlled cortical impact injury in mice.

Traumatic brain injury (TBI) can cause a broad array of behavioral problems including cognitive and emotional deficits. Human studies comparing neurobehavioral outcomes after TBI suggest that cognitive impairments increase with injury severity, but emotional problems such as anxiety and depression do not. To determine whether cognitive and emotional impairments increase as a function of injury severity we exposed mice to sham, mild, moderate, or severe controlled cortical impact (CCI) and evaluated performance on a variety of neurobehavioral tests in the same animals before assessing lesion volume as a histological measure of injury severity. Increasing cortical impact depth successfully produced lesions of increasing severity in our model. We found that cognitive impairments in the Morris water maze increased with injury severity, as did the degree of contralateral torso flexion, a measure of unilateral striatal damage. TBI also caused deficits in emotional behavior as quantified in the forced swim test, elevated-plus maze, and prepulse inhibition of acoustic startle, but these deficits were not dependent on injury severity. Stepwise regression analyses revealed that Morris water maze performance and torso flexion predicted the majority of the variability in lesion volume. In summary, we find that cognitive deficits increase in relation to injury severity, but emotional deficits do not. Our data suggest that the threshold for emotional changes after experimental TBI is low, with no variation in behavioral deficits seen between mild and severe brain injury.

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.

Neurotrophins modulate the expression of chemokine receptors in the brain.

In the central nervous system, chemokines are primarily mediators of inflammatory processes. Their receptors, in particular, CXCR4 and CCR5, serve as co-factors along with CD4 that permit Human immunodeficiency virus-1 (HIV) infection. Moreover, experimental evidence has shown that CXCR4 and CCR5 mediate the neurotoxic effects of the HIV envelope protein gp120, suggesting that these receptors could also promote the neuropathogenesis observed in HIV-positive individuals. Therefore, a better understanding of the molecular mechanisms governing the expression of chemokine receptors in the brain may lead to improved therapies that reduce HIV neurotoxicity. This study presents evidence that the expression of chemokine receptors in the brain is modulated by two neurotrophins in an area-specific manner. This new evidence suggests that the neurotrophins may be an adjunct therapy to reduce HIV-mediated neuronal injury evoked by chemokine receptor activation.

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.

Rapid microglial response around amyloid pathology after systemic anti-Abeta antibody administration in PDAPP mice.

Aggregation of amyloid-beta (Abeta) peptide in the brain in the form of neuritic plaques and cerebral amyloid angiopathy (CAA) is a key feature of Alzheimer's disease (AD). Microglial cells surround aggregated Abeta and are believed to play a role in AD pathogenesis. A therapy for AD that has entered clinical trials is the administration of anti-Abeta antibodies. One mechanism by which certain anti-Abeta 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-Abeta antibodies has not been fully assessed. We administered an anti-Abeta antibody (m3D6) that binds aggregated Abeta 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 Abeta. These findings demonstrate that some effects of antibodies that recognize aggregated Abeta are rapid, involve microglia, and provide insight into the mechanism of action of a specific passive immunotherapy for AD.

Overexpression of ABCA1 reduces amyloid deposition in the PDAPP mouse model of Alzheimer disease.

APOE genotype is a major genetic risk factor for late-onset Alzheimer disease (AD). ABCA1, a member of the ATP-binding cassette family of active transporters, lipidates apoE in the CNS. Abca1(-/-) mice have decreased lipid associated with apoE and increased amyloid deposition in several AD mouse models. We hypothesized that mice overexpressing ABCA1 in the brain would have increased lipidation of apoE-containing lipoproteins and decreased amyloid deposition. To address these hypotheses, we created PrP-mAbca1 Tg mice that overexpress mouse Abca1 throughout the brain under the control of the mouse prion promoter. We bred the PrP-mAbca1 mice to the PDAPP AD mouse model, a transgenic line overexpressing a mutant human amyloid precursor protein. PDAPP/Abca1 Tg mice developed a phenotype remarkably similar to that seen in PDAPP/Apoe(-/-) mice: there was significantly less amyloid beta-peptide (Abeta) deposition, a redistribution of Abeta to the hilus of the dentate gyrus in the hippocampus, and an almost complete absence of thioflavine S-positive amyloid plaques. Analyses of CSF from PrP-mAbca1 Tg mice and media conditioned by PrP-mAbca1 Tg primary astrocytes demonstrated increased lipidation of apoE-containing particles. These data support the conclusions that increased ABCA1-mediated lipidation of apoE in the CNS can reduce amyloid burden and that increasing ABCA1 function may have a therapeutic effect on AD.

Electromagnetic controlled cortical impact device for precise, graded experimental traumatic brain injury.

Genetically modified mice represent useful tools for traumatic brain injury (TBI) research and attractive preclinical models for the development of novel therapeutics. Experimental methods that minimize the number of mice needed may increase the pace of discovery. With this in mind, we developed and characterized a prototype electromagnetic (EM) controlled cortical impact device along with refined surgical and behavioral testing techniques. By varying the depth of impact between 1.0 and 3.0 mm, we found that the EM device was capable of producing a broad range of injury severities. Histologically, 2.0-mm impact depth injuries produced by the EM device were similar to 1.0-mm impact depth injuries produced by a commercially available pneumatic device. Behaviorally, 2.0-, 2.5-, and 3.0-mm impacts impaired hidden platform and probe trial water maze performance, whereas 1.5-mm impacts did not. Rotorod and visible platform water maze deficits were also found following 2.5- and 3.0-mm impacts. No impairment of conditioned fear performance was detected. No differences were found between sexes of mice. Inter-operator reliability was very good. Behaviorally, we found that we could statistically distinguish between injury depths differing by 0.5 mm using 12 mice per group and between injury depths differing by 1.0 mm with 7-8 mice per group. Thus, the EM impactor and refined surgical and behavioral testing techniques may offer a reliable and convenient framework for preclinical TBI research involving mice.

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 Alzheimer's 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.

Deletion of Abca1 increases Abeta deposition in the PDAPP transgenic mouse model of Alzheimer disease.

Apolipoprotein E (apoE) genotype has a major influence on the risk for Alzheimer disease (AD). Different apoE isoforms may alter AD pathogenesis via their interactions with the amyloid beta-peptide (Abeta). Mice lacking the lipid transporter ABCA1 were found to have markedly decreased levels and lipidation of apoE in the central nervous system. We hypothesized that if Abca1-/- mice were bred to the PDAPP mouse model of AD, PDAPP Abca1-/ mice would have a phenotype similar to that of PDAPP Apoe+/- and PDAPP Apoe-/- mice, which develop less amyloid deposition than PDAPP Apoe+/+ mice. In contrast to this prediction, 12-month-old PDAPP Abca -/- mice had significantly higher levels of hippocampal Abeta, and cerebral amyloid angiopathy was significantly more common compared with PDAPP Abca1+/+ mice. Amyloid precursor protein (APP) C-terminal fragments were not different between Abca1 genotypes prior to plaque deposition in 3-month-old PDAPP mice, suggesting that deletion of Abca1 did not affect APP processing or Abeta production. As expected, 3-month-old PDAPP Abca1-/- mice had decreased apoE levels, but they also had a higher percentage of carbonate-insoluble apoE, suggesting that poorly lipidated apoE is less soluble in vivo. We also found that 12-month-old PDAPP Abca1-/- mice had a higher percentage of carbonate-insoluble apoE and that apoE deposits co-localize with amyloid plaques, demonstrating that poorly lipidated apoE co-deposits with insoluble Abeta. Together, these data suggest that despite substantially lower apoE levels, poorly lipidated apoE produced in the absence of ABCA1 is strongly amyloidogenic in vivo.

P-glycoprotein deficiency at the blood-brain barrier increases amyloid-beta 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.

The low density lipoprotein receptor regulates the level of central nervous system human and murine apolipoprotein E but does not modify amyloid plaque pathology in PDAPP mice.

Apolipoprotein E (apoE), a chaperone for the amyloid beta (Abeta) peptide, regulates the deposition and structure of Abeta that deposits in the brain in Alzheimer disease (AD). The primary apoE receptor that regulates levels of apoE in the brain is unknown. We report that the low density lipoprotein receptor (LDLR) regulates the cellular uptake and central nervous system levels of astrocyte-derived apoE. Cells lacking LDLR were unable to appreciably endocytose astrocyte-secreted apoE-containing lipoprotein particles. Moreover, cells overexpressing LDLR showed a dramatic increase in apoE endocytosis and degradation. We also found that LDLR knock-out (Ldlr-/-) mice had a significant, approximately 50% increase in the level of apoE in the cerebrospinal fluid and extracellular pools of the brain. However, when the PDAPP mouse model of AD was bred onto an Ldlr-/- background, we did not observe a significant change in brain Abeta levels either before or after the onset of Abeta deposition. Interestingly, human APOE3 or APOE4 (but not APOE2) knock-in mice bred on an Ldlr-/- background had a 210% and 380% increase, respectively, in the level of apoE in cerebrospinal fluid. These results demonstrate that central nervous system levels of both human and murine apoE are directly regulated by LDLR. Although the increase in murine apoE caused by LDLR deficiency was not sufficient to affect Abeta levels or deposition by 10 months of age in PDAPP mice, it remains a possibility that the increase in human apoE3 and apoE4 levels caused by LDLR deficiency will affect this process and could hold promise for therapeutic targets in AD.

Human apolipoprotein E4 alters the amyloid-beta 40:42 ratio and promotes the formation of cerebral amyloid angiopathy in an amyloid precursor protein transgenic model.

Alzheimer's disease (AD) is characterized by the aggregation and deposition of the normally soluble amyloid-beta (Abeta) peptide in the extracellular spaces of the brain as parenchymal plaques and in the walls of cerebral vessels as cerebral amyloid angiopathy (CAA). CAA is a common cause of brain hemorrhage and is found in most patients with AD. As in AD, the epsilon4 allele of the apolipoprotein E (apoE) gene (APOE) is a risk factor for CAA. To determine the effect of human apoE on CAA in vivo, we bred human APOE3 and APOE4 "knock-in" mice to a transgenic mouse model (Tg2576) that develops amyloid plaques as well as CAA. The expression of both human apoE isoforms resulted in a delay in Abeta deposition of several months relative to murine apoE. Tg2576 mice expressing the more fibrillogenic murine apoE develop parenchymal amyloid plaques and CAA by 9 months of age. At 15 months of age, the expression of human apoE4 led to substantial CAA with very few parenchymal plaques, whereas the expression of human apoE3 resulted in almost no CAA or parenchymal plaques. Additionally, young apoE4-expressing mice had an elevated ratio of Abeta 40:42 in brain extracellular pools and a lower 40:42 ratio in CSF, suggesting that apoE4 results in altered clearance and transport of Abeta species within different brain compartments. These findings demonstrate that, once Abeta fibrillogenesis occurs, apoE4 favors the formation of CAA over parenchymal plaques and suggest that molecules or treatments that increase the ratio of Abeta 40:42 may favor the formation of CAA versus parenchymal plaques.

ApoAI deficiency results in marked reductions in plasma cholesterol but no alterations in amyloid-beta pathology in a mouse model of Alzheimer's disease-like cerebral amyloidosis.

Epidemiological studies suggest links between cholesterol metabolism and Alzheimer's disease (AD), with hypercholesterolemia associated with increased AD risk, and use of cholesterol-lowering drugs associated with decreased risk. Animal models using cholesterol-modifying dietary or pharmacological interventions demonstrate similar findings. Proposed mechanisms include effects of cholesterol on the metabolism of amyloid-beta (Abeta), the protein that deposits in AD brain. To investigate the effect of genetic alterations in plasma cholesterol on Abeta pathology, we crossed the PDAPP transgenic mouse model of AD-like cerebral amyloidosis to apolipoprotein AI-null mice that have markedly reduced plasma cholesterol levels due to a virtual absence of high density lipoproteins, the primary lipoprotein in mice. Interestingly and in contrast to models using non-physiological high fat diets or cholesterol-lowering drugs to modify plasma cholesterol, we observed no differences in Abeta pathology in PDAPP mice of the various apoAI genotypes despite robust differences in plasma cholesterol levels between the groups. Absence of apoAI also resulted in reductions in brain but not cerebrospinal fluid cholesterol, but had no effect on brain apolipoprotein E levels. These and other data suggest that it is perhaps the level of brain apolipoprotein E, not cholesterol per se, that plays a primary role in brain Abeta metabolism.