Aβ Efflux Research Articles

Cannabidiol (CBD) Improves Cognition and Decreases Anxiety in the SAMP8 Mouse Model of Alzheimer’s

The use of cannabidiol (CBD) has grown rapidly over the last few years. CBD has been purported to work on a range of conditions, including anxiety, epilepsy, pain, and psychosis. There has been recent interest in CBD as a treatment for age‐related cognitive disorders due to its anti‐inflammatory properties. Here, we tested the effects of chronic CBD administration on learning and memory in the SAMP8 mouse model of Alzheimer’s disease. SAMP8 mice develop an age‐related impairment in learning and memory which corresponds to an increase in amyloid‐β (Aβ), hyperphosphorylated tau, oxidative stress, impaired efflux of Aβ across the blood‐brain barrier and neuroinflammation. SAMP8 mice starting at 11 months of age were treated with CBD (0, 3 or 30 mg/kg) daily via oral administration for 60 days. A 3 month old SAMP8 group receiving vehicle served as an unimpaired control. After 30 days of treatment, learning and memory (T‐maze and novel object recognition), activity in open field, anxiety in the elevated plus maze, and strength were assessed. Mice which received CBD 30 mg/kg had significant improvement in learning and memory in the T‐maze, taking fewer trials to reach criterion than 12 month old vehicle treated mice. CBD improved object recognition memory of 12 month old SAMP8 mice, which spent significantly more time with the novel object in the 24‐hour retention test than the age‐matched vehicle treated controls. While there was no difference in total distance traveled measured by open field activity, the mice that that received CBD displayed decreased anxiety in the elevated + maze at both 3 and 30 mg/kg/day doses compared to the age‐matched control. The mice that received 3 mg/kg/day had significantly less anxiety than the young control group. CBD had no significant effects on the measures of strength in the aged mice. Our results indicate that CBD improves both spatial and recognition memory and decreases anxiety. CBD is a potential treatment for age‐related dementia.Support or Funding InformationPilot funding from the Edward A. Doisy Department of Biochemistry and Molecular Biology at Saint Louis University

5-Caffeoylquinic Acid Ameliorates Cognitive Decline and Reduces Aβ Deposition by Modulating Aβ Clearance Pathways in APP/PS2 Transgenic Mice.

The accumulation of amyloid β (Aβ) in the brain is a major pathological feature of Alzheimer’s disease (AD). In our previous study, we demonstrated that coffee polyphenols (CPP) prevent cognitive dysfunction and Aβ deposition in the brain of an APP/PS2 transgenic mouse AD model. The underlying mechanisms, however, remain to be elucidated. Here, we investigated the effects of the chronic administration of 5-caffeoylquinic acid (5-CQA), the most abundant component of CPP, on cognitive dysfunction in APP/PS2 mice to identify the role of CPP in Aβ elimination. Relative to the untreated controls, the mice fed a 5-CQA-supplemented diet showed significant improvements in their cognitive function assessed by Y-maze and novel object recognition tests. Histochemical analysis revealed that 5-CQA substantially reduced Aβ plaque formation and neuronal loss in the hippocampi. Moreover, 5-CQA upregulated the gene encoding low-density lipoprotein receptor-related protein 1, an Aβ efflux receptor, and normalized the perivascular localization of aquaporin 4, which facilitates Aβ clearance along the paravascular pathway. These results suggest that 5-CQA reduces Aβ deposition in the brain by modulating the Aβ clearance pathways and ameliorating cognitive decline and neuronal loss in APP/PS2 mice. Thus, 5-CQA may be effective in preventing cognitive dysfunction in AD.

Therapeutic Potential of Direct Clearance of the Amyloid-β in Alzheimer's Disease.

Alzheimer’s disease (AD) is characterized by deposition and accumulation of amyloid-β (Aβ) and its corresponding plaques within the brain. Although much debate exists whether these plaques are the cause or the effect of AD, the accumulation of Aβ is linked with the imbalance between the production and clearance of Aβ. The receptor for advanced glycation endproducts (RAGE) facilitates entry of free Aβ from the peripheral stream. Conversely, lipoprotein receptor-related protein 1 (LRP1), located in the abluminal side at the blood–brain barrier mediates the efflux of Aβ. Research on altering the rates of clearance of Aβ by targeting these two pathways has been extensively study. Additionally, a cerebrospinal fluid (CSF) circulation assistant device has also been evaluated as an approach to increase solute concentration in the CSF via mechanical drainage, to allow for removal of Aβ from the brain. Herein, we provide a brief review of these approaches that are designed to re-establish a homeostatic Aβ balance in the brain.

Ketone Bodies Promote Amyloid-β1-40 Clearance in a Human in Vitro Blood-Brain Barrier Model.

Alzheimer’s disease (AD) is characterized by the abnormal accumulation of amyloid-β (Aβ) peptides in the brain. The pathological process has not yet been clarified, although dysfunctional transport of Aβ across the blood–brain barrier (BBB) appears to be integral to disease development. At present, no effective therapeutic treatment against AD exists, and the adoption of a ketogenic diet (KD) or ketone body (KB) supplements have been investigated as potential new therapeutic approaches. Despite experimental evidence supporting the hypothesis that KBs reduce the Aβ load in the AD brain, little information is available about the effect of KBs on BBB and their effect on Aβ transport. Therefore, we used a human in vitro BBB model, brain-like endothelial cells (BLECs), to investigate the effect of KBs on the BBB and on Aβ transport. Our results show that KBs do not modify BBB integrity and do not cause toxicity to BLECs. Furthermore, the presence of KBs in the culture media was combined with higher MCT1 and GLUT1 protein levels in BLECs. In addition, KBs significantly enhanced the protein levels of LRP1, P-gp, and PICALM, described to be involved in Aβ clearance. Finally, the combined use of KBs promotes Aβ efflux across the BBB. Inhibition experiments demonstrated the involvement of LRP1 and P-gp in the efflux. This work provides evidence that KBs promote Aβ clearance from the brain to blood in addition to exciting perspectives for studying the use of KBs in therapeutic approaches.

Plasma amyloid beta level changes in aged mice with cognitive dysfunction following sevoflurane exposure.

Previous studies have stated that cognitive impairment induced by anesthetics was associated with amyloid beta (Aβ). However, few researchers have investigated the transport of Aβ inside and outside of the brain. We attempted to probe the effects of sevoflurane on cognitive functions, the plasma Aβ, and transporters of Aβ in aged mice. The receptor for advanced glycation end-products (RAGE) is an Aβ influx protein, and Low-density lipoprotein receptor-related protein-1 (LRP-1) is an Aβ efflux protein. Aged mice were divided into the control group and the sevoflurane group. The mice were exposed to 100% oxygen or 2.5% sevoflurane for 2 h. The abilities of spatial learning and memory in mice were tested using the Morris water maze. Aβ concentrations of plasma were measured with enzyme-linked immunosorbent assay kits. The RAGE and LRP-1 gene levels in the brain were assessed with quantitative polymerase chain reaction, and the protein levels were determined by western blot analysis. The locations of RAGE in the brain were confirmed via immunofluorescence. In the sevoflurane group mice, the escape latency was increased on the 5th day of training, and the time spent in the target quadrant was decreased on the 7th day after anesthesia. Sevoflurane reduced the concentration of plasma Aβ1-40. In addition, sevoflurane increased both gene and protein levels of RAGE in the brain, and increased RAGE proteins co-localized with the hippocampal vascular endothelial cells. RAGE over-expression in the hippocampal vascular endothelial cells possibly resulted in the excessive transport of the plasma Aβ1-40 into the brain after treatment with sevoflurane, which was associated with sevoflurane-induced cognitive dysfunction in aged mice.

Chronic cerebral hypoperfusion alters amyloid-β transport related proteins in the cortical blood vessels of Alzheimer’s disease model mouse

Abnormal accumulation of amyloid-β (Aβ) peptide defines progression of Alzheimer’s disease (AD) pathology in brain. Here, we investigated expressive changes of two main Aβ transport receptors low-density lipoprotein receptor related protein-1 (LRP1) and receptor for advanced glycation end products (RAGE) in a novel AD mice (APP23) with chronic cerebral hypoperfusion (CCH) model, moreover, examined a protective effect of a free radical scavenger edaravone (Eda). In contrast to wild type (WT) and APP23 mice, CCH strongly accelerated abnormal Aβ40 depositions and cerebral amyloid angiopathy (CAA) pathology, increased both LRP1 and RAGE expressions in brain parenchyma, while a decrease of LRP1 and an increase of RAGE were observed in vascular endothelial cells at age 12 months (M) of AD mice. Furthermore, CCH strongly increased expressions of two hypoxia-related proteins hypoxia inducible factor-1α (HIF-1α) and heme oxygenase-1 (HO-1), two oxidative-related proteins 4-hydroxy-2-nonenal (4-HNE) and 8-hydroxy-2′-deoxyguanosine (8-OHdG), and decreased both two vital nutrient transporter proteins major facilitator super family domain containing 2a (Mfsd2a) and glucose transporter 1 (Glut1) expressions. Such the above abnormal pathological changes were significantly ameliorated by edaravone treatment. The present study demonstrated that CCH strongly enhanced primary AD pathology causing double imbalances of Aβ efflux and influx transport related proteins in the cortical blood vessels in AD mice, and that such a neuropathologic abnormality was greatly ameliorated by Eda.

Liver Dysfunction as a Novel Player in Alzheimer's Progression: Looking Outside the Brain.

Alzheimer’s disease (AD) afflicts an estimated 20 million people worldwide and is the fourth-leading cause of death in the developed world. The most common cause of dementia in older individuals, AD is characterized by neuropathologies including synaptic and neuronal degeneration, amyloid plaques, and neurofibrillary tangles (NTFs). Amyloid plaques are primarily composed of amyloid-beta peptide (Aβ), which accumulates in the brains of patients with AD. Further, small aggregates termed Aβ oligomers are implicated in the synaptic loss and neuronal degeneration underlying early cognitive impairments. Whether Aβ accumulates in part because of dysregulated clearance from the brain remains unclear. The flow of substances (e.g., nutrients, drugs, toxins) in and out of the brain is mediated by the blood-brain-barrier (BBB). The BBB exhibits impairment in AD patients and animal models. The effect of BBB impairment on Aβ, and whether BBB function is affected by non-neurological pathologies that impair peripheral clearance requires further investigation. In particular, impaired peripheral clearance is a feature of nonalcoholic fatty liver disease (NAFLD), a spectrum of liver disorders characterized by accumulation of fat in the liver accompanied by varying degrees of inflammation and hepatocyte injury. NAFLD has reached epidemic proportions, with an estimated prevalence between 20% and 30% of the general population. This chronic condition may influence AD pathogenesis. This review article summarizes the current state of the literature linking NAFLD and AD, highlighting the role of the major Aβ efflux and clearance protein, the LRP-1 receptor, which is abundantly expressed in liver, brain, and vasculature.

The Extent of Human Apolipoprotein A-I Lipidation Strongly Affects the β-Amyloid Efflux Across the Blood-Brain Barrier in vitro.

Much evidence suggests a protective role of high-density lipoprotein (HDL) and its major apolipoprotein apoA-I, in Alzheimer’s disease (AD). The biogenesis of nascent HDL derived from a first lipidation of apoA-I, which is synthesized by the liver and intestine but not in the brain, in a process mediated by ABCA1. The maturation of nascent HDL in mature spherical HDL is due to a subsequent lipidation step, LCAT-mediated cholesterol esterification, and the change of apoA-I conformation. Therefore, different subclasses of apoA-I-HDL simultaneously exist in the blood circulation. Here, we investigated if and how the lipidation state affects the ability of apoA-I-HDL to target and modulate the cerebral β-amyloid (Aβ) content from the periphery, that is thus far unclear. In particular, different subclasses of HDL, each with different apoA-I lipidation state, were purified from human plasma and their ability to cross the blood-brain barrier (BBB), to interact with Aβ aggregates, and to affect Aβ efflux across the BBB was assessed in vitro using a transwell system. The results showed that discoidal HDL displayed a superior capability to promote Aβ efflux in vitro (9 × 10-5 cm/min), when compared to apoA-I in other lipidation states. In particular, no effect on Aβ efflux was detected when apoA-I was in mature spherical HDL, suggesting that apoA-I conformation, and lipidation could play a role in Aβ clearance from the brain. Finally, when apoA-I folded its structure in discoidal HDL, rather than in spherical ones, it was able to cross the BBB in vitro and strongly destabilize the conformation of Aβ fibrils by decreasing the order of the fibril structure (-24%) and the β-sheet content (-14%). These data suggest that the extent of apoA-I lipidation, and consequently its conformation, may represent crucial features that could exert their protective role in AD pathogenesis.

SU31 - ESTABLISHING A BLOOD-BRAIN BARRIER MODEL FOR THE ANALYSIS OF ALZHEIMER'S DISEASE USING PATIENT DERIVED INDUCED PLURIPOTENT STEM CELLS

Background Alzheimer's Disease (AD) is the most common form of dementia. One characteristic of AD is the accumulation of amyloid-β (Aβ) peptides in different regions of the brain caused by the sequential cleavage of the transmembrane amyloid precursor protein. These extracellular amyloid plaques are a result of a dysregulated Blood-Brain Barrier (BBB) and emerge either due to an augmented production or a decreased excretion through brain barriers. The BBB with its several transporter systems shows age dependent expression levels of unspecific Aβ transporters. Recently, 19 loci including ABCA7, an ABC transporter responsible for Aβ efflux at the BBB, were discovered showing genome-wide significant association with the sporadic, late-onset form of AD (LOAD). Together with other research groups we identified several mutations in ABCA7 in AD patients, which is why an analysis of mutated ABCA7 in AD-related BBB models should be considered. We established patient-specific pluripotent stem cells from AD patients and healthy donors to differentiate endothelial cells and other related BBB cells for the investigation of disease mechanisms related to ABCA7 in AD patients. Methods Episomal vectors were used for the generation of induced Pluripotent Stem (iPS) cell lines from AD-specific B-lymphoblastoid cell lines (B-LCLs) and control B-LCLs of healthy donors. Alkaline phosphatase staining, immunofluorescence analysis, transcript analysis, western blot analysis, and the induction of three germ layers verified pluripotency. Successfully established lines were applied for the generation of BBB cells and neighboring cells. Endothelial cells and astrocytes were characterized by transcript analysis and flow cytometry. The differentiation process was analyzed for the altered expression of ABCA7. Results AD-specific iPS cells were successfully generated from B-LCLs. We could show alkaline phosphatase activity and the nuclear localization of pluripotency-related transcription factors including OCT4, SOX2, and NANOG. Transcript analysis and western blot analysis verified permanent induction of pluripotency marker genes including OCT4. The spontaneous differentiation of iPS cells into derivatives of the three germ layers was demonstrated. The efficient differentiation of high quality endothelial cells was shown by the expression of endothelial marker genes including von Willebrand factor. Astrocyte-specific markers including EAAT1 demonstrated efficient astrocyte differentiation. Discussion Human iPS cells provide a powerful tool for the analysis of AD-associated and patient-specific mutations in ABCA7. We successfully established endothelial cells and astrocytes representing suitable to study disease mechanisms of the BBB. Accordingly, this approach is an excellent opportunity to analyze pathogenic phenotypes of AD in vitro towards the identification of potential therapeutic targets.

A new approach for Alzheimer's disease treatment through P-gp regulation via ibuprofen

This study was aimed to investigate whether ibuprofen could alter the P-glycoprotein expression and function under Alzheimer's Disease condition and whether this alteration was induced by the inhibition of inflammatory reaction. APP/PS1 mice were used as AD model mice and ibuprofen-treated AD mice were given ibuprofen for 5 months. Then, Abcb1a/1b mRNA levels and P-gp expression were evaluated by qRT-PCR and western blot. Abcb1 mRNA levels were significantly reduced in AD mice compared to control mice, and it could be restored by ibuprofen treatment. Meanwhile, P-gp expression result showed a similar trend. Aβ plaques in cerebral cortices and hippocampus were investigated via immunohistochemical, and the results revealed that Aβ plaques were reduced in ibuprofen-treated AD mice compared with the AD mice, indicated that P-gp function may be recovered by ibuprofen treatment. qRT-PCR and ELISA were used to determined TNF-α, IL-1β, IL-6 and NF-κB levels. The results demonstrated that TNF-α, IL-1β mRNA levels and NF-κB expression were all significantly upregulated in AD mice in comparison with the control mice, and ibuprofen treatment could suppress the increase of inflammatory factors. In conclusion, the P-gp expression and function were suppressed in AD condition by activating inflammatory reaction, and then causing the Aβ efflux decreased. However, upregulating P-gp could increase the Aβ efflux in further to treat AD via inhibiting the inflammatory factors expression.

The concerted amyloid-beta clearance of LRP1 and ABCB1/P-gp across the blood-brain barrier is linked by PICALM

The accumulation of neurotoxic amyloid-beta (Aβ) in the brain is a characteristic hallmark of Alzheimer’s disease (AD). The blood-brain barrier (BBB) provides a large surface area and has been shown to be an important mediator for removal of brain Aβ. Both, the ABC transporter P-glycoprotein (ABCB1/P-gp) and the receptor low-density lipoprotein receptor-related protein 1 (LRP1) have been implicated to play crucial roles in Aβ efflux from brain. Here, with immunoprecipitation experiments, co-immunostainings and dual inhibition of ABCB1/P-gp and LRP1, we show that both proteins are functionally linked, mediating a concerted transcytosis of Aβ through endothelial cells. Late-onset AD risk factor Phosphatidylinositol binding clathrin assembly protein (PICALM) is associated with both ABCB1/P-gp and LRP1 representing a functional link and guiding both proteins through the brain endothelium. Together, our results give more mechanistic insight on Aβ transport across the BBB and show that the functional interplay of different clearance proteins is needed for the rapid removal of Aβ from the brain.

A Small Molecule Inhibitor of Plasminogen Activator Inhibitor-1 Reduces Brain Amyloid-β Load and Improves Memory in an Animal Model of Alzheimer's Disease.

Alzheimer's disease (AD) is a major cause of dementia in the elderly with no effective treatment. Accumulation of amyloid-β peptide (Aβ) in the brain is a pathological hallmark of AD and is believed to be a central disease-causing and disease-promoting event. In a previous study, we showed that deletion of plasminogen activator inhibitor 1 (PAI-1), a primary inhibitor of tissue type and urokinase type plasminogen activators (tPA and uPA), significantly reduced brain Aβ load in APP/PS1 mice, an animal model of familial AD. In this study, we further show that oral administration of TM5275, a small molecule inhibitor of PAI-1, for a period of 6 weeks, inhibits the activity of PAI-1 and increases the activities of tPA and uPA as well as plasmin, which is associated with a reduction of Aβ load in the hippocampus and cortex and improvement of learning/memory function in APP/PS1 mice. Protein abundance of low density lipoprotein related protein-1 (LRP-1), a multi ligand endocytotic receptor involved in transporting Aβ out of the brain, as well as plasma Aβ42 are increased, whereas the expression and processing of full-length amyloid-β protein precursor is not affected by TM5275 treatment in APP/PS1 mice. In vitro studies further show that PAI-1 increases, whereas TM5275 reduces, Aβ40 level in the culture medium of SHSY5Y-APP neuroblastoma cells. Collectively, our data suggest that TM5275 improves memory function of APP/PS1 mice, probably by reducing brain Aβ accumulation through increasing plasmin-mediated degradation and LRP-1-mediated efflux of Aβ in the brain.

Catalpol provides a protective effect on fibrillary Aβ1-42 -induced barrier disruption in an in vitro model of the blood-brain barrier.

Excessive amyloid beta (Aβ) deposition in brain is mainly responsible for cell damage and blood-brain barrier (BBB) disruption in Alzheimer's disease (AD). Catalpol, an iridoid glucoside extracted from the root of Rehmannia glutinosa Libosch, has neuroprotective effect against AD. It is unclear whether catalpol has a protective effect on Aβ-induced BBB leakage. We employed an immortalized endothelial cell line (bEnd.3) and astrocytes co-culture to mimic a BBB model in vitro and investigated the effect of catalpol on BBB. We found that treatment with catalpol decreased BBB hyperpermeability induced by fibrillar Aβ1-42 . Data from western blotting showed that catalpol prevented fibrillar Aβ1-42 -induced bEnd.3 cell apoptosis through mitochondria-dependent and death receptor pathways; decreased the levels of matrix metalloproteinases (MMPs), MMP-2, MMP-9, and the receptor for advanced glycation end products; and increased the levels of tight junction proteins (ZO-1, occludin, and claudin-5), low-density lipoprotein receptor-related protein 1, and P-glycoprotein in fibrillar Aβ1-42 -treated bEnd.3 cells. Moreover, catalpol also enhanced soluble Aβ efflux across the fibrillar Aβ1-42 -treated bEnd.3 cells BBB monolayer model. Altogether, our results suggest that catalpol alleviate fibrillar Aβ1-42 -induced BBB disruption, enhance soluble Aβ clearance, and offer a feasible therapeutic application in AD treatment.

Magnesium Reduces Blood-Brain Barrier Permeability and Regulates Amyloid-β Transcytosis.

Poor Mg status is a risk factor for Alzheimer's disease (AD), and the underlying mechanisms remain elusive. Here, we provided the first evidence that elevated Mg levels significantly reduced the blood-brain barrier (BBB) permeability and regulated its function in vitro. Transient receptor potential melastatin 7 (TRPM7) and magnesium transporter subtype 1 (MagT1) were two major cellular receptors mediating entry of extracellular Mg2+ into the cells. Elevated Mg levels also induced an accelerated clearance of amyloid-β peptide (Aβ) from the brain to the blood side via BBB transcytosis through low-density lipoprotein receptor-related protein (LRP) and phosphatidylinositol binding clathrin assembly protein (PICALM), while reduced the influx of Aβ from the blood to the brain side involving receptor for advanced glycation end products (RAGE) and caveolae. Mg enhanced BBB barrier properties and overall expression of LRP1 and PICALM whereas reduced that of RAGE and caveolin-1. Apical-to-basolateral and vice versa steady-state Aβ flux achieved an equilibrium of 18 and 0.27fmol/min/cm2, respectively, about 30min after the initial addition of physiological levels of free Aβ. Knockdown of caveolin-1 or disruption of caveolae membrane microdomains reduced RAGE-mediated influx significantly, but not LRP1-mediated efflux of Aβ. Stimulating endothelial cells with vascular endothelial growth factor (VEGF) enhanced caveolin-1 phosphorylation and RAGE expression. Co-immunoprecipitation demonstrated that RAGE, but not LRP1, was physically associated with caveolin-1. Thus, Mg can reduce BBB permeability and promote BBB clearance of Aβ from the brain by increasing the expression of LRP1 and PICALM while reducing the level of RAGE and caveolin-1.

A novel monoclonal antibody against the N-terminus of Aβ1-42 reduces plaques and improves cognition in a mouse model of Alzheimer's disease.

Senile plaques consisting of Amyloid-beta (Aβ) peptides, in particular Aβ1–42, are the hallmark of Alzheimer’s disease (AD) and have been the primary therapeutic targets. Passive immunotherapy with monoclonal antibodies (mAbs) has shown initial success in mouse models of AD. However, the existing Aβ-directed mAbs mostly were tested on animal models or patients with advanced disease. The effects and mechanisms of mAbs on animals or human trial participants in the prodromal phase of AD are not fully clarified. In the current study, a novel mAb (3F5) directed against the 1–11 amino acids of Aβ1–42 was generated by immunizing mice with an emulsion of full length human Aβ1–42. The mAb (3F5) showed the ability to disrupt Aβ1–42 aggregation and prevent Aβ-mediated neurotoxicity in vitro. In a mouse model of AD, administration with 3F5 for 3 months in 6 months-old mice demonstrated that the mAb specifically bound with Aβ1–42 to promote the depolymerization of Aβ fibrils, facilitated endocytosis of Aβ1–42 by microglia, and attenuated the death and apoptosis of neuronal cells, accompanied by neurite outgrowth. APP/PS1 double-transgenic mice treated with 3F5 mAb showed reduced memory loss, cognitive decline, and decreased levels of amyloid deposits in the brain. Aβ1–42 levels in cerebral tissues were also significantly reduced, whereas serum Aβ1–42 was markedly increased. Interestingly, the concentration of 3F5 in peripheral circulation is much higher than that in the brain. These results indicate that 3F5 is able to cross the blood-brain barrier (BBB) to bind Aβ and initiates the phagocytosis of antibody/Aβ complexes by microglia in the amyloid depositing mice. 3F5 also promotes Aβ efflux from the brain. As a consequence, the antibody reduces plaques in the AD mouse brain, in association with reduction in the pathology of AD.

Transthyretin stability is critical in assisting beta amyloid clearance- Relevance of transthyretin stabilization in Alzheimer's disease.

The absence of transthyretin (TTR) in AD mice decreases brain Aβ clearance and reduces the low-density lipoprotein receptor-related protein 1 (LRP1). It is possible that neuroprotection by TTR is dependent on its tetramer structural stability, as studies using TTR mutants showed that unstable L55P TTR has low affinity for Aβ, and TTR tetrameric stabilizers such as iododiflunisal ameliorate AD features in vivo. We firstly investigated TTR folding status in human plasma measuring the resistance to urea denaturation. The importance of TTR stability on Aβ internalization was studied in human cerebral microvascular endothelial (hCMEC/D3) and hepatoma cells (HepG2), by flow cytometry. To investigate the fate of Aβ at the blood-brain barrier, Aβ efflux from hCMEC/D3 cells seeded on transwells was measured using ELISA. Further, to assess Aβ colocalization with lysosomes, Lysotracker was used. Moreover, levels of LRP1 were assessed in the liver and plasma of mice with different TTR backgrounds or treated with iododiflunisal. We showed that TTR stability is decreased in AD and that WT TTR and drug-stabilized L55P TTR are able to increase uptake of Aβ.Furthermore, measurement of Aβ efflux showed that stable or stabilized TTR increased Aβ efflux from the basolateral to the apical side. Moreover, HepG2 cells incubated with Aβ in the presence of WT TTR, but not L55P TTR, showed an increased number of lysosomes. Further, in the presence of WT TTR, Aβ peptide colocalized with lysosomes, indicating that only stable TTR assists Aβ internalization, leading to its degradation. Finally, we demonstrated that only stable TTR can increase LRP1 levels. TTR stabilization exerts a positive effect on Aβ clearance and LRP1 levels, suggesting that TTR protective role in AD is dependent on its stability. These results provide relevant information for the design of TTR-based therapeutic strategies for AD.

Piracetam Attenuates LPS-Induced Neuroinflammation and Cognitive Impairment in Rats.

The present study was performed to investigate the effect of piracetam on neuroinflammation induced by lipopolysaccharide (LPS) and resulting changes in cognitive behavior. Neuroinflammation was induced by a single dose of LPS solution infused into each of the lateral cerebral ventricles in concentrations of 1μg/μl, at a rate of 1μl/min over a 5-min period, with a 5-min waiting period between the two infusions. Piracetam in doses of 50, 100, and 200mg/kg i.p. was administered 30min before LPS infusion and continued for 9days. On ninth day, the behavioral test for memory and anxiety was done followed by blood collection and microdissection of the hippocampus (HIP) and prefrontal cortex brain regions. Piracetam attenuated the LPS-induced decrease in coping strategy to novel environment indicating anxiolytic activity. It also reversed the LPS-induced changes in the known arm and novel arm entries in the Y-maze test indicating amelioration of spatial memory impairment. Further, piracetam moderated LPS-induced decrease in the mitochondrial complex enzyme activities (I, II, IV, and V) and mitochondrial membrane potential. It ameliorated changes in hippocampal lipid peroxidation and nitrite levels including the activity of superoxide dismutase. Piracetam region specifically ameliorated LPS-induced increase in the level of IL-6 in HIP indicating anti-neuroinflammatory effect. Further, piracetam reduced HIP Aβ (1-40) and increased blood Aβ level suggesting efflux of Aβ from HIP to blood. Therefore, the present study indicates preclinical evidence for the use of piracetam in the treatment of neuroinflammatory disorders.

Amylin Enhances Amyloid-β Peptide Brain to Blood Efflux Across the Blood-Brain Barrier.

Findings from Alzheimer's disease (AD) mouse models showed that amylin treatment improved AD pathology and enhanced amyloid-β (Aβ) brain to blood clearance; however, the mechanism was not investigated. Using the Tg2576 AD mouse model, a single intraperitoneal injection of amylin significantly increased Aβ serum levels, and the effect was abolished by AC253, an amylin receptor antagonist, suggesting that amylin effect could be mediated by its receptor. Subsequent mechanistic studies showed amylin enhanced Aβ transport across a cell-based model of the blood-brain barrier (BBB), an effect that was abolished when the amylin receptor was inhibited by two amylin antagonists and by siRNA knockdown of amylin receptor Ramp3. To explain this finding, amylin effect on Aβ transport proteins expressed at the BBB was evaluated. Findings indicated that cells treated with amylin induced LRP1 expression, a major receptor involved in brain Aβ efflux, in plasma membrane fraction, suggesting intracellular translocation of LRP1 from the cytoplasmic pool. Increased LRP1 in membrane fraction could explain, at least in part, the enhanced uptake and transport of Aβ across the BBB. Collectively, our findings indicated that amylin induced Aβ brain to blood clearance through amylin receptor by inducing LRP1 subcellular translocation to the plasma membrane of the BBB endothelium.

Assessing the Impact of Lithium Chloride on the Expression of P-Glycoprotein at the Blood-Brain Barrier

In addition to extruding drugs from the brain, P-glycoprotein (P-gp) at the blood-brain barrier (BBB) facilitates the brain-to-blood clearance of beta-amyloid (Aβ) and is down-regulated in Alzheimer’s disease. Studies suggest that the mood-stabilizing drug lithium exerts a protective effect against Alzheimer’s disease. Although the mechanisms underlying this effect are not fully understood, evidence suggests that lithium chloride (LiCl) increases P-gp expression in vitro, albeit at concentrations substantially outside the therapeutic window. Therefore, we investigated the effects of pharmacologically-relevant concentrations of LiCl on P-gp expression using in vitro and in vivo approaches. Swiss outbred mice administered LiCl (300 mg/kg/day, 21 days) showed no change in brain microvascular P-gp protein expression. Furthermore, P-gp transcript and protein levels were unaltered by LiCl (1.25-5 mM, 24 h) in human immortalized brain endothelial cells, while both gene and protein expression were significantly enhanced by the P-gp up-regulator, SR12813 by 1.5-fold and 2.0-fold, respectively. P-gp efflux function was also unaffected by LiCl in vitro, by measuring accumulation of the fluorescent P-gp substrate rhodamine-123. This suggests therefore that LiCl is unlikely to affect the BBB efflux of Aβ or other P-gp substrates at pharmacologically-relevant concentrations, suggesting that the Aβ-lowering effects of LiCl are unrelated to elevated BBB P-gp expression.

In vivo Differential Brain Clearance and Catabolism of Monomeric and Oligomeric Alzheimer's Aβ protein.

Amyloid β (Aβ) is the major constituent of the brain deposits found in parenchymal plaques and cerebral blood vessels of patients with Alzheimer's disease (AD). Several lines of investigation support the notion that synaptic pathology, one of the strongest correlates to cognitive impairment, is related to the progressive accumulation of neurotoxic Aβ oligomers. Since the process of oligomerization/fibrillization is concentration-dependent, it is highly reliant on the homeostatic mechanisms that regulate the steady state levels of Aβ influencing the delicate balance between rate of synthesis, dynamics of aggregation, and clearance kinetics. Emerging new data suggest that reduced Aβ clearance, particularly in the aging brain, plays a critical role in the process of amyloid formation and AD pathogenesis. Using well-defined monomeric and low molecular mass oligomeric Aβ1-40 species stereotaxically injected into the brain of C57BL/6 wild-type mice in combination with biochemical and mass spectrometric analyses in CSF, our data clearly demonstrate that Aβ physiologic removal is extremely fast and involves local proteolytic degradation leading to the generation of heterogeneous C-terminally cleaved proteolytic products, while providing clear indication of the detrimental role of oligomerization for brain Aβ efflux. Immunofluorescence confocal microscopy studies provide insight into the cellular pathways involved in the brain removal and cellular uptake of Aβ. The findings indicate that clearance from brain interstitial fluid follows local and systemic paths and that in addition to the blood-brain barrier, local enzymatic degradation and the bulk flow transport through the choroid plexus into the CSF play significant roles. Our studies highlight the diverse factors influencing brain clearance and the participation of various routes of elimination opening up new research opportunities for the understanding of altered mechanisms triggering AD pathology and for the potential design of combined therapeutic strategies.