Markus Krohn

MDR1-P-Glycoprotein (ABCB1) Mediates Transport of Alzheimer’s Amyloid-β Peptides—Implications for the Mechanisms of Aβ Clearance at the Blood–Brain Barrier

Amyloid‐β (Aβ) is the major component of the insoluble amyloid plaques that accumulate intracerebrally in patients with Alzheimer’s disease (AD). It has been suggested that MDR1‐P‐glycoprotein (ABCB1, P‐gp) plays a substantial role in the elimination of Aβ from the brain. In the present study, MDR1‐transfected LLC cells growing in a polarized cell layer were used to characterize the interaction of Aβ1‐40/1‐42 with P‐gp. In this system, P‐gp‐mediated transport can be followed by the efflux of the fluorescent dye rhodamine‐123, or of Aβ itself from the cells into the apical extracellular space. Aβ significantly decreased the apical efflux of rhodamine‐123, and the transcellular transport of Aβ1‐40 and Aβ1‐42 into the apical chamber could be demonstrated using both ELISA and fluorescence (FITC)‐labeled peptides. This transport was inhibited by a P‐gp modulator. Furthermore, ATP‐dependent, P‐gp‐mediated transport of the fluorescence‐labeled peptides could be demonstrated in isolated, inside‐out membrane vesicles. Our data support the concept that P‐gp is important for the clearance of Aβ from brain, and thus may represent a target protein for the prevention and/or treatment of neurodegenerative disorders such as AD.

Cerebral amyloid-β proteostasis is regulated by the membrane transport protein ABCC1 in mice

In Alzheimer disease (AD), the intracerebral accumulation of amyloid-β (Aβ) peptides is a critical yet poorly understood process. Aβ clearance via the blood-brain barrier is reduced by approximately 30% in AD patients, but the underlying mechanisms remain elusive. ABC transporters have been implicated in the regulation of Aβ levels in the brain. Using a mouse model of AD in which the animals were further genetically modified to lack specific ABC transporters, here we have shown that the transporter ABCC1 has an important role in cerebral Aβ clearance and accumulation. Deficiency of ABCC1 substantially increased cerebral Aβ levels without altering the expression of most enzymes that would favor the production of Aβ from the Aβ precursor protein. In contrast, activation of ABCC1 using thiethylperazine (a drug approved by the FDA to relieve nausea and vomiting) markedly reduced Aβ load in a mouse model of AD expressing ABCC1 but not in such mice lacking ABCC1. Thus, by altering the temporal aggregation profile of Aβ, pharmacological activation of ABC transporters could impede the neurodegenerative cascade that culminates in the dementia of AD.

Alzheimer's disease and blood-brain barrier function - Why have anti-β-amyloid therapies failed to prevent dementia progression?

Proteopathies of the brain are defined by abnormal, disease-inducing protein deposition that leads to functional abrogation and death of neurons. Immunization trials targeting the removal of amyloid-beta plaques in Alzheimers disease have so far failed to stop the progression of dementia, despite autopsy findings of reduced plaque load. Here, we summarize current knowledge of the relationship between AD pathology and blood-brain barrier function, and propose that the activation of the excretion function of the blood-brain barrier might help to achieve better results in trials targeting the dissolution of cerebral amyloid-beta aggregates. We further discuss a possible role of oligomers in limiting the efficacy of immunotherapy.

Clinico-Pathologic Function of Cerebral ABC Transporters – Implications for the Pathogenesis of Alzheimer’s Disease

In recent years it has become evident that ABC transporters fulfill important barrier functions in normal organs and during disease processes. Most importantly, resistance to drugs in cancer cells led to intense oncological and pharmacological investigations in which researchers were able to highlight important pharmacological interactions of chemotherapeuticals with ABC transporter function. Recently, the development of neurodegenerative diseases and the maintenance of neuronal stem cells have been linked to the activity of ABC transporters. Here, we summarize findings from cell culture experiments, animal models and studies of patients with Alzheimers disease. Furthermore, we discuss pharmacological interactions and computational methods for risk assessment.

Mitochondrial DNA polymorphisms specifically modify cerebral β-amyloid proteostasis

Several lines of evidence link mutations and deletions in mitochondrial DNA (mtDNA) and its maternal inheritance to neurodegenerative diseases in the elderly. Age-related mutations of mtDNA modulate the tricarboxylic cycle enzyme activity, mitochondrial oxidative phosphorylation capacity and oxidative stress response. To investigate the functional relevance of specific mtDNA polymorphisms of inbred mouse strains in the proteostasis regulation of the brain, we established novel mitochondrial congenic mouse lines of Alzheimer’s disease (AD). We crossed females from inbred strains (FVB/N, AKR/J, NOD/LtJ) with C57BL/6 males for at least ten generations to gain specific mitochondrial conplastic strains with pure C57BL/6 nuclear backgrounds. We show that specific mtDNA polymorphisms originating from the inbred strains differentially influence mitochondrial energy metabolism, ATP production and ATP-driven microglial activity, resulting in alterations of cerebral β-amyloid (Aβ) accumulation. Our findings demonstrate that mtDNA-related increases in ATP levels and subsequently in microglial activity are directly linked to decreased Aβ accumulation in vivo, implicating reduced mitochondrial function in microglia as a causative factor in the development of age-related cerebral proteopathies such as AD.

Reduced Alzheimer’s disease pathology by St. John’s wort treatment is independent of hyperforin and facilitated by ABCC1 and microglia activation in mice

Soluble β-amyloid peptides (Aβ) and small Aβ oligomers represent the most toxic peptide moieties recognized in brains affected by Alzheimers disease (AD). Here we provide the first evidence that specific St. Johns wort (SJW) extracts both attenuate Aβ-induced histopathology and alleviate memory impairments in APP-transgenic mice. Importantly, these effects are attained independently of hyperforin. Specifically, two extracts characterized by low hyperforin content (i) significantly decrease intracerebral Aβ42 levels, (ii) decrease the number and size of amyloid plaques, (iii) rescue neocortical neurons, (iv) restore cognition to normal levels, and (iv) activate microglia in vitro and in vivo. Mechanistically, we reveal that the reduction of soluble Aβ42 species is the consequence of a highly increased export activity in the bloodbrain barrier ABCC1transporter, which was found to play a fundamental role in Aβ excretion into the bloodstream. These data (i) support the significant beneficial potential of SJW extracts on AD proteopathy, and (ii) demonstrate for the first time that hyperforin concentration does not necessarily correlate with their therapeutic effects. Hence, by activating ABC transporters, specific extracts of SJW may be used to treat AD and other diseases involving peptide accumulation and cognition impairment. We propose that the anti-depressant and anti-dementia effects of these hyperforin-reduced phytoextracts could be combined for treatment of the elderly, with a concomitant reduction in deleterious hyperforin-related side effects.

Impaired mitochondrial energy production and ABC transporter function – a crucial interconnection in dementing proteopathies of the brain

Ageing is the main risk factor for the development of dementing neurodegenerative diseases (NDs) and it is accompanied by the accumulation of variations in mitochondrial DNA. The resulting tissue-specific alterations in ATP production and availability cause deteriorations of cerebral clearance mechanisms that are important for the removal of toxic peptides and its aggregates. ABC transporters were shown to be the most important exporter superfamily for toxic peptides, e.g. β-amyloid and α-synuclein. Their activity is highly dependent on the availability of ATP and forms a directed energy-exporter network, linking decreased mitochondrial function with highly impaired ABC transporter activity and disease progression. In this paper, we describe a network based on interactions between ageing, energy metabolism, regeneration, accumulation of toxic peptides and the development of proteopathies of the brain with a focus on Alzheimers disease (AD). Additionally, we provide new experimental evidence for interactions within this network in regenerative processes in AD.

Automated detection of amyloid-β-related cortical and subcortical signal changes in a transgenic model of Alzheimer's disease using high-field MRI.

In vivo imaging of amyloid-β (Aβ) load as a biomarker of Alzheimers disease (AD) would be of considerable clinical relevance for the early diagnosis and monitoring of treatment effects. Here, we investigated automated quantification of in vivo T2 relaxation time as a surrogate measure of plaque load in the brains of ten AβPP/PS1 transgenic mice (age 20 weeks) using in vivo MRI acquisitions on a 7T Bruker ClinScan magnet. AβPP/PS1 mice present with rapid-onset cerebral β-amyloidosis, and were compared with eight age-matched, wild-type control mice (C57Bl/6J) that do not develop Aβ-deposition in brain. Data were analyzed with a novel automated voxel-based analysis that allowed mapping the entire brain for significant signal changes. In AβPP/PS1 mice, we found a significant decrease in T2 relaxation times in the deeper neocortical layers, caudate-putamen, thalamus, hippocampus, and cerebellum compared to wildtype controls. These changes were in line with the histological distribution of cerebral Aβ plaques and activated microglia. Grey matter density did not differ between wild-type mice and AβPP/PS1 mice, consistent with a lack of neuronal loss in histological investigations. High-field MRI with automated mapping of T2 time changes may be a useful tool for the detection of plaque load in living transgenic animals, which may become relevant for the evaluation of amyloid lowering intervention effects in future studies.

Determination of spatial and temporal distribution of microglia by 230nm-high-resolution, high-throughput automated analysis reveals different amyloid plaque populations in an APP/PS1 mouse model of Alzheimer's disease

One early and prominent pathologic feature of Alzheimers disease (AD) is the appearance of activated microglia in the vicinity of developing β-amyloid deposits. However, the precise role of microglia during the course of AD is still under discussion. Microglia have been reported to degrade and clear β-amyloid, but they also can exert deleterious effects due to overwhelming inflammatory reactions. Here, we demonstrate the occurrence of developing plaque populations with distinct amounts of associated microglia using time-dependent analyses of plaque morphology and the spatial distribution of microglia in an APP/PS1 mouse model. In addition to a population of larger plaques (>700µm(2)) that are occupied by a moderate contingent of microglial cells across the course of aging, a second type of small β-amyloid deposits develops (≤400µm(2)) in which the plaque core is enveloped by a relatively large number of microglia. Our analyses indicate that microglia are strongly activated early in the emergence of senile plaques, but that activation is diminished in the later stages of plaque evolution (>150 days). These findings support the view that microglia serve to restrict the growth of senile plaques, and do so in a way that minimizes local inflammatory damage to other components of the brain.

Chronic Toxoplasma gondii infection enhances β-amyloid phagocytosis and clearance by recruited monocytes

IntroductionAlzheimer’s disease (AD) is associated with the accumulation of β-amyloid (Aβ) as senile plaques in the brain, thus leading to neurodegeneration and cognitive impairment. Plaque formation depends not merely on the amount of generated Aβ peptides, but more importantly on their effective removal. Chronic infections with neurotropic pathogens, most prominently the parasite Toxoplasma (T.) gondii, are frequent in the elderly, and it has been suggested that the resulting neuroinflammation may influence the course of AD. In the present study, we investigated how chronic T. gondii infection and resulting neuroinflammation affect plaque deposition and removal in a mouse model of AD.ResultsChronic infection with T. gondii was associated with reduced Aβ and plaque load in 5xFAD mice. Upon infection, myeloid-derived CCR2hi Ly6Chi monocytes, CCR2+ Ly6Cint, and CCR2+ Ly6Clow mononuclear cells were recruited to the brain of mice. Compared to microglia, these recruited mononuclear cells showed highly increased phagocytic capacity of Aβ ex vivo. The F4/80+ Ly6Clow macrophages expressed high levels of Triggering Receptor Expressed on Myeloid cells 2 (TREM2), CD36, and Scavenger Receptor A1 (SCARA1), indicating phagocytic activity. Importantly, selective ablation of CCR2+ Ly6Chi monocytes resulted in an increased amount of Aβ in infected mice. Elevated insulin-degrading enzyme (IDE), matrix metalloproteinase 9 (MMP9), as well as immunoproteasome subunits β1i/LMP2, β2i/MECL-1, and β5i/LMP7 mRNA levels in the infected brains indicated increased proteolytic Aβ degradation. Particularly, LMP7 was highly expressed by the recruited mononuclear cells in the brain, suggesting a novel mechanism of Aβ clearance.ConclusionsOur results indicate that chronic Toxoplasma infection ameliorates β-amyloidosis in a murine model of AD by activation of the immune system, specifically by recruitment of Ly6Chi monocytes and by enhancement of phagocytosis and degradation of soluble Aβ. Our findings provide evidence for a modulatory role of inflammation-induced Aβ phagocytosis and degradation by newly recruited peripheral immune cells in the pathophysiology of AD.