Amyloid-Beta Peptides 40 and 42 Employ Distinct Molecular Pathways for Cell Entry and Intracellular Transit at the Blood-Brain Barrier Endothelium.

The blood-brain barrier (BBB) plays a critical role in maintaining the equilibrium between amyloid beta (Aß) levels in blood and the brain by regulating Aß transport. Our previous publications demonstrated that BBB trafficking of Aß42 and Aß40 is distinct and is disrupted under various pathophysiological conditions. However, the intracellular mechanisms that allow BBB endothelium to differentially handle Aß40 and Aß42 have not been clearly elucidated. In this study, we identified mechanisms of Aß endocytosis in polarized human cerebral microvascular endothelial cell monolayers. Our studies demonstrated that Aß peptides with fluorescent label (F-Aß) were internalized by BBB endothelial cells via energy, dynamin, and actin-dependent endocytosis. Interestingly, endocytosis of F-Aß40 but not F-Aß42 was substantially reduced by clathrin inhibition, whereas F-Aß42 but not F-Aß40 endocytosis was reduced by half after inhibiting the caveolae-mediated pathway. Following endocytosis, both isoforms were sorted by the endo-lysosomal system. Although Aß42 was shown to accumulate more in the lysosomes, which could lead to its higher degradation and/or aggregation at lower lysosomal pH, Aß40 demonstrated robust accumulation in recycling endosomes, which may facilitate its exocytosis by the endothelial cells. These results provide a mechanistic insight into the selective ability of BBB endothelium to transport Aß40 versus Aß42. This knowledge contributes to the understanding of molecular pathways underlying Aß accumulation in the BBB endothelium and associated BBB dysfunction. Moreover, it allows us to establish mechanistic rationale for altered Aß40:Aß42 ratios and anomalous amyloid deposition in the cerebral vasculature as well as brain parenchyma during Alzheimer's disease progression. SIGNIFICANCE STATEMENT: Differential interaction of Aß40 and Aß42 isoforms with the blood-brain barrier (BBB) endothelium may contribute to perturbation in Aß42:Aß40 ratio, which is associated with Alzheimer's disease (AD) progression and severity. The current study identified distinct molecular pathways by which Aß40 and Aß42 are trafficked at the BBB, which regulates equilibrium between blood and brain Aß levels. These findings provide molecular insights into mechanisms that engender BBB dysfunction and promote Aß accumulation in AD brain.

Differential Effects of Alzheimer's Disease Aß40 and 42 on Endocytosis and Intraneuronal Trafficking.

Anomalous neuronal accumulation of Aß peptides was shown to affect synaptic transmission and contribute to neurodegeneration in Alzheimer's disease (AD) brain. Neuronal cells internalize amyloid beta (Aß) peptides from the brain extracellular space even under normal physiological conditions, and these endocytotic pathways go awry during AD progression. We hypothesized that exposure to toxic Aß species accumulating in AD brain contributes to perturbations in neuronal endocytosis. We have shown substantial down-regulation of KEGG endocytotic pathway genes in AD patient brain regions that accumulate Aß compared to those in non-demented individuals. While both Aß40 and Aß42 perturbed endocytosis and intracellular trafficking in neuronal cells, Aß40 had a greater effect than Aß42. Moreover, Aß40 decreased the neuronal uptake and lysosomal accumulation of Aß42, which tends to oligomerize at low lysosomal pH. Hence, Aß40 may reduce the prevalence of stable Aß42 oligomers that are closely associated with neurodegeneration and are intercellularly propagated across the vulnerable brain regions to eventually nucleate as amyloid plaques. In conclusion, elevated brain Aß levels and Aß42:40 ratio apparent in the early stages of AD could perturb intraneuronal trafficking, augment the anomalous accumulation of amyloid peptides in AD brain, and drive AD pathogenesis.

Insulin differentially affects the distribution kinetics of amyloid beta 40 and 42 in plasma and brain.

Impaired brain clearance of amyloid-beta peptides (Aß) 40 and 42 across the blood-brain barrier (BBB) is believed to be one of the pathways responsible for Alzheimer's disease (AD) pathogenesis. Hyperinsulinemia prevalent in type II diabetes was shown to damage cerebral vasculature and increase Aß accumulation in AD brain. However, there is no clarity on how aberrations in peripheral insulin levels affect Aß accumulation in the brain. This study describes, for the first time, an intricate relation between plasma insulin and Aß transport at the BBB. Upon peripheral insulin administration in wild-type mice: the plasma clearance of Aß40 increased, but Aß42 clearance reduced; the plasma-to-brain influx of Aß40 increased, and that of Aß42 reduced; and the clearance of intracerebrally injected Aß40 decreased, whereas Aß42 clearance increased. In hCMEC/D3 monolayers (in vitro BBB model) exposed to insulin, the luminal uptake and luminal-to-abluminal permeability of Aß40 increased and that of Aß42 reduced; the abluminal-to-luminal permeability of Aß40 decreased, whereas Aß42 permeability increased. Moreover, Aß cellular trafficking machinery was altered. In summary, Aß40 and Aß42 demonstrated distinct distribution kinetics in plasma and brain compartments, and insulin differentially modulated their distribution. Cerebrovascular disease and metabolic disorders may disrupt this intricate homeostasis and aggravate AD pathology.

Ability of chitosan gels to disrupt bacterial biofilms and their applications in the treatment of bacterial vaginosis.

Recurrence of bacterial vaginosis is attributed to the inability of various formulations to disrupt bacterial biofilms. A negatively charged polysaccharide matrix coats the bacterial communities in the biofilm and restricts the penetration of antibiotics. Therefore, bacteria in the deeper segments of the biofilm persist and perpetuate the infection. In this study, we have tested the efficacy of two bioadhesive polymers, cationic chitosan and anionic polycarbophil, to disrupt Pseudomonas aeruginosa biofilms grown in the Center for Disease Control bioreactor as well as on the 96-well plates. The biofilms were treated with various concentrations of polycarbophil and chitosan at pH 4 or 6. Biofilm integrity following various treatments was evaluated by crystal violet stain and laser confocal microscopy employing Syto9 (live-cell stain) and propidium iodide (dead-cell stain). These studies demonstrated that chitosan gel disrupts the P. aeruginosa biofilm more effectively than does polycarbophil; and this effect is independent of the pH and charge densities on either polymers.

Differences in the cellular uptake and intracellular itineraries of amyloid beta proteins 40 and 42: ramifications for the Alzheimer's drug discovery.

Mounting evidence suggests that the pathological hallmarks of Alzheimer's disease (AD), neurofibrillary tangles and parenchymal amyloid plaques, are downstream reflections of neurodegeneration caused by the intraneuronal accumulation of amyloid-ß proteins (Aß), particularly Aß42 and Aß40. While the neurotoxicity of more amyloidogenic but less abundant Aß42 is well documented, the effect of Aß40 on neurons has been understudied. The Aß40 expression in the presymptomatic AD brain is ten times greater than that of Aß42. However, the Aß40:42 ratio decreases with AD progression and coincides with increased amyloid plaque deposition in the brain. Hence, it is thought that Aß40 protects neurons from the deleterious effects of Aß42. The pathophysiological pathways involved in the neuronal uptake of Aß40 or Aß42 have not been clearly elucidated. Lack of such critical information obscures therapeutic targets and thwarts rational drug development strategies aimed at preventing neurodegeneration in AD. The current study has shown that fluorescein labeled Aß42 (F-Aß42) is internalized by neurons via dynamin dependent endocytosis and is sensitive to membrane cholesterol, whereas the neuronal uptake of F-Aß40 is energy independent and nonendocytotic. Following their uptake, both F-Aß40 and F-Aß42 did not accumulate in early/recycling endosomes; F-Aß42 but not F-Aß40 accumulated in late endosomes and in the vesicles harboring caveolin-1. Furthermore, F-Aß42 demonstrated robust accumulation in the lysosomes and damaged their integrity, whereas F-Aß40 showed only a sparse lysosomal accumulation. Such regulated trafficking along distinct pathways suggests that Aß40 and Aß42 exercise differential effects on neurons. These differences must be carefully considered in the design of a pharmacological agent intended to block the neurodegeneration triggered by Aß proteins.