Targeting the Transferrin Receptor to Develop Erythropoietin for Alzheimer’s Disease

Alzheimer’s disease (AD) is the sixth leading cause of death in the United States with approximately 5.8 million Americans currently living with AD. Due to the lack of a disease modifying treatment for AD and the aging baby boomer generation, this number is projected to grow to 13.8 million by 2050 (Gaugler et al., 2019). Amyloid-beta (Aβ) plaque accumulation, one of the major pathological hallmarks of AD, can begin \u3e 20 years before clinical symptoms of AD. By the time AD is clinically diagnosed, neuronal loss and neuropathological lesions (Aβ plaques and tau tangles) have already occurred in many brain regions (Gaugler et al., 2019). AD dementia correlates highly with neuronal loss, and therefore, reduction of neuropathological lesions in the AD brain at the time of clinical diagnosis alone cannot reverse AD dementia. We propose that a therapy that combines a reduction of neuropathological lesions of AD along with neuronal repair and neurogenesis may be required to treat AD dementia

The Concentration of Brain Homogenates with the Amicon Ultra Centrifugal Filters

Accurately measuring the brain concentration of a neurotherapeutic is critical in determining its pharmacokinetic profile in vivo. Biologics are potential therapeutics for neurologic diseases and biologics fused to an antibody targeting a transcytosis receptor at the Blood-Brain Barrier, designated as antibody-biologic fusion proteins, are Blood-Brain Barrier penetrating neurotherapeutics. The use of sandwich immunosorbent assays to measure concentrations of antibody-biologic fusion proteins in brain homogenates has become increasingly popular. The raw brain homogenate contains many proteins and other macromolecules that can cause a matrix effect, potentially interfering with the limit of detection of such assays and reduce the overall sample signal. Further, the low sample loading volumes while running these assays can reduce the sample signal. Our aim was therefore to optimize the existing tissue sample preparation and processing to concentrate the sample to elevate the signal of the analyte. Here, we present a protocol for concentrating and increasing the signal of transferrin receptor antibody-biologic fusion proteins in mouse brain homogenates using the Amicon Ultra Centrifugal filters. • The presented method uses the Amicon Ultra Centrifugal filters to concentrate mouse brain tissue homogenates. • The concentrated brain tissue homogenates are then assayed using standard sandwich enzyme-linked immunosorbent assay (ELISA) protocols. • This method improves upon the traditional brain homogenization procedure and ELISA measurements for antibody-biologic fusion proteins by effectively concentrating brain tissue homogenates

Acute Depression of Energy Metabolism After Microdialysis Probe Implantation is Distinct from Ischemia-Induced Changes in Mouse Brain

The current study used measurements of metabolites and markers of membrane integrity to determine the most suitable time point for microdialysis experiments following probe implantation. Leakage of Evans blue and sodium fluorescein indicated increased BBB permeability only immediately (15 min), but not 1.5 and 24 h following probe implantation. Acute implantation decreased glucose and lactate levels relative to the levels after 24 h (to 13–37% and 25–60%, respectively). No change in extracellular levels of glutamate or glycerol was seen. In comparison to acute probe implantation, the pattern of damage under brain ischemia (middle cerebral artery occlusion) differed: While glucose levels dropped, lactate levels rose after ischemia, and glutamate (tenfold) and glycerol (eightfold) increased sharply. In conclusion, acute implantation of a microdialysis probe causes transient depression of the energy metabolites, glucose and lactate, likely due to injury-induced hypermetabolism. However, no massive tissue damage or severe ischemic conditions around the probe occur

Tumor Necrosis Factor α Inhibition for Alzheimer\u27s Disease

Tumor necrosis factor α (TNF-α) plays a central role in the pathophysiology of Alzheimer’s disease (AD). Food and Drug Administration–approved biologic TNF-α inhibitors are thus a potential treatment for AD, but they do not cross the blood-brain barrier. In this short review, we discuss the involvement of TNF-α in AD, challenges associated with the development of existing biologic TNF-α inhibitors for AD, and potential therapeutic strategies for targeting TNF-α for AD therapy

Unifying the Mathematical Modeling of \u3cem\u3ein vivo\u3c/em\u3e and \u3cem\u3ein vitro\u3c/em\u3e Microdialysis

A unifying approach is presented for developing mathematical models of microdialysis that are applicable to both in vitro and in vivo situations. Previous models for cylindrical probes have been limited by accommodating analyte diffusion through the surrounding medium in the radial direction only, i.e., perpendicular to the probe axis, or by incomplete incorporation of diffusion in the axial direction. Both radial and axial diffusion are included in the present work by employing two-dimensional finite element analysis. As in previous models, the nondimensional clearance modulus (Θ) represents the degree to which analyte clearance from the external medium influences diffusion through the medium for systems exhibiting analyte concentration linearity. Incorporating axial diffusion introduces a second dimensionless group, which is the length-to-radius aspect ratio of the membrane. These two parameter groups uniquely determine the external medium permeability, which is time dependent under transient conditions. At steady-state, the dependence of this permeability on the two groups can be approximated by an algebraic formula for much of the parameter ranges. Explicit steady-state expressions derived for the membrane and fluid permeabilities provide good approximations under transient conditions (quasi-steady-state assumption). The predictive ability of the unifying approach is illustrated for microdialysis of sucrose in vivo (brain) and inert media in vitro, under both well-stirred and quiescent conditions

Brain Protection from Stroke with Intravenous TNFα Decoy Receptor-Trojan Horse Fusion Protein

Tumor necrosis factor (TNF)-α is produced in brain in response to acute cerebral ischemia, and promotes neuronal apoptosis. Biologic TNF inhibitors (TNFIs), such as the etanercept, cannot be developed as new stroke treatments because these large molecule drugs do not cross the blood–brain barrier (BBB). A BBB-penetrating biologic TNFI was engineered by fusion of the type II human TNF receptor (TNFR) to each heavy chain of a genetically engineered chimeric monoclonal antibody (MAb) against the mouse transferrin receptor (TfR), designated as cTfRMAb-TNFR fusion protein. The cTfRMAb domain of the fusion protein acts as a molecular Trojan horse to deliver the fused TNFR across the BBB. Etanercept or the cTfRMAb-TNFR fusion protein (1 mg/kg) was administered intravenously in adult mice subjected to 1-hour reversible middle cerebral artery occlusion up to 90 minutes after the occlusion. Neuroprotection was assessed at 24 hours or 7 days after occlusion. The cTfRMAb-TNFR fusion protein treatment caused a significant 45%, 48%, 42%, and 54% reduction in hemispheric, cortical, and subcortical stroke volumes, and neural deficit, respectively. Intravenous etanercept had no therapeutic effect. Biologic TNFIs can be reengineered for BBB penetration, and the IgG-TNFR fusion protein is therapeutic after delayed intravenous administration in experimental stroke

Ginkgo Extract EGb761 Confers Neuroprotection by Reduction of Glutamate Release in Ischemic Brain

Purpose - Ginkgo extract EGb761 has shown anti-edema and anti-ischemic effects in various experimental models. In the present study, we demonstrate neuroprotective effects of EGb761 in experimental stroke while monitoring brain metabolism by microdialysis. Methods - We have used oxygen-glucose deprivation in brain slices in vitro and middle cerebral artery occlusion (MCAO) in vivo to induce ischemia in mouse brain. We used microdialysis in mouse striatum to monitor extracellular concentrations of glucose and glutamate. Results - In vitro, EGb761 reduced ischemia-induced cell swelling in hippocampal slices by 60%. In vivo, administration of EGb761 (300 mg/kg) reduced cell degeneration and edema formation after MCAO by 35-50%. Immediately following MCAO, striatal glucose levels dropped to 25% of controls, and this reduction was not significantly affected by EGb761. Striatal glutamate levels, in contrast, increased 15-fold after MCAO; after pretreatment with EGb761, glutamate levels only increased by 4-5fold. Conclusions - We show that pretreatment with EGb761 strongly reduces cellular edema formation and neurodegeneration under conditions of ischemia. The mechanism of action seems to be related to a reduction of excitotoxicity, because ischemia-induced release of glutamate was strongly suppressed. Ginkgo extracts such as EGb761 may be valuable to prevent ischemia-induced damage in stroke-prone patients. This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page

The Promises and Challenges of Erythropoietin for Treatment of Alzheimer\u27s Disease

Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder in the world, and intracellular neurofibrillary tangles and extracellular amyloid-beta protein deposits represent the major pathological hallmarks of the disease. Currently available treatments provide some symptomatic relief but fail to modify primary pathological processes that underlie the disease. Erythropoietin (EPO), a hematopoietic growth factor, acts primarily to stimulate erythroid cell production, and is clinically used to treat anemia. EPO has evolved as a therapeutic agent for neurodegeneration and has improved neurological outcomes and AD pathology in rodents. However, penetration of the blood–brain barrier (BBB) and negative hematopoietic effects are the two major challenges for the therapeutic development of EPO for chronic neurodegenerative diseases like AD. The transferrin receptors at the BBB, which are responsible for transporting transferrin-bound iron from the blood into the brain parenchyma, can be used to shuttle therapeutic molecules across the BBB. In this review, we discuss the role of EPO as a potential neurotherapeutic for AD, challenges associated with EPO development for AD, and targeting the BBB transferrin receptor for EPO brain delivery

Alcohol as a Modifiable Risk Factor for Alzheimer’s Disease—Evidence from Experimental Studies

Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by cognitive impairment and memory loss. Epidemiological evidence suggests that heavy alcohol consumption aggravates AD pathology, whereas low alcohol intake may be protective. However, these observations have been inconsistent, and because of methodological discrepancies, the findings remain controversial. Alcohol-feeding studies in AD mice support the notion that high alcohol intake promotes AD, while also hinting that low alcohol doses may be protective against AD. Chronic alcohol feeding to AD mice that delivers alcohol doses sufficient to cause liver injury largely promotes and accelerates AD pathology. The mechanisms by which alcohol can modulate cerebral AD pathology include Toll-like receptors, protein kinase-B (Akt)/mammalian target of rapamycin (mTOR) pathway, cyclic adenosine monophosphate (cAMP) response element-binding protein phosphorylation pathway, glycogen synthase kinase 3-β, cyclin-dependent kinase-5, insulin-like growth factor type-1 receptor, modulation of β-amyloid (Aβ) synthesis and clearance, microglial mediated, and brain endothelial alterations. Besides these brain-centric pathways, alcohol-mediated liver injury may significantly affect brain Aβ levels through alterations in the peripheral-to-central Aβ homeostasis. This article reviews published experimental studies (cell culture and AD rodent models) to summarize the scientific evidence and probable mechanisms (both cerebral and hepatic) by which alcohol promotes or protects against AD progression

Plasma Pharmacokinetics of High-Affinity Transferrin Receptor Antibody-Erythropoietin Fusion Protein is a Function of Effector Attenuation in Mice

Erythropoietin (EPO) is a potential therapeutic for Alzheimer’s disease (AD); however, limited blood–brain barrier (BBB) penetration reduces its applicability as a CNS therapeutic. Antibodies against the BBB transferrin receptor (TfRMAbs) act as molecular Trojan horses for brain drug delivery, and a fusion protein of EPO and TfRMAb, designated TfRMAb-EPO, is protective in a mouse model of AD. TfRMAbs have Fc effector function side effects, and removal of the Fc N-linked glycosylation site by substituting Asn with Gly reduces the Fc effector function. However, the effect of such Fc mutations on the pharmacokinetics (PK) of plasma clearance of TfRMAb-based fusion proteins, such as TfRMAb-EPO, is unknown. To examine this, the plasma PK of TfRMAb-EPO (wild-type), which expresses the mouse IgG1 constant heavy chain region and includes the Asn residue at position 292, was compared to the mutant TfRMAb-N292G-EPO, in which the Asn residue at position 292 is mutated to Gly. Plasma PK was compared following IV, IP, and SQ administration for doses between 0.3 and 3 mg/kg in adult male C57 mice. The results show a profound increase in clearance (6- to 8-fold) of the TfRMAb-N292G-EPO compared with the wild-type TfRMAb-EPO following IV administration. The clearance of both the wild-type and mutant TfRMAb-EPO fusion proteins followed nonlinear PK, and a 10-fold increase in dose resulted in a 7- to 11-fold decrease in plasma clearance. Following IP and SQ administration, the Cmax values of the TfRMAb-N292G-EPO mutant were profoundly (37- to 114-fold) reduced compared with the wild-type TfRMAb-EPO, owing to comparable increases in plasma clearance of the mutant fusion protein. The wild-type TfRMAb fusion protein was associated with reticulocyte suppression, and the N292G mutation mitigated this suppression of reticulocytes. Overall, the beneficial suppression of effector function via the N292G mutation may be offset by the deleterious effect this mutation has on the plasma levels of the TfRMAb-EPO fusion protein, especially following SQ administration, which is the preferred route of administration in humans for chronic neurodegenerative diseases including AD