Muthu Ramakrishnan

Development of a Smart Nano-vehicle to Target Cerebrovascular Amyloid Deposits and Brain Parenchymal Plaques Observed in Alzheimer’s Disease and Cerebral Amyloid Angiopathy

PurposeTo design a smart nano-vehicle (SNV) capable of permeating the blood-brain barrier (BBB) to target cerebrovascular amyloid formed in both Alzheimer’s disease (AD) and cerebrovascular amyloid angiopathy (CAA).MethodsSNV consists of a chitosan polymeric core prepared through ionic gelation with tripolyphosphate. A polyamine modified F(ab’) portion of IgG4.1, an anti-amyloid antibody, was coated as a biosensor on the SNV surface. A similar polymeric core coated with bovine serum albumin (BSA) served as a control nano-vehicle (CNV). The BBB uptake of 125I-SNVs and 125I-CNVs was evaluated in mice. The uptake and transcytosis of SNVs and CNVs across bovine brain microvascular endothelial cells (BBMECs) was evaluated using flow cytometry and confocal microscopy.ResultsPlasma clearance of 125I-SNVs was nine times higher than that of the 125I-CNVs. However, the uptake of 125I-SNVs in various brain regions was about 8 to 11 times higher than that of 125I-CNVs. The uptake of FITC-BSA loaded SNVs in BBMECs was twice the uptake of FITC-BSA loaded CNVs. Confocal micrographs demonstrated the uptake and transcytosis of Alexa Fluor 647 labeled SNVs, but not CNVs, across the BBMEC monolayer.ConclusionsSNVs are capable of carrying a payload of model protein across the BBB to target cerebral amyloid.

In vivo targeting of antibody fragments to the nervous system for Alzheimer’s disease immunotherapy and molecular imaging of amyloid plaques

Targeting therapeutic or diagnostic proteins to the nervous system is limited by the presence of the blood–brain barrier. We report that a F(ab′)2 fragment of a monoclonal antibody against fibrillar human Aβ42 that is polyamine (p)‐modified has increased permeability at the blood–brain barrier, comparable binding to the antigen, and comparable in vitro binding to amyloid plaques in Alzheimer’s disease (AD) transgenic mouse brain sections. Intravenous injection of the pF(ab′)24.1 in the AD transgenic mouse demonstrated efficient targeting to amyloid plaques throughout the brain, whereas the unmodified fragment did not. Removal of the Fc portion of this antibody derivative will minimize the inflammatory response and cerebral hemorrhaging associated with passive immunization and provide increased therapeutic potential for treating AD. Coupling contrast agents/radioisotopes might facilitate the molecular imaging of amyloid plaques with magnetic resonance imaging/positron emission tomography. The efficient delivery of immunoglobulin G fragments may also have important applications to other neurodegenerative disorders or for the generalized targeting of nervous system antigens.

Selective Contrast Enhancement of Individual Alzheimer’s Disease Amyloid Plaques Using a Polyamine and Gd-DOTA Conjugated Antibody Fragment Against Fibrillar Aβ42 for Magnetic Resonance Molecular Imaging

ABSTRACTPurposeThe lack of an in vivo diagnostic test for AD has prompted the targeting of amyloid plaques with diagnostic imaging probes. We describe the development of a contrast agent (CA) for magnetic resonance microimaging that utilizes the F(ab′)2 fragment of a monoclonal antibody raised against fibrillar human Aβ42MethodsThis fragment is polyamine modified to enhance its BBB permeability and its ability to bind to amyloid plaques. It is also conjugated with a chelator and gadolinium for subsequent imaging of individual amyloid plaquesResultsPharmacokinetic studies demonstrated this 125I-CA has higher BBB permeability and lower accumulation in the liver and kidney than F(ab′)2 in WT mice. The CA retains its ability to bind Aβ40/42 monomers/fibrils and also binds to amyloid plaques in sections of AD mouse brain. Intravenous injection of 125I-CA into the AD mouse demonstrates targeting of amyloid plaques throughout the cortex/hippocampus as detected by emulsion autoradiography. Incubation of AD mouse brain slices in vitro with this CA resulted in selective enhancement on T1-weighted spin-echo images, which co-register with individual plaques observed on spatially matched T2-weighted spin-echo imageConclusionsDevelopment of such a molecular probe is expected to open new avenues for the diagnosis of AD.

HH Domain of Alzheimer’s Disease Aβ Provides Structural Basis for Neuronal Binding in PC12 and Mouse Cortical/Hippocampal Neurons

A key question in understanding AD is whether extracellular Aβ deposition of parenchymal amyloid plaques or intraneuronal Aβ accumulation initiates the AD process. Amyloid precursor protein (APP) is endocytosed from the cell surface into endosomes where it is cleaved to produce soluble Aβ which is then released into the brain interstitial fluid. Intraneuronal Aβ accumulation is hypothesized to predominate from the neuronal uptake of this soluble extracellular Aβ rather than from ER/Golgi processing of APP. We demonstrate that substitution of the two adjacent histidine residues of Aβ40 results in a significant decrease in its binding with PC12 cells and mouse cortical/hippocampal neurons. These substitutions also result in a dramatic enhancement of both thioflavin-T positive fibril formation and binding to preformed Aβ fibrils while maintaining its plaque-binding ability in AD transgenic mice. Hence, alteration of the histidine domain of Aβ prevented neuronal binding and drove Aβ to enhanced fibril formation and subsequent amyloid plaque deposition - a potential mechanism for removing toxic species of Aβ. Substitution or even masking of these Aβ histidine residues might provide a new therapeutic direction for minimizing neuronal uptake and subsequent neuronal degeneration and maximizing targeting to amyloid plaques.

IC-P-037

patients (mild and moderate AD). We observed correlation between Naa/Cr and mI/Naa ratios in the posterior cingulate with the MMSE, being a positive correlation with Naa/Cr and negative correlation with mI/Naa. Conclusions: There was no advantage in performing MRS in the hippocampi instead of posterior cingulate, a technically challenging location, usually leading to a longer examination time, specially if taken into account the demential state of the patients, being desirable the shortest time of examination possible.

O1-03-02

formed. Results: Increased [11C](R)-PK11195 binding in AD was found in medial inferior frontal lobe, medial inferior temporal lobe, entorhinal cortex, posterior cingulate cortex and occipital lobe. In MCI the medial temporal lobe and, more specifically, the entorhinal cortex showed increased binding. However, BP did not allow for differentiation between converters or non-converters. SPM analysis showed increased binding in lateral temporal and occipital lobes in both MCI and AD. Discussion: The distribution of increased [11C](R)-PK11195 binding in MCI and AD agrees with areas known to be affected pathologically in AD and supports the theory that inflammation might be an early phenomenon in the etiology of AD. Although this ligand might lack sensitivity for diagnostic purposes in individual patients, it could be useful for addressing changes in [11C](R)-PK11195 binding (e.g., due to therapy) at a group level.