Development of a theranostic system for central nervous system based on superparamagnetic nanoparticles

Nanotechnology has revealed its potential in the biomedicine area with fundamental contributions to the imaging contrast agents field or to drug delivery systems development. Several studies have shown that the effect of drugs could be improved when linked to nanoparticles (NPs). A similar approach may be relevant to the treatment of central nervous system (CNS) diseases. In the last decades the knowledge of the brain has increased and the advance in technology has led to several biomedical applications to treat human disorders. However, diseases such as Alzheimer’s, Parkinson or cancer continue to be devastating and poorly treatable. There are several therapeutic molecules likely to treat these disorders, but more than 98% of all small molecules drugs, and ~100% of all large proteins are not able to cross the blood-brain-barrier (BBB) and get to the CNS. The BBB is formed by endothelial cells that are aligned with the capillaries to prevent unwanted substances crossing from blood to nervous tissue. One approach to overtake this difficulty is to find a controlled delivery system able to supply the drug to the affected tissue. However, such systems have the potential to affect the correct BBB behavior. An alternative method is the use of peptides with translocation capacity. These cell penetrating peptides (CPPs) have capacity to translocate various types of cargo molecules to the cells interior, e.g. low molecular weight drugs, liposomes, antibodies and NPs. CPPs are degraded in non-toxic compounds, they have low potential to drug-drug interactions and low probability to cause immunologic reactions when compared with larger proteins. Alzheimer’s disease is characterized by an accumulation of insoluble protein as -amyloid (A), senile plaques (SP) and neurofibrillary tangles (NFT). The accumulation of these aggregates leads to a loss of synapses and neurodegeneration resulting in memory impairment and cognitive decline. In the present work we have used iron oxide nanoparticles a dual functionalization, with a small peptide with translocation capacity and with therapeutic antibody against -amyloid peptides. The system has the ability to function as a theranostic system by taking advantage of the magnetic properties of the nanoparticles. The nanoparticles were characterized by several techniques at different phases of the functionalization process. The iron oxide nanoparticles revealed a simple cubic crystalline structure (by powder x-ray diffraction), a size of 9 nm (by transmission electron microscopy) and a hydrodynamic size of 32.4 ± 2.1 nm for the coated nanoparticles with dimercaptosuccinic acid (DMSA) (by dynamic light scattering). To mimic the BBB, a transwell in vitro system was used to study the translocation of functionalized nanoparticles across this barrier. After 6 h, 23.7 ± 3.7 % of functionalized nanoparticles were able to cross the BBB. In addition, to assess if the developed nanoparticle is able decrease or stop -amyloid aggregation, the SensoLyte Thioflavin-T Beta-Amyloid (1-42) aggregation assay was used. It was found that functionalized iron oxide nanoparticles were able to inhibit -amyloid aggregation when compared to a known inhibitor (morin)

MR-based protein imaging of the human brain by means of dualCEST

Chemical exchange saturation transfer (CEST) is an emerging magnetic resonance imaging (MRI) technique enabling indirect detection of low-concentration cellular compounds in living tissue by their magnetization transfer with water. In particular, protein-attributed CEST signals have been shown to provide valuable diagnostic information for various diseases. While conventional CEST approaches suffer from confounding signals from metabolites and macromolecules, the novel dual-frequency irradiation CEST (dualCEST) technique enables increased protein specificity by selectively detecting the intramolecular spin-diffusion. However, application of this technique has so far been limited to spectroscopic investigations of model solutions at ultrahigh magnetic field strengths. In this thesis, dualCEST was translated to a clinical whole-body MR scanner, enabling protein imaging of the human brain. To this end, several methodological developments were implemented and optimized: (i) improved dual-frequency pulses for signal preparation, (ii) a fast and robust volumetric image readout, (iii) a weighted acquisition scheme, and (iv) an adaptive denoising technique. The resulting improvements are not limited to dualCEST but are relevant for the research field of CEST-MRI in general. Extensive measurements of biochemical model solutions and volunteers demonstrated the protein specificity and reproducibility of dualCEST-MRI. The clinical applicability was verified in pilot studies with tumor and Alzheimer’s patients

Imaging mouse models of neurodegeneration using multi-parametric MRI

Alzheimer’s disease (AD) is a devastating condition characterised by significant cognitive impairment and memory loss. Transgenic mouse models are increasingly being used to further our knowledge of the cause and progression of AD, and identify new targets for therapeutic intervention. These mice permit the study of specific pathological hallmarks of the disease, including intracellular deposits of hyperphosphorylated tau protein and extracellular amyloid plaques. In order to characterise these transgenic mice, robust biomarkers are required to evaluate neurodegenerative changes and facilitate preclinical evaluation of emerging therapeutics. In this work, a platform for in vivo structural imaging of the rTg4510 mouse model of tauopathy was developed and optimised. This was combined with a range of other clinically relevant magnetic resonance imaging (MRI) biomarkers including: arterial spin labelling, diffusion tensor imaging and chemical exchange saturation transfer. These techniques were applied in a single time-point study of aged rTg4510 mice, as well as a longitudinal study to serially assess neurodegeneration in the same cohort of animals. Doxycycline was administered to a subset of rTg4510 mice to suppress the tau transgene; this novel intervention strategy permitted the evaluation of the sensitivity of MRI biomarkers to the accumulation and suppression of tau. Follow-up ex vivo scans were acquired in order to assess the sensitivity of in vivo structural MRI to the current preclinical gold standard. High resolution structural MRI, when used in conjunction with advanced computational analysis, yielded high sensitivity to pathological changes occurring in the rTg4510 mouse. Atrophy was reduced in animals treated with doxycycline. All other MRI biomarkers were able to discriminate between doxycycline-treated and untreated rTg4510 mice as well as wildtype controls, and provided insight into complimentary pathological mechanisms occurring within the disease process. In addition, this imaging protocol was applied to the J20 mouse model of familial AD. This mouse exhibits widespread plaque formation, enabling the study of amyloid-specific pathological changes. Atrophy and deficits in cerebral blood flow were observed; however, the changes occurring in this model were markedly less than those observed in the rTg4510 mouse. This study was expanded to investigate the early-onset AD observed in individuals with Down’s syndrome (DS) by breeding the J20 mouse with the Tc1 mouse model of DS, permitting the relationship between genetics and neurodegeneration to be dissected. This thesis demonstrates the application of in vivo multi-parametric MRI to mouse models of neurodegeneration. All techniques were sensitive to pathological changes occurring in the models, and may serve as important biomarkers in clinical studies of AD. In addition, in vivo multi-parametric MRI permits longitudinal studies of the same animal cohort. This experimental design produces more powerful results, whilst contributing to worldwide efforts to reduce animal usage with respect to the 3Rs principles

In-Vivo Detection of Cathepsin-D in Alzheimer’s Disease

Alzheimer’s disease is a neurodegenerative disease with no early diagnosis. Neuronal dysfunction is an early indication of Alzheimer’s disease which we can measure by examining the lysosomal protein Cathepsin-D. Molecular imaging of Cathepsin-D using Magnetic Resonance Imaging (MRI) contrast agents could enhance visualization of disease and provide contrast within tissues and cells that are morphologically similar but physiologically distinct. The purpose of our work was to evaluate a novel MRI/fluorescent contrast agent designed to detect Cathepsin-D in early Alzheimer’s disease. In-vitro MRI and fluorescent sensitivity were characterized in addition to cellular uptake in cells over-expressing Cathepsin-D. Cortical and hippocampal uptake was evaluated following in-vivo injection of contrast agent in mice. The contrast agent exhibited differences in retention and uptake between control and transgenic Alzheimer’s mice. The results demonstrate the potential utility of the contrast agent for in-vivo identification of cathepsin-D upregulation and will continue to be further evaluated and refined in future studies

Novel blood biomarkers that correlate with cognitive performance and hippocampal volumetry: Potential for early diagnosis of Alzheimer's disease

© 2019 - IOS Press and the authors. All rights reserved. Background: Differential diagnosis of people presenting with mild cognitive impairment (MCI) that will progress to Alzheimer's disease (AD) remains clinically challenging. Current criteria used to define AD include a series of neuropsychological assessments together with relevant imaging analysis such as magnetic resonance imaging (MRI). The clinical sensitivity and specificity of these assessments would be improved by the concomitant use of novel serum biomarkers. The branched chain aminotransferase proteins (BCAT) are potential candidates as they are significantly elevated in AD brain, correlate with Braak Stage, and may have a role in AD pathology. Objective: In this hypothesis-driven project, we aimed to establish if serum BCAT and its metabolites are significantly altered in AD participants and assess their role as markers of disease pathology. Methods: Serum amino acids were measured using a triple quadrupole mass spectrometer for tandem mass spectroscopy together with BCAT levels using western blot analysis, coupled with neuropsychological assessments and MRI. Results: We present data supporting a substantive mutually correlated system between BCAT and glutamate, neuropsychological tests, and MRI for the diagnosis of AD. These three domains, individually, and in combination, show good utility in discriminating between groups. Our model indicates that BCAT and glutamate accurately distinguish between control and AD participants and in combination with the neuropsychological assessment, MoCA, improved the overall sensitivity to 1.00 and specificity to 0.978. Conclusion: These findings indicate that BCAT and glutamate have potential to improve the clinical utility and predictive power of existing methods of AD assessment and hold promise as early indicators of disease pathology

Elucidating mechanisms of protein aggregation in Alzheimer’s Disease using antibody-based strategies.

Alzheimer’s Disease (AD) is a devastating neurodegenerative disorder. There are two characteristic histopathological hallmarks in the brain: senile plaques and neurofibrillary tangles, composed of insoluble aggregates of the amyloids Amyloid-β (Aβ) and tau protein, respectively. These diagnostic markers, though distinctive, are not apparent effectors of AD pathology. Evidence has mounted suggesting smaller soluble aggregates (oligomers) of Aβ or tau are the true drivers of disease progression. This dissertation presents several amyloid biophysics projects. Aggregate biophysical parameters such as weight, shape, and conformation were measured using a range of methodologies, including Multiangle Light Scattering, Dynamic Light Scattering, UV-Circular Dichroism, UV-Fluorescence, Scanning Electron Microscopy and immunochemistry. In the first, the behavior of 2N3R tau protein was examined in a seeded oligomerization paradigm, where preformed oligomer may act as a template for monomer aggregation. In comparison to unseeded controls an elution time shift in the seeded solution was noted, suggesting conformational change due to seeding. Interestingly, when the seeded solutions were probed with T22, a tau oligomer-specific antibody raised against 2N4R oligomers, no binding was detected, suggesting the seeded 2N3R oligomer conformation is not the same as the seeded 2N4R oligomer. The second project examined protofibril formation in Aβ42/Aβ40 monomer mixtures with variable isoform ratios. Novel methods were developed to generate pre-fibrillar soluble species from monomeric Aβ solutions, thus minimizing the influence of preformed aggregates on the monomer aggregation pathway. Mixed-monomer solutions displayed ratio-dependent aggregation profile changes. Biochemical analysis demonstrated a higher inclusion rate of Aβ42 inclusion into protofibrils. Furthermore, relative protofibril yield and β-sheet secondary structure were both reduced with decreased Aβ42/Aβ40 monomer ratio, which suggests an additional inhibitory effect from Aβ40. The final project presented is characterization of a novel conformation-specific anti-serum, AbSL, developed by the Nichols Lab against prefibrillar Aβ species. Testing demonstrated specificity for Aβ42 protofibrils over Aβ42 monomer or fibril, as well as over Aβ40 protofibrils. The AbSL specificity epitope was probed, and indicated to incorporate part of the N-terminal 1-16 region

APOE-e4-related differences in left thalamic microstructure in cognitively healthy adults

APOE-ε4 is a main genetic risk factor for developing late onset Alzheimer’s disease (LOAD) and is thought to interact adversely with other risk factors on the brain. However, evidence regarding the impact of APOE-ε4 on grey matter structure in asymptomatic individuals remains mixed. Much attention has been devoted to characterising APOE-ε4-related changes in the hippocampus, but LOAD pathology is known to spread through the whole of the Papez circuit including the limbic thalamus. Here, we tested the impact of APOE-ε4 and two other risk factors, a family history of dementia and obesity, on grey matter macro- and microstructure across the whole brain in 165 asymptomatic individuals (38–71 years). Microstructural properties of apparent neurite density and dispersion, free water, myelin and cell metabolism were assessed with Neurite Orientation Density and Dispersion (NODDI) and quantitative magnetization transfer (qMT) imaging. APOE-ε4 carriers relative to non-carriers had a lower macromolecular proton fraction (MPF) in the left thalamus. No risk effects were present for cortical thickness, subcortical volume, or NODDI indices. Reduced thalamic MPF may reflect inflammation-related tissue swelling and/or myelin loss in APOE-ε4. Future prospective studies should investigate the sensitivity and specificity of qMT-based MPF as a non-invasive biomarker for LOAD risk

THE PREBIOTIC INULIN BENEFICIALLY MODULATES THE GUT-BRAIN AXIS BY ENHANCING METABOLISM IN AN APOE4 MOUSE MODEL

Alzheimer’s disease (AD) is the most common form of dementia and a growing disease burden that has seen pharmacological interventions primarily fail. Instead, it has been suggested that preventative measures such as a healthy diet may be the best way in preventing AD. Prebiotics are one such potential measure and are fermented into metabolites by the gut microbiota and acting as gut-brain axis components, beneficially impact the brain. However, the impact of prebiotics in AD prevention is unknown. Here we show that the prebiotic inulin increased multiple gut-brain axis components such as scyllo-inositol and short chain fatty acids in the gut, periphery, and in the case of scyllo-inositol, the brain. We found in E3FAD and E4FAD mice fed either a prebiotic or control diet for 4-months, that the consumption of the prebiotic inulin can beneficially alter the gut microbiota, modulate metabolic function, and dramatically increase scyllo-inositol in the brain. This suggests that the consumption of prebiotics can beneficially impact the brain by enhancing metabolism, helping to decrease AD risk factors

Regulation of Alpha Synuclein Following Chronic Methamphetamine Administration in Guinea Pigs: Correlation with Memory and Synaptic Plasticity

Methamphetamine (METH) is a highly addictive drug of abuse that has a severe impact on neuronal changes in the brain including modulations of plasticity, cognitive dysfunction, as well as memory impairment. These changes can be seen as modifications in the expression of biochemical markers associated with synaptic plasticity. One such marker associated with memory impairment is alpha synuclein (α-syn). Alteration of α-syn expression has been linked to memory impairment in patients with Alzheimer’s disease (AD) and Parkinson’s disease (PD). Here we assess the effect of chronic METH treatment in correlation to cognitive functions. Twenty-nine guinea pigs (male, 150-250 g) were subcutaneously inserted with ALZET osmotic mini-pumps to deliver either a) saline (24 μl/day), b) METH (10 mg/kg) per day for 7 days or c) Post METH washout (10mg/kg) . On Day–7, the Novel Object Recognition test (NOR) was used to assess memory recall. Electrophysiological techniques were used to assess synaptic plasticity, in the hippocampus CA1 subfield, as it relates to learning and memory (n=6). Western blots were used to evaluate the expression of α-syn in the hippocampus. Saline treated animals (n=7) and the Post METH washout (n=12) showed a preference for the novel object as compared to the METH treated animals (n=10) that had a preference for the familiar object.even after 7 days post treatment (n=12). Molecular assays showed a down regulation of endogenous alpha synuclein protein levels in the hippocampus between subgroups. Furthermore, long-term potentiation (a cellular correlate of memory) was maintained in all subgroups. The results from this study warrant the conclusion, that METH affects some of the same mechanisms underlining memory function and the drug-induced activation lead to the memory impairments observed in METH addicts and the role of alpha synuclein in the memory function still remains unclear