Publications
Welcome to the publications page of our lab! Here, we showcase our latest research and contributions to the scientific community. From exploring the complexities of aging and neurodegeneration to innovative drug discovery methods using Drosophila melanogaster, our work aims to push the boundaries of knowledge and drive impactful solutions. Browse through our studies to learn more about our findings and ongoing endeavors.
The Drosophila wing is a high-throughput and versatile screening tool for Tau-mediated disease mechanisms and drug discovery
Miguel Ramirez-Moreno, Amber S. Cooper, Tianshun Lian, Jie Liu, Seyedehleila Abtahi, Efthimios M.C. Skoulakis, Lovesha Sivanantharajah, Douglas Watt Allan and Amrit Mudher
Tau protein contributes to microtubule stability, which is disrupted in Alzheimer's disease and other Tauopathies. In these diseases, Tau molecules become hyperphosphorylated, misfolded and aggregated, propagating pathology across the brain. Studies dissecting disease mechanisms or screening disease-modifying therapies rely on animal models that unveil pathogenic events in vivo but also take several weeks or months to complete. Here we describe a versatile experimental paradigm that yields results in days and yet offers all the advantages of a genetically tractable in vivo system: the Drosophila wing. Mimicking neurotoxicity, human Tau expression causes cell death in the wing disc leading to quantifiable phenotypes in the adult wing. The neuroprotective peptide NAP ameliorates Tau toxicity in this system, validating it as a cost-effective drug screening tool. Phenocopying adult neurons, Tau toxicity in the wing disc is exacerbated by simulating hyper-phosphorylation and prevented by suppressing aggregation. Additionally, we show that the wing disc can dissect disease mechanisms that underpin clinically relevant Tau variants. Thus, the Drosophila wing offers an in vivo experimental paradigm for fast and efficient exploration of disease mechanism and screening.
Are we missing a trick by not exploiting fruit flies in inflammation-led drug discovery for neurodegeneration?
Ray Price, Miguel Ramirez-Moreno, Amber Cooper, Rachita Singh, Yee Ming Khaw, Annastasiah Mudiwa Mhaka, Lovesha Sivanantharajah and Amrit Mudher
Introduction
Alzheimer’s disease (AD) remains a formidable challenge in neurodegeneration research, with limited therapeutic options despite decades of study. While Drosophila melanogaster has been instrumental in in modeling AD related Tau and amyloid beta toxicity, inflammation, a key driver of AD pathology, remains unexplored in fly models. Given the evolutionary conservation of innate immune pathways between flies and mammals, drosophila presents a powerful yet underutilized tool for inflammation led drug discovery in AD.
Areas covered
This perspective highlights the relevance of Drosophila in studying neuroinflammatory processes, including microglial-like glial activation, systemic inflammation and gut-brain axis interactions. It further explores how fly models can be leveraged to screen anti-inflammatory compounds and dissect immune related genetic factors implicated in AD.
Expert opinion
By integrating immune modulation in Drosophila-based drug discovery pipeline we can accelerate the identification of novel therapeutic strategies. Fully exploiting the potential of Drosophila in inflammation led drug screening may usher in a new era of AD therapeutics, bridging gaps between fundamental research and translational medicine.
Examining the vulnerability of adult neuron subtypes to tau-mediated toxicity in Drosophila
Lovesha Sivanantharajah, Amrit Mudher & David Shepherd
Selective vulnerability of nerve cells is a feature of neurodegenerative disease. To date, animal models have been limited to examining pathogenic protein expression in broad or heterogeneous neuronal populations. Consequently, noted pathological hallmarks represent an average of disease phenotypes over multiple neuron types, rather than exact measures of individual responses. Here we targeted gene expression to small, precisely defined and homogenous neuronal populations in the Drosophila melanogaster central nervous system (CNS), allowing dissection of selective vulnerability of single types of neurons with single-neuron resolution. Using cellular degeneration as a readout for vulnerability, we found while all neurons were affected by tau some neuron types were more affected (vulnerable) than others (resilient). The tau-mediated pathogenic effects fell on a spectrum, demonstrating that neurons in the fly CNS are differentially vulnerable to tau pathology. Mechanistically, total tau levels did not correlate with vulnerability; rather, the best correlatives of degeneration were significant age-dependent increases in phospho-tau levels in the same neuron type, and tau mislocalisation into dendrites. Lastly, we found that tau phosphorylation in vulnerable neuron types correlated with downstream vesicular and mitochondrial trafficking defects. However, all vulnerable neuron types did not show the same pattern, suggesting multiple paths to degeneration. Beyond highlighting the heterogeneity of neuronal responses to tau in determining vulnerability, this work provides a new, high-resolution, tractable model for studying the age-dependent effects of tau, or any pathogenic protein, on postmitotic neurons with sub-cellular resolution.
Optical biomarkers for Alzheimer's disease prediction in a heterogenous clinical cohort
George Devitt, Sofia Michopoulou, Angus Prosser, Boyd CP Ghosh, Amritpal Mudher, Christopher Kipps and Sumeet Mahajan
Background
Reported accuracies of clinical Alzheimer's Disease (AD) diagnosis vary widely due to diagnostic inconsistency and patient heterogeneity. Raman spectroscopy is a label-free, laser-based method that can rapidly provide chemically rich information from biofluids. Despite this, AD classification using Raman spectroscopy has not yet been tested in a cohort representative of the clinical setting with appropriate statistical power.
Method
Cerebrospinal fluid (CSF) samples were cross-sectionally assessed from a mixed clinical cohort of patients (N = 141) using Raman Spectroscopy. Raman spectra from AD (n = 66) and non-AD (n = 75) patients were divided into training at testing sets at a ratio of 80:20 and machine-learning (ML) models were trained, optimized and evaluated for AD classification. ML models included a support-vector machine (SVM) and a convolutional neural network (CNN). Area under the receiver operating characteristic curve (AUROC) analysis was used to assess classifier performance. To explain AD classification, key spectral features were extracted using the Mann–Whitney U test per Raman shift and spectral regions were integrated to provide univariate features that were corelated to ATN biomarker status.
Result
Optimized ML models generalized well in the AD cohort with 93% classification accuracy observed for the SVM model (AUROC = 0.92, sensitivity = 0.92, specificity = 0.93). There was a strong correlation between the classifier score and patient ATN biomarker status. Patients with other neurodegenerative diseases were correctly classified as non-AD group including corticobasal degeneration (CBD), dementia with Lewy bodies (DLB), frontotemporal dementia (FTD), and Parkinson's disease. Important features for AD classification primarily included protein-derived aromatic amino acids particularly phenylalanine and tyrosine. 9 features were extracted and moderate correlations were observed for each feature with ATN biomarker status demonstrating the importance of the composite spectrum for classification.
Conclusion
Our results demonstrate that Raman spectroscopy can accurately identify AD using CSF from a mixed clinical cohort in which other causes of dementia are prevalent. Although larger cross-sectional studies are required to assess whether accuracy is retained at a population level, establishing the utility of RS in a clinical population is an important first step towards the translation of optical biomarkers for supporting AD diagnosis in the future.
Classification of Alzheimer’s disease in a mixed clinical cohort using biofluid Raman spectroscopy
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George Devitt, Sofia K. Michopoulou, Latha Kadalayil, Niall Hanrahan, Angus Prosser, Boyd Ghosh, Amrit Mudher, Christopher M. Kipps and Sumeet Mahajan
There is a critical unmet need for scalable, accessible and objective diagnostic tests for stratification in dementia. Biofluid Raman spectroscopy (RS) due to its simplicity, holistic and label-free nature, is a powerful approach that has the potential to offer differential diagnosis across dementia types including Alzheimer’s disease (AD). RS is a laser-based optical method that can rapidly provide chemically rich information (‘spectral biomarkers’) from biofluids but its utility for AD diagnosis has not been established in a ‘real-world’ context, specifically from a clinically heterogenous cohort of patients. We carried out RS measurements on cerebrospinal fluid (CSF) samples of patients from a mixed clinical cohort (N = 143). All patients reported cognitive complaints and were clinically diagnosed over 2 years with conditions including AD and other neurodegenerative diseases, as well as developmental and long-term chronic conditions. Machine-learning algorithms were trained, optimised and evaluated on Raman spectra to classify AD from non-AD. AD was classified with 93% accuracy for patients in the testing set. Time from sample to classification was < 1 h. Spectral biomarkers explaining AD classification were identified and primarily assigned to protein-derived aromatic amino acids, representing a difference in proteome signature between AD and non-AD groups. Signals from a subset of spectral biomarkers directly correlated with pathological CSF biomarker concentrations including amyloid-β 42, phosphorylated-tau 181, and total tau. This pre-clinical study is a first step towards realising the real-world application of RS for dementia diagnosis. Compared to current and emerging methods, RS does not require sophisticated instrumentation or specialised labs. It is reagentless and simple, offering unprecedented rapidity, scalability, accessibility for dementia diagnosis.
Enhancing the specificity of Raman spectroscopy by using multiple lasers
​George Devitt, Niall Hanrahan, Simon Lane, Sofia K. Michopoulou, Angus Prosser, Latha Kadalayil, Jordi Mayneris-Perxachs, Jonathan Swann, Boyd Ghosh, Amrit Mudher, Christopher Kipps, Sumeet Mahajan
Raman spectroscopy is a powerful tool for molecular fingerprinting yet has been limited in clinical utility due to decrease in specificity when analyzing complex biological samples. Here we present MX-Raman, a multiexcitation Raman method that enhances molecular discrimination by fusing spectral data acquired with multiple laser wavelengths into a single, enhanced optical fingerprint. In this work, MX-Raman is applied to cerebrospinal fluid (CSF) samples from a heterogeneous dementia cohort, including Alzheimer’s disease (AD), frontotemporal dementia (FTD) spectrum disorders, and non-neurodegenerative cognitive conditions, to achieve improved clustering and disease stratification. Furthermore, we extend the MX-Raman approach to blood plasma, demonstrating potential for a rapid, label-free, and minimally invasive diagnostic test for dementia detection and stratification. Overall, MX-Raman offers a scalable, optical technology platform that underscores how photonic advancements can address real-world clinical challenges.
A Novel Spectral Barcoding and Classification Approach for Complex Biological Samples Using Multiexcitation Raman Spectroscopy (MX-Raman)
​George Devitt, Niall Hanrahan, Miguel Ramírez Moreno, Amrit Mudher, and Sumeet Mahajan
We report the development and application of a novel spectral barcoding approach that exploits our multiexcitation (MX) Raman spectroscopy-based methodology for improved label-free detection and classification of complex biological samples. To develop our improved MX-Raman methodology, we utilized post-mortem brain tissue from several neurodegenerative diseases (NDDs) that have considerable clinical overlap. For improving our methodology we used three sources of spectral information arising from distinct physical phenomena to assess which was most important for NDD classification. Spectral measurements utilized combinations of data from multiple, distinct excitation laser wavelengths and polarization states to differentially probe molecular vibrations and autofluorescence signals. We demonstrate that the more informative MX-Raman (532 nm–785 nm) spectra are classified with 96.7% accuracy on average, compared to conventional single-excitation Raman spectroscopy that resulted in 78.5% accuracy (532 nm) or 85.6% accuracy (785 nm) using linear discriminant analysis (LDA) on 5 NDD classes. By combining information from distinct laser polarizations we observed a nonsignificant increase in classification accuracy without the need of a second laser (785 nm–785 nm polarized), whereas combining Raman spectra with autofluorescence signals did not increase classification accuracy. Finally, by filtering out spectral features that were redundant for classification or not descriptive of disease class, we engineered spectral barcodes consisting of a minimal subset of highly disease-specific MX-Raman features that improved the unsupervised and cross-validated clustering of MX-Raman spectra. The results demonstrate that increasing spectral information content using our optical MX-Raman methodology enables enhanced identification and distinction of complex biological samples but only when that information is independent and descriptive of class. The future translation of such technology to biofluids could support diagnosis and stratification of patients living with dementia and potentially other clinical conditions such as cancer and infectious disease.
Aggregation promoting sequences rather than phosphorylation are essential for Tau-mediated toxicity in Drosophila
​Amber Cooper, Bradley Richardson, Eva Ruiz Ortega, Yongrui Zhang, Ben Batchelor, Aarya Vaikakkara Chithran, Jie Liu, Tianshun Lian, Miguel Ramírez Moreno, Benjamin Boehme, Leila Abtahi, George Devitt, Lovesha Sivanantharajah, Efthimios M. C. Skoulakis, Douglas W. Allan, and Amritpal Mudher
Background: Disease-modifying therapies for tauopathies like Alzheimer’s disease have targeted Tau hyperphosphorylation and aggregation, as both pathological manifestations are implicated in Tau-mediated toxicity. However, the relative contributions of these pathologylinked changes to Tau neurotoxicity remain unclear.
Methods: Leveraging the genetic tractability of Drosophila, we generated multiple inducible human Tau transgenes with altered phosphorylation status and/or aggregation propensity. Their individual and combined impact was tested in vivo by quantifying Tau accumulation and neurodegenerative phenotypes in the aging fly nervous system.
Results: We report that phospho-mimicking Tau (hTau2N4RE14) induced profound neurodegeneration, supporting a neurotoxic role for phosphorylation. However, when we rendered hTau2N4RE14 aggregation incompetent, by deleting the 306VQIVYK311 motif in the microtubule-binding region, neurotoxicity was abolished. Moreover, a peptide inhibitor targeting this motif efficaciously reduced Tau toxicity in aging Drosophila.
Conclusion: Neurodegeneration mediated by Tau hyperphosphorylation is gated via at least one aggregation-mediating motif on the protein. This highlights the primacy of blocking Tau aggregation in therapy, perhaps without the need to clear phosphorylated species.
A novel peptide-based tau aggregation inhibitor as a potential therapeutic for Alzheimer’s disease and other tauopathies.
Anthony Aggidis, George Devitt, Yongrui Zhang, Shreyasi Chatterjee, David Townsend, Nigel J. Fullwood, Eva Ruiz Ortega, Airi Tarutani, Masato Hasegawa, Amber Cooper, Philip Williamson, Ayde Mendoza-Oliva, Marc I. Diamond, Amritpal Mudher, David Allsop
INTRODUCTION: As aggregation underpins Tau toxicity, aggregation inhibitor peptides may have disease-modifying potential. They are therefore currently beingdesigned and target either the 306 VQIVYK311 aggregation-promoting hotspot found inall Tau isoforms or the 275 VQIINK280 aggregation-promoting hotspot found in 4R isoforms. However, for any Tau aggregation inhibitor to potentially be clinically relevantfor other tauopathies, it should target both hotspots to suppress aggregation of Tauisoforms, be stable, cross the blood-brain barrier, and rescue aggregation-dependentTau phenotypes in vivo.
METHODS: We developed a retro-inverso, stable D-amino peptide, RI-AG03 [Ac-rrrrrrrrGpkyk(ac)iqvGr-NH2], based on the 306 VQIVYK311 hotspots which exhibitthese disease-relevant attributes.
RESULTS: Unlike other aggregation inhibitors, RI-AG03 effectively suppresses aggregation of multiple Tau species containing both hotspots in vitro and in vivo, is non-toxic, and suppresses aggregation-dependent neurodegenerative and behavioralphenotypes.
DISCUSSION: RI-AG03 therefore meets many clinically relevant requirements foran anti-aggregation Tau therapeutic and should be explored further for its disease-modifying potential for Tauopathies.
How do neurons age? A focused review on the aging of the microtubular cytoskeleton
Brad Richardson, Thomas Goedert, Shmma Quraishe, Katrin Deinhardt, Amritpal Mudher
Aging is the leading risk factor for Alzheimer's disease and other neurodegenerative diseases. We now understand that a breakdown in the neuronal cytoskeleton, mainly underpinned by protein modifications leading to the destabilization of microtubules, is central to the pathogenesis of Alzheimer's disease. This is accompanied by morphological defects across the somatodendritic compartment, axon, and synapse. However, knowledge of what occurs to the microtubule cytoskeleton and morphology of the neuron during physiological aging is comparatively poor. Several recent studies have suggested that there is an age-related increase in the phosphorylation of the key microtubule stabilizing protein tau, a modification, which is known to destabilize the cytoskeleton in Alzheimer's disease. This indicates that the cytoskeleton and potentially other neuronal structures reliant on the cytoskeleton become functionally compromised during normal physiological aging. The current literature shows age-related reductions in synaptic spine density and shifts in synaptic spine conformation which might explain age-related synaptic functional deficits. However, knowledge of what occurs to the microtubular and actin cytoskeleton, with increasing age is extremely limited. When considering the somatodendritic compartment, a regression in dendrites and loss of dendritic length and volume is reported whilst a reduction in soma volume/size is often seen. However, research into cytoskeletal change is limited to a handful of studies demonstrating reductions in and mislocalizations of microtubule-associated proteins with just one study directly exploring the integrity of the microtubules. In the axon, an increase in axonal diameter and age-related appearance of swellings is reported but like the dendrites, just one study investigates the microtubules directly with others reporting loss or mislocalization of microtubule-associated proteins. Though these are the general trends reported, there are clear disparities between model organisms and brain regions that are worthy of further investigation. Additionally, longitudinal studies of neuronal/cytoskeletal aging should also investigate whether these age-related changes contribute not just to vulnerability to disease but also to the decline in nervous system function and behavioral output that all organisms experience. This will highlight the utility, if any, of cytoskeletal fortification for the promotion of healthy neuronal aging and potential protection against age-related neurodegenerative disease. This review seeks to summarize what is currently known about the physiological aging of the neuron and microtubular cytoskeleton in the hope of uncovering mechanisms underpinning age-related risk to disease.
Human Tau aggregates are permissive to protein synthesis-dependent memory in Drosophila tauopathy models.
Ergina Vourkou, Eva D. Rouiz Ortega, Sumeet Mahajan, Amrit Mudher and Efthimios M.C. Skoulakis
Tauopathies including Alzheimer's disease, are characterized by progressive cognitive decline, neurodegeneration, and intraneuronal aggregates comprised largely of the axonal protein Tau. It has been unclear whether cognitive deficits are a consequence of aggregate accumulation thought to compromise neuronal health and eventually lead to neurodegeneration. We use the Drosophila tauopathy model and mixed-sex populations to reveal an adult onset pan-neuronal Tau accumulation-dependent decline in learning efficacy and a specific defect in protein synthesis-dependent memory (PSD-M), but not in its protein synthesis-independent variant. We demonstrate that these neuroplasticity defects are reversible on suppression of new transgenic human Tau expression and surprisingly correlate with an increase in Tau aggregates. Inhibition of aggregate formation via acute oral administration of methylene blue results in re-emergence of deficient memory in animals with suppressed human Tau (hTau)0N4R expression. Significantly, aggregate inhibition results in PSD-M deficits in hTau0N3R-expressing animals, which present elevated aggregates and normal memory if untreated with methylene blue. Moreover, methylene blue–dependent hTau0N4R aggregate suppression within adult mushroom body neurons also resulted in emergence of memory deficits. Therefore, deficient PSD-M on human Tau expression in the Drosophila CNS is not a consequence of toxicity and neuronal loss because it is reversible. Furthermore, PSD-M deficits do not result from aggregate accumulation, which appears permissive, if not protective of processes underlying this memory variant.
Tau-mediated axonal degeneration is prevented by activation of the WldS pathway
Katy Stubbs , Ben Batchelor , Lovesha Sivanantharajah , Megan Sealey , Miguel Ramirez-Moreno , Eva Ruiz , Brad Richardson , Victor H Perry , Tracey A Newman , Amritpal Mudher
Tauopathy is characterized by neuronal dysfunction and degeneration occurring as a result of changes to the microtubule-associated protein tau. The neuronal changes evident in tauopathy bear striking morphological resemblance to those reported in models of Wallerian degeneration. The mechanisms underpinning Wallerian degeneration are not fully understood although it can be delayed by the expression of the slow Wallerian degeneration (WldS) protein, which has also been demonstrated to delay axonal degeneration in some models of neurodegenerative disease. Given the morphological similarities between tauopathy and Wallerian degeneration, this study investigated whether tau-mediated phenotypes can be modulated by co-expression of WldS. In a Drosophila model of tauopathy in which expression of human 0N3R tau protein leads to progressive age-dependent phenotypes, WldS was expressed with and without activation of the downstream pathway. The olfactory receptor neuron circuit OR47b was used for these studies in adults, and the larval motor neuron system was employed in larvae. Tau phenotypes studied included neurodegeneration, axonal transport, synaptic deficits and locomotor behaviour. Impact on total tau was ascertained by assessing total, phosphorylated and misfolded tau levels by immunohistochemistry. Activation of the pathway downstream of WldS completely suppressed tau-mediated degeneration. This protective effect was evident even if the pathway downstream of WldS was activated several weeks after tau-mediated degeneration had become established. Though total tau levels were not altered, the protected neurons displayed significantly reduced MC1 immunoreactivity suggestive of clearance of misfolded tau, as well as a trend for a decline in tau species phosphorylated at the AT8 and PHF1 epitopes. In contrast, WldS expression without activation of the downstream protective pathway did not rescue tau-mediated degeneration in adults or improve tau-mediated neuronal dysfunction including deficits in axonal transport, synaptic alterations and locomotor behaviour in tau-expressing larvae. This collectively implies that the pathway mediating the protective effect of WldS intersects with the mechanism(s) of degeneration initiated by tau and can effectively halt tau-mediated degeneration at both early and late stages. Understanding the mechanisms underpinning this protection could identify much-needed disease-modifying targets for tauopathies.