Biology subjects

Vadukul, D. M.

Publications and source records attributed to Vadukul, D. M..

10 recordsLinked to original sources

bioRxiv2025

Engineering a Dimeric Single-Domain Antibody for Improved Detection and neutralization of Amyloid-β oligomers

Soluble A{beta} oligomers are regarded as major neurotoxic agents in Alzheimers disease. Several monoclonal antibodies have been developed to target A{beta} oligomers, but most of them show limited specificity binding also to monomers and fibrils. To generate an antibody with high specificity for the oligomers, we aimed to increase the efficiency and sensitivity of a humanized A{beta}-oligomer-specific single domain antibody, called DesAb-O. We engineered a dimeric DesAb-O variant, DiDesAb-O, which showed a significantly improved binding properties for A{beta} oligomers as compared to the monomeric sdAb. Furthermore, DiDesAb-O detected A{beta}42 oligomers in cells, prevented their binding to cell membranes and the A{beta}42 oligomers-induced neurotoxicity from both synthetic A{beta}42 samples and cerebrospinal fluid of Alzheimers patients at lower concentrations compared to DesAb-O. Overall, our findings indicate that the rational engineering of dimeric sdAb variants is an effective strategy to improve their binding properties offering new opportunities for the development of clinical molecules in the early diagnosis and cure of Alzheimers disease. Significance StatementProtein aggregates, characterized by high structural heterogeneity, are central to numerous neurodegenerative diseases, yet the development of aggregate-specific molecular probes and therapeutic agents remains a significant challenge. We present a novel approach to enhance the binding of single-domain antibodies by engineering multivalent, specifically dimeric, molecules to exploit the avidity effect. As a proof of concept, we developed a dimeric version of a previously studied single-domain antibody named DesAb-O, which exhibits enhanced binding sensitivity and specificity to toxic amyloid-{beta} oligomers and greater efficacy in inhibiting their toxicity. These findings provide a robust foundation for creating next-generation antibody fragments with enhanced binding to heterogeneous protein aggregates, opening new avenues for innovative diagnostic tools and therapeutic strategies in neurodegenerative disease research and treatment.

biochemistry↗

bioRxiv2025

Multiparametric Correlative Topographical and Volumetric Fluorescence Microscopy

Live-cell imaging of cell surface topography and intracellular architecture is essential for understanding cellular function. However, conventional approaches often involve trade-offs between resolution, invasiveness, and volumetric coverage. Here, we present an integrated Scanning Ion Conductance Microscope and single-objective Oblique Plane Microscope (SICM-OPM) system that enables simultaneous non-contact topographical imaging and volumetric fluorescence imaging within the same live cell. Beyond correlative live imaging, the platform supports nanomechanical mapping with tens-of-nanometres resolution, fluorescence-guided localised molecular delivery via the SICM, and benefits from reduced photobleaching due to light-sheet excitation. We demonstrate this platforms capabilities by visualising imipramine-induced T-tubule remodelling in live cardiomyocytes, revealing subsurface detubulation while surface morphology remains preserved. Additionally, we show precision delivery of fluorescent cargos--including dextrans and -synuclein--into diatom and mammalian cells, alongside localised stiffness mapping to evaluate mechanical responses. We believe this technique opens new avenues for correlative structural, functional, and biophysical studies in live cells, with broad relevance to cell biology, neurodegeneration, and mechanobiology.

biophysics↗

bioRxiv2024

The N-terminal Region of alpha-Synuclein Controls Amyloid Nucleation at the Condensate Interface

-Synuclein self-assembles into amyloid fibrils during neurodegeneration. The protein can also self-assemble via liquid-liquid phase separation to form biomolecular condensates. The link between these processes is evident, as -synuclein condensates can mature into amyloids. However, the mechanisms driving this maturation remain largely unknown, particularly when incorporating pathological post-translational modifications known to affect -synuclein self-assembly in the absence of LLPS, such as N-terminal truncation. Moreover, condensates are primarily studied as isolated entities; however, it is increasingly evident that they interact with various cellular components and surfaces. Here, we developed a microscopy-based quantitative real-time imaging protocol to investigate how N-terminal truncation influences -synuclein condensate formation, well surface wetting, and maturation. We found that increasing -synuclein truncation, which reduces N-terminal hydrophobicity, inhibits condensate sedimentation, enhances surface wettability, and accelerates maturation. Additionally, by increasing well surface hydrophobicity we decreased -synuclein condensate wettability, delaying their maturation. Thus, we propose that enhanced wettability, which increases the condensate surface-to-volume ratio, promotes -synuclein nucleation at the condensate-bulk solution interface, thereby accelerating maturation. Our results reveal distinct mechanistic roles for -synuclein N-terminal residues and indicate that condensate wetting on cellular surfaces, such as synaptic vesicles, may drive toxic aggregate formation during neurodegeneration.

biochemistry↗

bioRxiv2022

Telomere-induced senescence increases aberrant intraneuronal amyloid-β accumulation by impairing autophagy in a mouse model of Alzheimer's disease

Aging is a well-known risk factor for Alzheimers disease (AD) and other neurodegenerative pathologies, but the molecular and cellular changes occurring in the aging brain are poorly understood. AD pathology seems to correlate with the appearance of cells that become senescent due to the progressive accumulation of cellular insults causing DNA damage. In this study, we investigated the role of cellular senescence on AD pathology by crossing an amyloid-{beta} (A{beta}) mouse model of AD (5xFAD) with a mouse model of senescence that is genetically deficient for the RNA component of the telomerase (Terc-/-). Our results show that accelerated senescence reduces amyloid plaque formation and A{beta} levels at an age when full-blown amyloid pathology is observed in 5xFAD mice. However, early and aberrant intraneuronal A{beta} accumulation is observed in the subiculum and cortical layer V of senescent mice. Selective neurodegeneration linked to telomere attrition and early intraneuronal A{beta} accumulation was observed in these particular regions. Finally, our results suggest that the effect of senescence on amyloid pathology might be mediated through an alteration in autophagy function. Altogether, these findings demonstrate the instrumental role of senescence in intraneuronal A{beta} accumulation associated to AD pathophysiology, and further support future approaches targeting these processes for therapeutic intervention.

neuroscience↗

bioRxiv2022

Amyloid-β Oligomers Serve as Nucleation Sites for α-Synuclein Aggregation

An increasing number of cases where amyloids of different proteins are found in the same patient are being reported. This observation complicates diagnosis and clinical intervention. Amyloids of the amyloid-{beta} peptide or the protein -synuclein are traditionally considered hallmarks of Alzheimers and Parkinsons diseases, respectively. However, the co-occurrence of amyloids of these proteins has also been reported in patients diagnosed with either disease. Here, we show that soluble species containing amyloid-{beta} can induce the aggregation of -synuclein. Fibrils formed under these conditions are solely composed of -synuclein to which amyloid-{beta} can be found associated, but not as part of the core of the fibrils. Importantly, by global kinetic analysis, we found that the aggregation of -synuclein under these conditions occurs via heterogeneous primary nucleation, triggered by soluble aggregates containing amyloid-{beta}.

biochemistry↗

bioRxiv2022

A Facile Method to Produce N-Terminally Truncated α-Synuclein

-Synuclein is a key protein of the nervous system, which regulates the release and recycling of neurotransmitters in the synapses. It is also involved in several neurodegenerative conditions, including Parkinsons disease and Multiple System Atrophy, where it forms toxic aggregates. The N-terminus of -synuclein is of particular interest as it has been linked to both the physiological and pathological functions of the protein and undergoes post-translational modification. One such modification, N-terminal truncation, affects the aggregation propensity of the protein in vitro and is also found in aggregates from patients brains. To date, our understanding of the role of this modification has been limited by the many challenges of introducing biologically relevant N-terminal truncations with no overhanging starting methionine. Here, we present a method to produce N-terminally truncated variants of -synuclein that do not carry extra terminal residues. We show that our method can generate highly pure protein to facilitate the study of this modification and its role in physiology and disease. Thanks to this method, we have determined that the first six residues of -synuclein play an important role in the formation of the amyloids.

biochemistry↗

bioRxiv2020

Mechanism of cellular production and in vivo seeding effects of hexameric β-amyloid assemblies

BackgroundThe {beta}-amyloid peptide (A{beta}) plays a key role in Alzheimers disease. After its production by catabolism of the amyloid precursor protein (APP) through the action of presenilin 1 (PS1)- or presenilin 2 (PS2)-dependent {gamma}-secretases, monomeric A{beta} can assemble in oligomers. In a pathological context, this eventually leads to the formation of fibrils, which deposit in senile plaques. Many studies suggest that A{beta} toxicity is related to its soluble oligomeric intermediates. Among these, our interest focuses on hexameric A{beta}, which acts as a nucleus for A{beta} self-assembly. MethodsBiochemical analyses were used to identify hexameric A{beta} in a wide range of models; cell lines, cerebrospinal fluid from cognitively impaired patients and transgenic mice exhibiting human A{beta} pathology (5xFAD). We isolated this assembly and assessed both its effect on primary neuron viability in vitro, and its contribution to amyloid deposition in vivo following intracerebral injection. In both cases, we used wild-type mice (C57BL/6) to mimic an environment where hexameric A{beta} is present alone and 5xFAD mice to incubate hexameric A{beta} in a context where human A{beta} species are pre-existing. Using CRISPR-Cas9, we produced stable knockdown human cell lines for either PS1 or PS2 to elucidate their contribution to the formation of hexameric A{beta}. ResultsIn WT mice, we found that neither in vitro or in vivo exposure to hexameric A{beta} was sufficient to induce cytotoxic effects or amyloid deposition. In 5xFAD mice, we observed a significant increase in neuronal death in vitro following exposure to 5M hexameric A{beta}, as well as a 1.47-fold aggravation of amyloid deposition in vivo. At the cellular level, we found hexameric A{beta} in extracellular vesicles and observed a strong decrease in its excretion when PS2 was knocked down by 60%. ConclusionsOur results indicate the absence of cytotoxic effects of cell-derived hexameric A{beta} by itself, but its capacity to aggravate amyloid deposition by seeding other A{beta} species. We propose an important role for PS2 in the formation of this particular assembly in vesicular entities, in line with previous reports linking the restricted location of PS2 in acidic compartments to the production of more aggregation-prone A{beta}.

neuroscience↗

bioRxiv2020

Cell-derived hexameric β-amyloid: a novel insight into composition, self-assembly and nucleating properties

A key hallmark of Alzheimers disease (AD) is the extracellular deposition of amyloid plaques composed primarily of the amyloidogenic amyloid-{beta} (A{beta}) peptide. The A{beta} peptide is a product of sequential cleavage of the Amyloid Precursor Protein (APP), the first step of which gives rise to a C-terminal Fragment (C99). Cleavage of C99 by {gamma}-secretase activity releases A{beta} of several lengths and the A{beta}42 isoform in particular has been identified as being neurotoxic. The misfolding of A{beta} leads to subsequent amyloid fibril formation by nucleated polymerisation. This requires an initial and critical nucleus for self-assembly. Here, we identify and characterise the composition and self-assembly properties of cell-derived hexameric A{beta}42 and show its nucleating properties which are dependent on the A{beta} monomer availability. Identification of nucleating assemblies that contribute to self-assembly in this way may serve as therapeutic targets to prevent the formation of toxic oligomers.

biochemistry↗

bioRxiv2020

Presenilin-deficient neurons and astrocytes display normal mitochondrial phenotypes

Presenilins 1 and 2 (PS1 and PS2) are predominantly known as the catalytic subunits of the {gamma}-secretase complex which generates the amyloid-{beta} (A{beta}) peptide, the major constituent of the senile plaques found in the brain of Alzheimers disease (AD) patients. Apart from their role in {gamma}-secretase activity, a growing number of cellular functions have been recently attributed to PSs. They are involved in synaptic transmission, endo-lysosomal function and calcium homeostasis. PSs were also found to be enriched in mitochondria-associated membranes (MAMs) where mitochondria and endoplasmic reticulum (ER) interact. PS2 was more specifically reported to regulate calcium shuttling between the ER and mitochondria by controlling the formation of functional MAMs through its interaction with the Mitofusin2 protein. We have previously demonstrated that the absence of PS2 (PS2KO) alters mitochondrial morphology and function. Indeed, a PS2KO cell line showed reduced mitochondrial respiration along with disrupted mitochondrial cristae and increased glycolysis. This phenotype is restored by the stable re-expression of human PS2. Still, all these results were obtained in immortalized Mouse Embryonic Fibroblasts (MEF) and one bottom-line question is to know whether these observations hold true for the Central Nervous System (CNS) cells, and in particular neurons and astrocytes. To that end, we carried out primary PS1KO, PS2KO and PS1/PS2KO (PSdKO) neuronal and astrocyte cultures. All the conditions were obtained in the same litter by crossing PS2 heterozygous and PS1 floxed (PS2+/-; PS1flox/flox) animals. Indeed, contrary to PS2KO mice, PS1KO are not viable and therefore require the use of the Cre-LoxP system to achieve gene deletion in vitro. Strikingly, we did not observe any mitochondrial phenotype in PS1KO, PS2KO or PSdKO primary cultures. Mitochondrial respiration and membrane potential were similar in all models, as were the glycolytic flux and NAD+/NADH ratio. We further investigated the discrepancies between these results and the ones previously reported in the MEF PS2KO cell line by analyzing PS2KO primary fibroblasts. No mitochondrial dysfunction was observed in this model, in line with observations in PS2KO primary neurons and astrocytes. These results indicate that the mitochondrial phenotype observed in immortalized PS2-deficient cell lines cannot be extrapolated to primary neurons, astrocytes and even to primary fibroblasts. The PS-dependent mitochondrial phenotype reported so far might be the consequence of a cell immortalization process and, therefore, should be critically reconsidered regarding its relevance to AD.

neuroscience↗

bioRxiv2020

Dimeric Transmembrane Orientations of APP/C99 Regulate γ-Secretase Processing Line Impacting Signaling and Oligomerization

Amyloid precursor protein (APP) cleavage by the {beta} secretase produces the C99 transmembrane (TM) protein, which contains three dimerization-inducing Gly-x-x-x-Gly motifs. We demonstrate that dimeric C99 TM orientations regulate the precise cleavage lines by {gamma}-secretase. Of all possible dimeric orientations imposed by a coiled coil to the C99 TM-cytosolic domain, the dimer (cc-del7) containing 33Gly-x-x-x-Gly37 in the interface promoted A{beta}42 processing line and AICD (APP Intracellular Domain)-dependent gene transcription, including BACE1 mRNA induction, enhancing amyloidogenic signaling. Another orientation exhibiting 25Gly-x-x-x-Gly29 in the interface (cc-del6) favored processing to A{beta}43/40, induced significantly less gene transcription, while promoting formation of SDS-resistant "A{beta}-like" oligomers, reminiscent of A{beta} peptide oligomers. These required both Val24 of a pro-{beta} motif and the 25Gly-x-x-x-Gly29 interface. Thus, crossing angles imposed by dimeric orientations give access to {gamma}-secretase at A{beta}48 or A{beta}49, linking the former to enhanced signaling and A{beta}42. We discuss avenues of blocking amyloidogenic processing.

neuroscience↗