Biology subjects

Mamand, D. R.

Publications and source records attributed to Mamand, D. R..

8 recordsLinked to original sources

bioRxiv2026

Brain mitovesicles undergo stage-specific remodelling during the progression of amyloid-β pathology

Mitochondrial dysfunction is an early feature of Alzheimer's disease that shows nonlinear changes as the disease progresses. However, how these changes are reflected in the mitochondrial material released by brain cells remains poorly understood. Mitovesicles are a recently described extracellular vesicle subpopulation of mitochondrial origin that provide a unique insight into mitochondrial alterations in the brain and could potentially show how nonlinear changes evolve across disease stages of amyloid-beta pathology. Here, we isolated forebrain-derived small extracellular vesicles and mitovesicles from wild-type and Amyloid precursor protein (App) knock-in (AppKI) mice, carrying familial AD mutations and progressively accumulating amyloid-beta plaques, at presymptomatic (2-month) and symptomatic (12-month) stages using high-resolution density gradient fractionation. Vesicle identity and composition were assessed by morphological, biophysical and quantitative proteomic analyses, and mitovesicle respiratory function was measured by extracellular flux analysis. We show that mitovesicles carry a distinct, predominantly mitochondrial inner membrane proteome that is remodelled by age and disease in a stage-specific manner. Compared with small extracellular vesicles, mitovesicle proteomic profiles captured disease progression more clearly, with symptomatic AppKI mice forming the most molecularly distinct group. At the presymptomatic stage, AppKI mitovesicles showed reduced mitochondrial protein representation, most notably of fatty acid {beta}-oxidation and lipid-metabolism proteins associated with astroglial and vascular signatures, preceding any detectable respiratory impairment. At the symptomatic stage this reversed: AppKI mitovesicles were enriched for oxidative phosphorylation, mitochondrial membrane and transporter proteins, yet showed reduced Complex IV-linked respiration, revealing a dissociation between mitochondrial cargo abundance and respiratory function. Selected cargo changes were not mirrored in whole-brain tissue, indicating selective remodelling of mitovesicle content. Comparison with mitovesicles from post-mortem human Alzheimer's disease frontotemporal cortex and hippocampus identified a conserved 62-protein mitovesicle core, dominated by oxidative phosphorylation subunits and mitochondrial carriers. In addition, 25 of these proteins were enriched in mitovesicles derived from Alzheimer's and AppKI forebrains, defining a conserved mitovesicular disease signature. Together, these findings establish forebrain-derived mitovesicles as a dynamically remodelled vesicle population that reflects progressive mitochondrial dysfunction in the Alzheimer's disease brain and provides a cross-species resource for future biomarker and mechanistic studies.

neuroscience↗

bioRxiv2026

Customizable bead-based platform for EV surface protein immunocapture and detection in plasma

Extracellular vesicles (EVs) are increasingly recognized as valuable biomarkers for liquid biopsy because they carry molecular signatures that reflect their cells of origin. However, EV isolation remains a challenge, and existing EV immunocapture platforms are constrained by nonspecific binding, limited flexibility, and insufficient sensitivity. Here, we present a customizable bead-based platform for the isolation and detection of specific EV populations directly from plasma. The platform combines bead-immobilized capture antibodies with fluorescent detection antibodies, enabling sensitive and multiplexed EV profiling. Optimization of bead conjugation chemistry improved antibody immobilization and reduced nonspecific background signals, enhancing assay specificity and performance. Using breast cancer-derived EVs expressing fluorescent protein, we demonstrate the detection of tumor-derived EVs in plasma at concentrations as low as 1E9 EVs/ml. Furthermore, the assay distinguished HER2- and TROP2-high from low EV populations, demonstrating its ability to detect clinically relevant differences in biomarker expression. The workflow is compatible with direct EV analysis in the unprocessed plasma samples, thereby reducing sample preparation requirements and assay complexity. These findings establish a sensitive, flexible, and scalable platform for EV biomarker analysis. The approach overcomes several limitations of current commercial bead-based technologies and may facilitate the development of customizable multiplexed EV-based assays for cancer detection, patient stratification, and disease monitoring.

cancer biology↗

bioRxiv2026

Quantitative Assessment of shRNA Loading and Delivery Efficiency of Engineered Extracellular Vesicles

RNA interference (RNAi) therapeutics enable selective silencing of disease-associated genes. Yet, their clinical application remains largely confined to the liver due to extrahepatic delivery constraints of current platforms such as GalNAc conjugates and lipid nanoparticles. Extracellular vesicles (EVs) offer an attractive alternative delivery strategy owing to their biocompatibility, ability to traverse biological barriers, and amenability to engineering. However, EV-mediated RNA delivery is limited by inefficient endogenous RNA loading and poor cytosolic release following uptake. Here, we establish a modular EV-based platform that addresses both challenges by integrating enhanced endogenous shRNA loading with fusogen-mediated cytosolic delivery. Using Argonaute 2 (AGO2)-assisted loading, we substantially increase shRNA copy numbers per vesicle (up to 3.7 copies/EV) and enable quantitative, molecule-resolved assessment of delivery potency. Engineered EVs achieve robust and reproducible shRNA-mediated gene silencing with picomolar IC50 values across multiple cell types and induce significant target knockdown in the mouse brain following intracerebral administration. Together, these findings demonstrate that coordinated engineering of shRNA loading and cytosolic release can overcome key limitations of EV-mediated small RNA delivery.

bioengineering↗

bioRxiv2026

Astrocyte-neuron mitochondrial transfer via mitoEVs supports neuronal energy metabolism and is impaired in early Alzheimer's disease

BackgroundMitochondrial dysfunction is an early and central feature of Alzheimers disease (AD). In particular, intercellular mitochondrial transfer has emerged as a mechanism of neuronal support in brain injury and neurodegeneration. However, pathways governing astrocyte-to-neuron transfer and its role in AD pathogenesis remain unknown. MethodsUsing the AppNL-G-F knock-in AD model, we combined high-resolution 4D live-cell imaging with quantitative fluorescence-based reporters to assess synaptic function and mitochondrial network dynamics in neurons and astrocytes. Direct and extracellular vesicle (EV)-restricted neuron-astrocyte co-culture systems were used to investigate bidirectional mitochondrial transfer. We performed the first in-depth structural, proteomic, and functional characterization of astrocyte-derived mitochondrial extracellular vesicles (mitoEVs) using cryo-electron microscopy, quantitative mass spectrometry, and bioenergetic analyses to define their cargo composition and metabolic effects. ResultsWe identified cell-type-specific mitochondrial remodeling in early AD, with compartmentalized synaptic energy deficits in neurons and hyperdynamic, less interconnected, yet metabolically preserved networks in astrocytes, preceding global bioenergetic decline. Bidirectional mitochondrial transfer between astrocytes and neurons, also at axonal terminals, was mediated by specialized mitoEVs but significantly reduced in the AppNL-G-Fmodel. Comprehensive proteomic and functional profiling revealed that WT astrocyte-derived mitoEVs are enriched in inner membrane and matrix proteins, supporting oxidative phosphorylation, lipid and amino acid metabolism, and redox homeostasis. In contrast, AppNL-G-F mitoEVs are selectively depleted of respiratory and fatty acid oxidation components and exhibit impaired respiration with reduced Complex IV activity. Functionally, WT mitoEVs promote mobilization of abnormal accumulation of lipid droplets in AppNL-G-Fneurons, restore fatty acid oxidation, and increase neuronal bioenergetics, including at the synapses. In contrast, disease-derived mitoEVs fail to engage these pathways. ConclusionsTogether, these findings identify mitoEV-mediated mitochondrial transfer as a glia-to-neuron metabolic pathway compromised in early AD and reveal a coordinated role for oxidative phosphorylation and fatty acid oxidation in supporting synaptic energy homeostasis.

neuroscience↗

bioRxiv2026

Bioengineered extracellular vesicles mitigate neuroinflammation by neutralizing pneumolysin and delaying disease onset in experimental pneumococcal meningitis

Bacterial meningitis is a life-threatening neurological disorder frequently caused by a Streptococcus pneumoniae (the pneumococcus) infection of the brain. Standard treatment consists of antibiotics to eliminate bacteria and dexamethasone to reduce inflammation. Despite this, mortality reaches 20% in treated individuals, and half of the survivors suffer long-term neurological sequelae. This is largely due to the poor capacity of antibiotics to reach the brain and the lack of antimicrobial treatment capable of neutralizing the pneumococcal toxin pneumolysin (Ply). To address these limitations, we isolated extracellular vesicles (EVs) derived from human HEK293T cells and evaluated their therapeutic potential in pneumococcal meningitis. Alongside wild-type EVs (WT.EVs), we bioengineered EVs to express RVG peptides (RVG.EV) for targeting neuronal acetylcholine receptors, signal incompetent IL-6 signal transducer (IL-6ST) decoy receptors (IL-6.EV) to block the pro-inflammatory signalling of IL-6, or EVs expressing both RVG peptides and IL-6ST (DB.EV). In vitro, all EVs reduced pneumococcal adhesion to neurons and mitigated cytotoxicity by binding and sequestering Ply. In a bacteremia-derived pneumococcal meningitis model, EV treatment significantly increased the survival of the mice without affecting bacterial load in the brain or the periphery. Among all groups, RVG.EV treatment was most effective in reducing pro-inflammatory cytokine release in the periphery and brain. These findings highlight the therapeutic potential of bioengineered EVs, particularly RVG peptides expressing EVs, as an adjunctive treatment for pneumococcal meningitis thanks to their (i) sequestration and neutralization of Ply, (ii) increased blood-brain barrier crossing, and (iii) dampening of inflammation.

neuroscience↗

bioRxiv2024

Surface adhesion and membrane fluctuations influence the elastic modulus of extracellular vesicles

Elastic properties of nanoscale extracellular vesicles (EVs) are believed to influence their cellular interactions, thus having a profound implication in intercellular communication. Yet, an accurate quantification of the elasticity of such small lipid vesicles is difficult even with AFM-based nanoindentation experiments as it crucially depends on the reliability of the theoretical interpretation of such measurements. Here we describe a complete method composed of theoretical framework, experimental procedure, and appropriate statistical approach for an accurate determination of bending modulus and effective elastic modulus of EVs. Further, we experimentally demonstrate that the quantification of EVs by the elastic modulus from AFM-based force spectroscopy measurement is marred by the interplay of their compositionally inhomogeneous fluid membrane with the adhesion forces from the substrate and thermal effects - two exquisite phenomena that could thus far only be theoretically predicted. The effects result in a large spreading of elastic modulus even for a single EV. Our unified model is then applied to genetically engineered classes of EVs to understand how the alterations in tetraspanin expression may influence their elastic modulus.

biophysics↗

bioRxiv2023

Multimodal engineering of extracellular vesicles for efficient intracellular protein delivery

Extracellular vesicles (EVs) are promising tools to transfer macromolecular therapeutic molecules to recipient cells, however, efficient functional intracellular protein delivery by EVs remains challenging. Here, we have developed novel and versatile systems that leverage selected molecular tools to engineer EVs for robust cytosolic protein delivery both in vitro and in vivo. These systems, termed VSV-G plus EV-sorting Domain-Intein-Cargo (VEDIC) and VSV-G-Foldon-Intein-Cargo (VFIC), exploit an engineered mini-intein (intein) protein with self-cleavage activity to link cargo to an EV-sorting domain and release it from the EV membrane inside the EV lumen. In addition, we utilize the fusogenic protein VSV-G to facilitate endosomal escape and cargo release from the endosomal system to the cytosol of recipient cells. Importantly, we demonstrate that the combination of the self-cleavage intein, fusogenic protein and EV-sorting domain are indispensable for efficient functional intracellular delivery of cargo proteins by engineered EVs. As such, nearly 100% recombination and close to 80% genome editing efficiency in reporter cells were observed by EV-transferred Cre recombinase and Cas9/sgRNA RNPs, respectively. Moreover, EV-mediated Cre delivery by VEDIC or VFIC engineered EVs resulted in significant in vivo recombination in Cre-LoxP R26-LSL-tdTomato reporter mice following both local and systemic injections. Finally, we applied these systems for improved treatment of LPS-induced systemic inflammation by delivering a super-repressor of NF-B activity. Altogether, this study describes a platform by which EVs can be utilized as a vehicle for the efficient intracellular delivery of macromolecular therapeutics for treatments of disease. Graphic summary: Development of VEDIC and VFIC systems for high-efficiency intracellular protein delivery in vitro and in vivo.Intein in tripartite fusion protein (EV-sorting Domain-Intein-Cargo) performs C-terminal cleavage during the process of EV-biogenesis, resulting in enriched free cargo proteins inside of vesicles. Together with fusogenic protein, VSV-G, these engineered EVs achieve high-efficiency intracellular delivery of cargo protein (Cre and super repressor of NF-B) or protein complex (Cas9/sgRNA RNPs) both in reporter cells and in mice models. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/535834v4_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@d0da37org.highwire.dtl.DTLVardef@1a6514corg.highwire.dtl.DTLVardef@235c9aorg.highwire.dtl.DTLVardef@194bbfa_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2021

Multiparametric Profiling of Single Nanoscale Extracellular Vesicles by Combined Atomic Force and Fluorescence Microscopy: Correlation and Heterogeneity in Their Molecular and Biophysical Features

Being a key player in intercellular communications, nanoscale extracellular vesicles (EVs) offer unique opportunities for both diagnostics and therapeutics. However, their cellular origin and functional identity remain elusive due to the high heterogeneity in their molecular and physical features. Here, for the first time, multiple EV parameters involving membrane protein composition, size and mechanical properties on single small EVs (sEVs) are simultaneously studied by combined fluorescence and atomic force microscopy. Furthermore, their correlation and heterogeneity in different cellular sources are investigated. The study, performed on sEVs derived from Human Embryonic Kidney 293, Cord Blood Mesenchymal Stromal and Human Acute Monocytic Leukemia cell lines, identifies both common and cell line-specific sEV subpopulations bearing distinct distributions of the common tetraspanins (CD9, CD63 and CD81) and biophysical properties. Although the tetraspanin abundances of individual sEVs are independent of their sizes, the expression levels of CD9 and CD63 are strongly correlated. A sEV population co-expressing all the three tetraspanins in relatively high abundance, however, having on average diameters <100 nm and relatively low Young moduli, is also found in all cell lines. Such a multiparametric approach is expected to provide new insights regarding EV biology and functions, potentially deciphering unsolved questions in this field.

bioengineering↗