Biology subjects

Shafiq, M.

Publications and source records attributed to Shafiq, M..

9 recordsLinked to original sources

bioRxiv2026

Astrocyte-driven small vessel disease is an early, amyloid-independent feature of PSEN1 E280A familial Alzheimer's disease

Cerebral Small vessel disease (cSVD) is a prevalent feature of Alzheimers disease (AD) pathology. Whether this pathology is a late consequence of amyloid and tau accumulation or an early, direct effect of PSEN1 dysfunction has remained unresolved. We found that it is more severe in familial AD (FAD) caused by E280A mutation in presenilin 1 (PSEN1). These cases present with a distinctive proteomic signature, associated with pathological features, more dysregulated in the occipital cortex (OC) compared to the frontal cortex (FC), and characterized by multiple dysregulated proteins involved in extracellular matrix (ECM) and RNA-associated processes. This proteomic fingerprint was associated with abnormal collagen build up, ECM disorganization, and signatures of aberrant angiogenesis. Six months old transgenic knock-in mice homozygous for Psen1 E280A mutation (PSEN1Ki) also showed a similar phenotype with microvascular tortuosity and proteomic changes. Critically, these mice develop neither A{beta} plaques nor tau tangles, indicating that the shared microvascular and RNA-associated changes are direct consequences of PSEN1 dysfunction rather than downstream effects of amyloid pathology. Remarkably, dysregulated RNA-associated protein networks overlapped between FAD and PSEN1Ki mice. Cerebral microvessels microstructure in PSEN1Ki mice at two months and six months showed abnormal astrocytic end-feet with lamellar deposits implicating blood-brain barrier damage. Finally, single nuclei transcriptomic analysis of AD patients and controls showed similar abnormal astrocytes in both sporadic and familial variants, but FAD astrocytes expressed dysregulated genes identified in the proteomic analyses, such as GLUL, APOE, and CLU. Our findings suggest that cSVD is an early pathological event in PSEN1 FAD and that is driven by abnormal RNA-associated processes and astrocytic dysfunction.

neuroscience↗

bioRxiv2025

Amyloid-β 42-carrying extracellular vesicles are associated with neurodegeneration and neuroinflammation in Alzheimer's disease

Alzheimers disease (AD) is the most common form of dementia, characterized by progressive amyloid-{beta} (A{beta}) accumulation and neurodegeneration. Extracellular vesicles (EVs) have been implicated in AD pathology, but their relationship to disease progression and the cerebrospinal fluid (CSF) proteome remains poorly understood. In this study, we analysed plasma-derived A{beta}42-containing EVs from participants of the Alzheimers Disease Neuroimaging Initiative (ADNI) and calculated the ratio between the percentage of A{beta}42-positive peripheral EVs and CSF A{beta}42 (rA{beta}42). This ratio was elevated in AD and positively correlated with amyloid pathology as measured by PET. Longitudinal analyses revealed that higher rA{beta}42 values predicted accelerated hippocampal atrophy and cognitive decline, independent of CSF A{beta}42 concentrations. Proteomic profiling of CSF using the SomaScan platform showed that rA{beta}42 was associated with an inflammatory signature characterized by myeloid activation and type II interferon-related signalling pathways. Together, these findings indicate that A{beta}42-carrying EVs reflect an active and inflammatory component of AD pathology and may represent a novel class of biomarkers for disease progression. Understanding the mechanisms that link A{beta}42-positive EVs to neuroinflammatory signalling could open new avenues for pathway-specific diagnostics and therapeutic strategies in AD.

neuroscience↗

bioRxiv2025

Multiplexed Brain and Visceral Two-Photon Imaging Using a Simulation-Guided Ultrafast Three-Color Fiber Laser

Multicolor two-photon microscopy is an essential tool in modern life sciences, enabling simultaneous, high-resolution imaging of multiple cellular structures and dynamic processes within complex biomedical systems. Realizing its full potential demands light sources that combine multiplexed spectral flexibility, high pulse quality, and practical implementation for efficient excitation of diverse cellular targets. Here, we present a novel ultrafast fiber laser platform that enables efficient three-color multiplexed two-photon imaging through numerically optimized nonlinear spectral shaping in a photonic crystal fiber (PCF). The system is driven by a nonlinear Yb-doped fiber amplifier with tailored dispersion and gain characteristics to generate clean sub-50 fs pulses at 1030 nm with over 40 nJ pulse energy. Subsequent, simulation-guided PCF-based spectral broadening enables controlled formation of three distinct high-energy bands centered at 940 nm, 1,080 nm, and 1,175 nm, overlapping with key fluorescent probes and biomolecular markers. The resulting pulses, isolated with high spectral and time-domain pulse quality, provide sub-115 fs duration and 2.5 - 6 nJ energy per channel. Multiplexed imaging is validated in labeled mouse brain, kidney, and liver tissue slices using spectrally independent multi-fluorophore targeting to visualize e.g., astrocytes, neuronal structures, and nuclei in triple-stained mouse hippocampus. The demonstrated fiber-optic laser platform provides a practical alternative to conventional single-color sources and more complex multi-laser systems, supporting robust and high-resolution three-color two-photon imaging for a range of biomedical applications.

biophysics↗

bioRxiv2025

Extracellular vesicles released from cortical neurons influence spontaneous activity of recipient neurons.

Extracellular vesicles (EVs) are membranous structures that cells release into the extracellular space. EVs carry various molecules such as proteins, lipids, and nucleic acids, and serve as specialized transporters to influence other cells. In the central nervous system, EVs have been linked to many important processes, including intercellular communication, but molecular details of their physiological functions are not fully understood. Our study aimed to investigate how EVs are released by neuronal cells, and how they affect the neuronal activity of other recipient neurons. We show that mature primary cortical neurons release EVs from both their soma and dendrites. EVs released from neurons closely resemble non-neuronal EVs regarding size and marker proteins and proteomic analyses showed that neuronally released EVs contain proteins typically acting in pre- and post-synaptic compartments. Interestingly, our analysis revealed that EVs alter spontaneous activity in target neurons by increasing the amplitude of postsynaptic potentials. In summary, our findings elaborates on the role of EVs in synaptic activity modulation in neurons mediated by glutamate receptors.

neuroscience↗

bioRxiv2025

Deep-Tissue Two-Photon Brain Imaging Enabled by a Tunable Fiber-Optic Dispersive Wave Generator

Here, we present a fiber-optic dispersive wave generator for highly-efficient, wavelength-tunable ultrashort pulse generation, enabling multicolor deep-tissue two-photon imaging of neuronal and vascular structures in labeled mouse brain. Guided by comprehensive numerical simulations, a compact Yb:fiber laser-driven system is constructed that utilizes precisely parameter- and phase-matching-controlled dispersive wave generation in a photonic crystal fiber. The system delivers sub-100 fs pulses with over [~]6.7 nJ of energy across a continuously tunable spectral range of 880-950 nm, achieving a record-high optical conversion efficiency of up to 65%. Optimizing the output for two-photon excitation of enhanced Green Fluorescent Protein and SYTOX Orange enables high-resolution structural imaging in mouse hippocampus and cerebellum at depths exceeding 450 {micro}m. This technique for wavelength-tunable, high-energy and ultrashort pulse generation with record optical efficiency represents a significant advancement in ultrafast fiber laser technology for versatile biomedical two-photon imaging applications.

bioengineering↗

bioRxiv2024

Efficient enzyme-free isolation of brain-derived extracellular vesicles

Extracellular vesicles (EVs) have gained significant attention as pathology mediators and potential diagnostic tools for neurodegenerative diseases. However, isolation of brain-derived EVs (BDEVs) from tissue remains challenging, often involving enzymatic digestion steps that may compromise the integrity of EV proteins and overall functionality. Here, we describe that collagenase digestion, commonly used for BDEV isolation, produces undesired protein cleavage of EV-associated proteins in brain tissue homogenates and cell-derived EVs. In order to avoid this effect, we studied the possibility of isolating BDEVs with a reduced amount of collagenase or without any protease. Characterization of the isolated BDEVs revealed their characteristic morphology and size distribution with both approaches. However, we revealed that even minor enzymatic digestion induces artificial proteolytic processing in key BDEV markers, such as Flotillin-1, CD81, and the cellular prion protein (PrPC), whereas avoiding enzymatic treatment completely preserves their integrity. We found no differences in mRNA and protein content between non-enzymatically and enzymatically isolated BDEVs, suggesting that we are purifying the same BDEV populations with both approaches. Intriguingly, the lack of Golgi marker GM130 signal, often referred to as contamination contamination-negative marker in EV preparations, seems to result from enzymatic digestion rather than from its actual absence in BDEV samples. Overall, we show that non-enzymatic isolation of EVs from brain tissue is possible and avoids artificial pruning of proteins while achieving a high BDEV yield and purity. This protocol will help to understand the functions of BDEV in a near-physiological setting, thus opening new research approaches.

neuroscience↗

bioRxiv2023

Cleavage site-directed antibodies reveal the prion protein in humans is shed by ADAM10 at Y226 and associates with misfolded protein deposits in neurodegenerative diseases

Proteolytic cell surface release ( shedding) of the prion protein (PrP), a broadly expressed GPI-anchored glycoprotein, by the metalloprotease ADAM10 impacts on neurodegenerative and other diseases in animal and in vitro models. Recent studies employing the latter also suggest shed PrP (sPrP) to be a ligand in intercellular communication and critically involved in PrP-associated physiological tasks. Although expectedly an evolutionary conserved event, and while soluble forms of PrP are present in human tissues and body fluids, neither proteolytic PrP shedding and its cleavage site nor involvement of ADAM10 or the biological relevance of this process have been demonstrated for the human body thus far. In this study, cleavage site prediction and generation (plus detailed characterization) of sPrP-specific antibodies enabled us to identify PrP cleaved at tyrosin 226 as the physiological and strictly ADAM10-dependent shed form in humans. Using cell lines, neural stem cells and brain organoids, we show that shedding of human PrP can be stimulated by PrP-binding ligands without targeting the protease, which may open novel therapeutic perspectives. Site-specific antibodies directed against human sPrP also detect the shed form in brains of cattle, sheep and deer, hence in all most relevant species naturally affected by fatal and transmissible prion diseases. In human and animal prion diseases, but also in patients with Alzheimers disease, sPrP relocalizes from a physiological diffuse tissue pattern to intimately associate with extracellular aggregates of misfolded proteins characteristic for the respective pathological condition. Findings and research tools presented here will accelerate novel insight into the roles of PrP shedding (as a process) and sPrP (as a released factor) in neurodegeneration and beyond.

neuroscience↗

bioRxiv2021

Biosorption potential and molecular characterization of metal resistant autochthonous microbes from tannery solid waste

This study encompasses isolation and screening of heavy metal-resistant fungal and bacterial strains from tannery solid waste (TSW). Twelve fungal strains and twenty-five bacterial strains were isolated from TSW. The growth of fungal strains was observed against different heavy metals ranging from 10 mg L-1 to 1050 mg L-1 and the growth of bacteria was observed in metal concentrations ranging from 10 mg L-1 to 1200 mg L-1. Five multi-metal resistant fungal isolates belonging to the genus Trichoderma and ten bacterial isolates belonging to the genus Bacillus showed good metal resistance and biosorption potential. They were identified through molecular techniques, fungi based on ITS region ribotyping, and bacteria based on 16S rRNA ribotyping. The fungal strains were characterized as T. hamatum (TSWF-06), T. harzianum (TSWF-11), T. lixii (TSWF-02) and T. pseudokoningii (TSWF-03, TSWF-10). The bacterial strains were characterized as Bacillus xiamenensis (TSW-02), B. velezensis (TSW-05), B. piscis (TSW-06), B. safensis (TSW-10), B. subtilis (TSW-14, TSW-15, TSW-17) B. licheniformis (TSW-19), B. cereus (TSW-20) and B. thuringiensis (TSW-22). The fungal strains namely, T. pseudokoningii (TSWF-03) and T. harzianum proved to be two multi-metal resistant strains with good biosorption efficiency. Unlike fungi, bacterial strains showed metal specific resistance. The strains Bacillus xiamenensis, B. subtilis (TSW-14) and B. subtilis (TSW-15) showed good biosorption efficiency against Cr, B. safensis against Cu, B. piscis and B. subtilis (TSW-17) against Pb and B. licheniformis and B. thuringiensis against Zn. The autochthonous fungal and bacterial strains can therefore be employed to clean metal contaminated environments.

microbiology↗

bioRxiv2021

Ligands binding to the cellular prion protein induce its protective proteolytic release with therapeutic potential in neurodegenerative proteinopathies

The cellular prion protein (PrPC) is a central player in neurodegenerative diseases caused by protein misfolding, such as prion diseases or Alzheimers disease (AD). Expression levels of this GPI-anchored glycoprotein, especially at the neuronal cell surface, critically correlate with various pathomechanistic aspects underlying these diseases, such as templated misfolding (in prion diseases) and neurotoxicity and, hence, with disease progression and severity. In stark contrast to cell-associated PrPC, soluble extracellular forms or fragments of PrP are linked with neuroprotective effects, which is likely due to their ability to interfere with neurotoxic disease-associated protein conformers in the interstitial fluid. Fittingly, the endogenous proteolytic release of PrPC by the metalloprotease ADAM10 ( shedding) was characterized as a protective mechanism. Here, using a recently generated cleavage-site specific antibody, we shed new light on earlier studies by demonstrating that shed PrP (sPrP) negatively correlates with conformational conversion (in prion disease) and is markedly redistributed in murine brain in the presence of prion deposits or AD-associated amyloid plaques indicating a blocking and sequestrating activity. Importantly, we reveal that administration of certain PrP-directed antibodies and other ligands results in increased PrP shedding in cells and organotypic brain slice cultures. We also provide mechanistic and structural insight into this shedding-stimulating effect. In addition, we identified a striking exception to this, as one particular neuroprotective antibody, due to its special binding characteristics, did not cause increased shedding but rather strong surface clustering followed by fast endocytosis and degradation of PrPC. Both mechanisms may contribute to the beneficial action described for some PrP-directed antibodies/ligands and pave the way for new therapeutic strategies against devastating and currently incurable neurodegenerative diseases.

neuroscience↗