Biology subjects

Boassa, D.

Publications and source records attributed to Boassa, D..

14 recordsLinked to original sources

bioRxiv2026

An age-preserving neuronal iSpheroid platform for modeling human aging and Alzheimer disease

Aging is the strongest risk factor for Alzheimer disease (AD), yet existing human neuronal models either erase age-associated features during reprogramming or fail to recapitulate three-dimensional organization of human neuronal tissue. Here, we establish directly induced neuronal spheroids (iSpheroids), a scalable three-dimensional platform generated by direct conversion of primary fibroblasts without pluripotency. iSpheroids generate long-lived, electrophysiologically active neurons from young, unimpaired aged, and sporadic AD donors while preserving donor-specific epigenetic age. Single-nucleus RNA sequencing revealed approximately 96% of cells acquired a neuronal identity and were distributed across multiple neuronal states, including a diversity of neurons. The platform supports incorporation of astrocytes and microglia and generation from non-human primate fibroblasts. AD iSpheroids spontaneously recapitulated neuronal degeneration, amyloid-beta; accumulation, progressive Tau pathology, and metabolic dysfunction. Integrated proteomic, metabolomic, and lipidomic profiling distinguished physiological aging from AD and revealed disease-associated metabolic remodeling. Oxaliplatin shifted AD iSpheroids toward oxidative metabolism, whereas a multi-compound screen identified trametinib as a modulator of inflammatory and age-associated programs.

neuroscience↗

bioRxiv2024

Glioma-Induced Alterations in Excitatory Neurons are Reversed by mTOR Inhibition

Gliomas are highly aggressive brain tumors characterized by poor prognosis and composed of diffusely infiltrating tumor cells that intermingle with non-neoplastic cells in the tumor microenvironment, including neurons. Neurons are increasingly appreciated as important reactive components of the glioma microenvironment, due to their role in causing hallmark glioma symptoms, such as cognitive deficits and seizures, as well as their potential ability to drive glioma progression. Separately, mTOR signaling has been shown to have pleiotropic effects in the brain tumor microenvironment, including regulation of neuronal hyperexcitability. However, the local cellular-level effects of mTOR inhibition on glioma-induced neuronal alterations are not well understood. Here we employed neuron-specific profiling of ribosome-bound mRNA via RiboTag, morphometric analysis of dendritic spines, and in vivo calcium imaging, along with pharmacological mTOR inhibition to investigate the impact of glioma burden and mTOR inhibition on these neuronal alterations. The RiboTag analysis of tumor-associated excitatory neurons showed a downregulation of transcripts encoding excitatory and inhibitory postsynaptic proteins and dendritic spine development, and an upregulation of transcripts encoding cytoskeletal proteins involved in dendritic spine turnover. Light and electron microscopy of tumor-associated excitatory neurons demonstrated marked decreases in dendritic spine density. In vivo two-photon calcium imaging in tumor-associated excitatory neurons revealed progressive alterations in neuronal activity, both at the population and single-neuron level, throughout tumor growth. This in vivo calcium imaging also revealed altered stimulus-evoked somatic calcium events, with changes in event rate, size, and temporal alignment to stimulus, which was most pronounced in neurons with high-tumor burden. A single acute dose of AZD8055, a combined mTORC1/2 inhibitor, reversed the glioma-induced alterations on the excitatory neurons, including the alterations in ribosome-bound transcripts, dendritic spine density, and stimulus evoked responses seen by calcium imaging. These results point to mTOR-driven pathological plasticity in neurons at the infiltrative margin of glioma - manifested by alterations in ribosome-bound mRNA, dendritic spine density, and stimulus-evoked neuronal activity. Collectively, our work identifies the pathological changes that tumor-associated excitatory neurons experience as both hyperlocal and reversible under the influence of mTOR inhibition, providing a foundation for developing therapies targeting neuronal signaling in glioma.

neuroscience↗

bioRxiv2023

Fe-TAMLs as a new class of small molecule peroxidase probes for correlated light and electron microscopy

We introduce Fe-TAML, a small molecule-based peroxidase as a versatile new member of the correlated fluorescence and electron microscopy toolkit. The utility of the probe is demonstrated by high resolution imaging of newly synthesized DNA (through biorthogonal labeling), genetically tagged proteins (using HaloTag), and untagged endogenous proteins (via immunostaining). EM visualization in these applications is facilitated by exploiting Fe-TAMLs catalytic activity for the deposition of localized osmiophilic precipitates based on polymerized 3,3-diaminobenzidine. Optimized conditions for synthesizing and implementing Fe-TAML based probes are also described. Overall, Fe-TAML is a new chemical biology tool that can be used to visualize diverse biomolecular species along nanometer and micron scales within cells.

cell biology↗

bioRxiv2023

CaBLAM! A high-contrast bioluminescent Ca2+ indicator derived from an engineered Oplophorus gracilirostris luciferase

Measuring ongoing cellular activity is essential to understanding the dynamic functions of biological organisms. The most popular current approach is imaging fluorescence-based genetically encoded Ca2+ indicators (GECIs). While fluorescent probes are useful in many contexts, bioluminescence-based GECIs--probes that generate light through oxidation of a small-molecule by a luciferase or photoprotein--have several distinct advantages. Because bioluminescent (BL) GECIs do not use the bright extrinsic excitation light required for fluorescence, BL GECIs do not photobleach, do not suffer from nonspecific autofluorescent background, and do not cause phototoxicity. Further, BL GECIs can be applied in contexts where directly shining photons on an imaging target is not possible. Despite these advantages, the use of BL GECIs has to date been limited by their small changes in bioluminescence intensity, high baseline signal at resting Ca2+ concentrations, and suboptimal Ca2+ affinities. Here, we describe a new BL GECI, CaBLAM (Ca2+ BioLuminescence Activity Monitor), that displays much higher dynamic range than previous BL GECIs and has a Ca2+ affinity suitable for capturing physiological changes in cytosolic Ca2+ concentration. With these improvements, CaBLAM captures single-cell and subcellular resolution activity at high frame rates in cultured neurons and in vivo, and allows multi-hour recordings in mice and behaving zebrafish. This new advance provides a robust alternative to traditional fluorescent GECIs that can enable or enhance imaging across many experimental conditions.

neuroscience↗

bioRxiv2023

Proximal Molecular Probe Transfer (PROMPT), a new approach for identifying sites of protein/nucleic acid interaction in cells by correlated light and electron microscopy

The binding and interaction of proteins with nucleic acids such as DNA and RNA constitutes a fundamental biochemical and biophysical process in all living organisms. Identifying and visualizing such temporal interactions in cells is key to understanding their function. To image sites of these events in cells across scales, we developed a method, named PROMPT for PROximal Molecular Probe Transfer, which is applicable to both light and correlative electron microscopy. This method relies on the transfer of a bound photosensitizer from a protein known to associate with specific nucleic acid sequence, allowing the marking of the binding site on DNA or RNA in fixed cells. The method produces a fluorescent mark at the site of their interaction, that can be made electron dense and reimaged at high resolution in the electron microscope. As proof of principle, we labeled in situ the interaction sites between the histone H2B and nuclear DNA. As an example of application for specific RNA localizations we labeled different nuclear and nucleolar fractions of the protein Fibrillarin to mark and locate where it associates with RNAs, also using electron tomography. While the current PROMPT method is designed for microscopy, with minimal variations, it can be potentially expanded to analytical techniques.

cell biology↗

bioRxiv2021

LRRK2 causes centrosomal deficits via phosphorylated Rab10 and RILPL1 at centriolar subdistal appendages

The Parkinsons disease-associated LRRK2 kinase phosphorylates multiple Rab GTPases including Rab8 and Rab10, which enhances their binding to RILPL1 and RILPL2. The nascent interaction between phospho-Rab10 and RILPL1 blocks ciliogenesis in vitro and in the intact brain, and interferes with the cohesion of duplicated centrosomes in dividing cells. We show here that various LRRK2 risk variants and all currently described regulators of the LRRK2 signaling pathway converge upon causing centrosomal cohesion deficits. The cohesion deficits do not require the presence of RILPL2 or of other LRRK2 kinase substrates including Rab12, Rab35 and Rab43. Rather, they depend on the RILPL1-mediated centrosomal accumulation of phosphorylated Rab10. RILPL1 localizes to the subdistal appendages of the mother centriole, followed by recruitment of the LRRK2-phosphorylated Rab protein to cause the centrosomal defects. These data reveal a common molecular pathway by which alterations in the LRRK2 kinase activity impact upon centrosome-related events.

neuroscience↗

bioRxiv2021

Unconventional tonicity-regulated nuclear trafficking of NFAT5 mediated by KPNB1, XPOT and RUVBL2

NFAT5 is the only known mammalian tonicity-responsive transcription factor functionally implicated in diverse physiological and pathological processes. NFAT5 activity is tightly regulated by extracellular tonicity but the underlying mechanisms remain elusive. We demonstrated that NFAT5 enters the nucleus via the nuclear pore complex. We also found that NFAT5 utilizes a non-canonical nuclear localization signal (NFAT5-NLS) for nuclear imports. siRNA screening revealed that karyopherin {beta}1 (KPNB1) drives nuclear import of NFAT5 via directly interacting with NFAT5-NLS. Proteomics analysis and siRNA screening further revealed that nuclear export of NFAT5 under hypotonicity is mediated by Exportin-T, and that it requires RuvB-Like AAA type ATPase 2 (RUVBL2) as an indispensable chaperone. Our findings have identified KPNB1 and RUVBL2 as key molecules responsible for the unconventional tonicity-regulated nucleocytoplasmic shuttling of NFAT5. These findings offer an opportunity for developing novel NFAT5 targeting strategies that are potentially useful for the treatment of diseases associated with NFAT5 dysregulation.

cell biology↗

bioRxiv2021

DOPAL initiates αSynuclein-mediated impaired proteostasis in neuronal projections leading to enhanced vulnerability in Parkinson's disease.

Dopamine dyshomeostasis has been acknowledged to be among the determinants of nigrostriatal neuron degeneration in Parkinsons disease (PD). Several studies in experimental models and postmortem PD patients underlined increasing levels of the aldehydic dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is highly reactive towards proteins. DOPAL has been shown to covalently modify the presynaptic protein Synuclein (Syn), whose misfolding and aggregation represent a major trait of PD pathology, triggering Syn oligomerization in dopaminergic neurons. Here, we demonstrated that DOPAL elicits Syn neuronal accumulation and hampers Syn clearance at synapses and the soma. By combining cellular and in vivo models, we provided evidence that DOPAL-induced Syn buildup lessens neuronal resilience, compromises synaptic integrity, and overwhelms protein quality control pathways, specifically at neuronal projections. The resulting progressive decline of neuronal homeostasis leads to dopaminergic neuron loss and motor impairment, corroborating the Syn-DOPAL interplay as an early event in PD neurodegeneration.

neuroscience↗

bioRxiv2020

Conformation and dynamics of the kinase domain drive subcellular location and activation of LRRK2

In a multi-tiered approach, we explored how Parkinsons Disease-related mutations hijack the finely tuned activation process of Leucine-Rich Repeat Kinase 2 (LRRK2) using a construct containing the ROC, Cor, Kinase and WD40 domains (LRRK2RCKW). We hypothesized that the N-terminal domains shield the catalytic domains in an inactive state. PD mutations, type-I LRRK2 inhibitors, or physiological Rab GTPases can unleash the catalytic domains while the active kinase conformation, but not kinase activity, is essential for docking onto microtubules. Mapping solvent accessible regions of LRRK2RCKW employing hydrogen-deuterium exchange mass spectrometry (HDX-MS) revealed how inhibitor binding is sensed by the entire protein. Molecular Dynamics simulations of the kinase domain elucidated differences in conformational dynamics between wt and mutants of the DYG{psi} motif. While all domains contribute to regulating kinase activity and spatial distribution, the kinase domain, driven by the DYG{psi} motif, coordinates domain crosstalk and serves as an intrinsic hub for LRRK2 regulation.

biochemistry↗

bioRxiv2020

CDeep3M-Preview: Online segmentation using the deep neural network model zoo

Sharing deep neural networks and testing the performance of trained networks typically involves a major initial commitment towards one algorithm, before knowing how the network will perform on a different dataset. Here we release a free online tool, CDeep3M-Preview, that allows end-users to rapidly test the performance of any of the pre-trained neural network models hosted on the CIL-CDeep3M modelzoo. This feature makes part of a set of complementary strategies we employ to facilitate sharing, increase reproducibility and enable quicker insights into biology. Namely we: (1) provide CDeep3M deep learning image segmentation software through cloud applications (Colab and AWS) and containerized installations (Docker and Singularity) (2) co-hosting trained deep neural networks with the relevant microscopy images and (3) providing a CDeep3M-Preview feature, enabling quick tests of trained networks on user provided test data or any of the publicly hosted large datasets. The CDeep3M-modelzoo and the cellimagelibrary.org are open for contributions of both, trained models as well as image datasets by the community and all services are free of charge.

neuroscience↗

bioRxiv2019

The in situ structure of Parkinson’s disease-linked LRRK2

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of familial Parkinsons disease. LRRK2 is a multi-domain protein containing a kinase and GTPase. Using in situ cryo-electron tomography and subtomogram averaging, we reveal a 14-[A] structure of LRRK2 bearing a pathogenic mutation that oligomerizes as a right-handed double-helix around microtubules, which are left-handed. Using integrative modeling, we determine the architecture of LRRK2, showing that the GTPase points towards the microtubule, while the kinase is exposed to the cytoplasm. We identify two oligomerization interfaces mediated by non-catalytic domains. Mutation of one of these abolishes LRRK2 microtubule-association. Our work demonstrates the power of cryo-electron tomography to obtain structures of previously unsolved proteins in their cellular environment and provides insights into LRRK2 function and pathogenicity.

biophysics↗

bioRxiv2018

Directed evolution of split APEX peroxidase

APEX is an engineered peroxidase that catalyzes the oxidation of a wide range of substrates, facilitating its use in a variety of applications, from subcellular staining for electron microscopy to proximity biotinylation for spatial proteomics and transcriptomics. To further advance the capabilities of APEX, we used directed evolution to engineer a split APEX tool (sAPEX). Twenty rounds of FACS-based selections from yeast-displayed fragment libraries, using three different yeast display configurations, produced a 200-amino acid N-terminal fragment (with 9 mutations relative to APEX2) called \"AP\" and a 50-amino acid C-terminal fragment called \"EX\". AP and EX fragments were each inactive on their own but reconstituted to give peroxidase activity when driven together by a molecular interaction. We demonstrate sAPEX reconstitution in the mammalian cytosol, on engineered RNA motifs within telomerase noncoding RNA, and at mitochondria-endoplasmic reticulum contact sites.

bioengineering↗

bioRxiv2018

CDeep3M - Plug-and-Play cloud based deep learning for image segmentation of light, electron and X-ray microscopy

As biological imaging datasets increase in size, deep neural networks are considered vital tools for efficient image segmentation. While a number of different network architectures have been developed for segmenting even the most challenging biological images, community access is still limited by the difficulty of setting up complex computational environments and processing pipelines, and the availability of compute resources. Here, we address these bottlenecks, providing a ready-to-use image segmentation solution for any lab, with a pre-configured, publicly available, cloud-based deep convolutional neural network on Amazon Web Services (AWS). We provide simple instructions for training and applying CDeep3M for segmentation of large and complex 2D and 3D microscopy datasets of diverse biomedical imaging modalities.

cell biology↗

bioRxiv2018

High-quality ultrastructural preservation using cryofixation for 3D electron microscopy of genetically labeled tissues

Electron microscopy (EM) offers unparalleled power to study cell substructures at the nanoscale. Cryofixation by high-pressure freezing offers optimal morphological preservation, as it captures cellular structures instantaneously in their near-native states. However, the applicability of cryofixation is limited by its incompatibilities with diaminobenzidine labeling using genetic EM tags and the high-contrast en bloc staining required for serial block-face scanning electron microscopy (SBEM). In addition, it is challenging to perform correlated light and electron microscopy (CLEM) with cryofixed samples. Consequently, these powerful methods cannot be applied to address questions requiring optimal morphological preservation and high temporal resolution. Here we developed an approach that overcomes these limitations; it enables genetically labeled, cryofixed samples to be characterized with SBEM and 3D CLEM. Our approach is broadly applicable, as demonstrated in cultured cells, Drosophila olfactory organ and mouse brain. This optimization exploits the potential of cryofixation, allowing quality ultrastructural preservation for diverse EM applications.

biophysics↗