Biology subjects

Butola, T.

Publications and source records attributed to Butola, T..

3 recordsLinked to original sources

bioRxiv2024

ClearScope: a fully integrated light sheet theta microscope for sub-cellular resolution imaging without lateral size constraints

Three-dimensional (3D) ex vivo imaging of cleared intact brains of animal models and large human and non-human primate postmortem brain specimens is important for understanding the physiological neural network connectivity patterns and the pathological alterations underlying neuropsychiatric and neurological disorders. Light-sheet microscopy has emerged as a highly effective imaging modality for rapid high-resolution imaging of large cleared samples. However, the orthogonal arrangements of illumination and detection optics in light sheet microscopy limits the size of specimen that can be imaged. Recently developed light sheet theta microscopy (LSTM) technology addressed this by utilizing a unique arrangement of two illumination light paths oblique to the detection light path, while allowing perpendicular arrangement of the detection light path relative to the specimen surface. Here, we report development of a next-generation, fully integrated, and user-friendly LSTM system for rapid sub-cellular resolution imaging uniformly throughout a large specimen without constraining the lateral (XY) size. In addition, we provide a seamlessly integrated workflow for image acquisition, data storage, pre- and post-processing, enhancement, and quantitative analysis. We demonstrate the system performance by high-resolution 3D imaging of intact mouse brains and human brain samples, and complete data analysis including digital neuron tracing, vessel reconstruction and design-based stereological analysis in 3D. This technically enhanced and user-friendly LSTM implementation will enable rapid quantitative mapping of molecular and cellular features of interests in diverse types of very large samples.

neuroscience↗

bioRxiv2024

Modeling and Correction of Protein Conformational Disease in iPSC-derived Neurons through Personalized Base Editing

AbstractAltered protein conformation can cause incurable neurodegenerative disorders. Mutations in SERPINI1, the gene encoding neuroserpin, can alter protein conformation resulting in cytotoxic aggregation leading to neuronal death. Familial encephalopathy with neuroserpin inclusion bodies (FENIB) is a rare autosomal dominant progressive myoclonic epilepsy that progresses to dementia and premature death. We developed HEK293T and induced pluripotent stem cell (iPSC) models of FENIB, harboring a patient-specific pathogenic SERPINI1 variant or stably overexpressing mutant neuroserpin fused to GFP (MUT NS-GFP). Here, we utilized a personalized adenine base editor (ABE)-mediated approach to correct the pathogenic variant efficiently and precisely to restore neuronal dendritic morphology. ABE-treated MUT NS-GFP cells demonstrated reduced inclusion size and number. Using an inducible MUT NS-GFP neuron system, we identified early prevention of toxic protein expression allowed aggregate clearance, while late prevention halted further aggregation. To address several challenges for clinical applications of gene correction, we developed a neuron-specific engineered virus-like particle to optimize neuronal ABE delivery, resulting in higher correction efficiency. Our findings provide a targeted strategy which may treat FENIB and potentially other neurodegenerative diseases due to altered protein conformation such as Alzheimers and Huntingtons diseases.

molecular biology↗

bioRxiv2021

RIM-Binding Protein 2 organizes Ca2+ channel topography and regulates release probability and vesicle replenishment at a fast central synapse

RIM-Binding Protein 2 (RIM-BP2) is a multi-domain protein of the presynaptic active zone (AZ). By binding to Rab-interacting protein (RIM), bassoon and voltage-gated Ca{superscript 2} channels (CaV), it is considered to be a central organizer of the topography of CaV and release sites of synaptic vesicles (SVs) at the AZ. Here, we investigated the role of RIM-BP2 at the endbulb of Held synapse of auditory nerve fibers with bushy cells of the cochlear nucleus, a fast relay of the auditory pathway with high release probability. Disruption of RIM-BP2 lowered release probability altering short-term plasticity and reduced evoked excitatory postsynaptic currents (EPSCs). Analysis of SV pool dynamics during high frequency train stimulation indicated a reduction of SVs with high release probability but an overall normal size of the readily releasable SV pool (RRP). The Ca2+-dependent fast component of SV replenishment after RRP depletion was slowed. Ultrastructural analysis by super-resolution light and electron microscopy revealed an impaired topography of presynaptic CaV and a reduction of docked and membrane-proximal SVs at the AZ. We conclude that RIM-BP2 organizes the topography of CaV, and promotes SV tethering and docking. This way RIM-BP2 is critical for establishing a high initial release probability as required to reliably signal sound onset information that we found to be degraded in bushy cells of RIM-BP2-deficient mice in vivo. Significance StatementRIM-binding proteins (RIM-BPs) are key organizers of the active zone (AZ). Using a multidisciplinary approach to the calyceal endbulb of Held synapse that transmit auditory information at rates of up to hundreds of Hertz with sub-millisecond precision we demonstrate a requirement for RIM-BP2 for normal auditory signaling. Endbulb synapses lacking RIM-BP2 show a reduced release probability despite normal whole-terminal Ca2+ influx and abundance of the key priming protein Munc13-1, a reduced rate of SV replenishment, as well as an altered topography of CaV2.1 Ca2+ channels, and fewer docked and membrane proximal synaptic vesicles. This hampers transmission of sound onset information likely affecting downstream neural computations such as of sound localization.

neuroscience↗