Biology subjects

Frank, R. A. W.

Publications and source records attributed to Frank, R. A. W..

5 recordsLinked to original sources

bioRxiv2025

Memory engram synapse 3D molecular architecture visualized by cryoCLEM-guided cryoET

Memory is incorporated into the brain as physicochemical changes to engram cells. These are neuronal populations that form complex neuroanatomical circuits, are modified by experiences to store information, and allow for memory recall. At the molecular level, learning modifies synaptic communication to rewire engram circuits, a mechanism known as synaptic plasticity. However, despite its functional role on memory formation, the 3D molecular architecture of synapses within engram circuits is unknown. Here, we demonstrate the use of engram labelling technology and cryogenic correlated light and electron microscopy (cryoCLEM)-guided cryogenic electron tomography (cryoET) to visualize the in-tissue 3D molecular architecture of engram synapses of a contextual fear memory within the CA1 region of the mouse hippocampus. Engram cells exhibited structural diversity of macromolecular constituents and organelles in both pre- and postsynaptic compartments and within the synaptic cleft, including in clusters of membrane proteins, synaptic vesicle occupancy, and F-actin copy number. This engram to tomogram approach, harnessing in vivo functional neuroscience and structural biology, provides a methodological framework for testing fundamental molecular plasticity mechanisms within engram circuits during memory encoding, storage and recall.

neuroscience↗

bioRxiv2023

In situ cryo-electron tomography of beta-amyloid and tau in post-mortem Alzheimer's disease brain

A defining pathological feature of most neurodegenerative diseases is the assembly of proteins into amyloid that form disease-specific structures. In Alzheimers disease (AD) this is characterised by the deposition of amyloid-{beta} (A{beta}) and tau with AD-specific conformations. The in situ structure of amyloid in the human brain is unknown. Here, using cryogenic fluorescence microscopy (cryoFM)-targeted cryo-sectioning, cryo-focused ion beam scanning electron microscopy (cryoFIB-SEM) liftout and cryo-electron tomography (cryoET), we determined the in-tissue structure of {beta}-amyloid and tau pathology in fresh post-mortem AD donor brain. {beta}-amyloid plaques contained a mixture of fibrils and protofilaments arranged in parallel arrays and lattice-like structures, some of which were branched. Extracellular vesicles, extracellular droplets and open lipid bilayer sheets defined non-amyloid constituents of amyloid plaques. In contrast, tau inclusions were characterised by clusters of unbranched filaments. Subtomogram averaging of filaments within each cluster revealed distinct structures including variably twisted paired helical filaments (PHF) and chronic traumatic encephalopathy (CTE)-like tau filaments that were situated [~]1 m apart within two microscopic regions of pathology. Filaments within a cluster were similar to each other, but different between clusters, showing that fibril heterogeneity is spatially organised and influenced by the subcellular tissue environment. The in situ structural approaches outlined here for targeting specific proteins within human donor tissues have applications to a broad range of neurodegenerative diseases.

pathology↗

bioRxiv2022

The in-tissue molecular architecture of β-amyloid pathology in the mammalian brain.

Amyloid plaques composed of extracellular focal deposition of A{beta} fibrils are a hallmark of Alzheimers disease (AD). Cryo-EM structures of A{beta} fibrils purified from human AD brain tissue post mortem have recently been determined. However, the molecular architecture of amyloid plaques in the context of fresh, unfixed mammalian brain tissue is unknown. Here, using cryogenic correlated light and electron tomography we report the native, in situ molecular architecture of A{beta} fibrils in the brain of a mouse model containing the Arctic familial AD mutation (AppNL-G-F) and an atomic model of Arctic A{beta} fibril purified from the brains of these animals. We show that in-tissue A{beta} fibrils are arranged in a lattice or in parallel bundles within a plaque, and are interdigitated by subcellular compartments, exosomes, extracellular droplets and extracellular multilamellar bodies. At the atomic level, the Arctic A{beta} fibril differs significantly from earlier structures of A{beta} amyloid extracted from AppNL-F mice models and human AD brain tissue, showing a striking effect of the Arctic mutation (E22G) on fibril structure. Cryo-electron tomography of ex vivo purified and in-tissue amyloid revealed an ensemble of additional fibrillar species, including thin protofilament-like rods and branched fibrils. Together, these results provide a structural model for the dense network architecture that characterises {beta}-amyloid plaque pathology.

neuroscience↗

bioRxiv2021

The molecular infrastructure of glutamatergic synapses in the mammalian forebrain

Glutamatergic synapses form the vast majority of connections within neuronal circuits, but how these subcellular structures are molecularly organized within the mammalian brain is poorly understood. Conventional electron microscopy using chemically fixed, metal- stained tissue has identified a proteinaceous, membrane-associated thickening called the postsynaptic density (PSD). Here, we combined mouse genetics and cryo-electron tomography to determine the 3D molecular architecture of fresh isolated and anatomically intact synapses in the adult forebrain. The native glutamatergic synapse did not consistently show a higher density of proteins at the postsynaptic membrane thought to be characteristic of the PSD. Instead, a synaptoplasm consisting of cytoskeletal elements, macromolecular complexes and membrane-bound organelles extended throughout the pre- and post- synaptic compartments. Snapshots of active processes gave insights into membrane remodeling processes. Clusters of up to 60 ionotropic glutamate receptors were positioned inside and outside the synaptic cleft. Together, these information-rich tomographic maps present a detailed molecular framework for the coordinated activity of synapses in the adult mammalian brain.

neuroscience↗

bioRxiv2018

Multidimensional dynamics of the proteome in the degenerative and ageing mammalian brain

The amount of any given protein in the brain is determined by the rates of its synthesis and destruction, which are regulated by different cellular mechanisms. Here, we combine metabolic labelling in live mice with global proteomic profiling to simultaneously quantify both the flux and amount of proteins in mouse models of neurodegeneration. In multiple models, protein turnover increases were associated with increasing pathology. This method distinguishes changes in protein expression mediated by synthesis from those mediated by degradation. In the AppNL-F knockin mouse model of Alzheimers disease increased turnover resulted from imbalances in both synthesis and degradation, converging on proteins associated with synaptic vesicle recycling (Dnm1, Cltc, Rims1) and mitochondria (Fis1, Ndufv1). In contrast to disease models, ageing in wildtype mice caused a widespread decrease in protein recycling associated with a decrease in autophagic flux. This simple multidimensional approach enables the comprehensive mapping of proteome dynamics and identifies affected proteins in mouse models of disease and other live animal test settings.

systems biology↗