Biology subjects

Grant, S. G. N.

Publications and source records attributed to Grant, S. G. N..

28 records · Page 2Linked to original sources

bioRxiv2022

Synaptic Expression of TAR-DNA-Binding Protein 43 in the Mouse Spinal Cord Determined Using Super-Resolution Microscopy

Cellular inclusions of hyperphosphorylated TAR-DNA-Binding Protein 43 (TDP-43) are a key hallmark of neurodegenerative diseases such as Amyotrophic Lateral Sclerosis (ALS). ALS is characterised by a loss of motor neurons in the brain and spinal cord that is preceded by early-stage changes in synaptic function that may be associated with TDP-43 pathology. However, there has been little characterisation of the synaptic expression of TDP-43 in spinal cord synapses. This study utilises a range of high-resolution and super-resolution microscopy techniques with immunolabelling, as well as an aptamer-based TDP-43 labelling strategy visualised with single-molecule localisation microscopy, to characterise and quantify the presence of phosphorylated TDP-43 (pTDP-43) in spinal cord synapses. We observe that TDP-43 is expressed in the majority of spinal cord synapses as nanoscale clusters as small as 60 nm in diameter. Synaptic TDP-43 expression is more frequently associated with presynaptic terminals than postsynaptic densities, and is more enriched in VGLUT1-associated synapses, compared to VGLUT2-associated synapses. Our nanoscopy techniques showed no difference in the subsynaptic expression of pTDP-43 in the ALS mouse model, SOD1G93a compared to healthy controls. This research characterizes the basic synaptic expression of TDP-43 with nanoscale precision and provides a framework with which to investigate the potential relationship between TDP-43 pathology and synaptic pathology in neurodegenerative diseases.

neuroscience↗

bioRxiv2022

Saturated reconstruction of living brain tissue

Complex wiring between neurons underlies the information-processing network enabling all brain functions, including cognition and memory. For understanding how the network is structured, processes information, and changes over time, comprehensive visualization of the architecture of living brain tissue with its cellular and molecular components would open up major opportunities. However, electron microscopy (EM) provides nanometre-scale resolution required for full in-silico reconstruction1-5, yet is limited to fixed specimens and static representations. Light microscopy allows live observation, with super-resolution approaches6-12 facilitating nanoscale visualization, but comprehensive 3D-reconstruction of living brain tissue has been hindered by tissue photo-burden, photobleaching, insufficient 3D-resolution, and inadequate signal-to-noise ratio (SNR). Here we demonstrate saturated reconstruction of living brain tissue. We developed an integrated imaging and analysis technology, adapting stimulated emission depletion (STED) microscopy6,13 in extracellularly labelled tissue14 for high SNR and near-isotropic resolution. Centrally, a two-stage deep-learning approach leveraged previously obtained information on sample structure to drastically reduce photo-burden and enable automated volumetric reconstruction down to single synapse level. Live reconstruction provides unbiased analysis of tissue architecture across time in relation to functional activity and targeted activation, and contextual understanding of molecular labelling. This adoptable technology will facilitate novel insights into the dynamic functional architecture of living brain tissue.

neuroscience↗

bioRxiv2021

Developmental resilience of synaptome architecture

Neurodevelopmental disorders of genetic origin delay the acquisition of normal abilities and cause disabling phenotypes. Spontaneous attenuation and even complete amelioration of symptoms in early childhood and adolescence occur in many disorders1-10, suggesting that brain circuits possess an intrinsic capacity to repair themselves. We examined the molecular composition of almost a trillion excitatory synapses on a brain-wide scale between birth and adulthood in mice carrying a mutation in the homeobox transcription factor Pax6, a neurodevelopmental disorder model11. Pax6 haploinsufficiency had no impact on total synapse number at any age. By contrast, the postnatal expansion of synapse diversity and acquisition of normal synaptome architecture were delayed in all brain regions, interfering with network and cognitive functions. Specific excitatory synapse types and subtypes were affected in two key developmental age-windows. These phenotypes were reversed within 2-3 weeks of onset, restoring synaptome architecture to its normal developmental trajectory. Synapse subtypes with high rates of protein turnover mediated these events. These results show synaptome remodelling confers resilience to neurodevelopmental disorders.

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bioRxiv2021

A brain atlas of synapse protein lifetime across the mouse lifespan.

Protein turnover is required for synapse maintenance and remodelling and may impact memory duration. We quantified the lifetime of postsynaptic protein PSD95 in individual excitatory synapses across the mouse brain and lifespan, generating the Protein Lifetime Synaptome Atlas. Excitatory synapses have a wide range of protein lifetimes that may extend from a few hours to several months, with distinct spatial distributions in dendrites, neuron types and brain regions. Short protein lifetime (SPL) synapses are enriched in developing animals and in regions controlling innate behaviors, whereas long protein lifetime (LPL) synapses accumulate during development, are enriched in the cortex and CA1 where memories are stored, and are preferentially preserved in old age. The protein lifetime synaptome architecture is disrupted in an autism model, with synapse protein lifetime increased throughout the brain. These findings add a further layer to synapse diversity in the brain and enrich prevailing concepts in behavior, development, ageing and brain repair.

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.

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bioRxiv2018

Synaptic combinatorial molecular mechanisms generate repertoires of innate and learned behavior

Although molecular mechanisms underpinning specific behaviors have been described, whether there are mechanisms that orchestrate a behavioral repertoire is unknown. To test if the postsynaptic proteome of excitatory synapses could impart such a mechanism we conducted the largest genetic study of mammalian synapses yet undertaken. A repertoire of sixteen innate and learned behaviors was assessed from 290,850 measures in 55 lines of mutant mice carrying targeted mutations in the principal classes of postsynaptic proteins. Each innate and learned behavior used a different combination of proteins. These combinations were comprised of proteins that amplified or attenuated the magnitude of each behavioral response. All behaviors required proteins found in PSD95 supercomplexes. We show the vertebrate increase in proteome complexity drove an expansion in behavioral repertoires and generated susceptibility to a wide range of diseases. Our results reveal a molecular mechanism that generates a versatile and complex behavioral repertoire that is central to human behavioral disorders.

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bioRxiv2018

A combinatorial postsynaptic molecular mechanism converts patterns of nerve impulses into the behavioral repertoire

How is the information encoded within patterns of nerve impulses converted into diverse behavioral responses? To address this question, we conducted the largest genetic study to date of the electrophysiological and behavioral properties of synapses. Postsynaptic responses to elementary patterns of activity in the hippocampal CA1 region were measured in 58 lines of mice carrying mutations in the principal classes of excitatory postsynaptic proteins. A combinatorial molecular mechanism was identified in which distinct subsets of proteins amplified or attenuated responses across timescales from milliseconds to an hour. The same mechanism controlled the diversity and magnitude of innate and learned behavioral responses. PSD95 supercomplex proteins were central components of this synaptic machinery. The capacity of vertebrate synapses to compute activity patterns increased with genome evolution and is impaired by disease-relevant mutations. We propose that this species-conserved molecular mechanism converts the temporally encoded information in nerve impulses into the repertoire of innate and learned behavior.

neuroscience↗

bioRxiv2018

Regional diversity in the postsynaptic proteome of the mouse brain

The proteome of the postsynaptic terminal of excitatory synapses comprises over one thousand proteins in vertebrate species and plays a central role in behavior and brain disease. The brain is organized into anatomically distinct regions and whether the synapse proteome differs across these regions is poorly understood. Postsynaptic proteomes were isolated from seven forebrain and hindbrain regions in mice and their composition determined using proteomic mass spectrometry. Seventy-four percent of proteins showed differential expression and each region displayed a unique compositional signature. These signatures correlated with the anatomical divisions of the brain and their embryological origins. Biochemical pathways controlling plasticity and disease, protein interaction networks and individual proteins involved with cognition all showed differential regional expression. Combining proteomic and connectomic data shows that interconnected regions have specific proteome signatures. Diversity in synapse proteome composition is key feature of mouse and human brain structure.

neuroscience↗

bioRxiv2018

Cell-type specific visualization and biochemical isolation of endogenous synaptic proteins in mice

In recent years, the remarkable molecular complexity of synapses has been revealed, with over 1000 proteins identified in the synapse proteome. Although it is known that different receptors and other synaptic proteins are present in different types of neurons and synapses, the extent of synapse diversity across the brain is largely unknown, mainly owing to technical limitations. Combining mouse genetics and proteomics we have previously reported highly efficient methods for purification of synaptic protein complexes under native conditions. In that approach, tandem affinity purification (TAP) tags were fused to the carboxyl terminus of PSD95 using gene targeting in mice. Here we report an approach that restricts tagging of endogenous PSD95 to cells expressing Cre recombinase. In addition, we developed a labelling strategy enabling visualization of endogenous PSD95 tagged by fluorescent proteins in Cre-expressing cells. We demonstrate the feasibility of proteomic characterisation of synapse proteomes and visualization of synapse proteins in specific cell types. We find that composition of PSD95 complexes purified from specific cell types differs from those extracted from tissues with diverse cellular composition. Therefore, these novel conditional PSD95 tagging lines will not only serve as powerful tools for precisely dissecting synapse diversity in specific subsets of regions/neuronal cells, but also provide an opportunity to better understand brain region-specific alterations associated with various psychiatric/neurological diseases. The newly developed conditional gene tagging methods can be applied to many different synaptic proteins and will thus facilitate research on the molecular complexity of synapses.

neuroscience↗

bioRxiv2016

TrkB signaling underlies the rapid antidepressant effects of isoflurane

A brief burst-suppressing isoflurane anesthesia has been shown to rapidly alleviate symptoms of depression in a subset of patients, but the neurobiological basis of these observations remains obscure. We show that a single isoflurane anesthesia produces antidepressant-like behavioural effects in the learned helplessness paradigm and regulates molecular events implicated in the mechanism of action of rapid-acting antidepressant ketamine: activation of brain-derived neurotrophic factor (BDNF) receptor TrkB, facilitation of mammalian target of rapamycin (mTOR) signaling pathway and inhibition of glycogen synthase kinase 3{beta} (GSK3{beta}). Moreover, isoflurane affected neuronal plasticity by facilitating long-term potentiation in the hippocampus. We also found that isoflurane increased activity of the parvalbumin interneurons, and facilitated GABAergic transmission in wild type mice but not in transgenic mice with reduced TrkB expression in parvalbumin interneurons. Our findings strengthen the role of TrkB signaling in the antidepressant responses and encourage further evaluation of isoflurane as a rapid-acting antidepressant devoid of the psychotomimetic effects and abuse potential of ketamine.

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