Biology subjects

Hardingham, G. E.

Publications and source records attributed to Hardingham, G. E..

10 recordsLinked to original sources

bioRxiv2026

Mononuclear myeloid cells mount an adaptive protective response to intracerebral haemorrhage by activation of NRF2

Following intracerebral haemorrhage (ICH), secondary injury contributes to negative outcomes. Here, we describe inter-dependent injurious and adaptive-protective pathways that form a therapeutically modifiable ICH response. In perihaematomal tissue from post-mortem ICH patients, transcriptomic analysis pointed to concurrent perihaematomal activation of the cytoprotective stress-induced transcription factor NRF2 and activation of interferon responses, with mononuclear myeloid cells (microglia and monocyte-derived cells, MMCs) and astrocytes contributing to this signature. In vitro, microglia-astrocyte-neuron co-culture studies revealed that microglial NRF2 activation was induced by clot-conditioned medium and could influence the interferon pathway in both microglia themselves as well as astrocytes. In vivo, MMC-specific deletion of NRF2 inhibited NRF2 target-gene induction, exacerbated interferon pathway activation following ICH, and impaired functional recovery. Pharmacological NRF2 activation suppressed interferon pathway induction post-ICH, and direct interferon pathway antagonism rescued neurological recovery deficits caused by MMC-specific NRF2 deficiency. Thus, augmenting NRF2- or inhibiting interferon-signalling may improve outcomes after ICH.

neuroscience↗

bioRxiv2026

Astrocytes transform the transcriptomic and epigenetic landscape of developing cortical neurons to promote activity via intrinsic excitability control

Astrocytes are implicated in neuronal development and function, particularly of synapses, but their genome-wide impact is unclear. We show that cortical astrocytes trigger epigenetic remodelling in developing cortical neurons and widespread transcriptomic changes distinct from neurons intrinsic developmental trajectory. These changes can cause an emergence of neuronal firing activity independent of functional excitatory synaptogenesis. The mechanism is via through astrocyte-derived signals causing the transcriptional repression of inwardly-rectifying K+-channels (Kirs), causing a pro-excitatory shift in intrinsic properties. This places neurons in a zone of excitability whereupon cell-autonomous homeostatic control was activated, also associated with Kir regulation. Astrocyte-induced neuronal genes were enriched for schizophrenia, epilepsy, ADHD and Alzheimers disease risk, disorders associated with circuit imbalance. Thus, astrocyte-to-neuron signaling can alter firing via intrinsic properties in addition to the classical synaptogenic mechanism. The opposing actions of astrocytes and neuronal activity in regulating neuronal intrinsic properties may prevent hyper- or hypo-activity in neuronal circuits

neuroscience↗

bioRxiv2025

Absence of short-term axon initial segment plasticity in human, mouse, and rat cortical circuits

Maintaining neuronal output with respect to input in the physiological range relies on the ability of neurons to update their responsiveness to inputs dependent on changing activity levels. Termed homeostatic plasticity, the mechanisms that neurons employ to control their responsiveness are varied, and proposed to include structural changes to a key neuronal structure - the axon initial segment (AIS). As the site of action potential initiation, the AIS has been postulated to rapidly change its length in response to increased or decreased cellular and circuit activity. To date, AIS structural plasticity has only been tested in tissue cultures and rodent models. In our current study, we assess the ability of neurons to alter their AIS length over a variety of timescales in ex vivo rodent and human brain slices, human neurons derived from induced pluripotent stem cells, and in mice dark-reared during early life; using a combination of electrophysiology and immunohistochemistry. We find no evidence for changes to AIS length following depolarisation for up to 3 hours, despite positive controls confirming modulated activity. However, we do find that neuronal physiological properties are altered by changes in activity - but these are largely independent of action potential initiation associated with the AIS. In summary, we find no evidence supporting a role for AIS structural plasticity in mouse, rat, or human cortical neurons.

neuroscience↗

bioRxiv2025

Altered GluN2A levels reduce the emergence of seizure-like events in a rat model of GRIN2A haploinsufficiency

GluN2A-containing NMDA receptors, encoded by the GRIN2A gene, are critical components of excitatory synaptic transmission and are essential for proper brain development and function. Dysregulation of NMDA receptor activity is strongly implicated in the pathophysiology of epilepsy. While excessive GluN2A-mediated signalling can lead to hyperexcitability and seizure generation, loss-of-function mutations in GRIN2A, which result in reduced GluN2A expression, have also been identified in patients with various forms of epilepsy, including focal epilepsies and epileptic encephalopathies. This paradox highlights the complexity of NMDA receptor contributions to network excitability and suggests that both gain- and loss-of-function mutations can be pathogenic. However, the mechanisms through which reduced GluN2A expression influences seizure susceptibility remains unclear. In this study, we examined the impact of reduced and absence of GluN2A on seizure-like activity using in vitro model system. Hippocampal slices were prepared from wild-type littermate controls (Grin2a+/+), heterozygous (Grin2a+/-) and homozygous (Grin2a-/-) transgenic rats and subjected to pro-convulsant conditions to elicit epileptiform-like events. Specifically, we employed three well established in vitro epilepsy models: (i) 4-aminopyridine (4-AP), (ii) zero-magnesium with elevated potassium (0Mg{superscript 2}/5K), and (iii) high-potassium (high-K+). Local field potentials were recorded from the CA1 pyramidal layer to quantify interictal and seizure-like activity, by measuring latency to onset, event frequency, and duration. Spectral analyses were also conducted to assess alterations in network dynamics. Our findings reveal that reduced GluN2A expression alters the susceptibility of hippocampal circuits to seizure-like activity in a model-dependent manner. In the 4-AP model, Grin2a-/- slices exhibited a significantly lower frequency of interictal events and a delayed onset of seizure-like activity relative to both wild-type and heterozygous slices. In the 0Mg{superscript 2}/5K model, both Grin2a+/- and Grin2a-/- slices displayed reduced seizure susceptibility compared to wild-type. Finally, in the high-K model, Grin2a-/- slices showed a reduced incidence of seizure-like events compared to wild-type controls when extracellular potassium concentration was increased to 7.5 mM, although this effect was less apparent at higher concentrations (9.5 mM). These results suggest that partial or complete loss of GluN2A-containing NMDA receptors reduces the propensity of hippocampal networks to develop epileptiform activity, potentially by altering intrinsic or synaptic excitability. Our findings provide mechanistic insights into how GRIN2A loss-of-function variants may contribute to epilepsy and highlight the need to consider developmental and circuit-level compensations when interpreting the impact of GluN2A disruption.

neuroscience↗

bioRxiv2025

Repopulation of the brain with microglia-like cells following intraperitoneal bone marrow cell transfer in microglia-deficient mice.

Germ-line deletion of a conserved enhancer (the Fms intrinsic regulatory element, FIRE) in the mouse Csf1r locus causes congenital absence of microglia. Homozygous FIRE deletion on a C57BL/6J background leads to perinatal lethality and hydrocephalus (HC) in surviving pups. We developed a congenic C57BL/6J line with defined regions of non-C57BL/6J genomic DNA, increased postnatal viability and reduced incidence of HC. Both perinatal lethality and HC were eliminated in F2 mice following outcross of the congenic line to CBA/J or BALBc/J backgrounds. To assess the impacts of microglial deficiency in postnatal neurodevelopment we analyzed deep total RNA-seq data from multiple brain regions of wild-type and Csf1r{Delta}FIRE/{Delta}FIRE mice. Aside from the loss of microglial-specific transcripts, we found no significant alterations in relative abundance of any cell-type or region-specific transcriptomic signature. Transcripts associated with endosome/lysosome function, which are enriched in microglia, were not affected, suggesting compensatory expression by other cell types. On the C57BL/6J x CBA/J F2 background, congenital absence of microglia did not affect motor activity, behavior or myelination up to 7 months of age but was associated with astrocytosis and calcification in the thalamus. In the congenic C57BL/6J Csf1r{Delta}FIRE/{Delta}FIRE mouse line, intraperitoneal transfer of wild-type bone marrow cells (BMT) at weaning led to complete repopulation of the brain with microglia-like cells without giving rise to monocytic intermediates. Our results suggest novel strategies for treatment of microglial deficiency.

neuroscience↗

bioRxiv2024

Typical development of synaptic and neuronal properties can proceed without microglia

Brain-resident macrophages, microglia, have been proposed to play an active role in synaptic refinement and maturation, influencing plasticity and circuit-level connectivity. Here we show that a variety of neurodevelopmental processes previously attributed to microglia can proceed without them. Using a genetically modified mouse which lacks microglia (Csf1r{Delta}FIRE/{Delta}FIRE) we find that intrinsic properties, synapse number and synaptic maturation are largely normal in the hippocampal CA1 region and somatosensory cortex at stages where microglia have been implicated. Seizure susceptibility and hippocampal-prefrontal cortex coherence in awake behaving animals, processes disrupted in mice deficient in microglia-enriched genes, are also normal. Similarly, eye-specific segregation of inputs into the lateral geniculate nucleus proceeds normally in the absence of microglia. Single cell/nucleus transcriptomic analyses of neurons and astrocytes did not uncover any substantial perturbation due to microglial absence. Thus, the brain possesses remarkable adaptability to execute developmental synaptic refinement, maturation and connectivity in the absence of microglia.

neuroscience↗

bioRxiv2024

Microglia determine beta-amyloid plaque burden but are non-essential for downstream pathology

Evidence points to a role for microglia in Alzheimers disease (AD) risk, although their position in the pathological cascade is incompletely understood, prompting us to generate a model of {beta}-amyloidopathy lacking microglia. We find evidence that microglia promote plaque formation and creation of an A{beta} fibril-rich zone surrounding the plaque core. However, plaque-proximal reactive astrogliosis, synapse loss, and neurite dystrophy are still observed in the absence of microglia.

neuroscience↗

bioRxiv2022

Evolutionary divergence of basal and activity-dependent exon splicing in cortical neurons

Alternative splicing of mRNA exons in mammalian neurons increases diversity of the proteome and is regulatable by signaling pathways. However, the degree of conservation of basal and signal-dependent exon usage between human neurons and those from experimental models such as mice is incompletely understood. We previously showed that cortical neuronal activity-dependent gene transcription exhibits human/mouse differences, driven by evolutionary divergence of cis-acting promoter elements (Qiu et al. 2016). Since alternative exon usage influences brain development and cognition, is controlled by neuronal activity, and is disturbed in brain disorders, we investigated human/mouse differences in exon usage in cortical neurons. Comparing orthologous exons, basal exon inclusion levels showed human-mouse conservation, but also significant differences determined by cis-acting sequences: human-mouse conservation and divergence in exon usage was recapitulated in neurons from Tc1 mice carrying human chromosome-21 (hCh21). Activity-dependent changes in exon usage also exhibited significant conservation: gene structure was more likely to be conserved in activity-regulated exons, and exons regulated in both human and mouse neurons were enriched in RBFOX and SAM68 targets, and genes were centred on cytoskeletal organisation, mRNA transcription/processing, and synaptic signaling. However, divergence was also evident, and human-specific activity-dependent exon usage was dominated by genes involved in lipid biosynthesis, signaling and trafficking. Notably, the pattern of activity-dependent usage of hCh21 exons in human neurons was not recapitulated in mouse Tc1 neurons. Thus, unlike species-specific differences in activity-dependent gene transcription, cis-acting DNA sequence divergence is insufficient to explain inter-species differences in activity-regulated exon usage. Trans-acting factors involved in activity-responsive splicing have likely also diverged.

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bioRxiv2022

Enhanced hippocampal LTP but typical NMDA receptor and AMPA receptor function in a novel rat model of CDKL5 deficiency disorder

Mutations in the X-linked gene cyclin-dependent kinase-like 5 (CDKL5) cause a severe neurological disorder characterised by early-onset epileptic seizures, autism and intellectual disability (ID). Impaired hippocampal function has been implicated in other models of monogenic forms of autism spectrum disorders and ID and is often linked to epilepsy and behavioural abnormalities. Many individuals with CDKL5 deficiency disorder (CDD) have null mutations and complete loss of CDKL5 protein, therefore in the current study we used a novel Cdkl5 KO rat model to elucidate the impact of CDKL5 loss on cellular excitability and synaptic function of CA1 pyramidal cells (PCs). We hypothesised abnormal pre and/or post synaptic function underlie the enhanced LTP we observe in the hippocampus of Cdkl5 KO rats. We tested this hypothesis using a combination of extracellular and whole-cell electrophysiological recordings, biochemistry, and histology. We show that NMDA receptor function and subunit expression are unaltered throughout development, and Ca2+ permeable AMPA receptor mediated currents are unchanged in Cdkl5 KO rats. We observe reduced mEPSC frequency accompanied by increased spine density in basal dendrites of CA1 PCs, however we find no evidence supporting an increase in silent synapses when assessed using a minimal stimulation protocol in slices. Additionally, we found no change in paired-pulse ratio, consistent with normal release probability in Cdkl5 KO rats and supported by typical expression of pre-synaptic proteins in synaptosome preparations. Together these data indicate a role for CDKL5 in hippocampal synaptic function and raise the possibility that altered intracellular signalling rather than synaptic deficits might contribute to the altered plasticity.

neuroscience↗

bioRxiv2018

Altered dendritic spine function and integration in a mouse model of Fragile X Syndrome

Cellular and circuit hyperexcitability are core features of Fragile X Syndrome and related autism spectrum disorder models. However, a synaptic basis for this hyperexcitability has proved elusive. We show in a mouse model of Fragile X Syndrome, glutamate uncaging onto individual dendritic spines yields stronger single-spine excitation than wild-type, with more silent spines. Furthermore, near-simultaneous uncaging at multiple spines revealed fewer spines are required to trigger an action potential. This arose, in part, from increased dendritic gain due to increased intrinsic excitability, resulting from reduced hyperpolarization-activated currents. Super-resolution microscopy revealed no change in dendritic spine morphology, pointing to an absence of a structure-function relationship. However, ultrastructural analysis revealed a 3-fold increase in multiply-innervated spines, accounting for the increased single-spine excitatory currents following glutamate uncaging. Thus, loss of FMRP causes abnormal synaptogenesis, leading to large numbers of poly-synaptic spines despite normal spine morphology, thus explaining the synaptic perturbations underlying circuit hyperexcitability.

neuroscience↗