Biology subjects

Graham, R. T.

Publications and source records attributed to Graham, R. T..

4 recordsLinked to original sources

bioRxiv2026

Large-Scale Assessment of NF1 Single Amino Acid Variants as HLA Class I Neoantigens

Neoantigens are cancer-specific antigens arising from genomic alterations. Single Amino Acid Variants (SAAVs) represent a primary class of these neoantigens. To evaluate the therapeutic potential of Neurofibromin 1 (NF1)-derived SAAVs - given that NF1 is frequently mutated in malignant brain tumors - we prioritized the 40 NF1 SAAVs determined to be HLA-A*02:01 binders using computational prediction coupled with experimental validation. To validate these predicted neoepitopes, we employed a two-tiered experimental approach in HLA-A*02:01 homozygous U87-MG cells. We first synthesized minigene constructs encoding the predicted neoepitopes, introduced them via lentiviral transfection and confirmed their expression by mass spectrometry (MS). Subsequently, we performed endogenous validation using pan-HLA immunoprecipitation mass spectrometry (IP-MS), confirming 4 (10 neoepitopes) of the 40 candidate SAAVs. We observed a discrepancy between in silico predictions and the observed sequences. Our endogenous peptidomics further revealed conserved peptide motifs and demonstrated that peptide selection for HLA presentation is transient. While our study substantiates the therapeutic feasibility of T-cell immunotherapies targeting NF1 mutations, these results underscore a limitation in current computational prediction. Our study highlights the necessity of experimental validation to refine neoantigen prioritization strategies.

immunology↗

bioRxiv2025

NDNF+ interneurons have a privileged role in regulating cortical excitability

Cortical processing depends critically on the precise modulation of excitatory activity by a diverse population of GABAergic interneurons. Among these, neuron-derived neurotrophic factor-expressing (NDNF+) interneurons have been proposed to be "master regulators" of cortical microcircuits. While their role in coordinating physiological cortical activity is beginning to be understood, their contribution to restraining hyperexcitability during epileptiform activity remains unexplored. To address this, we employed calcium imaging, optogenetics, and chemogenetics in an NDNF-Cre mouse line to investigate how NDNF+ interneurons influence cortical hyperactivity and spontaneous seizures. Our results demonstrate that NDNF+ interneurons are actively recruited during interictal spikes and focal seizures, although more slowly than parvalbumin-positive interneurons. Optogenetic hyperpolarization of NDNF+ interneurons exacerbates epileptic discharges, whereas their depolarization suppresses focal seizures, even when the optogenetic stimulus is delayed by several seconds from seizure onset. This effect is largely mediated by GABAB receptor signalling. Additionally, chemogenetic depolarization of NDNF+ interneurons has robust antiepileptic effects both ex vivo and in vivo. Collectively, these findings establish NDNF+ neurons as key regulators of cortical excitability and identify them as promising cellular targets for the development of anti-epileptic therapies.

neuroscience↗

bioRxiv2023

Anti-seizure Gene Therapy for Focal Cortical Dysplasia

Focal Cortical Dysplasias (FCDs) are a common subtype of malformation of cortical development, which frequently present with a spectrum of cognitive and behavioural abnormalities as well as pharmacoresistant epilepsy. FCD type II is typically caused by somatic mutations resulting in mTOR hyperactivity, and is the commonest pathology found in children undergoing epilepsy surgery. However, surgical resection does not always result in seizure freedom, and is often precluded by proximity to eloquent brain regions. Gene therapy is a promising potential alternative treatment and may be appropriate in cases that represent an unacceptable surgical risk. Here, we evaluated a gene therapy based on overexpression of the Kv1.1 potassium channel in a mouse model of frontal lobe FCD. An engineered potassium channel (EKC) transgene was placed under control of a human promoter that biases expression towards principal neurons (CAMK2A) and packaged in an adeno-associated viral vector (AAV9). We used an established FCD model generated by in utero electroporation of frontal lobe neural progenitors with a constitutively active human RHEB plasmid, an activator of mTOR Complex 1. First, we further characterised this by quantifying electrocorticograms and behavioural abnormalities, both in mice developing spontaneous generalised seizures and in mice only exhibiting abnormal interictal discharges. Then, using continuous video-electrocorticogram recordings from epileptic mice before and after injection of AAV9-CAMK2A-EKC in the dysplastic region, we observed a robust decrease in the frequency of seizures and in interictal activity, compared to mice injected with a control viral vector. Despite the robust anti-epileptic effect of the treatment, there was neither an improvement nor a worsening of performance in behavioural tests sensitive to frontal lobe function. AAV9-CAMK2A-EKC had no effect on interictal activity or behaviour in non-epileptic mice. AAV9-CAMK2A-EKC gene therapy is a promising therapy with translational potential to treat the epileptic phenotype of mTOR-related malformations of cortical development. Cognitive and behavioural co-morbidities may, however, resist an intervention aimed at reducing circuit excitability.

neuroscience↗

bioRxiv2021

Synergistic positive feedback underlying seizure initiation

Seizure onset is a critically important brain state transition that has proved very difficult to predict accurately from recordings of brain activity. Here we show that an intermittent, optogenetic, stimulation paradigm reveals a latent change in dendritic excitability that is tightly correlated to the onset of seizure activity. Our data show how the precipitous nature of the transition can be understood in terms of multiple, synergistic positive feedback mechanisms: raised intracellular Cl- and extracellular K+, coupled to a reduced threshold for dendritic plateau potentials, and which in turn leads to a switch to pyramidal burst firing. Notably, the stimulation paradigm also delays the evolving epileptic activity, meaning that not only can one monitor seizure risk safely, it may even have an additional anti-epileptic benefit. One Sentence SummaryRapid transitions into seizures arise from mutually accelerating feedback loops, involving changes in dendritic excitability

neuroscience↗