Biology subjects

Holt, M. G.

Publications and source records attributed to Holt, M. G..

7 recordsLinked to original sources

bioRxiv2024

Directed disruption of IL2 aggregation sites produces designer biologics with enhanced specificity coupled to improved production capacity

The pleotropic nature of interleukin-2 (IL2) has allowed it to be used as both a pro-inflammatory and anti-inflammatory therapeutic agent, through promotion of regulatory T cell (Treg) responses via the IL2RA receptor or promotion of CD8 T cell responses via the IL2RB receptor, respectively. However, the utility of IL2 as a treatment is limited by this same pleiotropy, and protein engineering to bias specificity towards either the regulatory T cell (Treg) or CD8 T cell lineage often requires a trade-off in protein production or total bioactivity. Here we use SolubiS, a computational algorithm-based method, to predict mutations within the IL2 structure to improve protein production yield while altering cellular selectivity, to generate a mutein with elevated therapeutic potential. The design and testing process identified the V126R (murine) / V111R (human) mutation as a Treg-enhancing mutein, creating a cation repulsion to inhibit primary binding to IL2RB, with a post-IL2RA confirmational shift enabling secondary IL2RB binding, and hence allowing the trimeric receptor complex to form. In human IL2, additional N110R T151R aggregation-protecting mutations could improve protein yield of the V111R mutation. The approach also generated novel CD8 T cell-promoting mutations. Y79K created a cation-cation repulsion with IL2RA, while Q50W enhanced CD8 T cell activity through potential {pi}-stacking enhancing binding to IL2RB, with the combination highly stimulatory for CD8 T cells. For human IL2, Y65K (homolog to murine Y79K) coupled with E82K prevented IL2RA binding, however it required the aggregation-protecting mutations of N110R T151R to rescue production. These muteins, designed with both cellular specificity and protein production features, have potential as both biological tools and therapeutics.

immunology↗

bioRxiv2023

The astrocyte alpha 1-adrenoreceptor is a key component of the neuromodulatory system in mouse visual cortex.

Noradrenaline (norepinephrine) is known to modulate many physiological functions and behaviors. In this study, we tested to what extent astrocytes, a type of glial cell, participate in noradrenergic signaling in mouse primary visual cortex (V1). Astrocytes are essential partners of neurons in the central nervous system. They are central to brain homeostasis, but also dynamically regulate neuronal activity, notably by relaying and regulating neuromodulator signaling. Indeed, astrocytes express receptors for multiple neuromodulators, including noradrenaline, but the extent to which astrocytes are involved in noradrenergic signaling remains unclear. To test whether astrocytes are involved in noradrenergic neuromodulation in mice, we knocked down the major noradrenaline receptor in astrocytes, the 1A- adrenoreceptor. Using this model, we found that the 1A-adrenoreceptor is involved in triggering intracellular calcium transients in astrocytes, which are generally thought to underlie astrocyte function. To test if impaired 1A-adrenoreceptor signaling in astrocytes affected the function of neuronal circuits in V1, we used electrophysiological measurements and found that noradrenergic signaling through astrocyte 1A-adrenoreceptor controls the basal level of inhibition and regulates plasticity in V1 by potentiating synaptic responses in circuits involved in visual information processing. MAIN POINTSO_LIThe 1A-adrenoreceptor (1A-NAR) is the major 1-NAR in primary visual cortex (V1) C_LIO_LI1A-NAR signaling triggers astrocyte Ca2+ responses in V1. C_LIO_LIAstrocyte 1A-NARs play a key role in regulating basal inhibitory transmission and are crucial for LTP induction in V1 C_LI

neuroscience↗

bioRxiv2021

A conformational switch controlling the toxicity of the prion protein

Prion infections cause conformational changes of PrPC and lead to progressive neurological impairment. Here we show that toxic, prion-mimetic ligands induce an intramolecular R208-H140 hydrogen bond ("H-latch") altering the flexibility of the 2-3 and {beta}2-2 loops of PrPC. Expression of a PrP2Cys mutant mimicking the H-latch was constitutively toxic, whereas a PrPR207A mutant unable to form the H-latch conferred resistance to prion infection. High-affinity ligands that prevented H-latch induction repressed prion-related neurodegeneration in organotypic cerebellar cultures. We then selected phage-displayed ligands binding wild-type PrPC, but not PrP2Cys. These binders depopulated H-latched conformers and conferred protection against prion toxicity. Finally, brain-specific expression of an antibody rationally designed to prevent H-latch formation, prolonged the life of prion-infected mice despite unhampered prion propagation, confirming that the H-latch is causally linked to prion neurotoxicity.

molecular biology↗

bioRxiv2019

AAV mediated delivery of a novel anti-BACE1 VHH reduces Abeta in an Alzheimer’s disease mouse model

Single domain antibodies (VHH) are potentially disruptive therapeutics, with important biological value for treatment of several diseases, including neurological disorders. However, VHH have not been widely used in the central nervous system (CNS), as it is hard to reach therapeutic levels, both because of their restricted blood-brain-barrier penetration and their apparent rapid clearance from the parenchyma. Here, we propose a gene transfer strategy based on adeno-associated virus (AAV)-based vectors to deliver VHH directly into the CNS, ensuring continuous production at therapeutic levels. As a proof-of-concept, we explored the potential of AAV-delivered VHH to inhibit BACE1, a well-characterized target in Alzheimers disease. First, we generated a panel of VHHs targeting BACE1. One of them, VHH-B9, showed high selectivity for BACE1 and efficacy in lowering BACE1 activity in vitro. We then went on to demonstrate significant reductions in amyloid beta (A{beta}) levels after AAV-based delivery of VHH-B9 into the CNS of a mouse model of cerebral amyloidosis. These results constitute a novel therapeutic approach for neurodegenerative diseases, which is applicable to a range of CNS disease targets.

neuroscience↗

bioRxiv2018

Flexible, fast and selective genetic manipulation of the vertebrate CNS with misPiggy

In utero electroporation is increasingly used for manipulation of gene expression in the mammalian central nervous system. Here we created a transposon-based single plasmid system, that allows long-term conditional loss- and gain-of-function experiments across major CNS cell types. The system is widely applicable across species, allowing fast, flexible and cheap cell manipulation in previously non-transgenic species.

neuroscience↗

bioRxiv2018

Astrocytes integrate local sensory and brain-wide neuromodulatory signals.

Astrocytes play multiple functions in the central nervous system, from control of blood flow through to modulation of synaptic activity. Transient increases in intracellular Ca2+ are thought to control these activities. The prevailing concept is that these Ca2+ transients are triggered by distinct pathways, with little mechanistic and functional overlap. Here we demonstrate that astrocytes in visual cortex of mice encode local visual signals in conjunction with arousal state, functioning as multi-modal integrators. Such activity adds an additional layer of complexity to astrocyte function and may enable astrocytes to specifically and subtly regulate local network activity and plasticity.

neuroscience↗

bioRxiv2018

Molecularly distinct astrocyte subpopulations spatially pattern the adult mouse brain

Astrocytes are a numerous cell type of the central nervous system. They perform many important functions in synapse formation and maintenance, control of local homeostasis and modulation of synaptic transmission. However, the degree to which specialist astrocyte subtypes fulfil these specific tasks is currently unclear. Here we use single cell transcriptomics to demonstrate that astrocytes, even those lying within the same region of adult mouse brain, show distinct molecular and spatial profiles, with profound implications for local CNS functions, including axon guidance, synaptogenesis and synaptic transmission.

neuroscience↗