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Biomedical subjects

M J Skynner

Publications and source records attributed to M J Skynner.

16 recordsLinked to original sources

Antibody-drug conjugates in selected solid tumours: a position statement update based on findings from the third workshop held by the ETOP IBCSG Partners Foundation.

The European Thoracic Oncology Platform (ETOP) International Breast Cancer Study Group (IBCSG) Partners Foundation initiated a series of workshops for experts to review current evidence and offer recommendations to guide future antibody-drug conjugate (ADC) research. Here, we summarise key findings from the third workshop, which included experts in various solid tumours, basic/translational research scientists and pharmaceutical industry representatives. Recent positive phase III trial data have further incorporated ADCs into the standard of care [e.g. lung: sacituzumab tirumotecan; breast: trastuzumab deruxtecan (T-DXd), sacituzumab govitecan, datopotamab deruxtecan; muscle-invasive bladder cancer: enfortumab vedotin; ovarian cancer: mirvetuximab soravastine; cervical cancer: tisotumab vedotin]. Thus, research priorities must be tailored according to tumour type, potentially focussing initially on settings where ADCs could replace chemotherapy. Many phase III ADC trials have been initiated based on positive phase I data and although these trials are larger than those conducted historically, prespecified criteria (e.g. patient numbers and magnitude of efficacy) should be met to justify proceeding directly to phase III. Importantly, although several ADCs have been successfully developed without mandatory biomarker selection, biomarker-driven ADC development enables rational patient selection, as illustrated by multiple ADCs (e.g. T-DXd, mirvetuximab soravtansine and telisotuzumab vedotin). The identification, development and validation of predictive biomarkers are therefore essential, particularly given several critical nuances, including the algorithms used to assess biomarker status and the type of specimen analysed, all of which may be influenced by temporal and spatial heterogeneity. Additional ADC research priorities include the optimisation of ADC constructs to enhance efficacy/tolerability and the identification of reliable ADC targets, including work to elucidate attributes of already-identified targets. Finally, considering the vast amount of ADC-related data being generated, artificial intelligence could be leveraged to analyse combined datasets and generate composite biomarkers, including tumour histology, optimal target expression thresholds, molecular alterations and activated pathways affecting payload activity and target function, to accelerate research.

Humans↗

Single cell expression analysis--pharmacogenomic potential.

A fundamental challenge in biology is to correlate physiology with gene expression in specific cell types. This can only be achieved by understanding gene expression at the level of the single cell because, in many systems, each cell has the capacity to express a unique set of genes. Therefore, each cell can be considered to be functionally distinct. A clearer understanding of gene expression differences at such a discrete level provides an opportunity to develop drugs with more targeted pharmacologies or with decreased side effects.

Animals↗

Molecular and cellular properties of GnRH neurons revealed through transgenics in the mouse.

Recent advances in the use of gonadotropin-releasing hormone (GnRH) promoter-driven transgenics in the mouse are beginning to open up the once elusive GnRH neuronal phenotype to detailed molecular and cellular investigation. This review highlights progress in the development of GnRH promoter transgenic constructs and the understanding of murine gene sequences required for the correct temporal and spatial targeting of transgenes to the GnRH phenotype in vivo. Strategies enabling the identification of single, living GnRH neurons in the acute brain slice preparation are allowing gene profiling and electrophysiological experiments to be undertaken. Results so far indicate that, like other neurons, GnRH cells express a variety of sodium, potassium and calcium channels as well as GABAergic and glutamatergic receptors which are responsible for determining the membrane properties and firing characteristics of the GnRH neuron. Many of these receptors and channels appear to be expressed heterogeneously within the GnRH phenotype. Furthermore, several display distinct postnatal developmental expression profiles which are likely to be of consequence to the development of synchronized, pulsatile GnRH secretion in the adult animal.

Animals↗

Conditional ablation of neurones in transgenic mice.

Conditional targeted ablation of specific cell populations in living transgenic animals is a very powerful strategy to determine cell functions in vivo. This approach would be of particular value to study the functions of distinct neuronal populations; however, the transgene of choice for conditional cell ablation studies in mice, the herpes simplex virus thymidine kinase gene, cannot be used to ablate neurones as its principal mode of action relies on cell proliferation. Here we report that expression of the E.coli nitroreductase gene (Ntr) and metabolism of the prodrug CB1954 (5-aziridin-1-yl-2-4-dinitrobenzamide) to its cytotoxic derivative can be used to conditionally and acutely ablate specific neuronal populations in vivo. As proof of principal, we have ablated olfactory and vomeronasal receptor neurones by expressing Ntr under the control of the olfactory marker protein (OMP) gene promoter. We demonstrate that following CB1954 administration, olfactory and vomeronasal receptor neurones expressing the transgene were selectively eliminated from the olfactory epithelium (OE), and projections to the olfactory bulb (OB) were lost. The functional efficacy of cell ablation was demonstrated using a highly sensitive behavioural test to show that ablated mice had lost the olfactory ability to discriminate distinct odors and were consequently rendered anosmic. Targeted expression of Ntr to specific neuronal populations using conventional transgenes, as described here, or by "knock-in" gene targeting using embryonic stem cells may be of significant value to address the functions of distinct neuronal populations in vivo.

Animals↗

Heterogeneity in the basic membrane properties of postnatal gonadotropin-releasing hormone neurons in the mouse.

The electrophysiological characteristics of unmodified, postnatal gonadotropin-releasing hormone (GnRH) neurons in the female mouse were studied using whole-cell recordings and single-cell RT-PCR methodology. The GnRH neurons of adult animals fired action potentials and exhibited distinguishable voltage-current relationships in response to hyperpolarizing and depolarizing current injections. On the basis of their patterns of inward rectification, rebound depolarization, and ability to fire repetitively, GnRH neurons in intact adult females were categorized into four cell types (type I, 48%; type II, 36%; type III, 11%; type IV, 5%). The GnRH neurons of juvenile animals (15-22 d) exhibited passive membrane properties similar to those of adult GnRH neurons, although only type I (61%) and type II (7%) cells were encountered, in addition to a group of "silent-type" GnRH neurons (32%) that were unable to fire action potentials. A massive, action potential-independent tonic GABA input, signaling through the GABA(A) receptor, was present at all ages. Afterdepolarization and afterhyperpolarization potentials (AHPs) were observed after single action potentials in subpopulations of each GnRH neuron type. Tetrodotoxin (TTX)-independent calcium spikes, as well as AHPs, were encountered more frequently in juvenile GnRH neurons compared with adults. These observations demonstrate the existence of multiple layers of functional heterogeneity in the firing properties of GnRH neurons. Together with pharmacological experiments, these findings suggest that potassium and calcium channels are expressed in a differential manner within the GnRH phenotype. This heterogeneity occurs in a development-specific manner and may underlie the functional maturation and diversity of this unique neuronal phenotype.

Action Potentials↗

Profiling gamma-aminobutyric acid (GABA(A)) receptor subunit mRNA expression in postnatal gonadotropin-releasing hormone (GnRH) neurons of the male mouse with single cell RT-PCR.

The present investigation has examined which subunits of the GABA(A) receptor are expressed by gonadotropin-releasing hormone (GnRH) neurons in the juvenile and adult male mouse. Cells of defined morphology, located in the medial septum (MS) and rostral preoptic area (POA), were patch-clamped in the acute brain slice preparation and their cell contents extracted. A reverse transcriptase polymerase chain reaction (RT-PCR) procedure using nested primers was used to establish individual GnRH mRNA-expressing cells which were then evaluated for eleven GABA(A) receptor (alpha1-5, beta1-3, gamma1-3) subunit transcripts. Single and multiple GABA(A) receptor subunit mRNAs were detected in approximately 70% of all GnRH neurons. A range of different subunit mRNAs (alpha1, alpha2, alpha5, beta1, beta2, beta3, gamma2) were found in juvenile GnRH neurons, with the alpha1gamma2 and alpha5gamma2 combinations encountered most frequently within individual cells. The expression profile in adult GnRH neurons was more extensive than that detected in juveniles with alpha1, alpha2, alpha3, alpha5, beta1, beta2, beta3, gamma1 and gamma2 subunits all being detected. The major difference in subunit profile between GnRH neurons located in the MS and POA involved the beta subunits. The principal postnatal developmental change was one of increasing overall subunit heterogeneity in maturing POA GnRH neurons. The profile of GABA(A) receptor subunit mRNAs detected in male GnRH neurons was quite different to that reported by us for female GnRH neurons in the mouse using the same RT-PCR approach. Together, these findings indicate that postnatal GnRH neurons are likely to express a range of GABA(A) receptor subunit mRNAs in a sexually dimorphic and developmentally-regulated manner.

Aging↗

Direct regulation of postnatal GnRH neurons by the progesterone derivative allopregnanolone in the mouse.

The mechanisms through which gonadal steroids exert critical feedback actions upon the activity of the GnRH neurons are not understood. We have examined here whether progesterone may modulate the electrical activity of the GnRH neurons following its rapid metabolism to the neuroactive steroid allopregnanolone within the brain. Using an acute brain slice preparation, whole-cell, patch-clamp recordings were made from GnRH neurons of juvenile (postnatal d 15-20) and adult (postnatal d 60-70) female mice in the presence of tetrodotoxin. Progesterone (1 microM) was not observed to have any actions (up to 5 min exposure) upon GnRH neurons. However, allopregnanolone (500 nM-1 microM) exerted rapid (<1 min) effects upon the baseline membrane potential of all GnRH neurons and also significantly (P < 0.01) enhanced their GABA responses by up to 4-fold. All GABA and allopregnanolone responses were abolished by the GABA(A) receptor antagonist bicuculline. No differences were detected in the allopregnanolone sensitivity of GnRH neurons recorded from juvenile and adult GnRH neurons. These results provide the first evidence for a direct action of the neurosteroid allopregnanolone on postnatal GnRH neurons and suggest a new mechanism through which fluctuating progesterone levels may influence the secretory activity of these important neurons in the female mouse.

Animals↗

Role of the GABA(A) receptor gamma2 subunit in the development of gonadotropin-releasing hormone neurons in vivo.

We have employed transgenic mouse models to examine the functional significance of the gamma2 subunit of the GABA(A) (gamma-aminobutyric acid) receptor to the correct development of gonadotropin-releasing hormone (GnRH) neurons in vivo. In the first experiment, the expression of gamma2 subunit protein by the GnRH phenotype was determined using transgenic mice in which GnRH gene sequences direct the expression of the LacZ reporter to the nucleus of the GnRH neurons. This greatly facilitates the immunocytochemical identification of non-nuclear-located antigens within GnRH neurons and revealed that approximately 25% of juvenile GnRH neurons were immunoreactive for the gamma2 subunit and that this increased to 40% in pubertal mice. In the second experiment, GnRH mRNA expression was examined in the brains of gamma2 subunit knockout mice (gamma2(0/0)) and their wild-type (gamma2+/+) littermates at embryonic day 15 and postnatal days (P) 0 and 11-16 using in situ hybridization. The distribution and numbers of cells expressing GnRH mRNA in gamma2+/+ and gamma2(0/0) mice were not found to differ at any age. However, the GnRH mRNA content of medial septal cells was significantly lower in gamma2(0/0) compared with gamma2+/+ mice at P11-16 (P<0.05) and the same trend was observed for preoptic area neurons. These results demonstrate that while the gamma2 subunit of the GABA(A) receptor is expressed by postnatal GnRH neurons, their embryonic development does not require a functional gamma2 subunit. In contrast, postnatal GnRH mRNA expression was found to be dependent upon signalling through the GABA(A) receptor.

Animals↗

Late postnatal reorganization of GABA(A) receptor signalling in native GnRH neurons.

The molecular and cellular characteristics of the gonadotropin-releasing hormone (GnRH) neurons have been difficult to ascertain due to their scattered distribution within the basal forebrain. Using morphological criteria coupled with single cell RT-PCR postidentification, we have developed a method for investigating native GnRH neurons in the mouse brain and used it to examine the development of GABA(A) receptor signalling in this phenotype. Following the harvesting of the cytoplasmic contents of individual GnRH neurons, single cell multiplex RT-PCR experiments demonstrated that GABAA receptor alpha1-5, beta1-3 and gamma2 & 3 subunit transcripts were expressed by both neonatal (postnatal day 5) and juvenile (day 15-20) GnRH neurons in a heterogeneous manner. Following puberty, this profile was reduced to a predominant alpha1, alpha5, beta1, gamma2 subunit complement in rostral preoptic area GnRH neurons of the adult female. Whole-cell patch-clamp recordings revealed little difference between juvenile and adult GnRH neurons in their resting membrane potential and spontaneous firing rates. All GnRH neurons were found to be subjected to a tetrodotoxin-insensitive, tonic GABAergic barrage signalling through the GABA(A) receptor. However, marked heterogeneity in the sensitivity of individual juvenile GnRH neurons to GABA was revealed and, in parallel with the change in subunit mRNA profile, this was dramatically reduced in the reproductively competent adult GnRH neurons. These findings provide the first electrical and molecular characterization of the GnRH phenotype and demonstrate a novel pattern of late postnatal reorganization of native GABA(A) receptor gene expression and signalling in the GnRH neuronal population.

Age Factors↗

Promoter transgenics reveal multiple gonadotropin-releasing hormone-I-expressing cell populations of different embryological origin in mouse brain.

Gonadotropin-releasing hormone-I (GnRH-I) is thought to be expressed by a single, highly spatially restricted group of neurons, which originate in the olfactory placode and migrate through the nose into the medial septum and hypothalamus from where they control fertility. Transgenic mice bearing a 13.5 kb GnRH-I-lacZ reporter construct were derived and found to express high levels of beta-galactosidase mRNA and protein within the septohypothalamic GnRH neurons in a correct temporal and spatial manner. Unexpectedly, low levels of beta-galactosidase were also present in three further populations of cells within the lateral septum, bed nucleus of the stria terminalis, and tectum. Analysis of wild-type mice with three different GnRH-I antibodies revealed distinct and transient patterns of GnRH-I peptide expression during development in all three of these populations revealed by transgenics. The synthesis of GnRH by cells of the lateral septum was the most persistent and remained until the third postnatal week. Embryonic "small eye" Pax-6 null mice, which fail to develop an olfactory placode, were also examined and shown to have equivalent populations of GnRH-I-immunoreactive cells in the lateral septum, tectum, and bed nucleus of the stria terminalis but none of the migrating cells that form the septohypothalamic GnRH population. These results prove that so-called "ectopic" expression in promoter transgenic lines can reflect authentic developmental patterns of gene expression. They further provide the first demonstration in mammalian brain that multiple neuronal populations of different embryological origin express GnRH-I peptide during embryonic and postnatal development.

Animals↗

Detection of estrogen receptor alpha and beta messenger ribonucleic acids in adult gonadotropin-releasing hormone neurons.

The behavior of the gonadotropin-releasing hormones (GnRH) neurons controlling fertility is dependent upon cyclic fluctuations in circulating concentrations of estrogen. However, the nature of estrogen action upon these cells has remained controversial due to their dispersed distribution within the brain, and evidence indicating that they do not express nuclear estrogen receptors (ERs) in vivo. We report here an acute brain slice preparation that enables individual living GnRH neurons to be identified within the mouse brain and show, using single cell multiplex RT-PCR, that the greater than 50% of GnRH neurons in adult and prepubertal females contain ERalpha messenger RNA. Approximately 10% of GnRH neurons contained ERbeta transcripts that were always coexistent with ERalpha. Single cell RT-PCR analysis of nonGnRH cells located in the medial preoptic area revealed a similar coexpression pattern of ERalpha and ERbeta transcripts. In contrast, single striatal cells were not found to contain ERbeta despite ERalpha being present in approximately 25% of cells. The analysis of single GnRH neurons in cycling female mice revealed that the detection of ERalpha and ERbeta transcripts was lowest on proestrus (ERalpha, 18% of all GnRH neurons; ERbeta, 0%) compared with diestrus (44% and 6%) and estrus (75% and 19%, respectively). Using a novel approach that enables single cell RT-PCR analysis of GnRH neurons, we present here evidence for the cyclic expression of ERalpha and ERbeta messenger RNAs within prepubertal and adult female GnRH neurons.

Animals↗

Transgenics identify distal 5'- and 3'-sequences specifying gonadotropin-releasing hormone expression in adult mice.

GnRH neurons play a critical role in regulating gonadotropin secretion, but their scattered distribution has prevented detailed understanding of their molecular and cellular properties in vivo. Using GnRH promoter-driven transgenics we have examined here the role of 5'- and 3'-murine GnRH sequences in specifying GnRH expression in the adult mouse. Transgenic mice bearing a lacZ construct incorporating 5.5 kb of 5'-, all the introns and exons, and 3.5 kb of 3'-murine GnRH sequence were found to express beta-galactosidase (betagal) immunoreactivity in approximately 85% of all GnRH neurons. Deletion of GnRH sequence 3' to exon II had no effect upon transgene expression in the GnRH population (89%) but resulted in the appearance of ectopic betagal immunoreactivity in several regions of the brain. The production of additional mice in which 5'-elements were deleted to leave only -2.1 kb of sequence resulted in an approximately 40% reduction in the number of GnRH neurons expressing betagal. Mice in which further deletion of 400 bp allowed only -1.7 kb of 5'-sequence to remain exhibited a complete absence of betagal immunoreactivity within GnRH and other neurons. These results suggest that elements 3' to exon II of the GnRH gene have little role in enabling GnRH expression within the GnRH phenotype but, instead, are particularly important in repressing the GnRH gene in non-GnRH neurons. In contrast, elements located between -2.1 and -1.7 kb of distal 5'-sequence appear to be critical for the in vivo activation of GnRH expression within GnRH neurons in the adult brain.

Alleles↗

Transgenic mice ubiquitously expressing human placental alkaline phosphatase (PLAP): an additional reporter gene for use in tandem with beta-galactosidase (lacZ).

A fundamental keystone of developmental biology has been the growing use of reporter genes in model transgenic systems. Their use has greatly facilitated investigations of cell lineage and cell fate in addition to aiding experiments aimed at determining patterns of gene expression, gene interaction and gene regulation. Through construction of transgenic mice, ubiquitously expressing human placental alkaline phosphatase (PLAP), we demonstrate the suitability of PLAP as a reporter gene for use in conjunction with, or as an alternative to, beta-galactosidase (lacZ). Our findings demonstrate that over-expression of PLAP has no adverse effects on mouse development or viability, despite a widespread pattern of expression. This technology provides a simple yet effective mechanism based on eukaryotic reporter gene technology to facilitate the identification of transgenic cells within complex in vivo systems.

Animals↗

Genetic mapping of the mouse neuromuscular mutation kyphoscoliosis.

The ky mouse mutant, kyphoscoliosis, exhibits a degenerative muscle disease resulting in chronic deformation of the spinal column. Using an interspecific backcross segregating the ky mutation, we have mapped the ky locus to a small region of mouse chromosome 9. ky is nonrecombinant with the microsatellites D9Mit24 and D9Mit169 and lies in a conserved linkage group that encompasses human chromosome 3. s-Laminin (LAMS) and the gene for dystrophin-associated glycoprotein 1 (DAG1), which map to human chromosome 3, are both recombinant with ky, ruling them out as candidates.

Animals↗

In situ localisation of single-stranded DNA breaks in nuclei of a subpopulation of cells within regenerating skeletal muscle of the dystrophic mdx mouse.

Degeneration of muscle fibres during the early stages of Duchenne Muscular Dystrophy (DMD) is accompanied by muscle fibre regeneration where cell division and myoblast fusion to form multinucleate myotubes within the lesions appear to recapitulate the events of normal muscle development. The mechanisms that govern the expression of genes regulating differentiation of myoblasts in regenerating skeletal muscle are of great interest for the development of future therapies designed to stimulate muscle regeneration. We show here that single-stranded breaks in DNA are localised in nuclei, using an exogenously applied medium containing labelled deoxynucleotides and the Klenow fragment of DNA polymerase I. The nuclei of a sub-population of cells lying in the inflammatory infiltrate of lesions in the skeletal muscle of the muscular dystrophic mouse (mdx), a genetic homologue of DMD, were labelled in this fashion. By contrast, labelled cells were completely absent from the muscles of normal non-myopathic animals (C57BL/10) and non-lesioned areas of mdx muscles. Cells expressing the muscle-specific regulatory gene, myogenin, were also found within mononucleate cells and myotubes within similar mdx muscle lesions. While we cannot yet say that the cells labelled by the DNA polymerase reaction are in fact differentiating, they were found only in significant numbers within mdx muscle lesions where new muscle fibres appear, providing strong circumstantial evidence that they are intimately associated with the regenerative process. Using a range of nucleases and different DNA polymerases, we show that the DNA polymerase-labelling reaction observed was DNA-dependent and most probably due to infilling of naturally occurring single-stranded gaps in DNA. Since the regenerative process in human Duchenne Muscular Dystrophy is apparently less effective than that seen in mdx mice, continued study of single-stranded DNA breaks may help to elucidate further the mechanisms controlling the expression of genes that characterise the myogenic process during skeletal muscle regeneration. Such findings might be applied in the development of future therapies designed to stimulate muscle regeneration in human dystrophies.

Animals↗

Localization of donor nuclei in skeletal muscle grafts by in situ hybridization to a cDNA probe.

The development of therapies, based upon implantation of normal muscle cell precursors, for the treatment of skeletal muscle diseases such as Duchenne Muscular Dystrophy is in its infancy. Detailed analysis of the genetic and phenotypic contribution made by donor myoblasts to the regenerated muscle is critical. Using non-radioactive in situ hybridization of a Y chromosome-specific DNA probe to sections of muscle, we have localized the position of male donor nuclei within female host muscles after myoblast implantation. These results were compared with the distribution of immunocytochemically-localized dystrophin and the expression of donor-specific glucose phosphate isomerase by isoelectric-focussing. We found consistent male-specific nuclear hybridization and a close spatial relationship between the distribution of male donor nuclei and dystrophin-positive muscle fibres within female, dystrophin-negative host muscles. This approach will be useful in the further analysis of myoblast implantation experiments.

Animals↗