Increased interaction between endoplasmic reticulum and mitochondria following sleep deprivation.

BACKGROUND: Prolonged cellular activity may overload cell function, leading to high rates of protein synthesis and accumulation of misfolded or unassembled proteins, which cause endoplasmic reticulum (ER) stress and activate the unfolded protein response (UPR) to re-establish normal protein homeostasis. Previous molecular work has demonstrated that sleep deprivation (SD) leads to ER stress in neurons, with a number of ER-specific proteins being upregulated to maintain optimal cellular proteostasis. It is still not clear which cellular processes activated by sleep deprivation lead to ER- stress, but increased cellular metabolism, higher request for protein synthesis, and over production of oxygen radicals have been proposed as potential contributing factors. Here, we investigate the transcriptional and ultrastructural ER and mitochondrial modifications induced by sleep loss. RESULTS: We used gene expression analysis in mouse forebrains to show that SD was associated with significant transcriptional modifications of genes involved in ER stress but also in ER-mitochondria interaction, calcium homeostasis, and mitochondrial respiratory activity. Using electron microscopy, we also showed that SD was associated with a general increase in the density of ER cisternae in pyramidal neurons of the motor cortex. Moreover, ER cisternae established new contact sites with mitochondria, the so-called mitochondria associated membranes (MAMs), important hubs for molecule shuttling, such as calcium and lipids, and for the modulation of ATP production and redox state. Finally, we demonstrated that Drosophila male mutant flies (elav > linker), in which the number of MAMs had been genetically increased, showed a reduction in the amount and consolidation of sleep without alterations in the homeostatic sleep response to SD. CONCLUSIONS: We provide evidence that sleep loss induces ER stress characterized by increased crosstalk between ER and mitochondria. MAMs formation associated with SD could represent a key phenomenon for the modulation of multiple cellular processes that ensure appropriate responses to increased cell metabolism. In addition, MAMs establishment may play a role in the regulation of sleep under baseline conditions.

Synergistic regulation of uterine radial artery adaptation to pregnancy by paracrine and hemodynamic factors.

Fetal growth throughout pregnancy relies on delivery of an increasing volume of maternal blood to the placenta. To facilitate this, the uterine vascular network adapts structurally and functionally, resulting in wider blood vessels with decreased flow-mediated reactivity. Impaired remodeling of the rate-limiting uterine radial arteries has been associated with fetal growth restriction. However, the mechanisms underlying normal or pathological radial artery remodeling are poorly understood. Here, we used pressure myography to determine the roles of hemodynamic (resistance, flow rate, shear stress) and paracrine [ß-estradiol, progesterone, placental growth factor (PlGF), vascular endothelial growth factor] factors on rat radial artery reactivity. We show that ß-estradiol, progesterone, and PlGF attenuate flow-mediated constriction of radial arteries from nonpregnant rats, allowing them to withstand higher flow rates in a similar manner to pregnant vessels. This effect was partly mediated by nitric oxide (NO) production. To better understand how the combination of paracrine factors and shear stress may impact human radial artery remodeling in the first half of gestation, computational models of uterine hemodynamics, incorporating physiological parameters for trophoblast plugging and spiral artery remodeling, were used to predict shear stress in the upstream radial arteries across the first half of pregnancy. Human microvascular endothelial cells subjected to these predicted shear stresses demonstrated higher NO production when paracrine factors were added. This suggests that synergistic effects of paracrine and hemodynamic factors induce uterine vascular remodeling and that alterations in this balance could impair radial artery adaptation, limiting blood flow to the placenta and negatively impacting fetal growth.NEW & NOTEWORTHY Placenta-specific paracrine factors ß-estradiol, progesterone, and placental growth factor attenuate flow-mediated constriction of the rate-limiting uterine radial arteries, enabling higher flow rates in pregnancy. These paracrine factors induce their actions in part via nitric oxide mediated mechanisms. A synergistic combination of paracrine factors and shear stress is likely necessary to produce sufficient levels of nitric oxide during early human pregnancy to trigger adequate uterine vascular adaptation.

Effects of Eph/ephrin signalling and human Alzheimer's disease-associated EphA1 on Drosophila behaviour and neurophysiology.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disease placing a great burden on people living with it, carers and society. Yet, the underlying patho-mechanisms remain unknown and treatments limited. To better understand the molecular changes associated with AD, genome-wide association studies (GWAS) have identified hundreds of candidate genes linked to the disease, like the receptor tyrosine kinase EphA1. However, demonstration of whether and how these genes cause pathology is largely lacking. Here, utilising fly genetics, we generated the first Drosophila model of human wild-type and P460L mutant EphA1 and tested the effects of Eph/ephrin signalling on AD-relevant behaviour and neurophysiology. We show that EphA1 mis-expression did not cause neurodegeneration, shorten lifespan or affect memory but flies mis-expressing the wild-type or mutant receptor were hyper-aroused, had reduced sleep, a stronger circadian rhythm and increased clock neuron activity and excitability. Over-expression of endogenous fly Eph and RNAi-mediated knock-down of Eph and its ligand ephrin affected sleep architecture and neurophysiology. Eph over-expression led to stronger circadian morning anticipation while ephrin knock-down impaired memory. A dominant negative form of the GTPase Rho1, a potential intracellular effector of Eph, led to hyper-aroused flies, memory impairment, less anticipatory behaviour and neurophysiological changes. Our results demonstrate a role of Eph/ephrin signalling in a range of behaviours affected in AD. This presents a starting point for studies into the underlying mechanisms of AD including interactions with other AD-associated genes, like Rho1, Ankyrin, Tau and APP with the potential to identify new targets for treatment.

Hereditary spastic paraparesis presenting as cerebral palsy due to ADD3 variant with mechanistic insight provided by a Drosophila γ-adducin model.

Cerebral palsy (CP) causes neurological disability in early childhood. Hypoxic-ischaemic injury plays a major role in its aetiology, nevertheless, genetic and epigenetic factors may contribute to the clinical presentation. Mutations in ADD3 (encoding γ-adducin) gene have been described in a monogenic form of spastic quadriplegic cerebral palsy (OMIM 601568). We studied a 16-year-old male with spastic diplegia. Several investigations including neurometabolic testing, brain and spine magnetic resonance imaging (MRI) and CGH-Array were normal. Further, clinical genetics assessment and whole exome sequencing (WES) gave the diagnosis. We generated an animal model using Drosophila to study the effects of γ-adducin loss and gain of function. WES revealed a biallelic variant in the ADD3 gene, NM_016824.5(ADD3): c.1100G > A, p.(Gly367Asp). Mutations in this gene have been described as an ultra-rare autosomal recessive, which is a known form of inherited cerebral palsy. Molecular modelling suggests that this mutation leads to a loss of structural integrity of γ-adducin and is therefore expected to result in a decreased level of functional protein. Pan-neuronal over-expression or knock-down of the Drosophila ortholog of ADD3 called hts caused a reduction of life span and impaired locomotion thereby phenocopying aspects of the human disease. Our animal experiments present a starting point to understand the biological processes underpinning the clinical phenotype and pathogenic mechanisms, to gain insights into potential future methods for treating or preventing ADD3 related spastic quadriplegic cerebral palsy.

The Drosophila ortholog of the schizophrenia-associated CACNA1A and CACNA1B voltage-gated calcium channels regulate memory, sleep and circadian rhythms.

Schizophrenia exhibits up to 80% heritability. A number of genome wide association studies (GWAS) have repeatedly shown common variants in voltage-gated calcium (Cav) channel genes CACNA1C, CACNA1I and CACNA1G have a major contribution to the risk of the disease. More recently, studies using whole exome sequencing have also found that CACNA1B (Cav2.2 N-type) deletions and rare disruptive variants in CACNA1A (Cav2.1 P/Q-type) are associated with schizophrenia. The negative symptoms of schizophrenia include behavioural defects such as impaired memory, sleep and circadian rhythms. It is not known how variants in schizophrenia-associated genes contribute to cognitive and behavioural symptoms, thus hampering the development of treatment for schizophrenia symptoms. In order to address this knowledge gap, we studied behavioural phenotypes in a number of loss of function mutants for the Drosophila ortholog of the Cav2 gene family called cacophony (cac). cac mutants showed several behavioural features including decreased night-time sleep and hyperactivity similar to those reported in human patients. The change in timing of sleep-wake cycles suggested disrupted circadian rhythms, with the loss of night-time sleep being caused by loss of cac just in the circadian clock neurons. These animals also showed a reduction in rhythmic circadian behaviour a phenotype that also could be mapped to the central clock. Furthermore, reduction of cac just in the clock resulted in a lengthening of the 24 h period. In order to understand how loss of Cav2 function may lead to cognitive deficits and underlying cellular pathophysiology we targeted loss of function of cac to the memory centre of the fly, called the mushroom bodies (MB). This manipulation was sufficient to cause reduction in both short- and intermediate-term associative memory. Memory impairment was accompanied by a decrease in Ca2+ transients in response to a depolarizing stimulus, imaged in the MB presynaptic terminals. This work shows loss of cac Cav2 channel function alone causes a number of cognitive and behavioural deficits and underlying reduced neuronal Ca2+ transients, establishing Drosophila as a high-throughput in vivo genetic model to study the Cav channel pathophysiology related to schizophrenia.

Impaired Pre-Motor Circuit Activity and Movement in a Drosophila Model of KCNMA1-Linked Dyskinesia.

BACKGROUND: Paroxysmal dyskinesias (PxDs) are characterized by involuntary movements and altered pre-motor circuit activity. Causative mutations provide a means to understand the molecular basis of PxDs. Yet in many cases, animal models harboring corresponding mutations are lacking. Here we utilize the fruit fly, Drosophila, to study a PxD linked to a gain-of-function (GOF) mutation in the KCNMA1/hSlo1 BK potassium channel. OBJECTIVES: We aimed to recreate the equivalent BK (big potassium) channel mutation in Drosophila. We sought to determine how this mutation altered action potentials (APs) and synaptic release in vivo; to test whether this mutation disrupted pre-motor circuit function and locomotion; and to define neural circuits involved in locomotor disruption. METHODS: We generated a knock-in Drosophila model using homologous recombination. We used electrophysiological recordings and calcium-imaging to assess AP shape, neurotransmission, and the activity of the larval pre-motor central pattern generator (CPG). We used video-tracking and automated systems to measure movement, and developed a genetic method to limit BK channel expression to defined circuits. RESULTS: Neuronal APs exhibited reduced width and an enhanced afterhyperpolarization in the PxD model. We identified calcium-dependent reductions in neurotransmitter release, dysfunction of the CPG, and corresponding alterations in movement, in model larvae. Finally, we observed aberrant locomotion and dyskinesia-like movements in adult model flies, and partially mapped the impact of GOF BK channels on movement to cholinergic neurons. CONCLUSION: Our model supports a link between BK channel GOF and hyperkinetic movements, and provides a platform to dissect the mechanistic basis of PxDs. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Drosophila Ionotropic Receptor 25a mediates circadian clock resetting by temperature.

Circadian clocks are endogenous timers adjusting behaviour and physiology with the solar day. Synchronized circadian clocks improve fitness and are crucial for our physical and mental well-being. Visual and non-visual photoreceptors are responsible for synchronizing circadian clocks to light, but clock-resetting is also achieved by alternating day and night temperatures with only 2-4 °C difference. This temperature sensitivity is remarkable considering that the circadian clock period (~24 h) is largely independent of surrounding ambient temperatures. Here we show that Drosophila Ionotropic Receptor 25a (IR25a) is required for behavioural synchronization to low-amplitude temperature cycles. This channel is expressed in sensory neurons of internal stretch receptors previously implicated in temperature synchronization of the circadian clock. IR25a is required for temperature-synchronized clock protein oscillations in subsets of central clock neurons. Extracellular leg nerve recordings reveal temperature- and IR25a-dependent sensory responses, and IR25a misexpression confers temperature-dependent firing of heterologous neurons. We propose that IR25a is part of an input pathway to the circadian clock that detects small temperature differences. This pathway operates in the absence of known 'hot' and 'cold' sensors in the Drosophila antenna, revealing the existence of novel periphery-to-brain temperature signalling channels.

Shaw and Shal voltage-gated potassium channels mediate circadian changes in Drosophila clock neuron excitability.

As in mammals, Drosophila circadian clock neurons display rhythms of activity with higher action potential firing rates and more positive resting membrane potentials during the day. This rhythmic excitability has been widely observed but, critically, its regulation remains unresolved. We have characterized and modelled the changes underlying these electrical activity rhythms in the lateral ventral clock neurons (LNvs). We show that currents mediated by the voltage-gated potassium channels Shaw (Kv3) and Shal (Kv4) oscillate in a circadian manner. Disruption of these channels, by expression of dominant negative (DN) subunits, leads to changes in circadian locomotor activity and shortens lifespan. LNv whole-cell recordings then show that changes in Shaw and Shal currents drive changes in action potential firing rate and that these rhythms are abolished when the circadian molecular clock is stopped. A whole-cell biophysical model using Hodgkin-Huxley equations can recapitulate these changes in electrical activity. Based on this model and by using dynamic clamp to manipulate clock neurons directly, we can rescue the pharmacological block of Shaw and Shal, restore the firing rhythm, and thus demonstrate the critical importance of Shaw and Shal. Together, these findings point to a key role for Shaw and Shal in controlling circadian firing of clock neurons and show that changes in clock neuron currents can account for this. Moreover, with dynamic clamp we can switch the LNvs between morning-like and evening-like states of electrical activity. We conclude that changes in Shaw and Shal underlie the daily oscillation in LNv firing rate.

Neuronal overexpression of Alzheimer's disease and Down's syndrome associated DYRK1A/minibrain gene alters motor decline, neurodegeneration and synaptic plasticity in Drosophila.

Down syndrome (DS) is characterised by abnormal cognitive and motor development, and later in life by progressive Alzheimer's disease (AD)-like dementia, neuropathology, declining motor function and shorter life expectancy. It is caused by trisomy of chromosome 21 (Hsa21), but how individual Hsa21 genes contribute to various aspects of the disorder is incompletely understood. Previous work has demonstrated a role for triplication of the Hsa21 gene DYRK1A in cognitive and motor deficits, as well as in altered neurogenesis and neurofibrillary degeneration in the DS brain, but its contribution to other DS phenotypes is unclear. Here we demonstrate that overexpression of minibrain (mnb), the Drosophila ortholog of DYRK1A, in the Drosophila nervous system accelerated age-dependent decline in motor performance and shortened lifespan. Overexpression of mnb in the eye was neurotoxic and overexpression in ellipsoid body neurons in the brain caused age-dependent neurodegeneration. At the larval neuromuscular junction, an established model for mammalian central glutamatergic synapses, neuronal mnb overexpression enhanced spontaneous vesicular transmitter release. It also slowed recovery from short-term depression of evoked transmitter release induced by high-frequency nerve stimulation and increased the number of boutons in one of the two glutamatergic motor neurons innervating the muscle. These results provide further insight into the roles of DYRK1A triplication in abnormal aging and synaptic dysfunction in DS.

Alzheimer's disease-associated tau alters Drosophila circadian activity, sleep and clock neuron electrophysiology.

Alzheimer's disease (AD) is the most common cause of dementia, which is associated with an enormous personal, social and economic burden worldwide. However, there are few current treatments with none of them targeting the underlying causes of the disease. Sleep and circadian rhythm defects are not only distressing symptoms of AD and other tauopathies and are a leading cause of care home admission but are also thought to accelerate AD pathology. Despite this, little is understood about the underlying causes of these behavioural changes. Expression of the 0N4R isoform of tau has been associated with AD pathology and we show that expressing it in the Drosophila clock network gives rise to circadian and sleep phenotypes which closely match the behavioural changes seen in human AD patients. Tauopathic flies exhibited greater locomotor activity throughout the day and night and displayed a loss of sleep, particularly at night. Under constant darkness, the locomotor behaviour of tau-expressing flies was less rhythmic than controls indicating a defect in their intrinsic circadian rhythm. Current clamp recordings from wake-promoting, pigment dispersing factor (PDF)-positive large lateral ventral clock neurons (l-LNvs) revealed elevated spontaneous firing throughout the day and night which likely underlies the observed hyperactive circadian phenotype. Interestingly, expression of tau in only the PDF-positive pacemaker neurons, which are thought to be the most important for behaviour under constant conditions, was not sufficient or even necessary to affect circadian rhythmicity. This work establishes Drosophila as a model to investigate interactions between human pathological versions of tau and the machinery that controls neuronal excitability, allowing the identification of underlying mechanisms of disease that may reveal new therapeutic targets.

CELLULAR IMAGING OF THE TAPETAL-LIKE REFLEX IN CARRIERS OF RPGR-ASSOCIATED RETINOPATHY.

PURPOSE: To examine the features of the tapetal-like reflex (TLR) in female carriers of RPGR-associated retinopathy by means of adaptive optics scanning light ophthalmoscopy (AOSLO) and spectral domain optical coherence tomography. METHODS: Nine molecularly confirmed RPGR carriers and three healthy controls underwent ocular examination and the following retinal imaging modalities: color photography, near-infrared reflectance, fundus autofluorescence, spectral domain optical coherence tomography, and AOSLO. After identifying TLR areas across all imaging modalities, normalized local contrast of outer retinal bands on spectral domain optical coherence tomography was calculated and AOSLO-acquired photoreceptor mosaic analysis was performed. RESULTS: Seven carriers had TLR areas, which colocalized with increased rod photoreceptor reflectivity on confocal AOSLO and reduced cone photoreceptor densities. Parafoveal TLR areas also exhibited reduced local contrast (i.e., increased reflectivity) of the outer retinal bands on spectral domain optical coherence tomography (inner segment ellipsoid zone and outer segment interdigitation zone). Healthy controls did not show TLR. CONCLUSION: The cellular resolution provided by AOSLO affords the characterization of the photoreceptor mosaic in RPGR carriers with a TLR. Features revealed include reduced cone density, increased cone inner segment diameter, and increased rod outer segment reflectivity.

Quasimodo mediates daily and acute light effects on Drosophila clock neuron excitability.

We have characterized a light-input pathway regulating Drosophila clock neuron excitability. The molecular clock drives rhythmic electrical excitability of clock neurons, and we show that the recently discovered light-input factor Quasimodo (Qsm) regulates this variation, presumably via an Na+, K+, Cl- cotransporter (NKCC) and the Shaw K+ channel (dKV3.1). Because of light-dependent degradation of the clock protein Timeless (Tim), constant illumination (LL) leads to a breakdown of molecular and behavioral rhythms. Both overexpression (OX) and knockdown (RNAi) of qsm, NKCC, or Shaw led to robust LL rhythmicity. Whole-cell recordings of the large ventral lateral neurons (l-LNv) showed that altering Qsm levels reduced the daily variation in neuronal activity: qsmOX led to a constitutive less active, night-like state, and qsmRNAi led to a more active, day-like state. Qsm also affected daily changes in K+ currents and the GABA reversal potential, suggesting a role in modifying membrane currents and GABA responses in a daily fashion, potentially modulating light arousal and input to the clock. When directly challenged with blue light, wild-type l-LNvs responded with increased firing at night and no net response during the day, whereas altering Qsm, NKKC, or Shaw levels abolished these day/night differences. Finally, coexpression of ShawOX and NKCCRNAi in a qsm mutant background restored LL-induced behavioral arrhythmicity and wild-type neuronal activity patterns, suggesting that the three genes operate in the same pathway. We propose that Qsm affects both daily and acute light effects in l-LNvs probably acting on Shaw and NKCC.

A third copy of the Down syndrome cell adhesion molecule (Dscam) causes synaptic and locomotor dysfunction in Drosophila.

Down syndrome (DS) is caused by triplication of chromosome 21 (HSA21). It is characterised by intellectual disability and impaired motor coordination that arise from changes in brain volume, structure and function. However, the contribution of each HSA21 gene to these various phenotypes and to the causal alterations in neuronal and synaptic structure and function are largely unknown. Here we have investigated the effect of overexpression of the HSA21 gene DSCAM (Down syndrome cell adhesion molecule), on glutamatergic synaptic transmission and motor coordination, using Drosophila expressing three copies of Dscam1. Electrophysiological recordings of miniature and evoked excitatory junction potentials at the glutamatergic neuromuscular junction of Drosophila larvae showed that the extra copy of Dscam1 changed the properties of spontaneous and electrically-evoked transmitter release and strengthened short-term synaptic depression during high-frequency firing of the motor nerve. Behavioural analyses uncovered impaired locomotor coordination despite preserved gross motor function. This work identifies DSCAM as a candidate causative gene in DS that is sufficient to modify synaptic transmission and synaptic plasticity and cause a DS behavioural phenotype.

State-dependent judgement bias in Drosophila: evidence for evolutionarily primitive affective processes.

Affective states influence decision-making under ambiguity in humans and other animals. Individuals in a negative state tend to interpret ambiguous cues more negatively than individuals in a positive state. We demonstrate that the fruit fly, Drosophila melanogaster, also exhibits state-dependent changes in cue interpretation. Drosophila were trained on a Go/Go task to approach a positive (P) odour associated with a sugar reward and actively avoid a negative (N) odour associated with shock. Trained flies were then either shaken to induce a purported negative state or left undisturbed (control), and given a choice between: air or P; air or N; air or ambiguous odour (1 : 1 blend of P : N). Shaken flies were significantly less likely to approach the ambiguous odour than control flies. This 'judgement bias' may be mediated by changes in neural activity that reflect evolutionarily primitive affective states. We cannot say whether such states are consciously experienced, but use of this model organism's versatile experimental tool kit may facilitate elucidation of their neural and genetic basis.

QUANTITATIVE ANALYSIS OF HYPERAUTOFLUORESCENT RINGS TO CHARACTERIZE THE NATURAL HISTORY AND PROGRESSION IN RPGR-ASSOCIATED RETINOPATHY.

PURPOSE: Quantitative analysis of hyperautofluorescent rings and progression in subjects with retinitis pigmentosa associated with retinitis pigmentosa GTPase regulator (RPGR) gene mutations. METHODS: Prospective observational study of 46 subjects. Ring area, horizontal and vertical diameter measurements taken from outer and inner ring borders. Intraobserver repeatability, baseline measurements, progression rates, interocular symmetry, and association with age and genotype were investigated. RESULTS: Baseline ring area was 11.8 ± 13.4 mm and 11.4 ± 13.2 mm for right and left eyes, respectively, with very strong interocular correlation (r = 0.9398; P < 0.0001). Ring area constriction was 1.5 ± 2.0 mm/year and 1.3 ± 1.9 mm/year for right and left eyes, respectively, with very strong interocular correlation (r = 0.878, P < 0.0001). Baseline ring area and constriction rate correlated negatively with age (r = -0.767; P < 0.0001 and r = -0.644, P < 0.0001, respectively). Constriction rate correlated strongly with baseline area (r = 0.850, P < 0.0001). Age, but not genotype, exerted a significant effect on constriction rates (P < 0.0001), with greatest rates of progression seen in younger subjects. An exponential decline overall was found. CONCLUSION: This study provides disease-specific baseline values and progression rates together with a repeatability assessment of fundus autofluorescence metrics. Our findings can guide future treatment trials and contribute to the clinical care of patients with RPGR-associated retinitis pigmentosa.

Drosophila PINK1 and parkin loss-of-function mutants display a range of non-motor Parkinson's disease phenotypes.

Parkinson's disease (PD) is more commonly associated with its motor symptoms and the related degeneration of dopamine (DA) neurons. However, it is becoming increasingly clear that PD patients also display a wide range of non-motor symptoms, including memory deficits and disruptions of their sleep-wake cycles. These have a large impact on their quality of life, and often precede the onset of motor symptoms, but their etiology is poorly understood. The fruit fly Drosophila has already been successfully used to model PD, and has been used extensively to study relevant non-motor behaviours in other contexts, but little attention has yet been paid to modelling non-motor symptoms of PD in this genetically tractable organism. We examined memory performance and circadian rhythms in flies with loss-of-function mutations in two PD genes: PINK1 and parkin. We found learning and memory abnormalities in both mutant genotypes, as well as a weakening of circadian rhythms that is underpinned by electrophysiological changes in clock neurons. Our study paves the way for further work that may help us understand the mechanisms underlying these neglected aspects of PD, thus identifying new targets for treatments to address these non-motor problems specifically and perhaps even to halt disease progression in its prodromal phase.

Isolation and characterisation of a novel trophoblast side-population from first trimester placentae.

The placenta is responsible for all nutrient and gas exchange between mother and baby during pregnancy. The differentiation of specialised placental epithelial cells called trophoblasts is essential for placental function, but we understand little about how these populations arise. Mouse trophoblast stem cells have allowed us to understand many of the factors that regulate murine trophoblast lineage development, but the human placenta is anatomically very different from the mouse, and it is imperative to isolate a human trophoblast stem cell to understand human placental development. Here we have developed a novel methodology to isolate a Hoechst side-population of trophoblasts from early gestation placentae and compared their transcriptome to differentiated trophoblast populations (cytotrophoblasts and extravillous trophoblasts) using microarray technology. Side-population trophoblasts clustered as a transcriptomically distinct population but were more closely related to cytotrophoblasts than extravillous trophoblasts. Side-population trophoblasts up-regulated a number of genes characteristic of trophectoderm and murine trophoblast stem cells in comparison to cytotrophoblasts or extravillous trophoblasts and could be distinguished from both of these more mature populations by a unique set of 22 up-regulated genes, which were enriched for morphogenesis and organ development and the regulation of growth functions. Cells expressing two of these genes (LAMA2 and COL6A3) were distributed throughout the cytotrophoblast layer at the trophoblast/mesenchymal interface. Comparisons to previously published trophoblast progenitor populations suggest that the side-population trophoblasts isolated in this work are a novel human trophoblast population. Future work will determine whether these cells exhibit functional progenitor/stem cell attributes.

The relationship between TGFß, low oxygen and the outgrowth of extravillous trophoblasts from anchoring villi during the first trimester of pregnancy.

BACKGROUND: During the first trimester of human pregnancy, specialised placental cells called extravillous trophoblasts (EVTs) grow out from anchoring villi, invade the maternal decidua and remodel the uterine spiral arteries. Inadequate EVT invasion is associated with pregnancy complications including intrauterine growth restriction (IUGR) and pre-eclampsia. During early pregnancy, the placenta exists in a physiologically normal low oxygen environment, which may regulate EVT outgrowth. One potential oxygen responsive regulator of EVTs is the transforming growth factor-beta (TGFß) family of cytokines. This work aimed to determine the role of TGFß1, ß2 and ß3 in regulating EVT outgrowth in the low oxygen environment of early pregnancy. RESULTS: Using a quantitative high-throughput first trimester villous explant model of EVT outgrowth we demonstrated no significant difference in the frequency of EVT outgrowth between explants treated with TGFß1, ß2 or ß3. However, explants treated with TGFß2, but not ß1 or ß3, produced EVT outgrowths with a significantly smaller area in comparison to untreated controls (p=0.03). When explants were cultured in 1.5% oxygen, TGFß2, but not ß1 or ß3, in the conditioned medium of explants that produced EVT outgrowth was significantly reduced in comparison to 8% oxygen (p<0.05). There was no significant difference in the concentration of TGFß2 or TGFß3 from isolated primary EVTs cultured in 1.5% or 8% oxygen. CONCLUSIONS: TGFß2 inhibits EVT outgrowth expansion from first trimester anchoring villi. As TGFß2 secretion from anchoring villi is down-regulated in low oxygen, these findings suggest that the low oxygen environment in early pregnancy may be important to allow EVT outgrowth expansion and promote adequate placentation.

Phase 1/2 AAV5-hRKp.RPGR (Botaretigene Sparoparvovec) Gene Therapy: Safety and Efficacy in RPGR-associated X-linked Retinitis Pigmentosa.

PURPOSE: To assess the safety and efficacy of AAV5-hRKp.RPGR in participants with retinitis pigmentosa GTPase regulator (RPGR)-associated X-linked retinitis pigmentosa (XLRP). DESIGN: Open-label, phase 1/2 dose escalation/expansion study (NCT03252847). METHODS: Males (≥5 years old) with XLRP-RPGR were evaluated. In the dose escalation phase, subretinal AAV5-hRKp.RPGR (low: 1.0×1011 vg/ml; intermediate: 2.0×1011 vg/ml; high: 4.0×1011 vg/ml) was administered to the poorer-seeing eye (n = 10). Dose confirmation (intermediate dose) was carried out in 3 pediatric participants. In the dose expansion phase, 36 participants were randomized 1:1:1 to immediate (low or intermediate dose) or deferred (control) treatment. The primary outcome was safety. Secondary efficacy outcomes included static perimetry, microperimetry, vision-guided mobility, best corrected visual acuity, and contrast sensitivity. Safety and efficacy outcomes were assessed for 52 weeks for immediate treatment participants and 26 weeks for control participants. RESULTS: AAV5-hRKp.RPGR was safe and well tolerated, with no reported dose-limiting events. Most adverse events (AEs) were transient and related to the surgical procedure, resolving without intervention. Two serious AEs were reported with immediate treatment (retinal detachment, uveitis). A third serious AE (increased intraocular pressure) was reported outside the reporting period. All ocular inflammation-related AEs responded to corticosteroids. Treatment with AAV5-hRKp.RPGR resulted in improvements in retinal sensitivity and functional vision compared with the deferred group at Week 26; similar trends were observed at Week 52. CONCLUSIONS: AAV5-hRKp.RPGR demonstrated an anticipated and manageable AE profile through 52 weeks. Safety and efficacy findings support investigation in a phase 3 trial.