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

BACKGROUND: 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 Cdkl5-/y 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 and plasticity would be observed in the hippocampus of Cdkl5-/y rats. METHODS: To allow cross-species comparisons of phenotypes associated with the loss of CDKL5, we generated a loss of function mutation in exon 8 of the rat Cdkl5 gene and assessed the impact of the loss of CDLK5 using a combination of extracellular and whole-cell electrophysiological recordings, biochemistry, and histology. RESULTS: Our results indicate that CA1 hippocampal long-term potentiation (LTP) is enhanced in slices prepared from juvenile, but not adult, Cdkl5-/y rats. Enhanced LTP does not result from changes in NMDA receptor function or subunit expression as these remain unaltered throughout development. Furthermore, Ca2+ permeable AMPA receptor mediated currents are unchanged in Cdkl5-/y 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 at Schaffer collateral to CA1 PC synapses. CONCLUSIONS: Our data indicate a role for CDKL5 in hippocampal synaptic function and raise the possibility that altered intracellular signalling rather than synaptic deficits contribute to the altered plasticity. LIMITATIONS: This study has focussed on the electrophysiological and anatomical properties of hippocampal CA1 PCs across early postnatal development. Studies involving other brain regions, older animals and behavioural phenotypes associated with the loss of CDKL5 are needed to understand the pathophysiology of CDD.

Efficiently recording and processing data from arbuscular mycorrhizal colonization assays using AMScorer and AMReader.

Arbuscular mycorrhizal (AM) fungi engage with land plants in a widespread, mutualistic endosymbiosis which provides their hosts with increased access to nutrients and enhanced biotic and abiotic stress resistance. The potential for reducing fertiliser use and improving crop resilience has resulted in rapidly increasing scientific interest. Microscopic quantification of the level of AM colonization is of fundamental importance to this research, however the methods for recording and processing these data are time-consuming and tedious. In order to streamline these processes, we have developed AMScorer, an easy-to-use Excel spreadsheet, which enables the user to record data rapidly during from microscopy-based assays, and instantly performs the subsequent data processing steps. In our hands, AMScorer has more than halved the time required for data collection compared to paper-based methods. Subsequently, we developed AMReader, a user-friendly R package, which enables easy visualization and statistical analyses of data from AMScorer. These tools require only limited skills in Excel and R, and can accelerate research into AM symbioses, help researchers with variable resources to conduct research, and facilitate the storage and sharing of data from AM colonization assays. They are available for download at https://github.com/EJarrattBarnham/AMReader, along with an extensive user manual.

Transfection in Primary Cultured Neuronal Cells.

Transfection allows the introduction of foreign nucleic acid into eukaryotic cells. It is an important tool in understanding the roles of NMDARs in neurons. Here we describe using lipofection-mediated transfection to introduce cDNA encoding NMDAR subunits into postmitotic rodent primary cortical neurons maintained in culture.

A receptor required for chitin perception facilitates arbuscular mycorrhizal associations and distinguishes root symbiosis from immunity.

Plants establish symbiotic associations with arbuscular mycorrhizal fungi (AMF) to facilitate nutrient uptake, particularly in nutrient-limited conditions. This partnership is rooted in the plant's ability to recognize fungal signaling molecules, such as chitooligosaccharides (chitin) and lipo-chitooligosaccharides. In the legume Medicago truncatula, chitooligosaccharides trigger both symbiotic and immune responses via the same lysin-motif-receptor-like kinases (LysM-RLKs), notably CERK1 and LYR4. The nature of plant-fungal engagement is opposite according to the outcomes of immunity or symbiosis signaling, and as such, discrimination is necessary, which is challenged by the dual roles of CERK1/LYR4 in both processes. Here, we describe a LysM-RLK, LYK8, that is functionally redundant with CERK1 for mycorrhizal colonization but is not involved in chitooligosaccharides-induced immunity. Genetic mutation of both LYK8 and CERK1 blocks chitooligosaccharides-triggered symbiosis signaling, as well as mycorrhizal colonization, but shows no further impact on immunity signaling triggered by chitooligosaccharides, compared with the mutation of CERK1 alone. LYK8 interacts with CERK1 and forms a receptor complex that appears essential for chitooligosaccharides activation of symbiosis signaling, with the lyk8/cerk1 double mutant recapitulating the impact of mutations in the symbiosis signaling pathway. We conclude that this novel receptor complex allows chitooligosaccharides activation specifically of symbiosis signaling and helps the plant to differentiate between activation of these opposing signaling processes.

Timely Questions Emerging in Chronobiology: The Circadian Clock Keeps on Ticking.

Chronobiology investigations have revealed much about cellular and physiological clockworks but we are far from having a complete mechanistic understanding of the physiological and ecological implications. Here we present some unresolved questions in circadian biology research as posed by the editorial staff and guest contributors to the Journal of Circadian Rhythms. This collection of ideas is not meant to be comprehensive but does reveal the breadth of our observations on emerging trends in chronobiology and circadian biology. It is amazing what could be achieved with various expected innovations in technologies, techniques, and mathematical tools that are being developed. We fully expect strengthening mechanistic work will be linked to health care and environmental understandings of circadian function. Now that most clock genes are known, linking these to physiological, metabolic, and developmental traits requires investigations from the single molecule to the terrestrial ecological scales. Real answers are expected for these questions over the next decade. Where are the circadian clocks at a cellular level? How are clocks coupled cellularly to generate organism level outcomes? How do communities of circadian organisms rhythmically interact with each other? In what way does the natural genetic variation in populations sculpt community behaviors? How will methods development for circadian research be used in disparate academic and commercial endeavors? These and other questions make it a very exciting time to be working as a chronobiologist.

Reflections on Several Landmark Advances in Circadian Biology.

Circadian Biology intersects with diverse scientific domains, intricately woven into the fabric of organismal physiology and behavior. The rhythmic orchestration of life by the circadian clock serves as a focal point for researchers across disciplines. This retrospective examination delves into several of the scientific milestones that have fundamentally shaped our contemporary understanding of circadian rhythms. From deciphering the complexities of clock genes at a cellular level to exploring the nuances of coupled oscillators in whole organism responses to stimuli. The field has undergone significant evolution lately guided by genetics approaches. Our exploration here considers key moments in the circadian-research landscape, elucidating the trajectory of this discipline with a keen eye on scientific advancements and paradigm shifts.

Laboratory Automated Interrogation of Data: an interactive web application for visualization of multilevel data from biological experiments.

A key step in understanding the results of biological experiments is visualization of the data. Many laboratory experiments contain a range of measurements that exist within a hierarchy of interdependence. An automated and facile way to visualize and interrogate such multilevel data, across many experimental variables, would (i) lead to improved understanding of the results, (ii) help to avoid misleading interpretation of statistics and (iii) easily identify outliers and sources of batch and confounding effects. While many excellent graphing solutions already exist, they are often geared towards the production of publication-ready plots and the analysis of a single variable at a time, require programming expertise or are unnecessarily complex for the task at hand. Here, we present Laboratory Automated Interrogation of Data (LAB-AID), an interactive tool specifically designed to automatically visualize and query hierarchical data resulting from biological experiments.

Circadian and daily rhythms of disease vector mosquitoes.

Mosquitoes express a rich repertoire of daily 24-hour rhythms in biochemistry, physiology, and behavior. The nocturnal Anopheles and Culex and diurnal Aedes mosquitoes are major vectors of human disease, transmitting parasites and arboviruses, such as malaria and dengue. In this review, we explore the role that 24-hour diel and circadian rhythms play in shaping the temporal life of the mosquito. We focus on recent advances in our understanding of behavioral rhythms, focusing on locomotor/flight activity, host-seeking, biting/blood feeding, and mating. We examine the molecular circadian clock, photocycle, and light signals, which in combination shape the mosquito 24-hour temporal program. We address species- and sex-specific differences and highlight important selective pressures from dynamically changing environments. This work also provides new insights into disease transmission, insect control, and future experimental design.

The peptide GOLVEN10 alters root development and noduletaxis in Medicago truncatula.

The conservation of GOLVEN (GLV)/ROOT MERISTEM GROWTH FACTOR (RGF) peptide encoding genes across plant genomes capable of forming roots or root-like structures underscores their potential significance in the terrestrial adaptation of plants. This study investigates the function and role of GOLVEN peptide-coding genes in Medicago truncatula. Five out of fifteen GLV/RGF genes were notably upregulated during nodule organogenesis and were differentially responsive to nitrogen deficiency and auxin treatment. Specifically, the expression of MtGLV9 and MtGLV10 at nodule initiation sites was contingent upon the NODULE INCEPTION transcription factor. Overexpression of these five nodule-induced GLV genes in hairy roots of M. truncatula and application of their synthetic peptide analogues led to a decrease in nodule count by 25-50%. Uniquely, the GOLVEN10 peptide altered the positioning of the first formed lateral root and nodule on the primary root axis, an observation we term 'noduletaxis'; this decreased the length of the lateral organ formation zone on roots. Histological section of roots treated with synthetic GOLVEN10 peptide revealed an increased cell number within the root cortical cell layers without a corresponding increase in cell length, leading to an elongation of the root likely introducing a spatiotemporal delay in organ formation. At the transcription level, the GOLVEN10 peptide suppressed expression of microtubule-related genes and exerted its effects by changing expression of a large subset of Auxin responsive genes. These findings advance our understanding of the molecular mechanisms by which GOLVEN peptides modulate root morphology, nodule ontogeny, and interactions with key transcriptional pathways.

Light-sensitive short hypocotyl genes confer symbiotic nodule identity in the legume Medicago truncatula.

Legumes produce specialized root nodules that are distinct from lateral roots in morphology and function, with nodules intracellularly hosting nitrogen-fixing bacteria. We have previously shown that a lateral root program underpins nodule initiation, but there must be additional developmental regulators that confer nodule identity. Here, we show two members of the LIGHT-SENSITIVE SHORT HYPOCOTYL (LSH) transcription factor family, predominantly known to define shoot meristem complexity and organ boundaries, function as regulators of nodule organ identity. In parallel to the root initiation program, LSH1/LSH2 recruit a program into the root cortex that mediates the divergence into nodules, in particular with cell divisions in the mid-cortex. This includes regulation of auxin and cytokinin, promotion of NODULE ROOT1/2 and Nuclear Factor YA1, and suppression of the lateral root program. A principal outcome of LSH1/LSH2 function is the production of cells able to accommodate nitrogen-fixing bacteria, a key feature unique to nodules.

Inhibiting CSF1R alleviates cerebrovascular white matter disease and cognitive impairment.

White matter abnormalities, related to poor cerebral perfusion, are a core feature of small vessel cerebrovascular disease, and critical determinants of vascular cognitive impairment and dementia. Despite this importance there is a lack of treatment options. Proliferation of microglia producing an expanded, reactive population and associated neuroinflammatory alterations have been implicated in the onset and progression of cerebrovascular white matter disease, in patients and in animal models, suggesting that targeting microglial proliferation may exert protection. Colony-stimulating factor-1 receptor (CSF1R) is a key regulator of microglial proliferation. We found that the expression of CSF1R/Csf1r and other markers indicative of increased microglial abundance are significantly elevated in damaged white matter in human cerebrovascular disease and in a clinically relevant mouse model of chronic cerebral hypoperfusion and vascular cognitive impairment. Using the mouse model, we investigated long-term pharmacological CSF1R inhibition, via GW2580, and demonstrated that the expansion of microglial numbers in chronic hypoperfused white matter is prevented. Transcriptomic analysis of hypoperfused white matter tissue showed enrichment of microglial and inflammatory gene sets, including phagocytic genes that were the predominant expression modules modified by CSF1R inhibition. Further, CSF1R inhibition attenuated hypoperfusion-induced white matter pathology and rescued spatial learning impairments and to a lesser extent cognitive flexibility. Overall, this work suggests that inhibition of CSF1R and microglial proliferation mediates protection against chronic cerebrovascular white matter pathology and cognitive deficits. Our study nominates CSF1R as a target for the treatment of vascular cognitive disorders with broader implications for treatment of other chronic white matter diseases.

General anesthesia alters CNS and astrocyte expression of activity-dependent and activity-independent genes.

General anesthesia represents a common clinical intervention and yet can result in long-term adverse CNS effects particularly in the elderly or dementia patients. Suppression of cortical activity is a key feature of the anesthetic-induced unconscious state, with activity being a well-described regulator of pathways important for brain health. However, the extent to which the effects of anesthesia go beyond simple suppression of neuronal activity is incompletely understood. We found that general anesthesia lowered cortical expression of genes induced by physiological activity in vivo, and recapitulated additional patterns of gene regulation induced by total blockade of firing activity in vitro, including repression of neuroprotective genes and induction of pro-apoptotic genes. However, the influence of anesthesia extended beyond that which could be accounted for by activity modulation, including the induction of non activity-regulated genes associated with inflammation and cell death. We next focused on astrocytes, important integrators of both neuronal activity and inflammatory signaling. General anesthesia triggered gene expression changes consistent with astrocytes being in a low-activity environment, but additionally caused induction of a reactive profile, with transcriptional changes enriched in those triggered by stroke, neuroinflammation, and Aß/tau pathology. Thus, while the effects of general anesthesia on cortical gene expression are consistent with the strong repression of brain activity, further deleterious effects are apparent including a reactive astrocyte profile.

"In the shape of a cooking pot over the fire": Records of solar prominences in the 1180s.

The second half of the 1180s witnessed an unusual number of solar eclipses visible within Europe in quick succession. These were recorded or referenced in a wide range of sources, from chronicles in Latin and Old Church Slavonic to the earliest epic poem from the medieval Rus'. A comparison between key elements of these accounts reveals several notable features. First, the identification of solar prominences. The account of the 1185 total eclipse from the Rus' Laurentian Chronicle is well established in this context as the first probable textual witness to the phenomenon in Europe. It may not be the only one, however. A similar identification can be made within a Latin chronicle from England, by Gervase, monk of the community at Christ Church Cathedral Priory, for the total eclipse of 1187. Second, the contemporaneous nature of the descriptions is noteworthy, and more so in comparison with other contemporary accounts. A third area for focus concerns the nature of the observations and a case-study of Gervase of Canterbury who, if not an eye-witness to what he records, includes generally accurate accounts. These make his occasional inaccuracies all the more intriguing. Fourth, the wider comparison highlights the importance of taking account historical records across the European medieval heritage, Slavic and Orthodox alongside Latin and Catholic traditions.

Human astrocytes and microglia show augmented ingestion of synapses in Alzheimer's disease via MFG-E8.

Synapse loss correlates with cognitive decline in Alzheimer's disease (AD). Data from mouse models suggests microglia are important for synapse degeneration, but direct human evidence for any glial involvement in synapse removal in human AD remains to be established. Here we observe astrocytes and microglia from human brains contain greater amounts of synaptic protein in AD compared with non-disease controls, and that proximity to amyloid-ß plaques and the APOE4 risk gene exacerbate this effect. In culture, mouse and human astrocytes and primary mouse and human microglia phagocytose AD patient-derived synapses more than synapses from controls. Inhibiting interactions of MFG-E8 rescues the elevated engulfment of AD synapses by astrocytes and microglia without affecting control synapse uptake. Thus, AD promotes increased synapse ingestion by human glial cells at least in part via an MFG-E8 opsonophagocytic mechanism with potential for targeted therapeutic manipulation.

The consequences of neurodegenerative disease on neuron-astrocyte metabolic and redox interactions.

Brain metabolic pathways relating to bioenergetic and redox homeostasis are closely linked, and deficits in these pathways are thought to occur in many neurodegenerative diseases. Astrocytes play important roles in both processes, and growing evidence suggests that neuron-astrocyte intercellular signalling ensures brain bioenergetic and redox homeostasis in health. Moreover, alterations to this crosstalk have been observed in the context of neurodegenerative pathology. In this review, we summarise the current understanding of how neuron-astrocyte interactions influence brain metabolism and antioxidant functions in health as well as during neurodegeneration. It is apparent that deleterious and adaptive protective responses alter brain metabolism in disease, and that knowledge of both may illuminate targets for future therapeutic interventions.

Differential splicing choices made by neurons and astrocytes and their importance when investigating signal-dependent alternative splicing in neural cells.

A variety of proteins can be encoded by a single gene via the differential splicing of exons. In neurons this form of alternative splicing can be controlled by activity-dependent calcium signaling, leading to the properties of proteins being altered, including ion channels, neurotransmitter receptors and synaptic cell adhesion molecules. The pre-synaptic cell adhesion molecule Neurexin 1 (Nrxn1) is alternatively spliced at splice-site 4 (SS4) which governs exon 22 inclusion (SS4+) and consequently postsynaptic NMDA receptor responses. Nrxn1 was reported to be subject to a delayed-onset shift in Nrxn1 SS4 splicing resulting in increased exon 22 inclusion, involving epigenetic mechanisms which, if disrupted, reduce memory stability. Exon inclusion at SS4 represented one of hundreds of exons reported to be subject to a genome-wide shift in fractional exon inclusion following membrane depolarization with high extracellular K+ that was delayed in onset. We report that high K+ does not increase the SS4+/SS4- ratio in cortical neurons, but does induce a delayed-onset NMDA receptor-dependent neuronal death. In mixed neuronal/astrocyte cultures this neuronal death results in an increase in the astrocyte: neuron ratio, and a misleading increase in SS4+/SS4- ratio attributable to astrocytes having a far higher SS4+/SS4- ratio than neurons, rather than any change in the neurons themselves. We reassessed the previously reported genome-wide delayed-onset shift in fractional exon inclusion after high K+ exposure. This revealed that the reported changes correlated strongly with differences in exon inclusion level between astrocytes and neurons, and was accompanied by a strong decrease in the ratio of neuron-specific: astrocyte-specific gene expression. As such, these changes can be explained by the neurotoxic nature of the stimulation paradigm, underlining the importance of NMDA receptor blockade when using high K+ depolarizing stimuli.

Functional roles of reactive astrocytes in neuroinflammation and neurodegeneration.

Despite advances in uncovering the mechanisms that underlie neuroinflammation and neurodegenerative disease, therapies that prevent neuronal loss remain elusive. Targeting of disease-defining markers in conditions such as Alzheimer disease (amyloid-ß and tau) or Parkinson disease (α-synuclein) has been met with limited success, suggesting that these proteins do not act in isolation but form part of a pathological network. This network could involve phenotypic alteration of multiple cell types in the CNS, including astrocytes, which have a major neurosupportive, homeostatic role in the healthy CNS but adopt reactive states under acute or chronic adverse conditions. Transcriptomic studies in human patients and disease models have revealed the co-existence of many putative reactive sub-states of astrocytes. Inter-disease and even intra-disease heterogeneity of reactive astrocytic sub-states are well established, but the extent to which specific sub-states are shared across different diseases is unclear. In this Review, we highlight how single-cell and single-nuclei RNA sequencing and other 'omics' technologies can enable the functional characterization of defined reactive astrocyte states in various pathological scenarios. We provide an integrated perspective, advocating cross-modal validation of key findings to define functionally important sub-states of astrocytes and their triggers as tractable therapeutic targets with cross-disease relevance.

Astrocyte-oligodendrocyte interaction regulates central nervous system regeneration.

Failed regeneration of myelin around neuronal axons following central nervous system damage contributes to nerve dysfunction and clinical decline in various neurological conditions, for which there is an unmet therapeutic demand. Here, we show that interaction between glial cells - astrocytes and mature myelin-forming oligodendrocytes - is a determinant of remyelination. Using in vivo/ ex vivo/ in vitro rodent models, unbiased RNA sequencing, functional manipulation, and human brain lesion analyses, we discover that astrocytes support the survival of regenerating oligodendrocytes, via downregulation of the Nrf2 pathway associated with increased astrocytic cholesterol biosynthesis pathway activation. Remyelination fails following sustained astrocytic Nrf2 activation in focally-lesioned male mice yet is restored by either cholesterol biosynthesis/efflux stimulation, or Nrf2 inhibition using the existing therapeutic Luteolin. We identify that astrocyte-oligodendrocyte interaction regulates remyelination, and reveal a drug strategy for central nervous system regeneration centred on targeting this interaction.

Potassium Phosphonate Induces Resistance in Sweet Chestnut against Ink Disease Caused by Phytophthora Species.

Ink disease, caused by Phytophthora spp., represents a serious threat to sweet chestnuts throughout their distribution area. Among the control strategies, new perspectives have been offered by using potassium phosphonate, which indirectly controls Phytophthora diseases by acting on both host physiology and host-pathogen interactions. In this study, we tested in planta the effectiveness of trunk injection with K-phosphonate against seven different Phytophthora species associated with ink disease. For the two most aggressive species, P. cinnamomi and P. ×cambivora, the treatments were repeated at two different environmental conditions (a mean temperature of 14.5 °C vs. 25 °C) and tree phenology stages. The results obtained in this study demonstrated that K-phosphonate could contain the development of Phytophthora infection in phloem tissues. However, its effectiveness varied based on the concentration applied and the Phytophthora species tested. A concentration of 280 g/L of K-phosphonate was the most effective, and in some cases, callus formation around the necrotic lesion was detected. Overall, this study broadens the knowledge of endotherapic treatments with K-phosphonate as an effective measure for managing chestnut ink disease. Interestingly, the increase in mean temperature had a positive impact on the development of P. cinnamomi lesions on chestnut phloem tissues.

Physiological and cannabinoid responses of hemp (Cannabis sativa) to rock phosphate dust under tropical conditions.

Growing a high-value crop such as industrial hemp (Cannabis sativa L.) in post-mining environments is economically and environmentally attractive but faces a range of biotic and abiotic challenges. An opportunity to investigate the cultivation of C. sativa presented itself as part of post-mining activities on Christmas Island (Australia) to profitably utilise disused phosphate (PS) quarries. Challenges to plant growth and cadmium (Cd) uptake were addressed in this study using potted plants under fully controlled conditions in a growth chamber. A complete nutritional spectrum, slow-release fertiliser was applied to all plants as a control treatment, and two levels of rock PS dust, a waste product of PS mining that contains 35% phosphorus (P) and 40ppm of naturally occurring Cd, were applied at 54 and 162gL-1 . After 12weeks, control plants (no PS dust) significantly differed in phenological development, with no flower production, lower aboveground biomass and reduced photosynthesis efficiency than those with P applied as rock dust. Compared with the controls, the 54gL-1 level of P dust increased shoot biomass by 38%, while 162gL-1 increased shoot biomass by 85%. The concentration of Δ9 -tetrahydrocannabinol also increased with the higher P levels. Cd uptake from PS dust by C. sativa was substantial and warrants further investigation. However, there was no increase in Cd content between the 54 and 162gL-1 application rates in seed and leaf. Results indicate that hemp could become a high-value crop on Christmas Island, with the readily available rock PS dust providing a source of P.