A Mouse Model with a Frameshift Mutation in the Nuclear Factor I/X (NFIX) Gene Has Phenotypic Features of Marshall-Smith Syndrome.

The nuclear factor I/X (NFIX) gene encodes a ubiquitously expressed transcription factor whose mutations lead to two allelic disorders characterized by developmental, skeletal, and neural abnormalities, namely, Malan syndrome (MAL) and Marshall-Smith syndrome (MSS). NFIX mutations associated with MAL mainly cluster in exon 2 and are cleared by nonsense-mediated decay (NMD) leading to NFIX haploinsufficiency, whereas NFIX mutations associated with MSS are clustered in exons 6-10 and escape NMD and result in the production of dominant-negative mutant NFIX proteins. Thus, different NFIX mutations have distinct consequences on NFIX expression. To elucidate the in vivo effects of MSS-associated NFIX exon 7 mutations, we used CRISPR-Cas9 to generate mouse models with exon 7 deletions that comprised: a frameshift deletion of two nucleotides (Nfix Del2); in-frame deletion of 24 nucleotides (Nfix Del24); and deletion of 140 nucleotides (Nfix Del140). Nfix +/Del2, Nfix +/Del24, Nfix +/Del140, Nfix Del24/Del24, and Nfix Del140/Del140 mice were viable, normal, and fertile, with no skeletal abnormalities, but Nfix Del2/Del2 mice had significantly reduced viability (p < 0.002) and died at 2-3 weeks of age. Nfix Del2 was not cleared by NMD, and NfixDel2/Del2 mice, when compared to Nfix +/+ and Nfix +/Del2 mice, had: growth retardation; short stature with kyphosis; reduced skull length; marked porosity of the vertebrae with decreased vertebral and femoral bone mineral content; and reduced caudal vertebrae height and femur length. Plasma biochemistry analysis revealed Nfix Del2/Del2 mice to have increased total alkaline phosphatase activity but decreased C-terminal telopeptide and procollagen-type-1-N-terminal propeptide concentrations compared to Nfix +/+ and Nfix +/Del2 mice. Nfix Del2/Del2 mice were also found to have enlarged cerebral cortices and ventricular areas but smaller dentate gyrus compared to Nfix +/+ mice. Thus, Nfix Del2/Del2 mice provide a model for studying the in vivo effects of NFIX mutants that escape NMD and result in developmental abnormalities of the skeletal and neural tissues that are associated with MSS. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

A First Principles Approach to Subjective Experience.

Understanding the neural bases of subjective experience remains one of the great challenges of the natural sciences. Higher-order theories of consciousness are typically defended by assessments of neural activity in higher cortical regions during perception, often with disregard to the nature of the neural computations that these regions execute. We have sought to refocus the problem toward identification of those neural computations that are necessary for subjective experience with the goal of defining the sorts of neural architectures that can perform these operations. This approach removes reliance on behaviour and brain homologies for appraising whether non-human animals have the potential to subjectively experience sensory stimuli. Using two basic principles-first, subjective experience is dependent on complex processing executing specific neural functions and second, the structure-determines-function principle-we have reasoned that subjective experience requires a neural architecture consisting of stacked forward models that predict the output of neural processing from inputs. Given that forward models are dependent on appropriately connected processing modules that generate prediction, error detection and feedback control, we define a minimal neural architecture that is necessary (but not sufficient) for subjective experience. We refer to this framework as the hierarchical forward models algorithm. Accordingly, we postulate that any animal lacking this neural architecture will be incapable of subjective experience.

Identifying gene expression profiles associated with neurogenesis and inflammation in the human subependymal zone from development through aging.

The generation of new neurons within the mammalian forebrain continues throughout life within two main neurogenic niches, the subgranular zone (SGZ) of the hippocampal dentate gyrus, and the subependymal zone (SEZ) lining the lateral ventricles. Though the SEZ is the largest neurogenic niche in the adult human forebrain, our understanding of the mechanisms regulating neurogenesis from development through aging within this region remains limited. This is especially pertinent given that neurogenesis declines dramatically over the postnatal lifespan. Here, we performed transcriptomic profiling on the SEZ from human post-mortem tissue from eight different life-stages ranging from neonates (average age ~ 2 months old) to aged adults (average age ~ 86 years old). We identified transcripts with concomitant profiles across these decades of life and focused on three of the most distinct profiles, namely (1) genes whose expression declined sharply after birth, (2) genes whose expression increased steadily with age, and (3) genes whose expression increased sharply in old age in the SEZ. Critically, these profiles identified neuroinflammation as becoming more prevalent with advancing age within the SEZ and occurring with time courses, one gradual (starting in mid-life) and one sharper (starting in old age).

Neural Design Principles for Subjective Experience: Implications for Insects.

How subjective experience is realized in nervous systems remains one of the great challenges in the natural sciences. An answer to this question should resolve debate about which animals are capable of subjective experience. We contend that subjective experience of sensory stimuli is dependent on the brain's awareness of its internal neural processing of these stimuli. This premise is supported by empirical evidence demonstrating that disruption to either processing streams or awareness states perturb subjective experience. Given that the brain must predict the nature of sensory stimuli, we reason that conscious awareness is itself dependent on predictions generated by hierarchically organized forward models of the organism's internal sensory processing. The operation of these forward models requires a specialized neural architecture and hence any nervous system lacking this architecture is unable to subjectively experience sensory stimuli. This approach removes difficulties associated with extrapolations from behavioral and brain homologies typically employed in addressing whether an animal can feel. Using nociception as a model sensation, we show here that the Drosophila brain lacks the required internal neural connectivity to implement the computations required of hierarchical forward models. Consequently, we conclude that Drosophila, and those insects with similar neuroanatomy, do not subjectively experience noxious stimuli and therefore cannot feel pain.

Abnormal Behavior and Cortical Connectivity Deficits in Mice Lacking Usp9x.

Genetic association studies have identified many factors associated with neurodevelopmental disorders such as autism spectrum disorder (ASD). However, the way these genes shape neuroanatomical structure and connectivity is poorly understood. Recent research has focused on proteins that act as points of convergence for multiple factors, as these may provide greater insight into understanding the biology of neurodevelopmental disorders. USP9X, a deubiquitylating enzyme that regulates the stability of many ASD-related proteins, is one such point of convergence. Loss of function variants in human USP9X lead to brain malformations, which manifest as a neurodevelopmental syndrome that frequently includes ASD, but the underlying structural and connectomic abnormalities giving rise to patient symptoms is unknown. Here, we analyzed forebrain-specific Usp9x knockout mice (Usp9x-/y) to address this knowledge gap. Usp9x-/y mice displayed abnormal communication and social interaction behaviors. Moreover, the absence of Usp9x culminated in reductions to the size of multiple brain regions. Diffusion tensor magnetic resonance imaging revealed deficits in all three major forebrain commissures, as well as long-range hypoconnectivity between cortical and subcortical regions. These data identify USP9X as a key regulator of brain formation and function, and provide insights into the neurodevelopmental syndrome arising as a consequence of USP9X mutations in patients.

Adult Neurogenesis in the Olfactory System: Improving Performance for Difficult Discrimination Tasks?

What is the function of new neurons entering the olfactory bulb? Many insights regarding the molecular control of adult neurogenesis have been uncovered, but the purpose of new neurons entering the olfactory bulb has been difficult to ascertain. Here, studies investigating the role of adult neurogenesis in olfactory discrimination in mice are reviewed. Studies in which adult neurogenesis is affected are highlighted, with a focus on the role of environment enrichment and what happens during ageing. There is evidence for a role of adult neurogenesis in fine discrimination tasks, as underscored by studies that enhance adult neurogenesis. This is also observed in ageing studies, where older mice with reduced levels of adult neurogenesis perform poorly in olfactory discrimination. Differences in methodology that could account for alternative conclusions, and the importance of specificity in methods being used to investigate the effect of adult neurogenesis in olfactory performance are emphasized.

Proportional Downscaling of Glutamatergic Release Sites by the General Anesthetic Propofol at Drosophila Motor Nerve Terminals.

Propofol is the most common general anesthetic used for surgery in humans, yet its complete mechanism of action remains elusive. In addition to potentiating inhibitory synapses in the brain, propofol also impairs excitatory neurotransmission. We use electrophysiological recordings from individual glutamatergic boutons in male and female larval Drosophila melanogaster motor nerve terminals to characterize this effect. We recorded from two bouton types, which have distinct presynaptic physiology and different average numbers of release sites or active zones. We show that a clinically relevant dose of propofol (3 µm) impairs neurotransmitter release similarly at both bouton types by decreasing the number of active release sites by half, without affecting release probability. In contrast, an analog of propofol has no effect on glutamate release. Coexpressing a truncated syntaxin1A protein in presynaptic boutons completely blocked this effect of propofol. Overexpressing wild-type syntaxin1A in boutons also conferred a level of resistance by increasing the number of active release sites to a physiological ceiling set by the number of active zones or T-bars, and in this way counteracting the effect of propofol. These results point to the presynaptic release machinery as a target for the general anesthetic. Proportionally equivalent effects of propofol on the number of active release sites across the different bouton types suggests that glutamatergic circuits that involve smaller boutons with fewer release sites may be more vulnerable to the presynaptic effects of the drug.

Investigating cortical features of Sotos syndrome using mice heterozygous for Nsd1.

Sotos syndrome is a developmental disorder characterized by a suite of clinical features. In children, the three cardinal features of Sotos syndrome are a characteristic facial appearance, learning disability and overgrowth (height and/or head circumference > 2 SDs above average). These features are also evident in adults with this syndrome. Over 90% of Sotos syndrome patients are haploinsufficient for the gene encoding nuclear receptor-binding Su(var)3-9, Enhancer-of-zesteand Trithorax domain-containing protein 1 (NSD1). NSD1 is a histone methyltransferase that catalyzes the methylation of lysine residue 36 on histone H3. However, although the symptomology of Sotos syndrome is well established, many aspects of NSD1 biology remain unknown. Here, we assessed the expression of Nsd1 within the mouse brain, and showed a predominantly neuronal pattern of expression for this histone-modifying factor. We also generated a mouse strain lacking one allele of Nsd1 and analyzed morphological and behavioral characteristics in these mice, showing behavioral characteristics reminiscent of some of the deficits seen in Sotos syndrome patients.

Common Regulatory Targets of NFIA, NFIX and NFIB during Postnatal Cerebellar Development.

Transcriptional regulation plays a central role in controlling neural stem and progenitor cell proliferation and differentiation during neurogenesis. For instance, transcription factors from the nuclear factor I (NFI) family have been shown to co-ordinate neural stem and progenitor cell differentiation within multiple regions of the embryonic nervous system, including the neocortex, hippocampus, spinal cord and cerebellum. Knockout of individual Nfi genes culminates in similar phenotypes, suggestive of common target genes for these transcription factors. However, whether or not the NFI family regulates common suites of genes remains poorly defined. Here, we use granule neuron precursors (GNPs) of the postnatal murine cerebellum as a model system to analyse regulatory targets of three members of the NFI family: NFIA, NFIB and NFIX. By integrating transcriptomic profiling (RNA-seq) of Nfia- and Nfix-deficient GNPs with epigenomic profiling (ChIP-seq against NFIA, NFIB and NFIX, and DNase I hypersensitivity assays), we reveal that these transcription factors share a large set of potential transcriptional targets, suggestive of complementary roles for these NFI family members in promoting neural development.

Syntaxin1A Neomorphic Mutations Promote Rapid Recovery from Isoflurane Anesthesia in Drosophila melanogaster.

BACKGROUND: Mutations in the presynaptic protein syntaxin1A modulate general anesthetic effects in vitro and in vivo. Coexpression of a truncated syntaxin1A protein confers resistance to volatile and intravenous anesthetics, suggesting a target mechanism distinct from postsynaptic inhibitory receptor processes. Hypothesizing that recovery from anesthesia may involve a presynaptic component, the authors tested whether syntaxin1A mutations facilitated recovery from isoflurane anesthesia in Drosophila melanogaster. METHODS: A truncated syntaxin1A construct was expressed in Drosophila neurons. The authors compared effects on isoflurane induction versus recovery in syntaxin1A mutant animals by probing behavioral responses to mechanical stimuli. The authors also measured synaptic responses from the larval neuromuscular junction using sharp intracellular recordings, and performed Western blots to determine whether the truncated syntaxin1A is associated with presynaptic core complexes. RESULTS: Drosophila expressing a truncated syntaxin1A (syx, n = 40) were resistant to isoflurane induction for a behavioral responsiveness endpoint (ED50 0.30 ± 0.01% isoflurane, P < 0.001) compared with control (0.240 ± 0.002% isoflurane, n = 40). Recovery from isoflurane anesthesia was also faster, with syx-expressing flies showing greater levels of responsiveness earlier in recovery (reaction proportion 0.66 ± 0.48, P < 0.001, n = 68) than controls (0.22 ± 0.42, n = 68 and 0.33 ± 0.48, n = 66). Measuring excitatory junction potentials of larvae coexpressing the truncated syntaxin1A protein showed a greater recovery of synaptic function, compared with controls (17.39 ± 3.19 mV and 10.29 ± 4.88 mV, P = 0.014, n = 8 for both). The resistance-promoting truncated syntaxin1A was not associated with presynaptic core complexes, in the presence or absence of isoflurane anesthesia. CONCLUSIONS: The same neomorphic syntaxin1A mutation that confers isoflurane resistance in cell culture and nematodes also produces isoflurane resistance in Drosophila. Resistance in Drosophila is, however, most evident at the level of recovery from anesthesia, suggesting that the syntaxin1A target affects anesthesia maintenance and recovery processes rather than induction. The absence of truncated syntaxin1A from the presynaptic complex suggests that the resistance-promoting effect of this molecule occurs before core complex formation.

Heterozygosity for Nuclear Factor One X in mice models features of Malan syndrome.

BACKGROUND: Nuclear Factor One X (NFIX) haploinsufficiency in humans results in Malan syndrome, a disorder characterized by overgrowth, macrocephaly and intellectual disability. Although clinical assessments have determined the underlying symptomology of Malan syndrome, the fundamental mechanisms contributing to the enlarged head circumference and intellectual disability in these patients remains undefined. METHODS: Here, we used Nfix heterozygous mice as a model to investigate these aspects of Malan syndrome. Volumetric magnetic resonance imaging (MRI) was used to calculate the volumes of 20 brain sub regions. Diffusion tensor MRI was used to perform tractography-based analyses of the corpus callosum, hippocampal commissure, and anterior commissure, as well as structural connectome mapping of the whole brain. Immunohistochemistry examined the neocortical cellular populations. Two behavioral assays were performed, including the active place avoidance task to assess spatial navigation and learning and memory function, and the 3-chambered sociability task to examine social behaviour. FINDINGS: Adult Nfix+/- mice exhibit significantly increased brain volume (megalencephaly) compared to wildtypes, with the cerebral cortex showing the highest increase. Moreover, all three forebrain commissures, in particular the anterior commissure, revealed significantly reduced fractional anisotropy, axial and radial diffusivity, and tract density intensity. Structural connectome analyses revealed aberrant connectivity between many crucial brain regions. Finally, Nfix+/- mice exhibit behavioral deficits that model intellectual disability. INTERPRETATION: Collectively, these data provide a significant conceptual advance in our understanding of Malan syndrome by suggesting that megalencephaly underlies the enlarged head size of these patients, and that disrupted cortical connectivity may contribute to the intellectual disability these patients exhibit. FUND: Australian Research Council (ARC) Discovery Project Grants, ARC Fellowship, NYSTEM and Australian Postgraduate Fellowships.

NFIX-Mediated Inhibition of Neuroblast Branching Regulates Migration Within the Adult Mouse Ventricular-Subventricular Zone.

Understanding the migration of newborn neurons within the brain presents a major challenge in contemporary biology. Neuronal migration is widespread within the developing brain but is also important within the adult brain. For instance, stem cells within the ventricular-subventricular zone (V-SVZ) and the subgranular zone of dentate gyrus of the adult rodent brain produce neuroblasts that migrate to the olfactory bulb and granule cell layer of the dentate gyrus, respectively, where they regulate key brain functions including innate olfactory responses, learning, and memory. Critically, our understanding of the factors mediating neuroblast migration remains limited. The transcription factor nuclear factor I X (NFIX) has previously been implicated in embryonic cortical development. Here, we employed conditional ablation of Nfix from the adult mouse brain and demonstrated that the removal of this gene from either neural stem and progenitor cells, or neuroblasts, within the V-SVZ culminated in neuroblast migration defects. Mechanistically, we identified aberrant neuroblast branching, due in part to increased expression of the guanylyl cyclase natriuretic peptide receptor 2 (Npr2), as a factor contributing to abnormal migration in Nfix-deficient adult mice. Collectively, these data provide new insights into how neuroblast migration is regulated at a transcriptional level within the adult brain.

Analysis of hippocampal-dependent learning and memory behaviour in mice lacking Nfix from adult neural stem cells.

OBJECTIVE: The active place avoidance task (APA) is a behavioural task used to assess learning and memory in rodents. This task relies on the hippocampus, a region of the cerebral cortex capable of generating new neurons from neural stem cells. In this study, to gain further insight into the behavioural phenotype of mice deficient in the transcription factor Nfix, a gene expressed by adult neural stem cells, we examined learning and memory parameters from the APA task that were not published in our original investigation. We analysed time to first and second shock, maximum path and time of shock avoidance, number of entries into the shock zone and time spent in the shock zone. We also assessed performance in the APA task based on sex. RESULTS: We found mice deficient in Nfix displayed decreased latency to second shock compared to the control mice. Nfix deficient mice entered the shock zone more frequently and also spent more time in the shock zone. Our data provides further insights into the memory deficits evident in Nfix mutant mice, indicating these mice have a memory retrieval problem and may employ a different navigation strategy in the APA task.

Correction to: BrdU/EdU dual labeling to determine the cell-cycle dynamics of defined cellular subpopulations.

In the original publication of the article, two errors were made in describing the equations for Tc.

Neurogenic differentiation by hippocampal neural stem and progenitor cells is biased by NFIX expression.

Our understanding of the transcriptional programme underpinning adult hippocampal neurogenesis is incomplete. In mice, under basal conditions, adult hippocampal neural stem cells (AH-NSCs) generate neurons and astrocytes, but not oligodendrocytes. The factors limiting oligodendrocyte production, however, remain unclear. Here, we reveal that the transcription factor NFIX plays a key role in this process. NFIX is expressed by AH-NSCs, and its expression is sharply upregulated in adult hippocampal neuroblasts. Conditional ablation of Nfix from AH-NSCs, coupled with lineage tracing, transcriptomic sequencing and behavioural studies collectively reveal that NFIX is cell-autonomously required for neuroblast maturation and survival. Moreover, a small number of AH-NSCs also develop into oligodendrocytes following Nfix deletion. Remarkably, when Nfix is deleted specifically from intermediate progenitor cells and neuroblasts using a Dcx-creERT2 driver, these cells also display elevated signatures of oligodendrocyte gene expression. Together, these results demonstrate the central role played by NFIX in neuroblasts within the adult hippocampal stem cell neurogenic niche in promoting the maturation and survival of these cells, while concomitantly repressing oligodendrocyte gene expression signatures.

BrdU/EdU dual labeling to determine the cell-cycle dynamics of defined cellular subpopulations.

Measuring the mean duration of synthesis-phase (Ts) and of the total cell-cycle (Tc) within progenitor cell populations can provide important insights into the biology governing these cells. Rather than a passive process that shows little variability across cellular contexts, the cell-cycle is instead highly regulated. For example, in the rodent forebrain, Ts is selectively lengthened in radial glial progenitor cells undergoing symmetric versus asymmetric division. This lengthening is thought to minimize the potential for copying errors that can occur during DNA replication. Manipulating cell-cycle duration can also affect cell fate, demonstrating that in certain circumstances cell-cycle duration is an instructive process. Currently, cell-cycle length is typically measured using either cumulative labeling with a single thymidine analogue, or via dual thymidine analogue labeling approaches. However, these methods are often time-consuming and inefficient. Here, using the embryonic mouse cerebral cortex as a model system, we describe a simplified dual thymidine analogue protocol using BrdU and EdU that can be used to measure Ts and Tc. The advantage of this protocol over cumulative labeling approaches is that only a single time-point is required for measurement. An additional benefit of this protocol over existing dual-analog approaches (CldU/IdU) is the antibody-free detection of EdU and the acid-free detection of BrdU, processes allowing for the parallel use of specific antibodies so as to measure the cell-cycle in immunologically defined cellular subpopulations.

Trapping of Syntaxin1a in Presynaptic Nanoclusters by a Clinically Relevant General Anesthetic.

Propofol is the most commonly used general anesthetic in humans. Our understanding of its mechanism of action has focused on its capacity to potentiate inhibitory systems in the brain. However, it is unknown whether other neural mechanisms are involved in general anesthesia. Here, we demonstrate that the synaptic release machinery is also a target. Using single-particle tracking photoactivation localization microscopy, we show that clinically relevant concentrations of propofol and etomidate restrict syntaxin1A mobility on the plasma membrane, whereas non-anesthetic analogs produce the opposite effect and increase syntaxin1A mobility. Removing the interaction with the t-SNARE partner SNAP-25 abolishes propofol-induced syntaxin1A confinement, indicating that syntaxin1A and SNAP-25 together form an emergent drug target. Impaired syntaxin1A mobility and exocytosis under propofol are both rescued by co-expressing a truncated syntaxin1A construct that interacts with SNAP-25. Our results suggest that propofol interferes with a step in SNARE complex formation, resulting in non-functional syntaxin1A nanoclusters.

A morphology independent approach for identifying dividing adult neural stem cells in the mouse hippocampus.

BACKGROUND: Type 1 adult hippocampal neural stem cells (AH-NSCs) continue to generate neurons throughout life, albeit at a very low rate. The relative quiescence of this population of cells has led to many studies investigating factors that may increase their division. Current methods of identifying dividing AH-NSCs in vivo require the identification and tracing of radial processes back to nuclei within the subgranular zone. However, caveats to this approach include the time-intensive nature of identifying AH-NSCs with such a process, as well as the fact that this approach ignores the relatively more active population of horizontally oriented AH-NSCs that also reside in the subgranular zone. RESULTS: Here we describe, and then verify using Hes5::GFP mice, that labeling for the cell cycle marker Ki67 and selection against the intermediate progenitor cell marker TBR2 (Ki67+ve ; TBR2-ve nuclei) is sufficient to identify dividing horizontally and radially oriented AH-NSCs in the adult mouse hippocampus. CONCLUSIONS: These findings provide a simple and accurate way to quantify dividing AH-NSCs in vivo using a morphology-independent approach that will facilitate studies into neurogenesis within the hippocampal stem cell niche of the adult brain. Developmental Dynamics 247:194-200, 2018. © 2017 Wiley Periodicals, Inc.

Combined allelic dosage of Nfia and Nfib regulates cortical development.

BACKGROUND: Nuclear factor I family members nuclear factor I A and nuclear factor I B play important roles during cerebral cortical development. Nuclear factor I A and nuclear factor I B regulate similar biological processes, as their expression patterns, regulation of target genes and individual knockout phenotypes overlap. We hypothesised that the combined allelic loss of Nfia and Nfib would culminate in more severe defects in the cerebral cortex than loss of a single member. METHODS: We combined immunofluorescence, co-immunoprecipitation, gene expression analysis and immunohistochemistry on knockout mouse models to investigate whether nuclear factor I A and nuclear factor I B function similarly and whether increasing allelic loss of Nfia and Nfib caused a more severe phenotype. RESULTS: We determined that the biological functions of nuclear factor I A and nuclear factor I B overlap during early cortical development. These proteins are co-expressed and can form heterodimers in vivo. Differentially regulated genes that are shared between Nfia and Nfib knockout mice are highly enriched for nuclear factor I binding sites in their promoters and are associated with neurodevelopment. We found that compound heterozygous deletion of both genes resulted in a cortical phenotype similar to that of single homozygous Nfia or Nfib knockout embryos. This was characterised by retention of the interhemispheric fissure, dysgenesis of the corpus callosum and a malformed dentate gyrus. Double homozygous knockout of Nfia and Nfib resulted in a more severe phenotype, with increased ventricular enlargement and decreased numbers of differentiated glia and neurons. CONCLUSION: In the developing cerebral cortex, nuclear factor I A and nuclear factor I B share similar biological functions and function additively, as the combined allelic loss of these genes directly correlates with the severity of the developmental brain phenotype.