Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by motor neuron degeneration, with alterations in neural excitability serving as key indicators. Recent advancements in induced pluripotent stem cell (iPSC) technology have enabled the generation of human iPSC-derived neuronal cultures, which, when combined with multi-electrode array (MEA) electrophysiology, provide rich spatial and temporal electrophysiological data. Traditionally, MEA data is analyzed using handcrafted features based on potentially imperfect domain knowledge, which while useful may not fully capture all useful characteristics inherent in the data. Machine learning, particularly deep learning, has the potential to automatically learn relevant characteristics from raw data without solely relying on handcrafted feature extraction. However, handcrafted features remain critical for encoding domain knowledge and improving interpretability, especially with limited or noisy data. This study introduces FRAME-C, a knowledge-augmented machine learning pipeline that combines domain knowledge, raw spike waveform data, and deep learning techniques to classify MEA signals and identify ALS-specific phenotypes. FRAME-C leverages deep learning to learn important features from spike waveforms while incorporating handcrafted features such as spike amplitude, inter-spike interval, and spike duration, preserving key spatial and temporal information. We validate FRAME-C on both simulated and real MEA data from human iPSC-derived neuronal cultures, demonstrating superior performance over existing classification methods. FRAME-C shows over 11 real data and up to 25 handcrafted feature importance, providing insights into ALS phenotypes.

INTRODUCTION:Sertraline is the frontline pharmacotherapy for the treatment of depression and anxiety during pregnancy. However, there is little evidence regarding the effects of sertraline on maternal behaviour or the maternal brain. Furthermore, the efficacy of non-pharmacological approaches to treatment in pregnancy, such as exercise, are unclear. Therefore, the aim of this study was to examine the effects of sertraline and exercise during pregnancy on maternal postpartum depressive-like, anxiety-like and associated behaviours, as well as litter characteristics, in a rat model of depression. We also investigated the effects of these treatments on the maternal brain, focusing initially on DNA methylation and glutamatergic markers, which have been implicated in depression. METHOD:Twenty-four female Wistar-Kyoto (WKY; strain that models depression and anxiety) rats were divided into three groups: 1. WKY-Sertraline; 2. WKY-Exercise, 3. WKY-Vehicle; Six female Wistar (WIS) rats were included as controls. Rats were treated with sertraline (10 mg/kg) or vehicle (33 % propylene glycol) twice/day, from gestational day (GD) 1 to postpartum day 14. The WKY-Exercise group were provided access to a running wheel during pregnancy for 3 h/day from GD1-18. Dam and litter characteristics, as well as pup ultrasonic vocalisations (USVs), were measured. Dams underwent behavioural testing at 5-weeks postpartum to assess depressive-, anxiety- and cognitive-like behaviours. Gene expression of DNA methylation markers (Dnmt1, Dnmt3a) and glutamate receptors (Grin1, Grin2a, Grin2b) were measured in the prefrontal cortex (PFC), using RT-qPCR. RESULT:The WKY-Sertraline group gained 39 % less weight in their first pregnancy week compared to all other groups (p < 0.05) and produced smaller litters compared to WIS controls (-43 %; p = 0.003) and WKY-Exercise (-38 %; p = 0.012); WKY-Sertraline pups had slightly smaller brain weights compared to WKY-Vehicle (p = 0.031). WKY-Vehicle pups showed reduced number of USV calls, call amplitude and call duration compared to WIS control (p < 0.001). The WKY-Exercise pups produced increased number of USVs, with increased call amplitude of USVs, at postnatal day (PN)7 compared to WKY-Vehicle (p < 0.01). Maternal sertraline treatment did not significantly affect dam postpartum behavioural measures, or maternal cortical gene expression. The WKY-Exercise group however showed reduced anxiety-like behaviours, spending more time in the open arms (620 %; p = 0.027) and less time in the closed arms (-22 %; p = 0.047) of the elevated plus maze (EPM) compared to WKY-Vehicle, and more time in the centre of the open field test (OFT) compared to WKY-Vehicle (132 %; p = 0.057). Furthermore, WKY-Exercise dams showed a 64 % increase in Dnmt3a mRNA levels in the PFC compared to WKY-Vehicle (p = 0.019), effectively reversing the 44 % reduction observed in WKY-Veh relative to WIS controls (p = 0.009). CONCLUSION:Voluntary exercise during pregnancy in the WKY rat model, reduced postpartum anxiety-like behaviour. This was accompanied by elevated DNMT3a gene expression in the PFC, suggesting this region may be sensitive to DNA methylation changes following maternal exercise. In contrast, maternal sertraline did not impact these behaviours or genes. Maintaining sertraline treatment beyond postpartum day 14, or trialling different doses, may have resulted in broader effects on postpartum behaviour, which should be explored further. Maternal sertraline did appear to have some adverse effects on the in-utero environment, evidenced by smaller litters, with slightly smaller pup brain weights, which should be investigated further. Our findings suggest a long-term beneficial effect of exercise during pregnancy and support future studies examining the effects of exercise in antenatal depression in the human population.

Long-term imaging formats are ideal for capturing dynamic neuronal network formation in vitro, yet fluorescent techniques are often constrained by the impact of phototoxicity on cell survival. Here we present a live-imaging protocol that was optimised via quantitative analysis of 3 target culturing conditions on neuromorphological health: extracellular matrix (human- versus murine-derived laminin), culture media (Neurobasal™ versus Brainphys™ Imaging media), and seeding density (1 × 105 versus 2 × 105 cells/cm2). A cortical neuron reporter line was differentiated from human embryonic stem cells by transduction of Neurogenin-2 and green fluorescent protein, then fluorescently imaged in 8 different microenvironments daily for 33 days. Alongside viability analysis by PrestoBlue assay and gene quantification by digital polymerase chain reaction, an automated image analysis pipeline was developed to characterise network morphology and organisation over time. Brainphys™ Imaging medium was observed to support neuron viability, outgrowth, and self-organisation to a greater extent than Neurobasal™ medium with either laminin type, while the combination of Neurobasal™ medium and human laminin reduced cell survival. Further, a higher seeding density fostered somata clustering, but did not significantly extend viability compared to low density. These findings suggest a synergistic relationship between species-specific laminin and culture media in phototoxic environments, which is positively mediated by light-protective compounds found in Brainphys™ Imaging medium.

The epigenetic regulator, histone deacetylase 2 (HDAC2), is dysregulated in Alzheimer’s disease (AD), resulting in disruption to neuronal dynamics, memory and cognition. The transcriptional repressor, REST corepressor 3 (CoREST3) has a potential binding site upstream of Hdac2 and therefore we hypothesised CoREST3 would directly regulate HDAC2 and that CoREST3 expression would be altered in the AD brain. CoREST3 exhibited three distinct bands at 70 kDa (band I), 60 kDa (band II) and 55 kDa (band III), which were consistent with CoREST3, as shRNA mediated knockdown reduced levels of all three bands. CoREST3 protein levels (band II) in AD post mortem brain tissue were significantly decreased in the superior temporal gyrus (STG), inferior temporal gyrus (ITG) and precuneus (PRE), whereas CoREST3 Band III was significantly increased in the PRE and primary visual cortex (PVC). Additionally, HDAC2 was significantly decreased by > 50

Mechanosensory neurons are a specialized class of neurons that detect mechanical stimuli elicited by external or internal body forces. Two major subclasses of mechanosensory neurons reside within the dorsal root ganglia; proprioceptor neurons (PN) that innervate muscle tissue and low threshold mechanoreceptor neurons (LTMR) that innervate skin. To date, the specific cellular neurophysiology of PN and LTMR subclasses are primarily defined by animal models due to the limited availability of human neural tissue. Here an efficient approach is described for generating PN and LTMR from human pluripotent stem cells (hPSC) by inducing co-expression of NGN2/RUNX3 or NGN2/SHOX2 in hPSC-derived neural crest, respectively. Molecular and functional mechanosensory profiles are validated in both populations. Of significance, functional interrogation of induced mechanosensory subtypes reveals their distinct responses to mechanical stimuli. Induced proprioceptor neurons produce scaled responses to increasing mechanical stimuli that can sustain repetition and result in action potential firing. In contrast, induced LTMRs desensitize upon repeated mechanical stimuli and display a lower mechanical threshold for action potential firing. Furthermore, both subtypes predominantly rely on PIEZO2 for mechanosensory function. These findings highlight the unique mechanically sensitive profiles and excitability properties that may distinguish human mechanosensory subtypes, distinct from the presence of end-organs.

Sensing mechanical stimuli is crucial for the function of internal and external tissues, such as the skin and muscles. Much of our understanding of mechanosensory physiology relies on rodent studies, which may not directly translate to humans. To address the knowledge gap in human mechanosensation, we developed distinct populations of human mechanosensory neuronal subtypes from human pluripotent stem cells (hPSC). By inducing co-expression of NGN2/RUNX3 or NGN2/SHOX2 in hPSC-derived migrating neural crest cells we directed their specification to proprioceptor and low-threshold mechanoreceptor neuronal subtypes, respectively. The induced neurons exhibited transcriptional profiles consistent with mechanosensory neurons and displayed functional responses to mechanical stimuli, such as stretch and submicrometer probe indentation to the soma. Notably, each subtype displayed unique mechanical thresholds and desensitization properties akin to proprioceptors and low-threshold mechanoreceptors and both induced neuronal subtypes fired action potentials in response to minute mechanical stimuli, predominantly relying on PIEZO2 for mechanosensory function. Collectively, this study provides a foundational model for exploring human neuronal mechanosensory biology.### Competing Interest StatementThe authors have declared no competing interest.

Repressor element-1 silencing transcription factor (REST) is a transcriptional repressor involved in neurodevelopment and neuroprotection. REST forms a complex with the REST corepressors, CoREST1, CoREST2, or CoREST3 (encoded by RCOR1, RCOR2, and RCOR3, respectively). Emerging evidence suggests that the CoREST family can target unique genes independently of REST, in various neural and glial cell types during different developmental stages. However, there is limited knowledge regarding the expression and function of the CoREST family in human neurodevelopment. To address this gap, we employed 2D and 3D human pluripotent stem cell (hPSC) models to investigate REST and RCOR gene expression levels. Our study revealed a significant increase in RCOR3 expression in glutamatergic cortical and GABAergic ventral forebrain neurons, as well as mature functional NGN2-induced neurons. Additionally, a simplified astrocyte transdifferentiation protocol resulted in a significant decrease in RCOR2 expression following differentiation. REST expression was notably reduced in mature neurons and cerebral organoids. In summary, our findings provide the first insights into the cell-type-specific expression patterns of RCOR genes in human neuronal and glial differentiation. Specifically, RCOR3 expression increases in neurons, while RCOR2 levels decrease in astrocytes. The dynamic expression patterns of REST and RCOR genes during hPSC neuronal and glial differentiation underscore the potential distinct roles played by REST and CoREST proteins in regulating the development of these cell types in humans.

Artificially generated induced pluripotent stem cells (iPSCs) from somatic cells play an important role for disease modeling and drug screening of neurodegenerative diseases. Astrocytes differentiated from iPSCs are important targets to investigate neuronal metabolism. The astrocyte differentiation progress can be monitored through the variations of morphology observed from microscopy images at different differentiation stages, then determined by molecular biology techniques upon maturation. However, the astrocytes usually ``perfectly'' blend into the background and some of them are covered by interference information (i.e., dead cells, media sediments, and cell debris), which makes astrocytes difficult to observe. Due to the lack of annotated datasets, the existing state-of-the-art deep learning approaches cannot be used to address this issue. In this paper, we introduce a new task named astrocyte segmentation with a novel dataset, called IAI704, which contains 704 images and their corresponding pixel-level annotation masks. Moreover, a novel frequency domain denoising network, named FDNet, is proposed for astrocyte segmentation. In detail, our FDNet consists of a contextual information fusion module (CIF), an attention block (AB), and a Fourier transform block (FTB). CIF and AB fuse multi-scale feature embeddings to localize the astrocytes. FTB transforms feature embeddings into the frequency domain and conducts a high-pass filter to eliminate interference information. Experimental results demonstrate the superiority of our proposed FDNet over the state-of-the-art substitutes in astrocyte segmentation, shedding insights for iPSC differentiation progress prediction.

Alzheimer’s disease (AD) is a devastating neurodegenerative condition that affects memory and cognition, characterized by neuronal loss and currently lacking a cure. Mutations in PSEN1 (Presenilin 1) are among the most common causes of early-onset familial AD (fAD). While changes in neuronal excitability are believed to be early indicators of AD progression, the link between PSEN1 mutations and neuronal excitability remains to be fully elucidated. This study examined iPSC-derived neurons (iNs) from fAD patients with PSEN1 mutations S290C or A246E, alongside CRISPR-corrected isogenic cell lines, to investigate early changes in excitability. Electrophysiological profiling revealed reduced excitability in both PSEN1 mutant iNs compared to their isogenic controls. Neurons bearing S290C and A246E mutations exhibited divergent passive membrane properties compared to isogenic controls, suggesting distinct effects of PSEN1 mutations on neuronal excitability. Additionally, both PSEN1 backgrounds exhibited higher current density of voltage-gated potassium (Kv) channels relative to their isogenic iNs, while displaying comparable voltage-gated sodium (Nav) channel current density. This suggests that the Nav/Kv imbalance contributes to impaired neuronal firing in fAD iNs. Deciphering these early cellular and molecular changes in AD is crucial for understanding disease pathogenesis.

Nitrosative stress is a feature of Alzheimer's disease (AD). Aims: We aimed to identify the cause underpinning increased nitric oxide (NO) in neurons and the impact of NO on neuronal function in AD. Results: We analyzed neuronal nitric oxide synthase (nNOS) protein levels in postmortem tissue and induced pluripotent stem cell (iPSC)-derived neurons from Alzheimer's patients and controls by immunohistochemistry and Western blots. Furthermore, we assessed the impact of modulating nNOS function or NO levels on neuronal glutamatergic signaling using calcium imaging. We show that nNOS protein levels are increased in early and severely affected brain regions of AD postmortem tissue, but not late and mildly affected regions, or cognitively normal individuals. The increased nNOS phenotype was also present in iPSC-derived neurons from late-onset Alzheimer's disease (LOAD) patients compared with controls, along with increased levels of nitrite, a stable marker of NO. Innovation: We observed a divergent functional impact of NO that included strengthening the calcium response in control neurons, while dysregulating calcium signaling and altering the amplitude and kinetics of the calcium responses to glutamate in the AD neurons. Pharmacological scavenging of NO or inhibition of nNOS prevented aberrant spontaneous calcium signaling in AD neurons. Conclusion: Together these data identify increases in nNOS protein in AD. Functional data suggest that NO modulation of glutamatergic calcium signaling is neuroprotective under nonpathogenic conditions, with increased nNOS and NO contributing to dysregulated spontaneous calcium signaling in AD neurons.

Diffuse Intrinsic Pontine Gliomas (DIPGs) are deadly brain cancers in children for which there is no effective treatment. This can partly be attributed to preclinical models that lack essential elements of the in vivo tissue environment, resulting in treatments that appear promising preclinically, but fail to result in effective cures. Recently developed co-culture models combining stem cell-derived brain organoids with brain cancer cells provide tissue dimensionality and a human-relevant tissue-like microenvironment. As these models are technically challenging, we aimed to establish whether interaction with the organoid influences DIPG biology and thus warrants their use. To address this question DIPG24 cells were cultured with pluripotent stem cell-derived cortical organoids. We created "mosaic" co-cultures enriched for tumour cell-neuronal cell interactions versus "assembloid" co-cultures enriched for tumour cell-tumour cell interactions. Sequential window acquisition of all theoretical mass spectra (SWATH-MS) was used to analyse the proteomes of DIPG fractions isolated by flow-assisted cell sorting. Control proteomes from DIPG spheroids were compared with DIPG cells isolated from mosaic and assembloid co-cultures. This suggested changes in cell interaction with the external environment reflected by decreased gene ontology terms associated with adhesion and extracellular matrix, and increased DNA synthesis and replication, in DIPG24 cells under either co-culture condition. By contrast, the mosaic co-culture was associated with neuron-specific brahma-associated factor (nBAF) complex signalling, a process associated with neuronal maturation. We propose that co-culture with brain organoids is a valuable tool to parse the contribution of the brain microenvironment to DIPG tumour biology.

Abstract Diffuse Intrinsic Pontine Gliomas (DIPGs) are deadly brain cancers in children for which there is currently no effective treatment. In part, this can be attributed to preclinical models that lack essential elements of the in vivo tissue environment, resulting in treatments that appear promising preclinically, but fail to result in effective cures. Recently developed co-culture models combining stem cell-derived brain organoids with brain cancer cells provide tissue dimensionality and a human-relevant tissue-like microenvironment. As these models are technically challenging and time consuming it is imperative to establish whether interaction with the organoid influences DIPG biology and thus warrants their use. To address this question, we cultured DIPG cells with GFP-expressing cortical organoids. We created “mosaic” co-cultures enriched for tumour cell-neuronal cell interactions, where disaggregated spheroids and organoids were mixed and allowed to reform, versus “assembloid” co-cultures enriched for tumour cell-tumour cell interactions, where preformed tumour spheroids and organoids were combined. Sequential window acquisition of all theoretical mass spectra (SWATH-MS) was used to analyse the proteomes of DIPG fractions isolated by flow-assisted cell sorting. Control proteomes from DIPG spheroids were compared with DIPG cells isolated from mosaic and assembloid co-cultures. This revealed that tumour cell adhesion was reduced, and DNA synthesis and replication were increased, in DIPG cells under either co-culture condition. By contrast, the mosaic co-culture was alone associated with pathways implicated in dendrite growth. We propose that co-culture with brain organoids is a valuable tool to parse the contribution of the brain microenvironment to DIPG tumour biology.

INTRODUCTION:Previous research has suggested that vanishing white matter disease (VWMD) astrocytes fail to fully differentiate and respond differently to cellular stresses compared to healthy astrocytes. However, few studies have investigated potential VWMD therapeutics in monoculture patient-derived cell-based models.METHODS:To investigate the impact of alterations in astrocyte expression and function in VWMD, astrocytes were differentiated from patient and control induced pluripotent stem cells and analyzed by proteomics, pathway analysis, and functional assays, in the absence and presence of stressors or potential therapeutics.RESULTS:Vanishing white matter disease astrocytes demonstrated significantly reduced expression of astrocyte markers and markers of inflammatory activation or cellular stress relative to control astrocytes. These alterations were identified both in the presence and absence of polyinosinic:polycytidylic acid stimuli, which is used to simulate viral infections. Pathway analysis highlighted differential signaling in multiple pathways in VWMD astrocytes, including eukaryotic initiation factor 2 (EIF2) signaling, oxidative stress, oxidative phosphorylation (OXPHOS), mitochondrial function, the unfolded protein response (UPR), phagosome regulation, autophagy, ER stress, tricarboxylic acid cycle (TCA) cycle, glycolysis, tRNA signaling, and senescence pathways. Since oxidative stress and mitochondrial function were two of the key pathways affected, we investigated whether two independent therapeutic strategies could ameliorate astrocyte dysfunction: edaravone treatment and mitochondrial transfer. Edaravone treatment reduced differential VWMD protein expression of the UPR, phagosome regulation, ubiquitination, autophagy, ER stress, senescence, and TCA cycle pathways. Meanwhile, mitochondrial transfer decreased VWMD differential expression of the UPR, glycolysis, calcium transport, phagosome formation, and ER stress pathways, while further modulating EIF2 signaling, tRNA signaling, TCA cycle, and OXPHOS pathways. Mitochondrial transfer also increased the gene and protein expression of the astrocyte marker, glial fibrillary acidic protein (GFAP) in VWMD astrocytes.CONCLUSION:This study provides further insight into the etiology of VWMD astrocytic failure and suggests edaravone and mitochondrial transfer as potential candidate VWMD therapeutics that can ameliorate disease pathways in astrocytes related to oxidative stress, mitochondrial dysfunction, and proteostasis.

Microglia have been implicated in Alzheimer's disease (AD) pathogenesis through the identification of risk factor genes that are specifically or predominantly expressed in this cell type. Additional evidence suggests that microglia undergo dramatic morphological and phenotypic state changes during AD progression, as observed in human post-mortem tissue and animal model research. Although valuable, these studies are often hampered by either representing one time point in human tissue (end point) or because of the lack of conservation between species of microglial transcriptomes, proteomes and cell states. Thus, the development and application of novel human model systems have been beneficial in the study of microglia in neurodegeneration. Recent innovations include the use of human pluripotent stem cell (hPSC)-derived microglia in 2D or 3D culture systems, the transdifferentiation of microglia from patient monocytes and the xenotransplantation of hPSC-derived microglia into mouse brains. This review summarizes the recent innovations that have advanced our understanding of microglia in AD, through the use of single-cell RNA sequencing, hPSC-derived microglia culture within brain organoids and xenotransplantation into mouse brain. Through outlining the strengths and limitations of these approaches, we provide recommendations that will aid future endeavours in advancing our understanding of the complex role of microglia in AD onset and progression.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders that share pathological features, including the aberrant accumulation of ubiquitinated protein inclusions within motor neurons. Previously, we have shown that the sequestration of ubiquitin (Ub) into inclusions disrupts Ub homeostasis in cells expressing ALS-associated variants superoxide dismutase 1 (SOD1), fused in sarcoma (FUS) and TAR DNA-binding protein 43 (TDP-43). Here, we investigated whether an ALS/FTD-linked pathogenic variant in the CCNF gene, encoding the E3 Ub ligase Cyclin F (CCNF), also perturbs Ub homeostasis. The presence of a pathogenic CCNF variant was shown to cause ubiquitin-proteasome system (UPS) dysfunction in induced pluripotent stem cell-derived motor neurons harboring the CCNF (S621G) mutation. The expression of the CCNFS621G variant was associated with an increased abundance of ubiquitinated proteins and significant changes in the ubiquitination of key UPS components. To further investigate the mechanisms responsible for this UPS dysfunction, we overexpressed CCNF in NSC-34 cells and found that the overexpression of both wild-type (WT) and the pathogenic variant of CCNF (CCNFS621G) altered free Ub levels. Furthermore, double mutants designed to decrease the ability of CCNF to form an active E3 Ub ligase complex significantly improved UPS function in cells expressing both CCNFWT and the CCNFS621G variant and were associated with increased levels of free monomeric Ub. Collectively, these results suggest that alterations to the ligase activity of the CCNF complex and the subsequent disruption to Ub homeostasis play an important role in the pathogenesis of CCNF-associated ALS/FTD.

A growing number of women experience depression and/or anxiety during pregnancy, however there is a large gap in our understanding of the efficacy of treatments (pharmacological and non-pharmacological) during pregnancy. This study examined the effects of sertraline and exercise during pregnancy on maternal anxiety-like and depressive-like behaviours, and gene expression in the frontal cortex and hippocampus.

Fig1_RT.xlsxOptimisations of thermocycler time and reaction mix Fig2_WB.svgCorrelation of chemiluminescent western blot signal to cell lysate loading. Human fibroblasts were lysed and processed by enhanced chemiluminescent blot probed for GADD34 (left). Cellular protein visualized by stain-free fluorescent imaging (right). Fig3_Applications.xlsxApplication of DL-RT-qPCR microplate assay to charactersiation of neural cell cultures, astrocyte IL6 activation, fibroblast IFNB1 and GADD34 activation Fig4_Differentiations.xlsxApplication of DL-RT-qPCR microplate assay to optimisation of astrocyte differentiation conditions based on S100B and GFAP expression, and neuronal differentiations by S100B, NEFH, SYN1 and VGLUT1 expression DL-RT-qPCR Extended Data.xlsxExtended Data

For neurological diseases, molecular and cellular research relies on the use of model systems to investigate disease processes and test potential therapeutics. The last decade has witnessed an increase in the number of studies using induced pluripotent stem cells to generate disease relevant cell types from patients. The reprogramming process permits the generation of a large number of cells but is potentially disadvantaged by introducing variability in clonal lines and the removal of phenotypes of aging, which are critical to understand neurodegenerative diseases. An under-utilized approach to disease modeling involves the transdifferentiation of aged cells from patients, such as fibroblasts or blood cells, into various neural cell types. In this review we discuss techniques used for rapid and efficient direct conversion to neural cell types. We examine the limitations and future perspectives of this rapidly advancing field that could improve neurological disease modeling and drug discovery.

Directed neuronal differentiation of human pluripotent stem cells (hPSCs), neural progenitors, or fibroblasts using transcription factors has allowed for the rapid and highly reproducible differentiation of mature and functional neurons. Exogenous expression of the transcription factor Neurogenin-2 (NGN2) has been widely used to generate different populations of neurons, which have been used in neurodevelopment studies, disease modeling, drug screening, and neuronal replacement therapies. Could NGN2 be a "one-glove-fits-all"approach for neuronal differentiations? This review summarizes the cellular roles of NGN2 and describes the applications and limitations of using NGN2 for the rapid and directed differentiation of neurons.

Abstract Background Nitrosative stress is a feature of Alzheimer’s disease, however the underlying mechanisms driving nitrosative stress and the impact of nitric oxide on neuronal function in Alzheimer’s disease is still largely unknown. Methods We analysed neuronal nitric oxide synthase (nNOS) protein levels in post mortem tissue and induced pluripotent stem cell (iPSC) derived neurons from Alzheimer’s patients and controls by immunohistochemistry and western blots. Furthermore, we assessed the impact of modulating nNOS function or nitric oxide levels on neuronal glutamatergic signalling using calcium imaging. Results We show that nNOS protein levels are increased in early and severely affected brain regions of late-onset Alzheimer’s disease post mortem tissue, but not late and mildly affected regions, or cognitively normal individuals. The increased nNOS phenotype was also present in iPSC-derived neurons from Alzheimer’s disease patients compared to controls, along with increased levels of nitrite, a stable marker of nitric oxide. We observed a divergent functional impact of nitric oxide that included strengthening the calcium response in control neurons, while dysregulating calcium signaling and altering the amplitude and kinetics of the calcium responses to glutamate in the Alzheimer’s disease neurons. Pharmacological modulation of nitric oxide levels or production prevented aberrant calcium signaling in Alzheimer’s disease neurons. Conclusions Together these data identify increases in nNOS protein in Alzheimer’s disease. Functional data suggest nitric oxide modulation of glutamatergic calcium signaling is neuroprotective under non-pathogenic conditions, with increased nNOS and nitric oxide contributing to pathogenic signaling changes during Alzheimer’s disease.