ALS-linked CCNF variant disrupts motor neuron ubiquitin homeostasis.

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.

Innovations advancing our understanding of microglia in Alzheimer's disease: From in vitro to in vivo models.

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.

Parsing the effect of co-culture with brain organoids on Diffuse Intrinsic Pontine Glioma (DIPG) using quantitative proteomics.

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.

REST and RCOR genes display distinct expression profiles in neurons and astrocytes using 2D and 3D human pluripotent stem cell models.

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.

Alzheimer's disease induced neurons bearing PSEN1 mutations exhibit reduced excitability.

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.

Edaravone and mitochondrial transfer as potential therapeutics for vanishing white matter disease astrocyte dysfunction.

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.

Making neurons, made easy: The use of Neurogenin-2 in neuronal differentiation.

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.

An Optimized Direct Lysis Gene Expression Microplate Assay and Applications for Disease, Differentiation, and Pharmacological Cell-Based Studies.

Routine cell culture reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) gene expression analysis is limited in scalability due to minimum sample requirement and multistep isolation procedures. In this study, we aimed to optimize and apply a cost-effective and rapid protocol for directly sampling gene expression data from microplate cell cultures. The optimized protocol involves direct lysis of microplate well population followed by a reduced thermocycler reaction time one-step RT-qPCR assay. In applications for inflammation and stress-induced cell-based models, the direct lysis RT-qPCR microplate assay was utilized to detect IFN1 and PPP1R15A expression by poly(I:C) treated primary fibroblast cultures, IL6 expression by poly(I:C) iPSC-derived astrocytes, and differential PPP1R15A expression by ER-stressed vanishing white-matter disease patient induced pluripotent stem cell (iPSC)-derived astrocytes. In application for neural differentiation medium recipe optimizations, conditions were screened for SYN1 and VGLUT1 in neuronal cultures, and S100B, GFAP and EAAT1 in astrocyte cultures. The protocol provides microplate gene expression results from cell lysate to readout within ~35 min, with comparable cost to routine RT-qPCR, and it may be utilized to support laboratory cell-based assays in basic and applied scientific and medical fields of research including stem-cell differentiation, cell physiology, and drug mechanism studies.

Transgene and Chemical Transdifferentiation of Somatic Cells for Rapid and Efficient Neurological Disease Cell Models.

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.

More than a Corepressor: The Role of CoREST Proteins in Neurodevelopment.

The molecular mechanisms governing normal neurodevelopment are tightly regulated by the action of transcription factors. Repressor element 1 (RE1) silencing transcription factor (REST) is widely documented as a regulator of neurogenesis that acts by recruiting corepressor proteins and repressing neuronal gene expression in non-neuronal cells. The REST corepressor 1 (CoREST1), CoREST2, and CoREST3 are best described for their role as part of the REST complex. However, recent evidence has shown the proteins have the ability to repress expression of distinct target genes in a REST-independent manner. These findings indicate that each CoREST paralogue may have distinct and critical roles in regulating neurodevelopment and are more than simply "REST corepressors," whereby they act as independent repressors orchestrating biological processes during neurodevelopment.

Molecular and Functional Characterization of Neurogenin-2 Induced Human Sensory Neurons.

Sensory perception is fundamental to everyday life, yet understanding of human sensory physiology at the molecular level is hindered due to constraints on tissue availability. Emerging strategies to study and characterize peripheral neuropathies in vitro involve the use of human pluripotent stem cells (hPSCs) differentiated into dorsal root ganglion (DRG) sensory neurons. However, neuronal functionality and maturity are limited and underexplored. A recent and promising approach for directing hPSC differentiation towards functionally mature neurons involves the exogenous expression of Neurogenin-2 (NGN2). The optimized protocol described here generates sensory neurons from hPSC-derived neural crest (NC) progenitors through virally induced NGN2 expression. NC cells were derived from hPSCs via a small molecule inhibitor approach and enriched for migrating NC cells (66% SOX10+ cells). At the protein and transcript level, the resulting NGN2 induced sensory neurons (NGN2iSNs) express sensory neuron markers such as BRN3A (82% BRN3A+ cells), ISLET1 (91% ISLET1+ cells), TRKA, TRKB, and TRKC. Importantly, NGN2iSNs repetitively fire action potentials (APs) supported by voltage-gated sodium, potassium, and calcium conductances. In-depth analysis of the molecular basis of NGN2iSN excitability revealed functional expression of ion channels associated with the excitability of primary afferent neurons, such as Nav1.7, Nav1.8, Kv1.2, Kv2.1, BK, Cav2.1, Cav2.2, Cav3.2, ASICs and HCN among other ion channels, for which we provide functional and transcriptional evidence. Our characterization of stem cell-derived sensory neurons sheds light on the molecular basis of human sensory physiology and highlights the suitability of using hPSC-derived sensory neurons for modeling human DRG development and their potential in the study of human peripheral neuropathies and drug therapies.

Particle-mediated delivery of frataxin plasmid to a human sensory neuronal model of Friedreich's ataxia.

Increasing frataxin protein levels through gene therapy is envisaged to improve therapeutic outcomes for patients with Friedreich's ataxia (FRDA). A non-viral strategy that uses submicrometer-sized multilayered particles to deliver frataxin-encoding plasmid DNA affords up to 27 000-fold increase in frataxin gene expression within 2 days in vitro in a stem cell-derived neuronal model of FRDA.

If Human Brain Organoids Are the Answer to Understanding Dementia, What Are the Questions?

Because our beliefs regarding our individuality, autonomy, and personhood are intimately bound up with our brains, there is a public fascination with cerebral organoids, the "mini-brain," the "brain in a dish". At the same time, the ethical issues around organoids are only now being explored. What are the prospects of using human cerebral organoids to better understand, treat, or prevent dementia? Will human organoids represent an improvement on the current, less-than-satisfactory, animal models? When considering these questions, two major issues arise. One is the general challenge associated with using any stem cell-generated preparation for in vitro modelling (challenges amplified when using organoids compared with simpler cell culture systems). The other relates to complexities associated with defining and understanding what we mean by the term "dementia." We discuss 10 puzzles, issues, and stumbling blocks to watch for in the quest to model "dementia in a dish."