Human Neurological Disorders Research Articles

Co-Transplantation-Based Human-Mouse Chimeric Brain Models to Study Human Glial-Glial and Glial-Neuronal Interactions.

Human-mouse chimeric brain models, generated by transplanting human induced pluripotent stem cell (hiPSC)-derived neural cells, are valuable for studying the development and function of human neural cells in vivo. Understanding glial-glial and glial-neuronal interactions is essential for unraveling the complexities of brain function and developing treatments for neurological disorders. To explore these interactions between human neural cells within an intact brain environment, we employe a co-transplantation strategy involving the engraftment of hiPSC-derived neural progenitor cells along with primitive macrophage progenitors into the neonatal mouse brain. This approach creates human-mouse chimeric brains containing human microglia, macroglia (astroglia and oligodendroglia), and neurons. Using super-resolution imaging and 3D reconstruction techniques, we examine the dynamics between human neurons and glia, unveiling human microglia engulfing immature human neurons, microglia pruning synapses of human neurons, and significant interactions between human oligodendrocytes and neurons. Single-cell RNA sequencing analysis of the chimeric brain uncovers a close recapitulation of the human glial progenitor cell population, along with a dynamic stage in astroglial development that mirrors the processes found in the human brain. Furthermore, cell-cell communication analysis highlights significant neuronal-glial and glial-glial interactions, especially the interaction between adhesion molecules neurexins and neuroligins. This innovative co-transplantation model opens up new avenues for exploring the complex pathophysiological mechanisms underlying human neurological diseases. It holds particular promise for studying disorders where glial-neuronal interactions and non-cell-autonomous effects play crucial roles.

A force-sensitive mutation reveals a non-canonical role for dynein in anaphase progression.

The diverse roles of the dynein motor in shaping microtubule networks and cargo transport complicate in vivo analysis of its functions significantly. To address this issue, we have generated a series of missense mutations in Drosophila Dynein heavy chain. We show that mutations associated with human neurological disease cause a range of defects, including impaired cargo trafficking in neurons. We also describe a novel microtubule-binding domain mutation that specifically blocks the metaphase-anaphase transition during mitosis in the embryo. This effect is independent from dynein's canonical role in silencing the spindle assembly checkpoint. Optical trapping of purified dynein complexes reveals that this mutation only compromises motor performance under load, a finding rationalized by the results of all-atom molecular dynamics simulations. We propose that dynein has a novel function in anaphase progression that depends on it operating in a specific load regime. More broadly, our work illustrates how in vivo functions of motors can be dissected by manipulating their mechanical properties.

Preclinical Evidence and Underlying Mechanisms of Polygonum multiflorum and Its Chemical Constituents Against Cognitive Impairments and Alzheimer's Disease.

Cognitive impairments, ranging from mild to severe, adversely affect daily functioning, quality of life, and work capacity. Despite significant efforts in the past decade, more than 200 promising drug candidates have failed in clinical trials. Herbal remedies are gaining interest as potential treatments for dementia due to their long history and safety, making them valuable for drug development. This review aimed to examine the mechanisms behind the effect of Polygonum multiflorum on cognitive function. This study focused primarily on the effects of Polygonum multiflorum and its chemical constituents on cognitive behavioral outcomes including the Morris water maze, the passive avoidance test, and the Y maze, as well as pathogenic targets of cognitive impairment and Alzheimer's disease (AD) like amyloid deposition, amyloid precursor protein, tau hyperphosphorylation, and cognitive decline. Additionally, a thorough evaluation of the mechanisms behind Polygonum multiflorum's impact on cognitive function was conducted. We reviewed the most recent data from preclinical research done on experimental models, particularly looking at Polygonum multiflorum's effects on cognitive decline and AD. According to recent research, Poligonum multiflorum and its bioactive components, stilbene, and emodin, influence cognitive behavioral results and regulate the pathological target of cognitive impairment and AD. Their mechanisms of action include reducing oxidative and mitochondrial damage, regulating neuroinflammation, halting apoptosis, and promoting increased neurogenesis and synaptogenesis. This review serves as a comprehensive compilation of current experiments on AD and other cognitive impairment models related to the therapeutic effects of Polygonum multiflorum. We believe that these findings can serve as a basis for future clinical trials and have potential applications in the treatment of human neurological disorders.

ROBUSTNESS EVALUATION OF MACHINE LEARNING ALGORITHMS FOR NEUROCOMPUTER INTERFACE SOFTWARE USING DISTRIBUTED AND PARALLEL COMPUTING

This article highlights the field of information technology for brain-computer interaction, and the main goal is to use it to determine patterns of human brain activity using electroencephalography (EEG) data. During the execution of the article, machine learning methods were used, namely such classifiers as Random Forest, Multi-Layer Perceptron (MLP), and Logistic Regression. The investigation begins with real-world experiments recording EEG signals during finger movement tasks, providing valuable insight into the complex dynamics of brain operation and interaction. Utilizing 10-fold cross-validation, the performance of each classifier is rigorously evaluated across various metrics, including accuracy, f1_weighted та roc_auc_ovr_weighted. Through this process, the robustness and consistency of classifier performance are assessed, with dispersion values computed to gauge variability across iterations. The findings reveal nuanced differences among the classifiers, with MLP demonstrating the highest robustness, followed by Logistic Regression and Random Forest. The main goal of the article was to find out the importance of such a classifier performance parameter as robustness. Software robustness is a key characteristic, especially in medical applications, where consistent and reliable performance of information technology is paramount. Neural interfaces offer many avenues for solving various limb problems, spinal cord injuries, and neurological diseases in humans. These devices contribute to improving the quality of life by minimizing these problems in people, which leads to increased mobility and functional capabilities of people. The article also emphasizes the potential associated with the transformation of neurointerface technologies in expanding human capabilities and revolutionizing human-machine interaction. In conclusion, the research contributes to advancing the field of brain-computer interaction by leveraging machine learning algorithms to decode neural signals and uncover hidden patterns within EEG data. By identifying the most stable classifier, the study lays the groundwork for the development of robust neurointerface technologies with practical applications in healthcare, rehabilitation, and beyond. Through interdisciplinary collaboration and innovative methodologies, the journey towards unlocking the full potential of brain-computer interaction continues, promising new horizons in human augmentation and technological innovation.

Microfluidic Interfaces for Chronic Bidirectional Access to the Brain.

Two-photon polymerization (TPP) is an additive manufacturing technique with micron-scale resolution that is rapidly gaining ground for a range of biomedical applications. TPP is particularly attractive for the creation of microscopic three-dimensional structures in biocompatible and noncytotoxic resins. Here, TPP is used to develop microfluidic interfaces which provide chronic fluidic access to the brain of preclinical research models. These microcatheters can be used for either convection-enhanced delivery (CED) or for the repeated collection of liquid biopsies. In a brain phantom, infusions with the micronozzle result in more localized distribution clouds and lower backflow compared to a control catheter. In mice, the delivery interface enables faster, more precise, and physiologically less disruptive fluid injections. A second microcatheter design enables repeated, longitudinal sampling of cerebrospinal fluid (CSF) over time periods as long as 250 days. Moreover, further in vivo studies demonstrate that the blood-CSF barrier is intact after chronic implantation of the sampling interface and that samples are suitable for downstream molecular analysis for the identification of nucleic acid- or peptide-based biomarkers. Ultimately, the versatility of this fabrication technique implies a great translational potential for simultaneous drug delivery and biomarker tracking in a range of human neurological diseases.

From Skin and Gut to the Brain: The Infectious Journey of the Human Commensal Fungus Malassezia and Its Neurological Consequences.

The human mycobiome encompasses diverse communities of fungal organisms residing within the body and has emerged as a critical player in shaping health and disease. While extensive research has focused on the skin and gut mycobiome, recent investigations have pointed toward the potential role of fungal organisms in neurological disorders. Among those fungal organisms, the presence of the commensal fungus Malassezia in the brain has created curiosity because of its commensal nature and primary association with the human skin and gut. This budding yeast is responsible for several diseases, such as Seborrheic dermatitis, Atopic dermatitis, Pityriasis versicolor, Malassezia folliculitis, dandruff, and others. However recent findings surprisingly show the presence of Malassezia DNA in the brain and have been linked to diseases like Alzheimer's disease, Parkinson's disease, Multiple sclerosis, and Amyotrophic lateral sclerosis. The exact role of Malassezia in these disorders is unknown, but its ability to infect human cells, travel through the bloodstream, cross the blood-brain barrier, and reside along with the lipid-rich neuronal cells are potential mechanisms responsible for pathogenesis. This also includes the induction of pro-inflammatory cytokines, disruption of the blood-brain barrier, gut-microbe interaction, and accumulation of metabolic changes in the brain environment. In this review, we discuss these key findings from studies linking Malassezia to neurological disorders, emphasizing the complex and multifaceted nature of these cases. Furthermore, we discuss potential mechanisms through which Malassezia might contribute to the development of neurological conditions. Future investigations will open up new avenues for our understanding of the fungal gut-brain axis and how it influences human behavior. Collaborative research efforts among microbiologists, neuroscientists, immunologists, and clinicians hold promise for unraveling the enigmatic connections between human commensal Malassezia and neurological disorders.

Tau, Glial Fibrillary Acidic Protein, and Neurofilament Light Chain as Brain Protein Biomarkers in Cerebrospinal Fluid and Blood for Diagnosis of Neurobiological Diseases.

Neurological damage is the pathological substrate of permanent disability in various neurodegenerative disorders. Early detection of this damage, including its identification and quantification, is critical to preventing the disease's progression in the brain. Tau, glial fibrillary acidic protein (GFAP), and neurofilament light chain (NfL), as brain protein biomarkers, have the potential to improve diagnostic accuracy, disease monitoring, prognostic assessment, and treatment efficacy. These biomarkers are released into the cerebrospinal fluid (CSF) and blood proportionally to the degree of neuron and astrocyte damage in different neurological disorders, including stroke, traumatic brain injury, multiple sclerosis, neurodegenerative dementia, and Parkinson's disease. Here, we review how Tau, GFAP, and NfL biomarkers are detected in CSF and blood as crucial diagnostic tools, as well as the levels of these biomarkers used for differentiating a range of neurological diseases and monitoring disease progression. We also discuss a biosensor approach that allows for the real-time detection of multiple biomarkers in various neurodegenerative diseases. This combined detection system of brain protein biomarkers holds significant promise for developing more specific and accurate clinical tools that can identify the type and stage of human neurological diseases with greater precision.

Monolayer, open-mesh, pristine PEDOT:PSS-based conformal brain implants for fully MRI-compatible neural interfaces

Understanding brain function is essential for advancing our comprehension of human cognition, behavior, and neurological disorders. Magnetic resonance imaging (MRI) stands out as a powerful tool for exploring brain function, providing detailed insights into its structure and physiology. Combining MRI technology with electrophysiological recording system can enhance the comprehension of brain functionality through synergistic effects. However, the integration of neural implants with MRI technology presents challenges because of its strong electromagnetic (EM) energy during MRI scans. Therefore, MRI-compatible neural implants should facilitate detailed investigation of neural activities and brain functions in real-time in high resolution, without compromising patient safety and imaging quality. Here, we introduce the fully MRI-compatible monolayer open-mesh pristine PEDOT:PSS neural interface. This approach addresses the challenges encountered while using traditional metal-based electrodes in the MRI environment such as induced heat or imaging artifacts. PEDOT:PSS has a diamagnetic property with low electrical conductivity and negative magnetic susceptibility similar to human tissues. Furthermore, by adopting the optimized open-mesh structure, the induced currents generated by EM energy are significantly diminished, leading to optimized MRI compatibility. Through simulations and experiments, our PEDOT:PSS-based open-mesh electrodes showed improved performance in reducing heat generation and eliminating imaging artifacts in an MRI environment. The electrophysiological recording capability was also validated by measuring the local field potential (LFP) from the somatosensory cortex with an in vivo experiment. The development of neural implants with maximized MRI compatibility indicates the possibility of potential tools for future neural diagnostics.

Rescue of impaired blood-brain barrier in tuberous sclerosis complex patient derived neurovascular unit

BackgroundTuberous sclerosis complex (TSC) is a multi-system genetic disease that causes benign tumors in the brain and other vital organs. The most debilitating symptoms result from involvement of the central nervous system and lead to a multitude of severe symptoms including seizures, intellectual disability, autism, and behavioral problems. TSC is caused by heterozygous mutations of either the TSC1 or TSC2 gene and dysregulation of mTOR kinase with its multifaceted downstream signaling alterations is central to disease pathogenesis. Although the neurological sequelae of the disease are well established, little is known about how these mutations might affect cellular components and the function of the blood–brain barrier (BBB).MethodsWe generated TSC disease-specific cell models of the BBB by leveraging human induced pluripotent stem cell and microfluidic cell culture technologies.ResultsUsing microphysiological systems, we demonstrate that a BBB generated from TSC2 heterozygous mutant cells shows increased permeability. This can be rescued by wild type astrocytes or by treatment with rapamycin, an mTOR kinase inhibitor.ConclusionOur results demonstrate the utility of microphysiological systems to study human neurological disorders and advance our knowledge of cell lineages contributing to TSC pathogenesis and informs future therapeutics.

Effective cryopreservation of human brain tissue and neural organoids

Human brain tissue models and organoids are vital for studying and modeling human neurological disease. However, the high cost of long-term cultured organoids inhibits their wide-ranging application. It is therefore urgent to develop methods for the cryopreservation of brain tissue and organoids. Here, we establish a method using methylcellulose, ethylene glycol, DMSO, and Y27632 (termed MEDY) for the cryopreservation of cortical organoids without disrupting the neural cytoarchitecture or functional activity. MEDY can be applied to multiple brain-region-specific organoids, including the dorsal/ventral forebrain, spinal cord, optic vesicle brain, and epilepsy patient-derived brain organoids. Additionally, MEDY enables the cryopreservation of human brain tissue samples, and pathological features are retained after thawing. Transcriptomic analysis shows that MEDY can protect synaptic function and inhibit the endoplasmic reticulum-mediated apoptosis pathway. MEDY will enable the large-scale and reliable storage of diverse neural organoids and living brain tissue and will facilitate wide-ranging research, medical applications, and drug screening.

Lysophosphatidylserine: A Signaling Lipid with Implications in Human Diseases.

Lysophosphatidylserine (lyso-PS) has emerged as yet another important signaling lysophospholipid in mammals, and deregulation in its metabolism has been directly linked to an array of human autoimmune and neurological disorders. It has an indispensable role in several biological processes in humans, and therefore, cellular concentrations of lyso-PS are tightly regulated to ensure optimal signaling and functioning in physiological settings. Given its biological importance, the past two decades have seen an explosion in the available literature toward our understanding of diverse aspects of lyso-PS metabolism and signaling and its association with human diseases. In this Review, we aim to comprehensively summarize different aspects of lyso-PS, such as its structure, biodistribution, chemical synthesis, and SAR studies with some synthetic analogs. From a biochemical perspective, we provide an exhaustive coverage of the diverse biological activities modulated by lyso-PSs, such as its metabolism and the receptors that respond to them in humans. We also briefly discuss the human diseases associated with aberrant lyso-PS metabolism and signaling and posit some future directions that may advance our understanding of lyso-PS-mediated mammalian physiology.

An adapted protocol to derive microglia from stem cells and its application in the study of CSF1R-related disorders

BackgroundInduced pluripotent stem cell-derived microglia (iMGL) represent an excellent tool in studying microglial function in health and disease. Yet, since differentiation and survival of iMGL are highly reliant on colony-stimulating factor 1 receptor (CSF1R) signaling, it is difficult to use iMGL to study microglial dysfunction associated with pathogenic defects in CSF1R.MethodsSerial modifications to an existing iMGL protocol were made, including but not limited to changes in growth factor combination to drive microglial differentiation, until successful derivation of microglia-like cells from an adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) patient carrying a c.2350G > A (p.V784M) CSF1R variant. Using healthy control lines, the quality of the new iMGL protocol was validated through cell yield assessment, measurement of microglia marker expression, transcriptomic comparison to primary microglia, and evaluation of inflammatory and phagocytic activities. Similarly, molecular and functional characterization of the ALSP patient-derived iMGL was carried out in comparison to healthy control iMGL.ResultsThe newly devised protocol allowed the generation of iMGL with enhanced transcriptomic similarity to cultured primary human microglia and with higher scavenging and inflammatory competence at ~ threefold greater yield compared to the original protocol. Using this protocol, decreased CSF1R autophosphorylation and cell surface expression was observed in iMGL derived from the ALSP patient compared to those derived from healthy controls. Additionally, ALSP patient-derived iMGL presented a migratory defect accompanying a temporal reduction in purinergic receptor P2Y12 (P2RY12) expression, a heightened capacity to internalize myelin, as well as heightened inflammatory response to Pam3CSK4. Poor P2RY12 expression was confirmed to be a consequence of CSF1R haploinsufficiency, as this feature was also observed following CSF1R knockdown or inhibition in mature control iMGL, and in CSF1RWT/KO and CSF1RWT/E633K iMGL compared to their respective isogenic controls.ConclusionsWe optimized a pre-existing iMGL protocol, generating a powerful tool to study microglial involvement in human neurological diseases. Using the optimized protocol, we have generated for the first time iMGL from an ALSP patient carrying a pathogenic CSF1R variant, with preliminary characterization pointing toward functional alterations in migratory, phagocytic and inflammatory activities.Graphical

Altered circular RNA expressions in extracellular vesicles from bronchoalveolar lavage fluids in mice after bacterial infections.

Circular RNAs (circRNAs) are a class of transcripts that often are generated by back-splicing that covalently connects the 3'end of the exon to the 5'end. CircRNAs are more resistant to nuclease and more stable than their linear counterparts. One of the well-recognized roles of circRNAs is the miRNA sponging effects that potentially lead to the regulation of downstream proteins. Despite that circRNAs have been reported to be involved in a wide range of human diseases, including cancers, cardiovascular, and neurological diseases, they have not been studied in inflammatory lung responses. Here, we analyzed the circRNA profiles detected in extracellular vesicles (EVs) obtained from the broncho-alveolar lavage fluids (BALF) in response to LPS or acid instillation in mice. Next, we validated two specific circRNAs in the BALF-EVs and BALF cells in response to endotoxin by RT-qPCR, using specific primers targeting the circular form of RNAs rather than the linear host RNAs. The expression of these selected circRNAs in the BALF inflammatory cells, alveolar macrophages (AMs), neutrophils, and lung tissue were analyzed. We further predicted the potential miRNAs that interact with these circRNAs. Our study is the first report to show that circRNAs are detectable in BALF EVs obtained from mice. The EV-cargo circRNAs are significantly altered by the noxious stimuli. The circRNAs identified using microarrays may be validated by RT-qPCR using primers specific to the circular but not the linear form. Future studies to investigate circRNA expression and function including miRNA sponging in lung inflammation potentially uncover novel strategies to develop diagnostic/therapeutic targets.

Exploiting fly models to investigate rare human neurological disorders.

Rare neurological diseases, while individually are rare, collectively impact millions globally, leading to diverse and often severe neurological symptoms. Often attributed to genetic mutations that disrupt protein function or structure, understanding their genetic basis is crucial for accurate diagnosis and targeted therapies. To investigate the underlying pathogenesis of these conditions, researchers often use non-mammalian model organisms, such as Drosophila (fruit flies), which is valued for their genetic manipulability, cost-efficiency, and preservation of genes and biological functions across evolutionary time. Genetic tools available in Drosophila, including CRISPR-Cas9, offer a means to manipulate gene expression, allowing for a deep exploration of the genetic underpinnings of rare neurological diseases. Drosophila boasts a versatile genetic toolkit, rapid generation turnover, and ease of large-scale experimentation, making it an invaluable resource for identifying potential drug candidates. Researchers can expose flies carrying disease-associated mutations to various compounds, rapidly pinpointing promising therapeutic agents for further investigation in mammalian models and, ultimately, clinical trials. In this comprehensive review, we explore rare neurological diseases where fly research has significantly contributed to our understanding of their genetic basis, pathophysiology, and potential therapeutic implications. We discuss rare diseases associated with both neuron-expressed and glial-expressed genes. Specific cases include mutations in CDK19 resulting in epilepsy and developmental delay, mutations in TIAM1 leading to a neurodevelopmental disorder with seizures and language delay, and mutations in IRF2BPL causing seizures, a neurodevelopmental disorder with regression, loss of speech, and abnormal movements. And we explore mutations in EMC1 related to cerebellar atrophy, visual impairment, psychomotor retardation, and gain-of-function mutations in ACOX1 causing Mitchell syndrome. Loss-of-function mutations in ACOX1 result in ACOX1 deficiency, characterized by very-long-chain fatty acid accumulation and glial degeneration. Notably, this review highlights how modeling these diseases in Drosophila has provided valuable insights into their pathophysiology, offering a platform for the rapid identification of potential therapeutic interventions. Rare neurological diseases involve a wide range of expression systems, and sometimes common phenotypes can be found among different genes that cause abnormalities in neurons or glia. Furthermore, mutations within the same gene may result in varying functional outcomes, such as complete loss of function, partial loss of function, or gain-of-function mutations. The phenotypes observed in patients can differ significantly, underscoring the complexity of these conditions. In conclusion, Drosophila represents an indispensable and cost-effective tool for investigating rare neurological diseases. By facilitating the modeling of these conditions, Drosophila contributes to a deeper understanding of their genetic basis, pathophysiology, and potential therapies. This approach accelerates the discovery of promising drug candidates, ultimately benefiting patients affected by these complex and understudied diseases.

A guide to selecting high-performing antibodies for Secreted frizzled-related protein 1 (sFRP-1) for use in Western Blot and immunoprecipitation.

Secreted frizzled-related protein 1 (sFRP-1) is a secreted protein, belonging to the secreted glycoprotein SFRP family. As a modulator of the Wnt/β-catenin signalling pathway, sFRP-1 has implications in human cancers and neurological diseases. If the community had access to well-characterized anti-sFRP-1 antibodies, the reproducibility of sFRP-1 research would be enhanced. In this study, we characterized 11 sFRP-1 commercial antibodies for Western Blot and immunoprecipitation, using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address the antibody reproducibility issue by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

Single-cell RNA-seq data analysis reveals functionally relevant biomarkers of early brain development and their regulatory footprints in human embryonic stem cells (hESCs).

The complicated process of neuronal development is initiated early in life, with the genetic mechanisms governing this process yet to be fully elucidated. Single-cell RNA sequencing (scRNA-seq) is a potent instrument for pinpointing biomarkers that exhibit differential expression across various cell types and developmental stages. By employing scRNA-seq on human embryonic stem cells, we aim to identify differentially expressed genes (DEGs) crucial for early-stage neuronal development. Our focus extends beyond simply identifying DEGs. We strive to investigate the functional roles of these genes through enrichment analysis and construct gene regulatory networks to understand their interactions. Ultimately, this comprehensive approach aspires to illuminate the molecular mechanisms and transcriptional dynamics governing early human brain development. By uncovering potential links between these DEGs and intelligence, mental disorders, and neurodevelopmental disorders, we hope to shed light on human neurological health and disease. In this study, we have used scRNA-seq to identify DEGs involved in early-stage neuronal development in hESCs. The scRNA-seq data, collected on days 26 (D26) and 54 (D54), of the in vitro differentiation of hESCs to neurons were analyzed. Our analysis identified 539 DEGs between D26 and D54. Functional enrichment of those DEG biomarkers indicated that the up-regulated DEGs participated in neurogenesis, while the down-regulated DEGs were linked to synapse regulation. The Reactome pathway analysis revealed that down-regulated DEGs were involved in the interactions between proteins located in synapse pathways. We also discovered interactions between DEGs and miRNA, transcriptional factors (TFs) and DEGs, and between TF and miRNA. Our study identified 20 significant transcription factors, shedding light on early brain development genetics. The identified DEGs and gene regulatory networks are valuable resources for future research into human brain development and neurodevelopmental disorders.

Current status and progress of research on the ADP-dependent glucokinase gene.

ADP-dependent glucokinase (ADPGK) produces glucose-6-phosphate with adenosine diphosphate (ADP) as the phosphate group donor, in contrast to ATP-dependent hexokinases (HKs). Originally found in archaea, ADPGK is involved in glycolysis. However, its biological function in most eukaryotic organisms is still unclear, and the molecular mechanism of action requires further investigation. This paper provides a concise overview of ADPGK's origin, biological function and clinical application. It aims to furnish scientific information for the diagnosis and treatment of human metabolic diseases, neurological disorders, and malignant tumours, and to suggest new strategies for the development of targeted drugs.

Abstract 3334: Combined treatment with PG3 and Nelfinavir induced synergistic apoptosis though sustained activation of integrated stress response

Abstract Integrated stress response (ISR) activation has been linked to many human diseases, including cancer and neurological diseases. The ISR controls protein synthesis and proteostasis. ISR main effector ATF4 transcriptional factor regulates the balance between survival and cell death. Under acute ISR, ATF4 promotes cell adaptation and survival. Sustained ISR leads to ATF4-mediated apoptosis. In many cancers, mutations or overexpression of oncogenes, results in enhanced cell proliferation and increased protein synthesis, which activate the ISR. Therefore, protein translation is reduced to maintain proteostasis. In other words, many cancers are primed by the ISR. Therefore, additional push by small molecule ISR-inducers will disrupt the balance and force cancer cells to enter apoptosis. Nelfinavir activates ATF4 through inhibition of eIF2α specific phosphatases CReP and GADD34. PG3 upregulates ATF4 through the HRI/eIF2α/ATF4 pathway. We hypothesize that combined treatment of PG3 and Nelfinavir will sustain upregulation of ATF4, and lead to ATF4-mediated cell apoptosis. Cell viability assays indicated that the combined treatment potently and synergistically inhibited cell viability in colorectal cancer cell lines HT29, SW480 and HCT116 p53−/−, osteosarcoma cell line U2OS and breast cancer cell lines MCF7 and T47D. The combined treatment sustained the induction of ATF4 and enhanced the expression of its proapoptotic target genes, CHOP and PUMA, and cell apoptosis. We confirmed that the enhanced induction of ATF4, CHOP and PUMA is through the ISR because ISRIB (ISR inhibitor) blocked the combined treatment-induced upregulation of ATF4, CHOP and PUMA. Citation Format: Xiaobing Tian, Wafik S. El-Deiry. Combined treatment with PG3 and Nelfinavir induced synergistic apoptosis though sustained activation of integrated stress response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3334.

Brain organoid protocols and limitations.

Stem cell-derived organoid technology is a powerful tool that revolutionizes the field of biomedical research and extends the scope of our understanding of human biology and diseases. Brain organoids especially open an opportunity for human brain research and modeling many human neurological diseases, which have lagged due to the inaccessibility of human brain samples and lack of similarity with other animal models. Brain organoids can be generated through various protocols and mimic whole brain or region-specific. To provide an overview of brain organoid technology, we summarize currently available protocols and list several factors to consider before choosing protocols. We also outline the limitations of current protocols and challenges that need to be solved in future investigation of brain development and pathobiology.

An in vivo BSL-2 model for henipavirus infection based on bioluminescence imaging of recombinant Cedar virus replication in mice.

Henipaviruses are enveloped single-stranded, negative-sense RNA viruses of the paramyxovirus family. Two henipaviruses, Nipah virus and Hendra virus, cause a systemic respiratory and/or neurological disease in humans and ten additional species of mammals, with a high fatality rate. Because of their highly pathogenic nature, Nipah virus and Hendra virus are categorized as BSL-4 pathogens, which limits the number and scope of translational research studies on these important human pathogens. To begin to address this limitation, we are developing a BSL-2 model of authentic henipavirus infection in mice, using the non-pathogenic henipavirus, Cedar virus. Notably, wild-type mice are highly resistant to Hendra virus and Nipah virus infection. However, previous work has shown that mice lacking expression of the type I interferon receptor (IFNAR-KO mice) are susceptible to both viruses. Here, we show that luciferase-expressing recombinant Cedar virus (rCedV-luc) is also able to replicate and establish a transient infection in IFNAR-KO mice, but not in wild-type mice. Using longitudinal bioluminescence imaging (BLI) of luciferase expression, we detected rCedV-luc replication as early as 10 h post-infection. Viral replication peaks between days 1 and 3 post-infection, and declines to levels undetectable by BLI by 7 days post-infection. Immunohistochemistry is consistent with viral infection and replication in endothelial cells and other non-immune cell types within tissue parenchyma. Serology analyses demonstrate significant IgG responses to the Cedar virus surface glycoprotein with potent neutralizing activity in IFNAR-KO mice, whereas antibody responses in wild-type animals were non-significant. Overall, these data suggest that rCedV-luc infection of IFNAR-KO mice represents a viable platform for the study of in vivo henipavirus replication, anti-henipavirus host responses and henipavirus-directed therapeutics.