Human Primary Neurons Research Articles

Pharmacology and Mechanism of Action of Suzetrigine, a Potent and Selective NaV1.8 Pain Signal Inhibitor for the Treatment of Moderate to Severe Pain.

There is a high unmet need for safe and effective non-opioid medicines to treat moderate to severe pain without risk of addiction. Voltage-gated sodium channel 1.8 (NaV1.8) is a genetically and pharmacologically validated pain target that is selectively expressed in peripheral pain-sensing neurons and not in the central nervous system (CNS). Suzetrigine (VX-548) is a potent and selective inhibitor of NaV1.8, which has demonstrated clinical efficacy and safety in multiple acute pain studies. Our study was designed to characterize the mechanism of action of suzetrigine and assess both nonclinical and clinical data to test the hypothesis that selective NaV1.8 inhibition translates into clinical efficacy and safety, including lack of addictive potential. Preclinical pharmacology and mechanism of action studies were performed in vitro using electrophysiology and radiolabeled binding methods in cells recombinantly expressing human NaV channels, human proteins, and primary human dorsal root ganglion (DRG) sensory neurons. Safety and addictive potential assessments included in vitro secondary pharmacology studies, nonclinical repeat-dose toxicity and dependence studies in rats and/or monkeys, and a systematic analysis of adverse event data generated from 2447 participants from phase 3 acute pain studies of suzetrigine. Suzetrigine is selective against all other NaV subtypes (≥ 31,000-fold) and 180 other molecular targets. Suzetrigine inhibits NaV1.8 by binding to the protein's second voltage sensing domain (VSD2) to stabilize the closed state of the channel. This novel allosteric mechanism results in tonic inhibition of NaV1.8 and reduces pain signals in primary human DRG sensory neurons. Nonclinical and clinical safety assessments with suzetrigine demonstrate no adverse CNS, cardiovascular or behavioral effects and no evidence of addictive potential or dependence. The comprehensive pharmacology assessment presented here indicates that suzetrigine represents the first in a new class of non-opioid analgesics that are selective NaV1.8 pain signal inhibitors acting in the peripheral nervous system to safely treat pain without addictive potential.

Linear ubiquitination at damaged lysosomes induces local NFKB activation and controls cell survival

ABSTRACT Lysosomes are the major cellular organelles responsible for nutrient recycling and degradation of cellular material. Maintenance of lysosomal integrity is essential for cellular homeostasis and lysosomal membrane permeabilization (LMP) sensitizes toward cell death. Damaged lysosomes are repaired or degraded via lysophagy, during which glycans, exposed on ruptured lysosomal membranes, are recognized by galectins leading to K48- and K63-linked poly-ubiquitination (poly-Ub) of lysosomal proteins followed by recruitment of the macroautophagic/autophagic machinery and degradation. Linear (M1) poly-Ub, catalyzed by the linear ubiquitin chain assembly complex (LUBAC) E3 ligase and removed by OTULIN (OTU deubiquitinase with linear linkage specificity) exerts important functions in immune signaling and cell survival, but the role of M1 poly-Ub in lysosomal homeostasis remains unexplored. Here, we demonstrate that L-leucyl-leucine methyl ester (LLOMe)-damaged lysosomes accumulate M1 poly-Ub in an OTULIN- and K63 Ub-dependent manner. LMP-induced M1 poly-Ub at damaged lysosomes contributes to lysosome degradation, recruits the NFKB (nuclear factor kappa B) modulator IKBKG/NEMO and locally activates the inhibitor of NFKB kinase (IKK) complex to trigger NFKB activation. Inhibition of lysosomal degradation enhances LMP- and OTULIN-regulated cell death, indicating pro-survival functions of M1 poly-Ub during LMP and potentially lysophagy. Finally, we demonstrate that M1 poly-Ub also occurs at damaged lysosomes in primary mouse neurons and induced pluripotent stem cell-derived primary human dopaminergic neurons. Our results reveal novel functions of M1 poly-Ub during lysosomal homeostasis, LMP and degradation of damaged lysosomes, with important implications for NFKB signaling, inflammation and cell death. Abbreviation: ATG: autophagy related; BafA1: bafilomycin A1; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CRISPR: clustered regularly interspaced short palindromic repeats; CHUK/IKKA: component of inhibitor of nuclear factor kappa B kinase complex; CUL4A-DDB1-WDFY1: cullin 4A-damage specific DNA binding protein 1-WD repeat and FYVE domain containing 1; DGCs: degradative compartments; DIV: days in vitro; DUB: deubiquitinase/deubiquitinating enzyme; ELDR: endo-lysosomal damage response; ESCRT: endosomal sorting complex required for transport; FBXO27: F-box protein 27; GBM: glioblastoma multiforme; IKBKB/IKKB: inhibitor of nuclear factor kappa B kinase subunit beta; IKBKG/NEMO: inhibitor of nuclear factor kappa B kinase regulatory subunit gamma; IKK: inhibitor of NFKB kinase; iPSC: induced pluripotent stem cell; KBTBD7: kelch repeat and BTB domain containing 7; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LCD: lysosomal cell death; LGALS: galectin; LMP: lysosomal membrane permeabilization; LLOMe: L-leucyl-leucine methyl ester; LOP: loperamide; LUBAC: linear ubiquitin chain assembly complex; LRSAM1: leucine rich repeat and sterile alpha motif containing 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; NBR1: NBR1 autophagy cargo receptor; NFKB/NF-κB: nuclear factor kappa B; NFKBIA/IĸBα: nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha; OPTN: optineurin; ORAS: OTULIN-related autoinflammatory syndrome; OTULIN: OTU deubiquitinase with linear linkage specificity; RING: really interesting new gene; RBR: RING-in-between-RING; PLAA: phospholipase A2 activating protein; RBCK1/HOIL-1: RANBP2-type and C3HC4-type zinc finger containing 1; RNF31/HOIP: ring finger protein 31; SHARPIN: SHANK associated RH domain interactor; SQSTM1/p62: sequestosome 1; SR-SIM: super-resolution-structured illumination microscopy; TAX1BP1: Tax1 binding protein 1; TBK1: TANK binding kinase 1; TH: tyrosine hydroxylase; TNF/TNFα: tumor necrosis factor; TNFRSF1A/TNFR1-SC: TNF receptor superfamily member 1A signaling complex; TRIM16: tripartite motif containing 16; Ub: ubiquitin; UBE2QL1: ubiquitin conjugating enzyme E2 QL1; UBXN6/UBXD1: UBX domain protein 6; VCP/p97: valosin containing protein; WIPI2: WD repeat domain, phosphoinositide interacting 2; YOD1: YOD1 deubiquitinase.

Profiling Human iPSC-Derived Sensory Neurons for Analgesic Drug Screening Using a Multi-Electrode Array.

Chronic pain affects approximately 1.5 billion people worldwide, yet effective treatments remain elusive. A significant barrier to progress in analgesic drug discovery is the limited translation of preclinical findings to human clinical outcomes. Traditional rodent models, although widely used, often fail to accurately predict human responses, while human primary tissues are limited by scarcity, technical difficulties, and ethical concerns. Recent advancements have identified human induced pluripotent stem cell (hiPSC)-derived nociceptors as promising alternatives; however, current differentiation protocols produce cells with inconsistent and physiologically questionable phenotypes.To address these challenges, our study introduces a novel high-content screening (HCS) platform using hiPSC-derived nociceptors cultured on multi-well micro-electrode arrays (MEAs). The "Anatomic" protocol, used to generate these nociceptors, ensures cells with transcriptomic profiles closely matching human primary sensory neurons. Our platform achieves nearly 100% active electrode yield within two weeks and demonstrates sustained, stable activity over time. Additionally, robust Z' factor analysis (exceeding 0.5) confirms the platform's reliability, while pharmacological validation establishes the functional expression of critical analgesic targets. This innovative approach improves both the efficiency and clinical relevance of analgesic drug screening, potentially bridging the translational gap between preclinical studies and human clinical trials, and offering new hope for effective pain management.

Modeling extrahepatic hepatitis E virus infection in induced human primary neurons

Hepatitis E virus (HEV) infections are one of the most common causes of acute viral hepatitis, annually causing over 3 million symptomatic cases and 70,000 deaths worldwide. Historically, HEV was described as a hepatotropic virus, but has recently been associated with various extrahepatic manifestations including neurological disorders such as Guillain-Barré syndrome and neuralgic amyotrophy. However, the underlying pathogenesis of these neurological diseases remains largely unknown. The aim of this study was to investigate extrahepatic HEV manifestations in a neuronal model system using human-induced primary neurons (iPNs). Renal epithelial cells from human urine were reprogrammed to induced pluripotent stem cells to generate neuronal progenitor cells, which were subsequently differentiated into iPNs over 21 d. These iPNs supported HEV infection preferentially in neurite-bearing cells. Transcriptional profiling of the neuronal development process as well as viral infection dynamics in iPNs uncovered a lack of antiviral innate immune responses to HEV infection with only an intrinsic expression of distinct interferon-regulated genes and signaling molecules. Viral open reading frame 2 encoded capsid protein could be visualized by volumetric three-dimensional reconstitution within the neurites, which were reduced in length in an HEV inoculation-dependent manner. In conclusion, this neuron-derived human model system provides a powerful tool for studying extrahepatic manifestations of HEV infection. It further indicates a potential mechanism of pathogenesis driven by the interaction between host and viral factors.

The Parkinson's disease DJ-1/PARK7 gene controls peripheral neuronal excitability and painful neuropathy.

Parkinson's disease is a progressive neurodegenerative disease with well-documented motor symptoms as well as less recognised, but significant, non-motor symptoms. These non-motor symptoms include prodromal pain and peripheral neuropathy, the causes of which are unknown. We investigated the role of DJ-1/PARK7, a Parkinson's disease-associated gene, in prodromal pain and peripheral neuropathy. Using DJ-1 deficient mice, we conducted comprehensive sensory tests, cutaneous staining, molecular analyses and electrophysiological studies on mouse and human primary sensory neurons from dorsal root ganglia. We found that these mice exhibited cold hypersensitivity, oxidative stress, and neuropathy of the cutaneous fibres of primary sensory neurones before any motor impairments were observed. Mechanistically, DJ-1 in primary sensory neurones regulated this hypersensitivity and neuropathy via TRPA1 signalling. Interestingly, we discovered that DJ-1 also plays a role in the progression of chemotherapy-induced peripheral neuropathies. Pain and mechanisms associated with these neuropathies were exacerbated in DJ-1 deficient mice but were significantly reduced by the pharmacological activation of DJ-1. Importantly, we also confirmed the expression of DJ-1 and its therapeutic potential in human primary sensory neurons. Thus, we uncover a peripheral mechanism of DJ-1 and propose that it may serve as a new target for developing therapeutic approaches for Parkinson's disease-linked and other painful neuropathies.

Biochemical Properties of Synaptic Proteins Are Dependent on Tissue Preparation: NMDA Receptor Solubility Is Regulated by the C-Terminal Tail.

Synaptic proteins are essential for neuronal development, synaptic transmission, and synaptic plasticity. The postsynaptic density (PSD) is a membrane-associated structure at excitatory synapses, which is composed of a huge protein complex. To understand the interactions and functions of PSD proteins, researchers have employed a variety of imaging and biochemical approaches including sophisticated mass spectrometry. However, the field is lacking a systematic comparison of different experimental conditions and how they might influence the study of the PSD interactome isolated from various tissue preparations. To evaluate the efficiency of several common solubilization conditions, we isolated receptors, scaffolding proteins, and adhesion molecules from brain tissue or primary cultured neurons or human forebrain neurons differentiated from induced pluripotent stem cells (iPSCs). We observed some striking differences in solubility. We found that N-methyl-d-aspartate receptors (NMDARs) and PSD-95 are relatively insoluble in brain tissue, cultured neurons, and human forebrain neurons compared to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic receptors (AMPARs) or SAP102. In general, synaptic proteins were more soluble in primary neuronal cultures and human forebrain neurons compared to brain tissue. Interestingly, NMDARs are relatively insoluble in HEK293T cells suggesting that insolubility does not directly represent the synaptic fraction but rather it is related to a detergent-insoluble fraction such as lipid rafts. Surprisingly, truncation of the intracellular carboxyl-terminal tail (C-tail) of NMDAR subunits increased NMDAR solubility in HEK293T cells. Our findings show that detergent, pH, and temperature are important for protein preparations to study PSD protein complexes, and NMDAR solubility is regulated by its C-tail, thus providing a technical guide to study synaptic interactomes and subcellular localization of synaptic proteins.

The V-ATPase/ATG16L1 axis is controlled by the V1H subunit

Defects in organellar acidification indicate compromised or infected compartments. Recruitment of the autophagy-related ATG16L1 complex to pathologically neutralized organelles targets ubiquitin-like ATG8 molecules to perturbed membranes. How this process is coupled to proton gradient disruption is unclear. Here, we reveal that the V1H subunit of the vacuolar ATPase (V-ATPase) proton pump binds directly to ATG16L1. The V1H/ATG16L1 interaction only occurs within fully assembled V-ATPases, allowing ATG16L1 recruitment to be coupled to increased V-ATPase assembly following organelle neutralization. Cells lacking V1H fail to target ATG8s during influenza infection or after activation of the immune receptor stimulator of interferon genes (STING). We identify a loop within V1H that mediates ATG16L1 binding. A neuronal V1H isoform lacks this loop and is associated with attenuated ATG8 targeting in response to ionophores in primary murine and human iPSC-derived neurons. Thus, V1H controls ATG16L1 recruitment following proton gradient dissipation, suggesting that the V-ATPase acts as a cell-intrinsic damage sensor.

Integration of Functional Human Auditory Neural Circuits Based on a 3D Carbon Nanotube System.

The physiological interactions between the peripheral and central auditory systems are crucial for auditory information transmission and perception, while reliable models for auditory neural circuits are currently lacking. To address this issue, mouse and human neural pathways are generated by utilizing a carbon nanotube nanofiber system. The super-aligned pattern of the scaffold renders the axons of the bipolar and multipolar neurons extending in a parallel direction. In addition, the electrical conductivity of the scaffold maintains the electrophysiological activity of the primary mouse auditory neurons. The mouse and human primary neurons from peripheral and central auditory units in the system are then co-cultured and showed that the two kinds of neurons form synaptic connections. Moreover, neural progenitor cells of the cochlea and auditory cortex are derived from human embryos to generate region-specific organoids and these organoids are assembled in the nanofiber-combined 3D system. Using optogenetic stimulation, calcium imaging, and electrophysiological recording, it is revealed that functional synaptic connections are formed between peripheral neurons and central neurons, as evidenced by calcium spiking and postsynaptic currents. The auditory circuit model will enable the study of the auditory neural pathway and advance the search for treatment strategies for disorders of neuronal connectivity in sensorineural hearing loss.

Secondary progressive MS cerebrospinal fluid induces axonal degeneration in human primary neurons (P7-6.018)

Secondary progressive MS cerebrospinal fluid induces axonal degeneration in human primary neurons (P7-6.018)

Mitophagy Upregulation Occurs Early in the Neurodegenerative Process Mediated by α-Synuclein.

Parkinson's disease (PD) is a progressive neurogenerative movement disorder characterized by dopaminergic cell death within the substantia nigra pars compacta (SNpc) due to the aggregation-prone protein α-synuclein. Accumulation of α-synuclein is implicated in mitochondrial dysfunction and disruption of the autophagic turnover of mitochondria, or mitophagy, which is an essential quality control mechanism proposed to preserve mitochondrial fidelity in response to aging and stress. Yet, the precise relationship between α-synuclein accumulation, mitochondrial autophagy, and dopaminergic cell loss remains unresolved. Here, we determine the kinetics of α-synuclein overexpression and mitophagy using the pH-sensitive fluorescent mito-QC reporter. We find that overexpression of mutant A53T α-synuclein in either human SH-SY5Y cells or rat primary cortical neurons induces mitophagy. Moreover, the accumulation of mutant A53T α-synuclein in the SNpc of rats results in mitophagy dysregulation that precedes the onset of dopaminergic neurodegeneration. This study reveals a role for mutant A53T α-synuclein in inducing mitochondrial dysfunction, which may be an early event contributing to neurodegeneration.

Effects of Prolactin on Brain Neurons under Hypoxia.

The levels and potential role of prolactin (PRL) in the brain under conditions of acute systemic hypoxia were examined, focusing on the accumulation of PRL in cerebrospinal fluid (CSF) and its effects on neuronal activity and injury. The amount of PRL in the brain was investigated using brain tissues from forensic autopsy cases. We counted the number of neurites that formed in human primary neurons (HNs) after the addition of PRL. Furthermore, HNs supplemented with PRL or triiodothyronine (T3) were exposed to hypoxic conditions, and the dead cells were counted. The results showed correlations between brain PRL and CSF PRL levels. Additionally, PRL accumulation in the brain was observed in cases of asphyxia. In vitro experimental findings indicated increased neurite formation in the HNs treated with PRL. Moreover, both PRL and T3 demonstrated neuroprotective effects against hypoxia-induced neuronal cell death, with PRL showing stronger neuroprotective potential than T3. These results suggest that PRL accumulates in the brain during hypoxia, potentially influences neuronal activity, and exhibits neuroprotective properties against hypoxia-induced neuronal injury.

Axonal Lysosomal Assays for Characterizing the Effects of LRRK2 G2019S.

The degeneration of axon terminals before the soma, referred to as "dying back", is a feature of Parkinson's disease (PD). Axonal assays are needed to model early PD pathogenesis as well as identify protective therapeutics. We hypothesized that defects in axon lysosomal trafficking as well as injury repair might be important contributing factors to "dying back" pathology in PD. Since primary human PD neurons are inaccessible, we developed assays to quantify axonal trafficking and injury repair using induced pluripotent stem cell (iPSC)-derived neurons with LRRK2 G2019S, which is one of the most common known PD mutations, and isogenic controls. We observed a subtle axonal trafficking phenotype that was partially rescued by a LRRK2 inhibitor. Mutant LRRK2 neurons showed increased phosphorylated Rab10-positive lysosomes, and lysosomal membrane damage increased LRRK2-dependent Rab10 phosphorylation. Neurons with mutant LRRK2 showed a transient increase in lysosomes at axotomy injury sites. This was a pilot study that used two patient-derived lines to develop its methodology; we observed subtle phenotypes that might correlate with heterogeneity in LRRK2-PD patients. Further analysis using additional iPSC lines is needed. Therefore, our axonal lysosomal assays can potentially be used to characterize early PD pathogenesis and test possible therapeutics.

Directed Differentiation of Neurons from Human iPSCs for Modeling Neurological Disorders.

Human-induced pluripotent stem cell (hiPSC) technology has enabledcomprehensive human cell-based disease modeling in vitro. Due to limited accessibility of primary human neurons as well as species-specific divergence between human and rodent brain tissues, hiPSC-derived neurons have become a popular tool for studying neuronal biology in a dish. Here, we provide methods for transcription factor-driven directed differentiation of neurons from hiPSCs via a neural progenitor cell (NPC) intermediate. Doxycycline-inducible expression of neuron fate-determining transcription factors neurogenin 2 (NGN2) and achaete-scute homolog 1 (ASCL1) enables rapid and controllable differentiation of human neurons for disease modeling applications. The provided method is also designed to improve the reproducibility of human neuron differentiation by reducing the batch-to-batch variation of NPC differentiation and lentiviral transduction.

Nuclear Shuttling of IGF1R/INSR in Alzheimer’s disease and its corresponding role in regulation of neurodegeneration

AbstractBackgroundAlzheimer’s disease (AD) has been long associated with ageing and Insulin dysregulation. The receptors and ligands of the Insulin family including Insulin, IGF1, INSR, and IGF1R mainly has been explored over the years. However, their differential distribution in terms of compartmentalization and trafficking requires investigation.MethodWe started this study with SHSY5Y cells and checked for differential distribution in nucleus and cytoplasm in case of AD model. We repeated the study in N2A cells. To understand the nuclear shuttling better, we extended our study in differentiated SHSY5Y (into the Acetylcholinergic lineage) and exploited super resolution microscopy for imaging and trafficking. We fractionated the cellular compartments for biochemical evidence. To ascertain the partners and forms of protein that shuttles, antibody based assays were performed. For validation of this phenomena as of uniform occurrence, mice brain tissues and human primary neurons were utilized for imaging studies.ResultLigand independent shuttling into the nucleus occurs in case of AD models in presence of amyloid beta (1‐42). There is a 6.4 fold increase in phosphorylation in cytosolic pIGF1R/INSR in case of AD and a 2.5 fold increase in nuclear fractions. The occurrence correlated with the increase in Cyclin D1 production as suggested by ChIP studies. Upon treatment with Sumoylation inhibitor which blocks IGF1R transit into the nucleus, Cyclin D1 levels lowered validating transcriptional control of Cyclin D1 by IGF1R in AD scenario. Thus cell cycle can be directly influenced through interaction of IGF1R with binding partners LEF and TCF in AD.ConclusionCanonical ligand independent shuttling of phosphorylated IGF1R/INSR influences the cell cycle in AD thus triggering cell cycle through a direct control mechanism.

Negative modulation of mitochondrial calcium uniporter complex protects neurons against ferroptosis

Ferroptosis is an iron- and reactive oxygen species (ROS)-dependent form of regulated cell death, that has been implicated in Alzheimer’s disease and Parkinson’s disease. Inhibition of cystine/glutamate antiporter could lead to mitochondrial fragmentation, mitochondrial calcium ([Ca2+]m) overload, increased mitochondrial ROS production, disruption of the mitochondrial membrane potential (ΔΨm), and ferroptotic cell death. The observation that mitochondrial dysfunction is a characteristic of ferroptosis makes preservation of mitochondrial function a potential therapeutic option for diseases associated with ferroptotic cell death. Mitochondrial calcium levels are controlled via the mitochondrial calcium uniporter (MCU), the main entry point of Ca2+ into the mitochondrial matrix. Therefore, we have hypothesized that negative modulation of MCU complex may confer protection against ferroptosis. Here we evaluated whether the known negative modulators of MCU complex, ruthenium red (RR), its derivative Ru265, mitoxantrone (MX), and MCU-i4 can prevent mitochondrial dysfunction and ferroptotic cell death. These compounds mediated protection in HT22 cells, in human dopaminergic neurons and mouse primary cortical neurons against ferroptotic cell death. Depletion of MICU1, a [Ca2+]m gatekeeper, demonstrated that MICU is protective against ferroptosis. Taken together, our results reveal that negative modulation of MCU complex represents a therapeutic option to prevent degenerative conditions, in which ferroptosis is central to the progression of these pathologies.

Classification of T lymphocyte motility behaviors using a machine learning approach.

T lymphocytes migrate into organs and interact with local cells to perform their functions. How human T lymphocytes communicate with organ-specific cells and participate in pathobiological processes remains unresolved. Brain infiltration of T lymphocytes is associated with multiple neurological disorders. Thus, to characterize the behavior of human T lymphocytes reaching the human brain, we performed time-lapse microscopy on human CD8+ T lymphocytes co-cultured with either primary human astrocytes or neurons. Using traditional manual and visual assessment of microscopy data, we identified distinct CD8+ T lymphocyte motility behaviors. However, such characterization is time and labor-intensive. In this work, we trained and validated a machine-learning model for the automated classification of behaviors of CD8+ T lymphocytes interacting with astrocytes and neurons. A balanced random forest was trained for the binary classification of established classes of cell behaviors (synapse vs. kinapse) as well as visually identified behaviors (scanning, dancing, and poking). Feature selection was performed during 3-fold cross-validation using the minimum redundancy maximum relevance algorithm. Results show promising performances when tested on a held-out dataset of CD8+ T lymphocytes interacting with astrocytes with a new experimenter and a held-out independent dataset of CD8+ T lymphocytes interacting with neurons. When tested on the independent CD8+ T cell-neuron dataset, the final model achieved a binary classification accuracy of 0.82 and a 3-class accuracy of 0.79. This novel automated classification approach could significantly reduce the time required to label cell motility behaviors while facilitating the identification of interactions of T lymphocytes with multiple cell types.

Methylglyoxal-induced neurotoxic effects in primary neuronal-like cells transdifferentiated from human mesenchymal stem cells: Impact of low concentrations.

In the last decades, advanced glycation end-products (AGEs) have aroused the interest of the scientific community due to the increasing evidence of their involvement in many pathophysiological processes including various neurological disorders and cognitive decline age related. Methylglyoxal (MG) is one of the reactive dicarbonyl precursors of AGEs, mainly generated as a by-product of glycolysis, whose accumulation induces neurotoxicity. In our study, MG cytotoxicity was evaluated employing a human stem cell-derived model, namely, neuron-like cells (hNLCs) transdifferentiated from mesenchymal stem/stromal cells, which served as a source of human based species-specific "healthy" cells. MG increased ROS production and induced the first characteristic apoptotic hallmarks already at low concentrations (≥10μM), decreased the cell growth (≥5-10μM) and viability (≥25 μM), altered Glo-1 and Glo-2 enzymes (≥25 μM), and markedly affected the neuronal markers MAP-2 and NSE causing their loss at low MG concentrations (≥10μM). Morphological alterations started at 100 μM, followed by even more marked effects and cell death after few hours (5h) from 200 μM MG addition. Substantially, most effects occurred as low as 10μM, concentration much lower than that reported from previous observations using different in vitro cell-based models (e.g., human neuroblastoma cell lines, primary animal cells, and human iPSCs). Remarkably, this low effective concentration approaches the level range measured in biological samples of pathological subjects. The use of a suitable cellular model, that is, human primary neurons, can provide an additional valuable tool, mimicking better the physiological and biochemical properties of brain cells, in order to evaluate the mechanistic basis of molecular and cellular alterations in CNS.

Using Live Cell STED Imaging to Visualize Mitochondrial Inner Membrane Ultrastructure in Neuronal Cell Models.

Mitochondria play many essential roles in the cell, including energy production, regulation of Ca2+ homeostasis, lipid biosynthesis, and production of reactive oxygen species (ROS). These mitochondria-mediated processes take on specialized roles in neurons, coordinating aerobic metabolism to meet the high energy demands of these cells, modulating Ca2+ signaling, providing lipids for axon growth and regeneration, and tuning ROS production for neuronal development and function. Mitochondrial dysfunction is therefore a central driver in neurodegenerative diseases. Mitochondrial structure and function are inextricably linked. The morphologically complex inner membrane with structural infolds called cristae harbors many molecular systems that perform the signature processes of the mitochondrion. The architectural features of the inner membrane are ultrastructural and therefore, too small to be visualized by traditional diffraction-limited resolved microscopy. Thus, most insights on mitochondrial ultrastructure have come from electron microscopy on fixed samples. However, emerging technologies in super-resolution fluorescence microscopy now provide resolution down to tens of nanometers, allowing visualization of ultrastructural features in live cells. Super-resolution imaging therefore offers an unprecedented ability to directly image fine details of mitochondrial structure, nanoscale protein distributions, and cristae dynamics, providing fundamental new insights that link mitochondria to human health and disease. This protocol presents the use of stimulated emission depletion (STED) super-resolution microscopy to visualize the mitochondrial ultrastructure of live human neuroblastoma cells and primary rat neurons. This procedure is organized into five sections: (1) growth and differentiation of the SH-SY5Y cell line, (2) isolation, plating, and growth of primary rat hippocampal neurons, (3) procedures for staining cells for live STED imaging, (4) procedures for live cell STED experiments using a STED microscopefor reference, and (5) guidance for segmentation and image processing using examples to measure and quantify morphological features of the inner membrane.

Pooled analysis of frontal lobe transcriptomic data identifies key mitophagy gene changes in Alzheimer's disease brain.

The growing prevalence of Alzheimer's disease (AD) is becoming a global health challenge without effective treatments. Defective mitochondrial function and mitophagy have recently been suggested as etiological factors in AD, in association with abnormalities in components of the autophagic machinery like lysosomes and phagosomes. Several large transcriptomic studies have been performed on different brain regions from AD and healthy patients, and their data represent a vast source of important information that can be utilized to understand this condition. However, large integration analyses of these publicly available data, such as AD RNA-Seq data, are still missing. In addition, large-scale focused analysis on mitophagy, which seems to be relevant for the aetiology of the disease, has not yet been performed. In this study, publicly available raw RNA-Seq data generated from healthy control and sporadic AD post-mortem human samples of the brain frontal lobe were collected and integrated. Sex-specific differential expression analysis was performed on the combined data set after batch effect correction. From the resulting set of differentially expressed genes, candidate mitophagy-related genes were identified based on their known functional roles in mitophagy, the lysosome, or the phagosome, followed by Protein-Protein Interaction (PPI) and microRNA-mRNA network analysis. The expression changes of candidate genes were further validated in human skin fibroblast and induced pluripotent stem cells (iPSCs)-derived cortical neurons from AD patients and matching healthy controls. From a large dataset (AD: 589; control: 246) based on three different datasets (i.e., ROSMAP, MSBB, & GSE110731), we identified 299 candidate mitophagy-related differentially expressed genes (DEG) in sporadic AD patients (male: 195, female: 188). Among these, the AAA ATPase VCP, the GTPase ARF1, the autophagic vesicle forming protein GABARAPL1 and the cytoskeleton protein actin beta ACTB were selected based on network degrees and existing literature. Changes in their expression were further validated in AD-relevant human in vitro models, which confirmed their down-regulation in AD conditions. Through the joint analysis of multiple publicly available data sets, we identify four differentially expressed key mitophagy-related genes potentially relevant for the pathogenesis of sporadic AD. Changes in expression of these four genes were validated using two AD-relevant human in vitro models, primary human fibroblasts and iPSC-derived neurons. Our results provide foundation for further investigation of these genes as potential biomarkers or disease-modifying pharmacological targets.

Impedance spectroscopy of the cell/nanovolcano interface enables optimization for electrophysiology

Volcano-shaped microelectrodes have demonstrated superior performance in measuring attenuated intracellular action potentials from cardiomyocyte cultures. However, their application to neuronal cultures has not yet yielded reliable intracellular access. This common pitfall supports a growing consensus in the field that nanostructures need to be pitched to the cell of interest to enable intracellular access. Accordingly, we present a new methodology that enables us to resolve the cell/probe interface noninvasively through impedance spectroscopy. This method measures changes in the seal resistance of single cells in a scalable manner to predict the quality of electrophysiological recordings. In particular, the impact of chemical functionalization and variation of the probe’s geometry can be quantitatively measured. We demonstrate this approach on human embryonic kidney cells and primary rodent neurons. Through systematic optimization, the seal resistance can be increased by as much as 20-fold with chemical functionalization, while different probe geometries demonstrated a lower impact. The method presented is therefore well suited to the study of cell coupling to probes designed for electrophysiology, and it is poised to contribute to elucidate the nature and mechanism of plasma membrane disruption by micro/nanostructures.