Knock-Down of HDAC2 in Human Induced Pluripotent Stem Cell Derived Neurons Improves Neuronal Mitochondrial Dynamics, Neuronal Maturation and Reduces Amyloid Beta Peptides.

Histone deacetylase 2 (HDAC2) is a major HDAC protein in the adult brain and has been shown to regulate many neuronal genes. The aberrant expression of HDAC2 and subsequent dysregulation of neuronal gene expression is implicated in neurodegeneration and brain aging. Human induced pluripotent stem cell-derived neurons (hiPSC-Ns) are widely used models for studying neurodegenerative disease mechanisms, but the role of HDAC2 in hiPSC-N differentiation and maturation has not been explored. In this study, we show that levels of HDAC2 progressively decrease as hiPSCs are differentiated towards neurons. This suppression of HDAC2 inversely corresponds to an increase in neuron-specific isoforms of Endophilin-B1, a multifunctional protein involved in mitochondrial dynamics. Expression of neuron-specific isoforms of Endophilin-B1 is accompanied by concomitant expression of a neuron-specific alternative splicing factor, SRRM4. Manipulation of HDAC2 and Endophilin-B1 using lentiviral approaches shows that the knock-down of HDAC2 or the overexpression of a neuron-specific Endophilin-B1 isoform promotes mitochondrial elongation and protects against cytotoxic stress in hiPSC-Ns, while HDAC2 knock-down specifically influences genes regulating mitochondrial dynamics and synaptogenesis. Furthermore, HDAC2 knock-down promotes enhanced mitochondrial respiration and reduces levels of neurotoxic amyloid beta peptides. Collectively, our study demonstrates a role for HDAC2 in hiPSC-neuronal differentiation, highlights neuron-specific isoforms of Endophilin-B1 as a marker of differentiating hiPSC-Ns and demonstrates that HDAC2 regulates key neuronal and mitochondrial pathways in hiPSC-Ns.

Dynamic behaviors of α-synuclein and tau in the cellular context: New mechanistic insights and therapeutic opportunities in neurodegeneration.

α-Synuclein (αS) and tau have a lot in common. Dyshomeostasis and aggregation of both proteins are central in the pathogenesis of neurodegenerative diseases: Parkinson's disease, dementia with Lewy bodies, multi-system atrophy and other 'synucleinopathies' in the case of αS; Alzheimer's disease, frontotemporal dementia, progressive supranuclear palsy and other 'tauopathies' in the case of tau. The aggregated states of αS and tau are found to be (hyper)phosphorylated, but the relevance of the phosphorylation in health or disease is not well understood. Both tau and αS are typically characterized as 'intrinsically disordered' proteins, while both engage in transient interactions with cellular components, thereby undergoing structural changes and context-specific folding. αS transiently binds to (synaptic) vesicles forming a membrane-induced amphipathic helix; tau transiently interacts with microtubules forming an 'extended structure'. The regulation and exact nature of the interactions are not fully understood. Here we review recent and previous insights into the dynamic, transient nature of αS and tau with regard to the mode of interaction with their targets, the dwell-time while bound, and the cis and trans factors underlying the frequent switching between bound and unbound states. These aspects are intimately linked to hypotheses on how subtle changes in the transient behaviors may trigger the earliest steps in the pathogenesis of the respective brain diseases. Based on a deeper understanding of transient αS and tau conformations in the cellular context, new therapeutic strategies may emerge, and it may become clearer why existing approaches have failed or how they could be optimized.

Novel interactions between erythroblast macrophage protein and cell migration.

Erythroblast macrophage protein is a novel protein known to mediate attachment of erythroid cells to macrophages to form erythroblastic islands in bone marrow during erythropoiesis. Emp-null macrophages are small with round morphologies, and lack cytoplasmic projections which imply immature structure. The role of Emp in macrophage development and function is not fully elucidated. Macrophages perform varied functions (e.g. homeostasis, erythropoiesis), and are implicated in numerous pathophysiological conditions such as cellular malignancy. The objective of the current study is to investigate the interaction of Emp with cytoskeletal- and cell migration-associated proteins involved in macrophage functions. A short hairpin RNA lentiviral system was use to down-regulate the expression of Emp in macrophage cells. A cell migration assay revealed that the relocation of macrophages was significantly inhibited when Emp expression was decreased. To further analyze changes in gene expression related to cell motility, PCR array was performed by down-regulating Emp expression. The results indicated that expression of mitogen-activated protein kinase 1 and thymoma viral proto-oncogene 1 were significantly higher when Emp was down-regulated. The results implicate Emp in abnormal cell motility, thus, warrants to assess its role in cancer where tumor cell motility is required for invasion and metastasis.

Ultra-fast genetically encoded sensor for precise real-time monitoring of physiological and pathophysiological peroxide dynamics.

Hydrogen Peroxide (H2O2) is a central oxidant in redox biology due to its pleiotropic role in physiology and pathology. However, real-time monitoring of H2O2 in living cells and tissues remains a challenge. We address this gap with the development of an optogenetic hydRogen perOxide Sensor (oROS), leveraging the bacterial peroxide binding domain OxyR. Previously engineered OxyR-based fluorescent peroxide sensors lack the necessary sensitivity and response speed for effective real-time monitoring. By structurally redesigning the fusion of Escherichia coli (E. coli) ecOxyR with a circularly permutated green fluorescent protein (cpGFP), we created a novel, green-fluorescent peroxide sensor oROS-G. oROS-G exhibits high sensitivity and fast on-and-off kinetics, ideal for monitoring intracellular H2O2 dynamics. We successfully tracked real-time transient and steady-state H2O2 levels in diverse biological systems, including human stem cell-derived neurons and cardiomyocytes, primary neurons and astrocytes, and mouse brain ex vivo and in vivo. These applications demonstrate oROS's capabilities to monitor H2O2 as a secondary response to pharmacologically induced oxidative stress and when adapting to varying metabolic stress. We showcased the increased oxidative stress in astrocytes via Aß-putriscine-MAOB axis, highlighting the sensor's relevance in validating neurodegenerative disease models. Lastly, we demonstrated acute opioid-induced generation of H2O2 signal in vivo which highlights redox-based mechanisms of GPCR regulation. oROS is a versatile tool, offering a window into the dynamic landscape of H2O2 signaling. This advancement paves the way for a deeper understanding of redox physiology, with significant implications for understanding diseases associated with oxidative stress, such as cancer, neurodegenerative, and cardiovascular diseases.

Structure-guided engineering of a fast genetically encoded sensor for real-time H2O2 monitoring.

Hydrogen Peroxide (H2O2) is a central oxidant in redox biology due to its pleiotropic role in physiology and pathology. However, real-time monitoring of H2O2 in living cells and tissues remains a challenge. We address this gap with the development of an optogenetic hydRogen perOxide Sensor (oROS), leveraging the bacterial peroxide binding domain OxyR. Previously engineered OxyR-based fluorescent peroxide sensors lack the necessary sensitivity or response speed for effective real-time monitoring. By structurally redesigning the fusion of Escherichia coli (E. coli) ecOxyR with a circularly permutated green fluorescent protein (cpGFP), we created a novel, green-fluorescent peroxide sensor oROS-G. oROS-G exhibits high sensitivity and fast on-and-off kinetics, ideal for monitoring intracellular H2O2 dynamics. We successfully tracked real-time transient and steady-state H2O2 levels in diverse biological systems, including human stem cell-derived neurons and cardiomyocytes, primary neurons and astrocytes, and mouse neurons and astrocytes in ex vivo brain slices. These applications demonstrate oROS's capabilities to monitor H2O2 as a secondary response to pharmacologically induced oxidative stress, G-protein coupled receptor (GPCR)-induced cell signaling, and when adapting to varying metabolic stress. We showcased the increased oxidative stress in astrocytes via Aß-putriscine-MAOB axis, highlighting the sensor's relevance in validating neurodegenerative disease models. oROS is a versatile tool, offering a window into the dynamic landscape of H2O2 signaling. This advancement paves the way for a deeper understanding of redox physiology, with significant implications for diseases associated with oxidative stress, such as cancer, neurodegenerative disorders, and cardiovascular diseases.

A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate.

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR malfunction, expressed as either over- or under-activity caused by mutations, altered expression, trafficking, or localization, can contribute to numerous diseases, especially in the central nervous system. Therefore, understanding the receptor's biology as well as facilitating the discovery of compounds and small molecules is crucial in ongoing efforts to combat neurological diseases. Current approaches to studying the receptor have limitations including low throughput, high cost, and the inability to study its functional abilities due to the necessary presence of channel blockers to prevent NMDAR-mediated excitotoxicity. Additionally, the existing assay systems are sensitive to stimulation by glutamate only and lack sensitivity to stimulation by glycine, the other co-ligand of the NMDAR. Here, we present the first plate-based assay with high-throughput power to study an NMDA receptor with sensitivity to both co-ligands, glutamate and D-serine/glycine. This approach allows the study of different NMDAR subunit compositions and allows functional studies of the receptor in glycine- and/or glutamate-sensitive modes. Additionally, the method does not require the presence of inhibitors during measurements. The effects of positive and negative allosteric modulators can be detected with this assay and the known pharmacology of NMDAR has been replicated in our system. This technique overcomes the limitations of existing methods and is cost-effective. We believe that this novel technique will accelerate the discovery of therapies for NMDAR-mediated pathologies.

A NMDA-receptor calcium influx assay sensitive to stimulation by glutamate and glycine/D-serine.

N-methyl-D-aspartate-receptors (NMDARs) are ionotropic glutamate receptors that function in synaptic transmission, plasticity and cognition. Malfunction of NMDARs has been implicated in a variety of nervous system disorders, making them attractive therapeutic targets. Overexpression of functional NMDAR in non-neuronal cells results in cell death by excitotoxicity, hindering the development of cell-based assays for NMDAR drug discovery. Here we report a plate-based, high-throughput approach to study NMDAR function. Our assay enables the functional study of NMDARs with different subunit composition after activation by glycine/D-serine or glutamate and hence presents the first plate-based, high throughput assay that allows for the measurement of NMDAR function in glycine/D-serine and/or glutamate sensitive modes. This allows to investigate the effect of small molecule modulators on the activation of NMDARs at different concentrations or combinations of the co-ligands. The reported assay system faithfully replicates the pharmacology of the receptor in response to known agonists, antagonists, positive and negative allosteric modulators, as well as the receptor's sensitivity to magnesium and zinc. We believe that the ability to study the biology of NMDARs rapidly and in large scale screens will enable the identification of novel therapeutics whose discovery has otherwise been hindered by the limitations of existing cell based approaches.