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

Abstract

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.

AD Informer Set: Chemical tools to facilitate Alzheimer’s disease drug discovery

“…With this concept in mind, the same nine compounds were preliminarily analyzed by the Young lab in a panel of AD-relevant assays. iPSC-derived neurons were differentiated and plated for assays as we have previously described [35,36]. Neurons were treated with either 0.1 or 1 μM of compound for 24h or 48h, and media, cell lysates, and RNA were harvested.…”

Reductions in Hydrogen Sulfide Precede Onset of Mitochondrial Quality Control in the Corneal Kindled Mouse Model of Epilepsy

“…Mitochondria fulfill their diverse functions partly through constant remodeling via fission and fusion. Dysregulation of mitochondrial fission and fusion contributes to numerous types of nervous system disease [29]. Mitochondrial dysfunction contributes to the neurodegeneration in epilepsy, including in animal models [49].…”

“…Immunoblot: Protein extracts for western blot analysis were prepared as previously reported [27]. Primary antibodies used were: mouse monoclonal Endophilin (Novus; NBP2-24733; 1:500); rabbit polyclonal Mff (Proteintech; 17090-1-AP, 1:2000); rabbit monoclonal Mfn2 (Abcam; ab124773, 1:2000); mouse monoclonal HDAC2 (Sigma; H2663, 1:5000); mouse monoclonal Opa-1 (clone 18/OPA1, 1: 5000; BD Biosciences); mouse monoclonal Drp1 (clone 8/DLP1, 1:1000; BD Biosciences); and mouse monoclonal ꞵ-actin (clone AC-15, 1:10,000; Sigma-Aldrich), as previously reported [29]. Horseradish peroxidase-conjugated secondary antibodies (1:2000) were from GE Healthcare.…”

“…The neuron-specific isoform, Endo-B1-B/C participates in the trafficking of the TrkA receptor, promoting receptor internalization and recycling in early endosomes and preventing endosome-lysosome fusion [28]. Further, histone deacetylase 2 (HDAC2) regulates the expression of Endo-B1, particularly neuron-specific isoforms B/C [26,27], and is thus a non-mitochondrial marker of mitochondrial function that may prevent to neuronal pathology [29]. However, how these molecular regulators are dynamically altered during the development of chronic seizures or epilepsy in various animal models has yet to be established.…”

Reductions in Hydrogen Sulfide and Changes in Mitochondrial Quality Control Proteins Are Evident in the Early Phases of the Corneally Kindled Mouse Model of Epilepsy

“…The neuron-specific isoforms Endo-B1-B/C participates in the trafficking of the TrkA receptor, promoting receptor internalization and recycling in early endosomes and preventing endosome-lysosome fusion [28]. Furthermore, histone deacetylase 2 (HDAC2) regulates the expression of Endo-B1, particularly neuron-specific isoforms B/C [26,27], and is thus a non-mitochondrial marker of mitochondrial function, which may prevent neuronal pathology [29]. However, how these molecular regulators are dynamically altered during the development of chronic seizures or epilepsy in various animal models has yet to be established.…”