Hesperetin Ameliorates Inhibition of Neuronal and Oligodendroglial Cell Differentiation Phenotypes Induced by Knockdown of Rab2b, an Autism Spectrum Disorder-Associated Gene Product

Abstract

Autism spectrum disorder (ASD) is a central nervous system (CNS) neurodevelopmental disorder that includes autism, pervasive developmental disorder, and Asperger’s syndrome. ASD is characterized by repetitive behaviors and social communication deficits. ASD is thought to be a multifactorial disorder with a range of genetic and environmental factors/candidates. Among such factors is the rab2b gene, although it remains unclear how Rab2b itself is related to the CNS neuronal and glial developmental disorganization observed in ASD patients. Rab2 subfamily members regulate intracellular vesicle transport between the endoplasmic reticulum and the Golgi body. To the best of our knowledge, we are the first to report that Rab2b positively regulates neuronal and glial cell morphological differentiation. Knockdown of Rab2b inhibited morphological changes in N1E-115 cells, which are often used as the neuronal cell differentiation model. These changes were accomplished with decreased expression levels of marker proteins in neuronal cells. Similar results were obtained for FBD-102b cells, which are used as the model of oligodendroglial cell morphological differentiation. In contrast, knockdown of Rab2a, which is another Rab2 family member not known to be associated with ASD, affected only oligodendroglial and not neuronal morphological changes. In contrast, treatment with hesperetin, a citrus flavonoid with various cellular protective effects, in cells recovered the defective morphological changes induced by Rab2b knockdown. These results suggest that knockdown of Rab2b inhibits differentiation in neuronal and glial cells and may be associated with pathological cellular phenotypes in ASD and that hesperetin can recover their phenotypes at the in vitro level at least.

Rab11a Controls Cell Shape via C9orf72 Protein: Possible Relationships to Frontotemporal Dementia/Amyotrophic Lateral Sclerosis (FTDALS) Type 1

“…These effects are supported by the results of phosphorylation states of mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/ERK) [27][28][29]. Also, the knockdown of Rab11a or Rab11b affects morphological changes using FBD-102b cells as oligodendroglial differentiation model cells [29,30]. These studies are expected to contribute to our understanding of the potential molecular and cellular pathological mechanisms underlying FTDALSs and the functional deficiency of Rab protein.…”

“…Their decreased morphological changes by Rab11a knockdown are recovered by treatment with hesperetin, a flavonoid with neuronal-protective effects [23][24][25][26]. These effects are supported by the results of phosphorylation states of mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/ERK) [27][28][29]. Also, the knockdown of Rab11a or Rab11b affects morphological changes using FBD-102b cells as oligodendroglial differentiation model cells [29,30].…”

“…Mouse neuronal N1E-115 (JCRB Cell Bank of Japan Health Sciences Foundation) were cultured on Nunc's cell culture dishes (ThermoFisher Scientific, Waltham, MA, USA) in high-glucose Dulbecco's modified Eagle medium (DMEM; Nacalai Tesque) containing 10% heat-inactivated fetal bovine serum (ThermoFisher Scientific) and PenStrep (ThermoFisher Scientific) in 5% CO 2 at 37 • C. To induce differentiation, N1E-115 cells were cultured in DMEM and 1% fetal bovine serum containing PenStrep in the presence or absence of 15 micromolar concentration of hesperetin (Fujifilm), excluding research on its concentration in 5% CO 2 at 37 • C for 0 or 3 days. Cells with processes of more than three cell bodies in lengths were considered to be process-bearing cells (i.e., differentiated cells) [21,22,29]. Under these conditions, attached cells incorporating trypan blue (Nacalai Tesque) were estimated to be less than 5% in each experiment [29].…”

Investigating the Protective Effects of a Citrus Flavonoid on the Retardation Morphogenesis of the Oligodendroglia-like Cell Line by Rnd2 Knockdown

“…Hesperetin, a citrus flavonoid, has protective effects on both neuronal and glial cells [ 43 , 44 ]. As we previously reported, hesperetin has the ability to recover certain inflammatory cytokine- or disease-related gene-induced retardation of oligodendroglial cell-like morphological differentiation [ 45 , 46 ]. First, to confirm that hesperetin can induce morphological differentiation in control knockdown experiments, we transfected control siRNA into cells and treated them with hesperetin or a control vehicle.…”

“…Observation after several days indicates that knockdown results in a slowing of differentiation, rather than complete inhibition; (3) At day 0, the FBD-102b cells display spindle-like phenotypes, as seen in primary oligodendroglial precursor cells [ 26 , 27 ], and cell morphologies at day 0 are similar regardless of control- and target signaling molecule-siRNA or gRNA transfection. As the experiment proceeds following the induction of differentiation, the cells exhibit oligodendroglial cell-like differentiated morphology, in which branches extend from the cell body and the cytoplasmic regions slowly expand [ 45 , 46 ]. Differentiation retardation occurs only in the target signaling molecule knockdown cells, not the control knockdown cells.…”