Autism Spectrum Disorder- and/or Intellectual Disability-Associated Semaphorin-5A Exploits the Mechanism by Which Dock5 Signalosome Molecules Control Cell Shape.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that includes autism, Asperger's syndrome, and pervasive developmental disorder. Individuals with ASD may exhibit difficulties in social interactions, communication challenges, repetitive behaviors, and restricted interests. While genetic mutations in individuals with ASD can either activate or inactivate the activities of the gene product, impacting neuronal morphogenesis and causing symptoms, the underlying mechanism remains to be fully established. Herein, for the first time, we report that genetically conserved Rac1 guanine-nucleotide exchange factor (GEF) Dock5 signalosome molecules control process elongation in the N1E-115 cell line, a model line capable of achieving neuronal morphological changes. The increased elongation phenotypes observed in ASD and intellectual disability (ID)-associated Semaphorin-5A (Sema5A) Arg676-to-Cys [p.R676C] were also mediated by Dock5 signalosome molecules. Indeed, knockdown of Dock5 using clustered regularly interspaced short palindromic repeat (CRISPR)/CasRx-based guide(g)RNA specifically recovered the mutated Sema5A-induced increase in process elongation in cells. Knockdown of Elmo2, an adaptor molecule of Dock5, also exhibited similar recovery. Comparable results were obtained when transfecting the interaction region of Dock5 with Elmo2. The activation of c-Jun N-terminal kinase (JNK), one of the primary signal transduction molecules underlying process elongation, was ameliorated by either their knockdown or transfection. These results suggest that the Dock5 signalosome comprises abnormal signaling involved in the process elongation induced by ASD- and ID-associated Sema5A. These molecules could be added to the list of potential therapeutic target molecules for abnormal neuronal morphogenesis in ASD at the molecular and cellular levels.

Extracellular HSPA5 is autocrinally involved in the regulation of neuronal process elongation.

The molecular mechanisms by which neuronal processes grow are extremely complicated, involving fine-tuned regulation of extracellular and intracellular signals. It remains to be elucidated which molecules are contained in the regulation. Herein, we report for the first time that heat shock protein family A member 5 (HSPA5, also called immunoglobulin heavy chain binding endoplasmic reticulum [ER] protein [BiP]) is secreted from mouse primary dorsal neuronal ganglion (DRG) cells or neuronal cell line N1E-115, a frequently used neuronal differentiation model. Supporting these results, HSPA5 protein was co-localized not only with ER antigen KDEL but also with intracellular vesicles such as Rab11-positive secretory vesicles. Unexpectedly, addition of HSPA5 inhibited elongation of neuronal processes, whereas neutralization of extracellular HSPA5 with the antibodies elongated processes, characterizing extracellular HSPA5 as a negative regulator of neuronal differentiation. Treatment of cells with neutralizing antibodies for low-density lipoprotein receptor (LDLR) did not have significant effects on process elongation, whereas LDLR-related protein 1 (LRP1) antibodies promoted differentiation, implying that LRP1 may act as a receptor candidate for HSPA5. Interestingly, extracellular HSPA5 was greatly decreased following treatment with tunicamycin, an ER stress inducer, illustrating that the ability to form neuronal processes could be preserved, even under stress. These results suggest that neuronal HSPA5 itself is secreted to contribute to inhibitory effects on neuronal cell morphological differentiation and can be included on the list of extracellular signaling molecules negatively controlling differentiation.

Knockdown of Rab7B, But Not of Rab7A, Which Antagonistically Regulates Oligodendroglial Cell Morphological Differentiation, Recovers Tunicamycin-Induced Defective Differentiation in FBD-102b Cells.

In the central nervous system (CNS), insulative myelin sheaths are generated from the differentiated plasma membranes of oligodendrocytes (oligodendroglial cells) and surround neuronal axons to achieve saltatory conduction. Despite the functional involvement of myelin sheaths in the CNS, the molecular mechanism by which oligodendroglial cells themselves undergo differentiation of plasma membranes remains unclear. It also remains to be explored whether their signaling mechanisms can be applied to treating diseases of the oligodendroglial cells. Here, we describe that Rab7B of Rab7 subfamily small GTPases negatively regulates oligodendroglial cell morphological differentiation using FBD-102b cells, which are model cells undergoing differentiation of oligodendroglial precursors. Knockdown of Rab7B or Rab7A by the respective specific siRNAs in cells positively or negatively regulated morphological differentiation, respectively. Consistently, these changes were supported by changes on differentiation- and myelination-related structural protein and protein kinase markers. We also found that knockdown of Rab7B has the ability to recover inhibition of morphological differentiation following tunicamycin-induced endoplasmic reticulum (ER) stress, which mimics one of the major molecular pathological causes of hereditary hypomyelinating disorders in oligodendroglial cells, such as Pelizaeus-Merzbacher disease (PMD). These results suggest that the respective molecules among very close Rab7 homologues exhibit differential roles in morphological differentiation and that knocking down Rab7B can recover defective differentiating phenotypes under ER stress, thereby adding Rab7B to the list of molecular therapeutic cues taking advantage of signaling mechanisms for oligodendroglial diseases like PMD.

Hypomyelinating Leukodystrophy 10 (HLD10)-Associated Mutations of PYCR2 Form Large Size Mitochondria, Inhibiting Oligodendroglial Cell Morphological Differentiation.

Hypomyelinating leukodystrophy 10 (HLD10) is an autosomal recessive disease related to myelin sheaths in the central nervous system (CNS). In the CNS, myelin sheaths are derived from differentiated plasma membranes of oligodendrocytes (oligodendroglial cells) and surround neuronal axons to achieve neuronal functions. Nucleotide mutations of the pyrroline-5-carboxylate reductase 2 (PYCR2) gene are associated with HLD10, likely due to PYCR2's loss-of-function. PYCR2 is a mitochondrial residential protein and catalyzes pyrroline-5-carboxylate to an amino acid proline. Here, we describe how each of the HLD10-associated missense mutations, Arg119-to-Cys [R119C] and Arg251-to-Cys [R251C], lead to forming large size mitochondria in the FBD-102b cell line, which is used as an oligodendroglial cell differentiation model. In contrast, the wild type proteins did not participate in the formation of large size mitochondria. Expression of each of the mutated R119C and R251C proteins in cells increased the fusion abilities in mitochondria and decreased their fission abilities relatively. The respective mutant proteins, but not wild type proteins also decreased the activities of mitochondria. While cells expressing the wild type proteins exhibited differentiated phenotypes with widespread membranes and increased expression levels of differentiation marker proteins following the induction of differentiation, cells harboring each of the mutant proteins did not. Taken together, these results indicate that an HLD10-associated PYCR2 mutation leads to the formation of large mitochondria with decreased activities, inhibiting oligodendroglial cell morphological differentiation. These results may reveal some of the pathological mechanisms in oligodendroglial cells underlying HLD10 at the molecular and cellular levels.

Hyaluronic acid and its receptor CD44, acting through TMEM2, inhibit morphological differentiation in oligodendroglial cells.

Hyaluronic acid is a main extracellular matrix component in the central nervous system (CNS), which provides structural support under physical and physiological conditions to maintain cellular homeostasis. However, hyaluronic acid and its degradation products are present within focal demyelinating lesions in multiple sclerosis (MS) patients and autoimmune encephalomyelitis (EAE) mouse models. Differentiated plasma membranes called myelin membranes are generated by oligodendrocytes (also called oligodendroglial cells), which are glial cells that wrap neuronal axons in the CNS. Despite these positive or negative relationships of hyaluronic acid with oligodendroglial cell differentiation and/or myelination, it remains unclear whether and how hyaluronic acid affects oligodendroglial cells. Here, we showed that hyaluronic acid and the cognate receptor CD44 are directly involved in inhibiting morphological differentiation in FBD-102b cells, which are differentiation models of oligodendroglial precursor cells, and primary oligodendroglial precursor cells. Their phenotype changes were supported by decreased oligodendroglial cell differentiation, myelin marker protein expression levels, and Akt kinase phosphorylation levels as a marker kinase. Furthermore, the effects of hyaluronic acid required transmembrane protein 2 (TMEM2), a cell surface hyaluronidase. These results suggest that hyaluronic acid and the CD44 receptor, acting through TMEM2, contribute to inhibiting morphological differentiation of oligodendroglial cells, providing a mechanism underlying cell physiological and possible pathological effects responsible for hyaluronic acid.

Hesperetin, a Citrus Flavonoid, Ameliorates Inflammatory Cytokine-Mediated Inhibition of Oligodendroglial Cell Morphological Differentiation.

Oligodendrocytes (oligodendroglial cells) are glial cells that wrap neuronal axons with their differentiated plasma membranes called myelin membranes. In the pathogenesis of inflammatory cytokine-related oligodendroglial cell and myelin diseases such as multiple sclerosis (MS), typical inflammatory cytokines tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) are thought to contribute to the degeneration and/or progression of the degeneration of oligodendroglial cells and, in turn, the degeneration of naked neuronal cells in the central nervous system (CNS) tissues. Despite the known involvement of these inflammatory cytokines in disease progression, it has remained unclear whether and how TNFα or IL-6 affects the oligodendroglial cells themselves or indirectly. Here we show that TNFα or IL-6 directly inhibits morphological differentiation in FBD-102b cells, which are differentiation models of oligodendroglial cells. Their phenotype changes were supported by the decreased expression levels of oligodendroglial cell differentiation and myelin marker proteins. In addition, TNFα or IL-6 decreased phosphorylation levels of Akt kinase, whose upregulation has been associated with promoting oligodendroglial cell differentiation. Hesperetin, a flavonoid mainly contained in citrus fruit, is known to have neuroprotective effects. Hesperetin might also be able to resolve pre-illness conditions, including the irregulated secretion of cytokines, through diet. Notably, the addition of hesperetin into cells recovered TNFα- or IL-6-induced inhibition of differentiation, as supported by increased levels of marker protein expression and phosphorylation of Akt kinase. These results suggest that TNFα or IL-6 itself contributes to the inhibitory effects on the morphological differentiation of oligodendroglial cells, possibly providing information not only on their underlying pathological effects but also on flavonoids with potential therapeutic effects at the molecular and cellular levels.

The Antiepileptic Valproic Acid Ameliorates Charcot-Marie-Tooth 2W (CMT2W) Disease-Associated HARS1 Mutation-Induced Inhibition of Neuronal Cell Morphological Differentiation Through c-Jun N-terminal Kinase.

Hereditary peripheral neuropathies called Charcot-Marie-Tooth (CMT) disease affect the sensory nerves as well as motor neurons. CMT diseases are composed of a heterogeneous group of diseases. They are characterized by symptoms such as muscle weakness and wasting. Type 2 CMT (CMT2) disease is a neuropathy with blunted or disrupted neuronal morphological differentiation phenotypes including process formation of peripheral neuronal axons. In the early stages of CMT2, demyelination that occurs in Schwann cells (glial cells) is rarely observed. CMT2W is an autosomal-dominant disease and is responsible for the gene encoding histidyl-tRNA synthetase 1 (HARS1), which is a family molecule of cytoplasmic aminoacyl-tRNA synthetases and functions by ligating histidine to its cognate tRNA. Despite increasing knowledge of the relationship of mutations on responsible genes with diseases, it still remains unclear how each mutation affects neuronal differentiation. Here we show that in neuronal N1E-115 cells, a severe Asp364-to-Tyr (D364Y) mutation of HARS1 leads to formation of small aggregates of HARS1 proteins; in contrast, wild type proteins are distributed throughout cell bodies. Expression of D364Y mutant proteins inhibited process formation whereas expression of wild type proteins possessed the normal differentiation ability to grow processes. Pretreatment with the antiepileptic valproic acid recovered inhibition of process formation by D364Y mutant proteins through the c-Jun N-terminal kinase signaling pathway. Taken together, these results indicate that the D364Y mutation of HARS1 causes HARS1 proteins to form small aggregates, inhibiting process growth, and that these effects are recovered by valproic acid. This could be a potential therapeutic drug for CMT2W at the cellular levels.

The Infantile Leukoencephalopathy-Associated Mutation of C11ORF73/HIKESHI Proteins Generates de novo Interactive Activity with Filamin A, Inhibiting Oligodendroglial Cell Morphological Differentiation.

Genetic hypomyelinating diseases are a heterogeneous group of disorders involving the white matter. One infantile hypomyelinating leukoencephalopathy is associated with the homozygous variant (Cys4-to-Ser (C4S)) of the c11orf73 gene. Methods: We observed that in mouse oligodendroglial FBD-102b cells, the C4S mutant proteins but not the wild type ones of C11orf73 are microscopically localized in the lysosome. And, they downregulate lysosome-related signaling in an immunoblotting technique. Results: The C4S mutant proteins specifically interact with Filamin A, which is known to anchor transmembrane proteins to the actin cytoskeleton; the C4S mutant proteins and Filamin A are also observed in the lysosome fraction. While parental FBD-102b cells and cells harboring the wild type constructs exhibit morphological differentiation, cells harboring C4S mutant constructs do not. It may be that morphological differentiation is inhibited because expression of these C4S mutant proteins leads to defects in the actin cytoskeletal network involving Filamin A. Conclusions: The findings that leukoencephalopathy-associated C11ORF73 mutant proteins specifically interact with Filamin A, are localized in the lysosome, and inhibit morphological differentiation shed light on the molecular and cellular pathological mechanisms that underlie infantile hypomyelinating leukoencephalopathy.