Skip to Content
MDPIMDPI

Advanced

IJMSInternational Journal of Molecular Sciences
PDF
  • Article
  • Open Access

18 February 2026

18 Pages

Hesperetin, Acting Through Inhibition of c-Jun Signaling, Mitigates Hypomyelinating Disease-Associated Stop-Loss Claudin-11-Induced Defective Morphogenesis in Oligodendroglial FBD-102b Cells

,
,
,
,
,
,
and
1
Department of Pharmacology, National Research Institute for Child Health and Development, Tokyo 157-8535, Japan
2
Department of Molecular Life Sciences, Tokyo University of Pharmacy and Life Sciences, Tokyo 192-0392, Japan
3
TSUMURA Advanced Technology Research Laboratories, TSUMURA & Co., Ibaraki 300-1192, Japan
4
Department of Biological Science, Tokyo College of Biotechnology, Tokyo 144-0032, Japan
Int. J. Mol. Sci.2026, 27(4), 1956;https://doi.org/10.3390/ijms27041956
This article belongs to the Section Molecular Neurobiology
Version Notes
Order Reprints

Abstract

Hypomyelinating leukodystrophies (HLDs) are a group of hereditary CNS disorders characterized by hypomyelination and, sometimes, repeated cycles of demyelination and remyelination. In HLDs, various genetic mutations in the responsible genes disrupt the morphogenesis of oligodendrocytes (oligodendroglial cells), which wrap neuronal axons with their differentiated myelin sheaths. A stop-loss mutation (c.622T-C or c.622T-G) in the gene encoding claudin family tetraspan plasma membrane protein claudin-11 (CLDN11) is associated with HLD22, which is characterized by incomplete differentiation and hypomyelination or delayed myelination in the brain. Herein, we describe for the first time that a CLDN11 mutant protein with an additional amino acid sequence due to the stop-loss mutation, but not the wild-type protein, leads to decreased expression of oligodendroglial differentiation marker proteins in the FBD-102b oligodendroglial progenitor cell line, the model undergoing its differentiation, at both the molecular and morphological levels. Consistently, mutant CLDN11 exhibited decreased morphological differentiation with a reduced ability to extend processes. These cells contained punctate structures that were partially localized in the endoplasmic reticulum (ER) and stimulated phosphorylation of c-Jun N-terminal kinase (JNK) and eukaryotic translation initiation factor 2A (eIF2A) kinase, ER stress-responsible kinases. Hesperetin, a neuroprotective flavonoid that can downregulate ER stress, recovered the differentiation abilities of these cells. Notably, the effects were related to decreased phosphorylation of ER stress-responsible kinases. JNK was found to be present in a co-precipitate with the hesperetin core, whereby hesperetin inhibited signaling through c-Jun as a negative regulator of differentiation. These findings indicate that the HLD22-associated mutant protein can cause an ER stress response, decreasing cell morphological differentiation. In addition, this study offers possible therapeutic implications for the as-yet-unexplored mechanisms involved in HLD22, at least at the molecular and cellular levels.

1. Introduction

In the development of the central nervous system (CNS), intricate interactions between neuronal cells and glial cells, including oligodendrocytes (oligodendroglial cells), are essential for the formation of mature brain and spinal cord tissues [1,2,3,4]. Oligodendroglial cells play a critical role by enveloping neuronal axons with myelin sheaths, specialized structures derived from differentiated plasma membranes [1,2,3,4]. These myelin sheaths facilitate saltatory conduction, significantly increasing the speed of nerve impulse propagation, and provide crucial protection against physical and physiological stresses. They also deliver essential nutrients to neuronal cells [1,2]. Disruptions in the precise differentiation of oligodendrocytes, rather than other CNS cell types, can impede proper myelination and overall CNS development [5,6]. Abnormalities due to inflammation and genetic causes result in severe neurological diseases such as multiple sclerosis and hypomyelinating leukodystrophies (HLDs), highlighting the importance of proper oligodendrocyte function [5,6,7,8,9,10]. These diseases ultimately have an impact on neuronal cells, leading to their degeneration; however, there are no specific and effective treatments developed specially for HLDs [6,9,10].
The prototypic hereditary HLDs, Pelizaeus-Merzbacher disease and Pelizaeus-Merzbacher-like disease, are now classified as HLD types 1 and 2 (HLD1 and HLD2). They are associated with genes encoding the tetraspan membrane proteins proteolipid protein 1 (PLP1) and gap junction protein 2 (GJC2), respectively [6]. Rapidly increasing classified HLDs are syndromes characterized by white matter hypoplasia caused by insufficient oligodendroglial cell differentiation or abnormal myelin development in the CNS [6,9,10]. Symptoms of HLDs typically include nystagmus and brain developmental delay beginning at birth, causing spastic quadriplegia and ataxia. Despite these severe symptoms, current treatments are limited to symptomatic treatments such as anticonvulsants and muscle relaxants [6,9,10] and do not address the underlying causes of HLDs. A major challenge in developing targeted therapies is our incomplete understanding of the molecular and cellular mechanisms underlying HLDs [6,9,10].
The human claudin-11 (CLDN11) gene encodes a member of the claudin tetraspan membrane protein family that makes up tight junctions [11,12]. It is believed that CLDN11 is involved in the component of fully differentiated myelin sheaths [11,12]. CLDN11 is the third most abundant protein identified in CNS myelin, accounting for approximately 7%, following the major myelin proteins PLP1 and myelin basic protein (MBP) [11,12]. CLDN11 with a stop-loss mutation, either c.622T-C or c.622T-G, is associated with HLD22, with both mutations resulting in the same C-terminal 39-amino acid extension [13,14].
The aim of this study was to investigate whether mutations in CLDN11 lead to defects in oligodendroglial cell morphogenesis, potentially revealing molecular mechanisms underlying impaired morphological differentiation. To address this, we used the oligodendroglial progenitor cell line FBD-102b, a well-established model for analyzing oligodendroglial morphological differentiation at both molecular and cellular levels [15,16,17,18]. We provide insight into the molecular pathology of the CLDN11 stop-loss variant by demonstrating that they disrupt cell morphogenesis through a mechanism that is distinct from the major causes of the endoplasmic reticulum (ER) stress responses previously reported for PLP1- and GJC2-associated HLDs [5,6,7,8,9,10]. In addition, we examined whether hesperetin (an aglycone form of hesperidin [vitamin P]), a neuroprotective flavonoid known to alleviate various forms of organelle stress [19,20,21,22,23,24,25,26], could mitigate the molecular and cellular phenotypes induced by mutant CLDN11. Furthermore, we explored the potential involvement of hesperetin in modulating c-Jun N-terminal kinase (JNK) and the c-Jun signaling pathway [27,28]. Collectively, our findings raise the possibility that, in addition to ER stress-related mechanisms, signaling through JNK/c-Jun contributes to the molecular pathogenesis of HLD22 caused by CLDN11 mutation.

2. Results

2.1. Mutated CLDN11 Protein Decreases Cell Morphological Changes

To investigate whether HLD22-associated CLDN11 protein with an additional amino acid sequence due to a stop-loss mutation [13,14] affects differentiation in oligodendroglial cells, we transfected mammalian cell expression plasmids encoding stop-loss CLDN11 or wild-type CLDN11 into oligodendroglial FBD-102b cells and allowed the cells to differentiate in low serum conditions. Cells harboring wild-type CLDN11 extended processes and underwent morphological differentiation, whereas those harboring mutated CLDN11 failed to do so sufficiently (Figure 1A,B). In the same culture conditions, the percentages of morphological differentiation in cells harboring the wild type were comparable to that of the parental cells (Figure S1).
Figure 1. Mutated CLDN11 decreases cell morphogenesis. (A,B) FBD-102b cells expressing wild-type CLDN11 (WT) or mutated CLDN11 (mutant) were allowed to differentiate for 0 or 5 days. The square fields a and b indicated by the dotted lines in the left panels are magnified in the right panels (a,b). Following the induction of differentiation, percentages of cells displaying secondary branches extending from primary branches or widespread membranes were statistically depicted (**, p < 0.01; n = 10 fields). (C,D) Following the induction of differentiation, the lysates of the respective cells were immunoblotted with an antibody against PLP1, MBP, or the internal control actin. Expression levels (immunoreactive bands) were normalized to that of control actin bands and depicted as percentages (**, p < 0.01 or *, p < 0.05; n = 3 blots).
Additionally, the results in mutated CLDN11 were consistent with decreased expression of oligodendroglial cell differentiation marker proteins PLP1 and MBP (Figure 1C,D). Taken together, these findings indicate that mutated CLDN11 impairs morphological differentiation.

2.2. Mutated CLDN11 Protein Stimulates ER Stress Signaling

Because other tetraspan proteins with HLD-related mutations stimulate two major ER stress signaling kinases, protein kinase R-like ER kinase (PERK) and JNK [29,30,31,32], we investigated whether mutated CLDN11 protein exerted a similar effect. Phosphorylation of PERK (activation of PERK) was detected in cells harboring mutated CLDN11 (Figure 2A,B). In the same cells, active phosphorylated c-Jun as the JNK substrate was increased relative to that in cells harboring wild-type CLDN11 (Figure 2C,D), indicating that ER stress signaling was stimulated in the presence of mutated CLDN11. Thus, we asked whether mutated CLDN11 protein is actually localized in the ER. Confocal microscopic images illustrate that mutated CLDN11 protein was, at least in part, localized in some intracellular organelles positive for the ER antigen and the lysosomal antigen, whereas wild-type CLDN11 protein was distributed throughout cytoplasmic regions (Figures S2 and S3). We then explored whether other ER stress markers were upregulated following expression of mutated CLDN11 [31,32]. Expression levels of ER stress markers CCAAT/enhancer-binding protein-homologous protein (CHOP) and heat shock protein family A member 5 protein (HSPA5) were increased in cells harboring mutated CLDN11 protein (Figure S4). Collectively, the expression of mutated CLDN11 protein in cells can stimulate ER stress signaling.
Figure 2. Mutated CLDN11 decreases increased ER stress signaling kinase. (A,B) The lysates of the respective FBD-102b cells following the induction of differentiation were immunoblotted with an antibody against phosphorylated eIF2alpha (active p-eIF2alpha), eIF2alpha, or PERK. The immunoreactive bands were normalized to control bands and depicted as percentages (**, p < 0.01; n = 3 blots). (C,D) The lysates of the respective cells following the induction of differentiation were immunoprecipitated with an anti-JNK antibody. The precipitates were incubated with a recombinant c-Jun (a JNK substrate) and ATP for 0.5 h at 37 °C and immunoblotted with an antibody against active phosphorylated c-Jun (p-c-Jun), c-Jun, or JNK. The immunoreactive bands were normalized to control bands and depicted as percentages (**, p < 0.01; n = 3 blots).

2.3. Hesperetin Recovers Phenotypes Induced by Mutated CLDN11 Protein

Hesperetin is a citrus flavonoid, characterized by a flavanone backbone with neuroprotective and ER stress-relieving abilities [23,24]. We tested the possibility that hesperetin can retain cell phenotypes induced by mutated CLDN11. As expected, treatment with hesperetin recovered cell morphologies (Figure 3A,B). Similar effects were obtained in expression levels of PLP1 and MBP (Figure 3C,D)
Figure 3. Hesperetin recovers phenotypes resulting from mutated CLDN11. (A,B) FBD-102b cells expressing mutated CLDN11 were allowed to differentiate in the presence (Hes) or absence (its vehicle dimethyl sulfoxide [DMSO]) of 0.01 mM hesperetin for 0 or 5 days. The square fields (a,b) indicated by the dotted lines in the left panels are magnified in the right panels a and b. Following the induction of differentiation, percentages of cells displaying secondary branches extending from primary branches or widespread membranes were statistically depicted (**, p < 0.01; n = 10 fields). (C,D) Their cell lysates were immunoblotted with an antibody against PLP1, MBP, or actin. Expression levels (immunoreactive bands) were normalized to that of control bands and depicted as percentages (**, p < 0.01 or *, p < 0.05; n = 3 blots).
We also checked whether hesperetin could recover ER stress signaling. Treatment of cells harboring mutated CLDN11 with hesperetin led to decreasing phosphorylation levels of PERK (Figure 4A,B) and c-Jun (Figure 4C,D), indicating that hesperetin can recover cell phenotypes at the molecular and cellular levels. Consistently, expression levels of ER stress markers CHOP and HSPA5 were decreased (Figure S4). In summary, expression of mutated CLDN11 protein in cells can stimulate ER stress signaling, and hesperetin is able to recover this signaling.
Figure 4. Hesperetin recovers ER stress signaling resulting from mutated CLDN11. (A,B) The lysates of FBD-102b cells expressing mutated CLDN11 following the induction of differentiation were immunoblotted with an antibody against phosphorylated eIF2alpha (p-eIF2alpha), eIF2alpha, or PERK. The immunoreactive bands were normalized to control bands and depicted as percentages (**, p < 0.01; n = 3 blots). (C,D) Their cell lysates were immunoprecipitated with an anti-JNK antibody. The precipitates were incubated with a recombinant c-Jun (a JNK substrate) and ATP and immunoblotted with an antibody against phosphorylated c-Jun (p-c-Jun), c-Jun, or JNK. The immunoreactive bands were normalized to another set of bands and depicted as percentages (**, p < 0.01; n = 3 blots).

2.4. Hesperetin co-Precipitates with JNK and Decreases c-Jun Phosphorylation

We then determined whether PERK or JNK signaling is inhibited by hesperetin. First, we carried out co-precipitation of the monoglycosylated form of hesperidin (a glycosylated hesperetin) using FBD-102b cell lysates. Precipitates with monoglycosylated hesperidin specifically contained an immunoreactive band representing JNK but not PERK (Figure 5A). Then, we examined the effects of hesperetin on c-Jun phosphorylation by monoglycosylated hesperidin precipitates. Hesperetin inhibited c-Jun phosphorylation in a concentration-dependent manner (Figure 5B,C). Collectively, hesperetin decreases signaling through both PERK and JNK, and a protein complex involved in the upstream regulation of c-Jun may constitute one of the molecular targets of hesperetin.
Figure 5. Hesperetin specifically forms a complex with JNK and inhibits the activities. (A) The lysates of FBD-102b cells transfected with mutated CLDN11 were incubated with 0.1 mM monoglycosylated hesperidin for 1.5 h at 4 °C. Then, cells were mixed with concanavalin A (plus ConA) coupled to Sepharose 4B resin or the control Sepharose 4B resin (minus ConA) for 1.5 h at 4 °C and in turn collected by centrifugation. The precipitates or total lysates were immunoblotted with an antibody against JNK or PERK. (B,C) The solution eluted from monoglycosylated hesperidin precipitates was dialyzed and incubated with a recombinant c-Jun protein and ATP for 0.5 h at 37 °C in the presence of indicated concentrations of hesperetin. The reaction was stopped by adding sample buffers and immunoblotted with an antibody against phosphorylated c-Jun (p-c-Jun). The c-Jun used for incubation and JNK in the lysates were also detected by immunoblotting. The immunoreactive bands were normalized to control bands and depicted as percentages (**, p < 0.01; n = 3 blots).
Because the hesperidin precipitates affected c-Jun phosphorylation, we next examined whether they contained other members of the mitogen-activated protein kinase (MAPK) superfamily [29]. However, the precipitates did not exhibit kinase activity toward ribosomal protein S6 kinase A1 (RPS6KA1) or MAPK-activated protein kinase 2 (MAPKAPK2), which are downstream substrates of MAPK/extracellular signal-regulated kinase (ERK) and p38 MAPK pathways, respectively (Figure S5). These results suggest that the hesperetin-associated protein complex is selectively involved in the upstream regulation of c-Jun, rather than in broader MAPK signaling pathways.

2.5. Signaling Through c-Jun Negatively Regulates Oligodendroglial Cell Differentiation

The data described above suggest that signaling pathways involving activated c-Jun may inhibit morphological differentiation. Thus, we introduced the c-Jun active form (63rd Ser-to-Glu and 73rd Ser-to-Glu [S63E and S73E] of c-Jun) [29] or control c-Jun into cells to examine whether differentiation would be blocked. Transfection of active c-Jun or c-Jun into cells led to decreasing differentiation marker proteins PLP1 and MBP (Figure 6A–D).
Figure 6. c-Jun decreases morphological differentiation. (A,B) FBD-102b cells were transfected with a plasmid encoding active c-Jun (phosphorylation-mimicking c-Jun EE with 63rd Ser-to-Glu and 73rd Ser-to-Glu [S63E and S73E]). Cells were allowed to differentiate for 2 days. The lysates were immunoblotted with an antibody against PLP1, MBP, or actin. Expression levels (immunoreactive bands) were normalized to that of control bands and depicted as percentages (**, p < 0.01; n = 3 blots). (C,D) Cells were transfected with the plasmid encoding c-Jun and cells were allowed to differentiate for 2 days. The lysates were immunoblotted with an antibody against PLP1, MBP, or actin. Expression levels (immunoreactive bands) were normalized to that of control bands and depicted as percentages (**, p < 0.01; n = 3 blots).
To confirm that this observed effect was not limited to immortalized cell lines, we examined the effect of active c-Jun in primary oligodendrocyte precursor cells. Consistent with the results obtained in the oligodendroglial cell line, expression of the c-Jun active form decreased morphological differentiation and reduced the expression of CC1 and MBP markers in primary cells (Figure S6). Collectively, these results demonstrate that c-Jun signaling acts as a negative regulator of oligodendroglial cell differentiation and that inhibition of this pathway can contribute to the pro-differentiation effect of hesperetin.

3. Discussion

Throughout development, expression of CLDN11 protein with a tetraspan membrane protein structure is restricted to the oligodendroglial cell lineage, primarily including oligodendrocyte precursor cells (OPCs) and immature and mature oligodendroglial cells [13,14]. Studies using knockout mice illustrate abnormal myelin sheath structures, reduced nerve conduction velocity, and impaired motor function, highlighting its key role in mature developmental stages [11]. Electron microscopy analyses indicate that CLDN11 is localized to components between myelin layers, suggesting normal nerve conduction by regulating ion diffusion and myelin permeability [11,13,14]. Similar to other tetraspan membrane proteins expressed in myelin sheaths, CLDN11 is a key protein that supports the structure and function of myelin sheaths [3,4,13,14].
In contrast, stop-loss mutations (c.622T-C or c.622T-G) resulting in the same C-terminal extension of 39 amino acids are responsible for HLD22. HLD22 appears to cause underdevelopment or absence of myelin structures [11,12]. Although HLD22-associated mutations do not lead to deficient expression of CLDN11, its severe symptoms are somewhat reminiscent of the relationship between an oligodendroglial cell pathological state and organelle stress, like ER stress, caused by other HLD-associated tetraspan membrane proteins [5,6,7,8,9,10]. In this study, we found that HLD22-associated stop-loss CLDN11 led to decreased morphological differentiation with reduced expression levels of oligodendrocyte differentiation/myelin marker proteins in oligodendroglial FBD-102b cells. It is thought that the HLD22-associated CLDN11 mutant protein could cause an undifferentiated state in oligodendroglial cells. Additionally, CLDN11 mutant protein, but not wild-type protein, was partly co-stained with KDEL antigen and stimulated ER stress signals. It is conceivable that a certain stress signal is linked to an undifferentiated state in cells. Previous reports on HLD1 and HLD2 have emphasized ER stress as a central pathogenic mechanism. Our results indicate that the CLDN11 stop-loss variants impair oligodendroglial cell differentiation primarily via altered JNK/c-Jun signaling, rather than classical ER stress pathways. Also, the ability of hesperetin to rescue these defects can establish JNK/c-Jun modulation as a relevant regulator of oligodendroglial cell morphogenesis.
Hesperetin appears to exert two major types of organelle stress-protective effects [23,24]. First, like other flavonoids, hesperetin possesses potent antioxidative properties. Protein aggregation within the ER leads to increased intracellular oxidative stress, which in turn causes cellular damage and ultimately cell degeneration. Flavonoids such as hesperetin confer cytoprotection through their intrinsic antioxidant activity in various cell types, potentially including glial cells [23,24,33,34]. Second, hesperetin activates intracellular signaling pathways with neuroprotective functions, although its direct molecular target has not yet been clearly identified [23,24,33,34]. For example, hesperetin enhances the phosphorylation and activation of MAPK/ERK in some types of cells [33,34]. These kinases exert protective effects in many cell types, not only neuronal cells, by directly phosphorylating transcription factors and cytoskeletal proteins. Also, hesperetin is known to increase the phosphorylation and activation of Akt kinase. Akt not only promotes cell survival but also serves as a central regulator of metabolic and catabolic signaling cascades [33,34]. Taken together, these findings suggest that hesperetin acts at convergent upstream nodes of these kinase pathways.
We have identified components of the JNK signaling cascade as molecular targets of hesperetin. Treatment with hesperetin suppressed phosphorylation of c-Jun in a clear concentration-dependent manner, indicating effective inhibition of JNK pathway activity. Although activation of the JNK pathway alone does not necessarily trigger cell states such as cell cycle arrest, it is known to perturb cellular homeostasis and often exerts effects antagonistic to those mediated by MAPK/ERK signaling in many cellular contexts [33,34]. Importantly, the biological functions of JNK are highly context-dependent and vary according to the cellular environment, the nature of extracellular stimuli, and the intensity and duration of pathway activation [35,36]. Our data suggest that JNK signaling acts as a negative regulator of glial cell differentiation, whereas hesperetin counteracts this inhibitory influence. This interpretation is supported by our observation that forced activation of the JNK pathway, particularly at the level of c-Jun, markedly inhibits the course of differentiation. Therefore, identification of the precise molecular target of hesperetin within the JNK signaling cascade is expected to reveal the critical node responsible for restraining cell differentiation. In Schwann cells, the myelinating glial cells of the peripheral nervous system, JNK signaling has likewise been shown to negatively regulate both differentiation and myelination [37,38]. These findings raise the possibility that a shared JNK-dependent regulatory mechanism operates in differentiating glial cells of both the central and peripheral nervous systems. Thus, future studies aimed at pinpointing the direct target(s) of hesperetin in glial cells may uncover a common signaling module that governs glial differentiation and myelin formation across the nervous system.
In addition to activation of the JNK/c-Jun pathway, our data indicate induction of the PERK branch of the unfolded protein response (UPR). This suggests that CLDN11 mutant protein-induced stress engages a broader UPR network rather than a single linear pathway [29,30,31,32]. Although we cannot currently establish the temporal order of PERK and JNK signaling, it is possible that PERK contributes upstream to JNK/c-Jun, or alternatively, that PERK is engaged secondarily in response to JNK-mediated cellular signaling. Additional time-course and loss-of-function studies in cells are needed to clarify the causal relationship between these pathways.
In very early developmental stages, CLDN11 protein is found in mesenchymal cells near areas rich in meningeal cartilage [13,14]. If CLDN11 mutant protein causes ER stress signals in vivo, stress signals may arise in these cells, potentially hindering mesenchymal cell differentiation; however, it has not been reported that mesenchymal cells undergo abnormal differentiation. This could be because CLDN11 protein, even in mutated form, does not significantly influence mesenchymal cell morphogenesis, since the expression level of CLDN11 is relatively lower in mesenchymal cell differentiation than in oligodendroglial lineage cells [13,14]. Alternatively, this could be because expression of CLDN11 is observed over a limited period. In either case, CLDN11 is presumed to play a less central role in mesenchymal cells than in oligodendroglial cells.
We previously reported that shroom family member 2 (SHRM2, [39]) specifically binds to a PSD-95/Dlg1/ZO-1 (PDZ)-ligand of CLDN11 at its C-terminus with micromole orders, implicating the binding abilities of the normal PDZ ligand and PDZ domain interaction [40]. CLDN11 mutant protein fails to interact with SHRM2 protein. The interaction contributes to morphological differentiation, being reminiscent of that HLD22-associated mutation is loss-of-function in cells. Taken together with the results of the present study, these findings suggests that, at least at the molecular and cellular levels, CLDN11 mutant protein may have both loss-of-function and possibly toxic gain-of-function properties. The latter is that the stop-loss mutation of CLDN11 is a type of mutation that can induce ER stress.
Although hesperetin showed protective effects in our cellular model, its potential as a therapeutic agent for HLD22 must be interpreted with caution. Hesperetin is known to have limited oral bioavailability and rapid metabolism, which may restrict its effective concentrations in vivo. Moreover, while some flavonoids can cross the blood–brain barrier to varying extents, the brain permeability of hesperetin remains incompletely characterized and may be insufficient for targeting central nervous system pathology. Therefore, substantial pharmacokinetic optimization and in vivo validation will be required before any translational application can be considered.
In the present study, we showed that expression of a stop-loss CLDN11 mutant protein was associated with decreased morphological differentiation and increased ER stress signaling. Notably, these effects were alleviated by treatment with hesperetin. We also found that JNK signaling molecules form a complex with hesperetin and that hesperetin inhibits JNK phosphorylation in a concentration-dependent manner. Further studies would help to clarify not only the detailed molecular mechanisms by which the CLDN11 mutant protein impairs differentiation, but also whether similar effects are observed in genetically modified mice and clustered regularly interspaced short palindromic repeats (CRISPR)-manipulated cells. Using a chemical biology approach [41,42], it is important to identify the cellular target(s) of hesperetin in systems including induced pluripotent stem cells derived from a patient with HLD22 and from patients with other forms of leukodystrophy [43,44], as well as in mice. Identifying the as-yet-unknown molecular target of hesperetin may provide important insight into the fundamental mechanisms regulating oligodendroglial differentiation and myelination. In particular, it may help define how the balance between pro-differentiation and anti-differentiation signals is controlled.
Ultimately, these studies may help pave the way toward improved and more targeted approaches for HLD22 and related disorders. Future in vivo studies would be important to examine whether hesperetin can alleviate hypomyelination and associated neurological phenotypes in CLDN11 mutant models of HLD22. In parallel, further dissection of downstream molecular pathways, including both JNK-dependent and JNK-independent signaling, may help clarify how ER stress responses and differentiation cues are integrated in oligodendroglial cells. Such mechanistic insights are expected to provide a stronger biological basis for the rational development of disease-modifying strategies for HLD22 and related leukodystrophies.

4. Limitation of This Study

Although the present study employed a widely used oligodendroglial mouse cell line to assess morphological differentiation, future studies using mouse or cell models and advanced genetic engineering approaches are necessary to better reflect physiological conditions in vivo. In addition, because human cells or tissues were not examined, further work is required to bridge the translational gap between mouse and human systems; therefore, the direct applicability of our findings to human HLD22 pathology or therapy remains uncertain at this stage. Nevertheless, the biochemical interactions identified here are unlikely to be strongly species dependent and thus provide a solid foundation for subsequent mechanistic investigations. Overall, although our findings are based on a mouse oligodendroglial cell line, they offer valuable biochemical insights that can guide future studies in more physiologically relevant and human-based experimental models.

5. Materials and Methods

5.1. Key Materials

The key antibodies and plasmids used in this study are listed in Table 1.
Table 1. Key materials used in this study.

5.2. Cell Line Culture

The FBD-102b cell line (Cell ID: RCB4965; RIKEN, Saitama, Japan), a mouse oligodendroglial precursor cell line, was cultured on cell and tissue culture dishes (Nunc, Thermo Fisher Scientific, Waltham, MA, USA) in Dulbecco’s modified Eagle’s medium/F-12 nutrient mixture medium (DMEM/F-12; Fujifilm, Tokyo, Japan; Nacalai Tesque, Kyoto, Japan) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotic PenStrep mixture (both from Thermo Fisher Scientific) at 37 °C in a humidified atmosphere containing 5% CO2. To induce differentiation, cells were seeded on poly-L-lysine–coated culture dishes (Fujifilm; Nacalai Tesque) to provide a positively charged surface. Differentiation was induced by culturing the cells in medium containing 0.5% FBS for several days under the same conditions (37 °C, 5% CO2). Cells exhibiting secondary branches extending from primary processes or broad, myelin-like membranous structures were defined as differentiated phenotypes [17,18]. Under these conditions, trypan blue–positive adherent cells accounted for less than 5% of the total population [17,18]. Cell morphology was visualized using a microscope system equipped with the i-NTER LENS set (Micronet, Inc., Saitama, Japan) and i-NTER SHOT software (ver. 2; Micronet, Inc.). Representative images from multiple independent samples were analyzed using Fiji software (ver. Java 8; https://imagej.nih.gov/).

5.3. Isolation of Primary Oligodendrocytes

Primary oligodendrocyte precursor cells were isolated from embryonic rat brains [17]. Briefly, embryonic brains were dissected and mechanically dissociated. The dissected cells were then plated onto culture flasks and expanded in precursor cell growth medium containing appropriate mitogens such as platelet-derived growth factor-AA (PDGF-AA) and basic fibroblast growth factor (bFGF, both from PeproTech, Rosemont, IL, USA) to enrich for precursor cells. Following several rounds of expansion, precursor cells were collected and replated onto poly-L-lysine (Nacalai Tesque)-coated dishes for use in assays [17].

5.4. Transfection

Cells were transfected with the indicated plasmids using ScreenFect A (Fujifilm) following the manufacturer’s protocol. The culture medium was replaced 4 h after transfection, and cells were subjected to biochemical analyses more than 48 h post-transfection [17,18]. Stable cell lines were established by selection with 0.8 mg/mL G418 (Nacalai Tesque) under standard conditions. Under these conditions, trypan blue–positive adherent cells represented less than 5% of the total population in each experiment [17,18].
For primary oligodendrocyte precursor cells, retrovirus-mediated gene transfer was carried out using the Retro-X Universal Packaging System (Takara Bio, Kyoto, Japan), according to the manufacturer’s protocol and as previously described [17].

5.5. Immunofluorescence

Transfected cells grown on coverslips were fixed with 4% paraformaldehyde (Nacalai Tesque) or cold 100% methanol (Nacalai Tesque), blocked with Blocking One (Nacalai Tesque), and incubated with primary antibodies followed by fluorescent dye–conjugated secondary antibodies. Samples were mounted using Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA). Fluorescence images were acquired using FV3000 or FV4000 laser-scanning confocal microscope systems (Olympus, Tokyo, Japan) and processed with Fiji software. Representative merged images are shown in the figures.

5.6. Cell Lysis, Immunoprecipitation, Affinity-Precipitation, and Phosphorylation in Cell-Free System

Cells were lysed in ice-cold buffer composed of 50 mM HEPES–NaOH (pH 7.5), 150 mM NaCl, 3 mM MgCl2, 1 mM DTT, 1 mM PMSF, 2 mM leupeptin, 1 mM EDTA, 1 mM Na3VO4, 10 mM NaF, and 0.5% NP-40. The lysates were centrifuged to collect their supernatants [17,18]. Unless otherwise indicated, all experiments were carried out at 4 °C.
For immunoprecipitation or affinity precipitation, centrifuged collected cell supernatants (2 mg per sample) were mixed and gently rotated with protein G resin (0.5%, GE Healthcare, Chicago, IL, USA) conjugated to the respective antibodies (0.5 microgram/mL) or monoglycosylated hesperidin (0.1 mM, Fujifilm) and concanavalin A (ConA) resin (5%, GE Healthcare) for several hours at 4 °C. The immunoprecipitated or affinity-precipitated complexes containing protein G resin or ConA resin were washed with lysis buffer and were denatured in sample buffers for protein denaturation (Fujifilm).
Elution from affinity-precipitated complexes was performed using 1 M NaCl and 0.5 mM EDTA, and the eluates were dialyzed against reaction buffer (50 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 10 mM MgCl2, 0.1 mM MnCl2, 1 mM dithiothreitol). The kinase reaction was started by adding a reaction solution containing 1 mM ATP plus 0.1 mg/mL of a recombinant c-Jun protein (GST-tagged N-terminal portion of c-Jun) or FLAG-tagged RPS6KA1 (401-735 amino acids) or MAPKAPK2 (21-353 amino acids) [27,28] to eluates or immunoprecipitated complexes in the presence or absence of hesperetin. The kinase reaction was carried out at 30 °C for 30 min and stopped by adding premade denaturation sample buffer (Nacalai Tesque; Fujifilm).

5.7. Polyacrylamide Gel Electrophoresis and Immunoblotting

Proteins denatured in sample buffer were resolved by SDS–PAGE using precast polyacrylamide gels (Nacalai Tesque; Fujifilm). Following electrophoresis, proteins were transferred to PVDF membranes (Nacalai Tesque), blocked with Blocking One solution, and probed with specific primary antibodies, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies. Immunoreactive bands on an X-ray film (Fujifilm) or TMB Solution (Nacalai Tesque)-treated immunoreactive bands were scanned using CanoScan LiDE 400 (Canon, Tokyo, Japan) and CanoScan LiDE software (ver. OS14, Canon). Multiple experiments were conducted for immunoblotting studies. Using Fiji software, immunoreactive bands were provided for quantification and normalized to another control sample’s band set.

5.8. Statistical Analysis

Quantitative data are presented as means ± standard deviation (SD) from at least three independent experiments. Statistical analyses were carried out using unpaired t-tests (Student’s or Welch’s correction, depending on variance homogeneity) in Microsoft Excel (Redmond, WA, USA). For multiple comparisons, a one-way ANOVA followed by Tukey’s honest significant difference (HSD) test was conducted using the StatPlus add-in (version 2021; AnalystSoft, Alexandria, VA, USA). Statistical significance was defined as p < 0.05. All statistical evaluations were performed under blinded conditions.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms27041956/s1.

Author Contributions

Conceptualization, J.Y.; methodology, Y.M. (Yuki Miyamoto), T.O., Y.M. (Yuta Muraki), M.Y. (Moeri Yagi), M.Y. (Masahiro Yamamoto), A.N. and H.Y.; software, Y.M. (Yuki Miyamoto), T.O., Y.M. (Yuta Muraki), M.Y. (Moeri Yagi), M.Y. (Masahiro Yamamoto), A.N. and H.Y.; validation, H.Y.; formal analysis, H.Y.; investigation, Y.M. (Yuki Miyamoto), T.O., Y.M. (Yuta Muraki), M.Y. (Moeri Yagi), M.Y. (Masahiro Yamamoto), A.N. and H.Y.; resources, Y.M. (Yuki Miyamoto), T.O., Y.M. (Yuta Muraki), M.Y. (Moeri Yagi), M.Y. (Masahiro Yamamoto), A.N. and H.Y.; data curation, H.Y.; writing—original draft preparation, Y.M. (Yuki Miyamoto) and J.Y.; writing—review and editing, Y.M. (Yuki Miyamoto) and J.Y.; visualization, Y.M. (Yuki Miyamoto), T.O., Y.M. (Yuta Muraki), M.Y. (Moeri Yagi), M.Y. (Masahiro Yamamoto), A.N. and H.Y.; supervision, J.Y.; project administration, J.Y.; funding acquisition, Y.M. (Yuki Miyamoto), H.Y. and J.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by Grants-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT) and Grants-in-Aid for Medical Scientific Research from the Japanese Ministry of Health, Labor and Welfare (MHLW). This work was also supported by Core Research for Evolutional Science and Technology (CREST) of Japan Science and Technology Agency (JST), Daiichi Sankyo Science Foundation, Japan Foundation for Pediatric Research. Mishima Kaiun Memorial Foundation, Mitsubishi Tanabe Science Foundation, Otsuka Science Foundation, and Takeda Science Foundation.

Institutional Review Board Statement

All cell experiments were conducted in accordance with the protocol reviewed and approved by the Tokyo University of Pharmacy and Life Sciences Cell, Animal Care, Use, and Gene Committee (Approval No. L25-10; date: 1 April 2025). Gene-related experiments were specifically conducted according to the protocol reviewed and approved by the Tokyo University of Pharmacy and Life Sciences Gene Committee (Approval No. LSR3-01; date: 1 April 2025).

Data Availability Statement

The datasets used and/or analyzed for the current study are available from the corresponding author upon reasonable request.

Acknowledgments

We thank Takako Morimoto and Yoichi Seki for the insightful comments they provided throughout this study.

Conflicts of Interest

Y.M. (Yuta Muraki), M.Y. (Moeri Yagi), M.Y. (Masahiro Yamamoto), and A.N. are employees of TSUMURA & Co., Ltd. and the study was conducted using internal research funding from the company. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

References

  1. Nave, K.A.; Werner, H.B. Myelination of the nervous system: Mechanisms and functions. Annu. Rev. Cell Dev. Biol. 2014, 30, 503–533. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  2. Stadelmann, C.; Timmler, S.; Barrantes-Freer, A.; Simons, M. Myelin in the central nervous system: Structure, function, and pathology. Physiol. Rev. 2019, 99, 1381–1431. [Google Scholar] [CrossRef] [Scilit]
  3. Peles, E.; Salzer, J.L. Molecular domains of myelinated axons. Curr. Opin. Neurobiol. 2000, 10, 558–565. [Google Scholar] [CrossRef] [Scilit]
  4. Knowles, J.K.; Batra, A.; Xu, H.; Monje, M. Adaptive and maladaptive myelination in health and disease. Nat. Rev. Neurol. 2022, 18, 735–746. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  5. Garbern, J.; Cambi, F.; Shy, M.; Kamholz, J. The molecular pathogenesis of Pelizaeus-Merzbacher disease. Arch. Neurol. 1999, 56, 1210–1214. [Google Scholar] [CrossRef] [Scilit]
  6. Wolf, N.I.; Ffrench-Constant, C.; van der Knaap, M.S. Hypomyelinating leukodystrophies-unravelling myelin biology. Nat. Rev. Neurol. 2021, 17, 88–103. [Google Scholar] [CrossRef] [Scilit]
  7. Khalaf, G.; Mattern, C.; Begou, M.; Boespflug-Tanguy, O.; Massaad, C.; Massaad-Massade, L. Mutation of proteolipid protein 1 gene: From severe hypomyelinating leukodystrophy to inherited spastic paraplegia. Biomedicines 2022, 10, 1709. [Google Scholar] [CrossRef] [Scilit]
  8. Pouwels, P.J.; Vanderver, A.; Bernard, G.; Wolf, N.I.; Dreha-Kulczewksi, S.F.; Deoni, S.C.L.; Bertini, E.; Kohlschutter, A.; Richardson, W.; Ffrench-Constant, C.; et al. Hypomyelinating leukodystrophies: Translational research progress and prospects. Ann. Neurol. 2014, 76, 5–19. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  9. Inoue, K. Pelizaeus-Merzbacher disease: Molecular and cellular pathologies and associated phenotypes. Adv. Exp. Med. Biol. 2019, 1190, 201–216. [Google Scholar]
  10. Torii, T.; Yamauchi, J. Molecular pathogenic mechanisms of hypomyelinating leukodystrophis (HLDs). Neurol. Int. 2023, 15, 1155–1173. [Google Scholar] [CrossRef] [Scilit]
  11. Gow, A.; Southwood, C.M.; Li, J.S.; Pariali, M.; Riordan, G.P.; Brodie, S.E.; Danias, J.; Bronstein, J.M.; Kachar, B.; Lazzarini, R.A. CNS myelin and Sertoli cell tight junction strands are absent in Osp/claudin-11 null mice. Cell 1999, 99, 649–659. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  12. Chow, E.; Mottahedeh, J.; Prins, M.; Ridder, W.; Nusinowitz, S.; Bronstein, J.M. Disrupted compaction of CNS myelin in an OSP/Claudin-11 and PLP/DM20 double knockout mouse. Mol. Cell Neurosci. 2005, 29, 405–413. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  13. Riedhammer, K.M.; Stockler, S.; Ploski, R.; Wenzel, M.; Adis-Dutschmann, B.; Ahting, U.; Alhaddad, B.; Blaschek, A.; Haack, T.B.; Kopajtich, R.; et al. De novo stop-loss variants in CLDN11 cause hypomyelinating leukodystrophy. Brain 2021, 144, 411–419, Corrigendum in Brain 2021, 144, e48. [Google Scholar] [CrossRef] [Scilit]
  14. Gjervan, S.C.; Ozgoren, O.K.; Gow, A.; Stockler-Ipsiroglu, S.; Pouladi, M.A. Claudin-11 in health and disease: Implications for myelin disorders, hearing, and fertility. Front. Cell Neurosci. 2024, 17, 1344090. [Google Scholar] [CrossRef] [Scilit]
  15. Horiuchi, M.; Tomooka, Y. An oligodendroglial progenitor cell line FBD-102b possibly secretes a radial glia-inducing factor. Neurosci. Res. 2006, 56, 213–219. [Google Scholar] [CrossRef] [Scilit]
  16. Okada, A.; Tomooka, Y. A role of Sema6A expressed in oligodendrocyte precursor cells. Neurosci. Lett. 2013, 539, 48–53. [Google Scholar] [CrossRef] [Scilit]
  17. Miyamoto, Y.; Yamauchi, J.; Chan, J.R.; Okada, A.; Tomooka, Y.; Hisanaga, S.; Tanoue, A. Cdk5 regulates differentiation of oligodendrocyte precursor cells through the direct phosphorylation of paxillin. J. Cell Sci. 2007, 120, 4355–4366. [Google Scholar] [CrossRef] [Scilit]
  18. Fukushima, N.; Miyamoto, Y.; Yamauchi, J. CRISPR/CasRx-mediated knockdown of Rab7B restores incomplete cell shape induced by Pelizaeus-Merzbacher disease-associated PLP1 p.Ala243Val. Neurosci. Insights 2024, 19, 26331055241276873. [Google Scholar] [CrossRef] [Scilit]
  19. Li, C.; Schluesener, H. Health-promoting effects of the citrus flavanone hesperidin. Crit. Rev. Food Sci. Nutr. 2017, 57, 613–631. [Google Scholar] [CrossRef] [Scilit]
  20. Spagnuolo, C.; Moccia, S.; Russo, G.L. Anti-inflammatory effects of flavonoids in neurodegenerative disorders. Eur. J. Med. Chem. 2018, 153, 105–115. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  21. Hwang, S.L.; Yen, G.C. Effect of hesperetin against oxidative stress via ER- and TrkA-mediated actions in PC12 cells. J. Agric. Food Chem. 2011, 59, 5779–5785. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  22. Hussain, Y.; Khan, H.; Efferth, T.; Alam, W. Regulation of endoplasmic reticulum stress by hesperetin: Focus on antitumor and cytoprotective effects. Phytomedicine 2022, 100, 153985. [Google Scholar] [CrossRef] [Scilit]
  23. Khan, A.; Ikram, M.; Hahm, J.R.; Kim, M.O. Antioxidant and anti-inflammatory effects of citrus flavonoid hesperetin: Special focus on neurological disorders. Antioxidants 2020, 9, 609. [Google Scholar] [CrossRef] [Scilit]
  24. Lai, M.-C.; Liu, W.-Y.; Liou, S.-S.; Liu, I.-M. The citrus flavonoid hesperetin encounters diabetes-mediated Alzheimer-type neuropathologic changes through relieving advanced glycation end-products inducing endoplasmic reticulum stress. Nutrients 2022, 14, 745. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  25. Bandiwadekar, A.; Jose, J.; Khayatkashani, M.; Habtemariam, S.; Khayat Kashani, H.R.; Nabavi, S.M. Emerging novel approaches for the enhanced delivery of natural products for the management of neurodegenerative diseases. J. Mol. Neurosci. 2022, 72, 653–676. [Google Scholar] [CrossRef] [Scilit]
  26. Li, D.; Fan, J.; Du, L.; Ren, G. Prenylated flavonoid fractions from Glycyrrhiza glabra alleviate insulin resistance in HepG2 cells by regulating the ERK/IRS-1 and PI3K/Akt signaling pathways. Arch. Pharm. Res. 2024, 47, 127–145. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  27. Yamauchi, J.; Kawano, T.; Nagao, M.; Kaziro, Y.; Itoh, H. Gi-dependent activation of c-Jun N-terminal kinase in human embryonal kidney 293 cells. J. Biol. Chem. 2000, 275, 7633–7640. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  28. Németh, M.; Brandt, B.; Les, H.; Kele-Morvai, P.; Maifeld, A.; Rauch, T.A.; Schwartz, K.; Pap, M. Role of the transcription factor CREB in ethanol-induced endoplasmic reticulum stress and apoptosis in PC12 cells. Biology 2025, 14, 1277. [Google Scholar] [CrossRef] [Scilit]
  29. Kim, E.K.; Choi, E.J. Pathological roles of MAPK signaling pathways in human diseases. Biochim. Biophys. Acta 2010, 1802, 396–405. [Google Scholar] [CrossRef] [Scilit]
  30. Walter, P.; Ron, D. The unfolded protein response: From stress pathway to homeostatic regulation. Science 2011, 334, 1081–1086. [Google Scholar] [CrossRef] [Scilit]
  31. Hetz, C. The unfolded protein response: Controlling cell fate decisions under ER stress and beyond. Nat. Rev. Mol. Cell Biol. 2012, 13, 89–102. [Google Scholar] [CrossRef] [Scilit]
  32. Agalakova, N.I. Modulation of endoplasmic reticulum stress in experimental anti-cancer therapy. Int. J. Mol. Sci. 2025, 26, 6407. [Google Scholar] [CrossRef] [Scilit]
  33. Jomova, K.; Raptova, R.; Alomar, S.Y.; Alwasel, S.H.; Nepovimova, E.; Kuca, K.; Valko, M. Reactive oxygen species, toxicity, oxidative stress, and antioxidants: Chronic diseases and aging. Arch. Toxicol. 2023, 97, 2499–2574. [Google Scholar] [CrossRef] [Scilit]
  34. Averill-Bates, D. Reactive oxygen species and cell signaling. Biochim. Biophys. Acta. Mol. Cell Res. 2024, 1871, 119573. [Google Scholar] [CrossRef] [Scilit]
  35. Weston, C.R.; Davis, R.J. The JNK signal transduction pathway. Curr. Opin. Cell Biol. 2007, 19, 142–149. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  36. de Los Reyes Corrales, T.; Losada-Pérez, M.; Casas-Tintó, S. JNK pathway in CNS pathologies. Int. J. Mol. Sci. 2021, 22, 3883. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  37. Miyamoto, Y.; Yamauchi, J. Cellular signaling of Dock family proteins in neural function. Cell. Signal. 2010, 22, 175–182. [Google Scholar] [CrossRef] [Scilit]
  38. Yamauchi, J.; Miyamoto, Y.; Hamasaki, H.; Sanbe, A.; Kusakawa, S.; Nakamura, A.; Tsumura, H.; Maeda, M.; Nemoto, N.; Kawahara, K.; et al. The atypical guanine-nucleotide exchange factor, Dock7, negatively regulates Schwann cell differentiation and myelination. J. Neurosci. 2011, 31, 12579–12592. [Google Scholar] [CrossRef] [Scilit]
  39. Boëda, B.; Michel, V.; Etournay, R.; England, P.; Rigaud, S.; Mary, H.; Gobaa, S.; Etienne-Manneville, S. SCRIB controls apical contractility during epithelial differentiation. J. Cell Biol. 2023, 222, e202211113. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  40. Kobayashi, S.; Yokoi, T.; Omata, T.; Yako, H.; Miyamoto, Y.; Yamauchi, J. Claudin-11, a hypomyelinating leukodystrophy 22 (HLD22)-responsible protein, uniquely interacts with shroom-2 to change cell phenotypes. BBA Adv. 2025, 7, 100159. [Google Scholar] [CrossRef] [Scilit]
  41. Baradaran, S.; Moghaddam, A.H.; Ghasemi-Kasman, M. Hesperetin reduces myelin damage and ameliorates glial activation in lysolecithin-induced focal demyelination model of rat optic chiasm. Life Sci. 2018, 207, 471–479. [Google Scholar] [CrossRef] [Scilit]
  42. Baradaran, S.; Ghasemi-Kasman, M.; Moghaddam, A.H. Nano-hesperetin enhances the functional recovery and endogenous remyelination of the optic pathway in focal demyelination model. Brain Res. Bull. 2020, 164, 392–399. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  43. Ozgoren, O.K.; Sequiera, G.L.; Ferrari Bardile, C.; Gjervan, S.C.; Salman, A.; Lehman, A.; Turvey, S.E.; Ross, C.J.D.; Stockler, S.; Pouladi, M.A. Generation of a human induced pluripotent stem cell line from a patient with hypomyelinating leukodystrophy 22 (HLD22). Stem Cell Res. 2023, 71, 103174. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  44. Saleh, M.A.; Boichuck, M.; Ottolenghi, A.; Rabinski, T.; Goldenthal, O.; Sanchez, D.S.; Fattal-Valevski, A.; Heimer, G.; Ben-Shachar, S.; Libzon, S.; et al. Novel pathogenic variant identified in HIKESHI-related hypomyelinating leukodystrophy disrupts heat shock response in iPSCs. Int. J. Mol. Sci. 2025, 26, 6037. [Google Scholar] [CrossRef] [Scilit] [PubMed]
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Article Metrics

Citations

Article Access Statistics

Journal Statistics
Multiple requests from the same IP address are counted as one view.
Usnic Acid Derivatives as Inhibitors of Mycobacterium tuberculosis Uracil–DNA Glycosylase
Previous
Methodological and Pathophysiological Considerations in Obesity-Associated Thrombosis
Next