QKI-V5 is downregulated in CNS inflammatory demyelinating diseases

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QKI-5 is downregulated in CNS inflammatory demyelinating diseases Iris Lavon ConceptualizationMethodologyFormal analysisWriting - Original DraftSupervision , Ina leykin , Hanna Charbit ValidationWriting - Original Draft , Orli Binyamin , Livnat Brill , Haim Ovadia , Adi Vaknin-Dembinsky ConceptualizationMethodologyWriting - Original DraftSupervision PII: DOI: Reference:

S2211-0348(19)30952-6 https://doi.org/10.1016/j.msard.2019.101881 MSARD 101881

To appear in:

Multiple Sclerosis and Related Disorders

Received date: Revised date: Accepted date:

12 September 2019 25 November 2019 30 November 2019

Please cite this article as: Iris Lavon ConceptualizationMethodologyFormal analysisWriting - Original DraftSupervisi Ina leykin , Hanna Charbit ValidationWriting - Original Draft , Orli Binyamin , Livnat Brill , Haim Ovadia , Adi Vaknin-Dembinsky ConceptualizationMethodologyWriting - Original DraftSupervision , QKI-5 is downregulated in CNS inflammatory demyelinating diseases, Multiple Sclerosis and Related Disorders (2019), doi: https://doi.org/10.1016/j.msard.2019.101881

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Highlights 

QKI gene encodes a RNA-binding-protein that plays a central role in myelination.



Expression of QKI-V5 is decreased in the blood of NMO and MS patients.



Sera of NMO patients reduced the expression of QKI-V5 in PBMCs or glial cells.



Blood and brain of EAE mice exhibited a corresponding decreased QKI-V1 expression.



It can be concluded that QKI might play a role in these diseases.

QKI-5 is downregulated in CNS inflammatory demyelinating diseases

Iris Lavon, PhD 1,2, Ina leykin, MSc 1,2, Hanna Charbit, MSc 1,2, Orli Binyamin MSc 1, Livnat Brill1, MSc, Haim Ovadia, PhD 1 and Adi Vaknin-Dembinsky, MD, PhD1

1

Department of Neurology, the Agnes-Ginges Center for Neurogenetics, Hadassah-

Medical Center, Hebrew university, Jerusalem, Israel. 2

Leslie and Michael Center for Neuro-oncology, Hadassah- Medical Center,

Jerusalem, Israel

Key words: QKI variants, Multiple sclerosis (MS), Neuromyelitis optica (NMO), Astrocytes, Demyelination

Corresponding author:

Iris Lavon, PhD E-Mail: [email protected] Neurology Department Hadassah Medical Center, Ein Karem P.O.B. 12000, Jerusalem 91120, ISRAEL Tel: 972-2-677-6939 Fax: 972-2-642-6741

Abstract

Background: Neuromyelitis-optica (NMO) and multiple-sclerosis (MS) are inflammatory- demyelinating-diseases of the central-nervous-system (CNS). In a previous study, we identified 17 miRNAs that were significantly upregulated in the peripheral blood of patients with NMO, relative to healthy controls (HCs). Target gene analysis have demonstrated that QKI is targeted by 70% of the upregulated miRNAs. QKI gene encodes for a RNA-binding-protein that plays a central role in myelination and QKI variants 5, 6, 7 (QKI-V5, QKI-V6, QKI-V7) are generated via alternative splicing. Given the role played by QKI in myelination we aimed to study the expression levels of QKI variants in the circulation of patients with NMO and MS and in the brain tissue and the circulation of mice-model to CNS-inflammatorydemyelinating-disease. Methods: RNA and protein expression levels of QKI variants QKI-V5, QKI-V6 and QKI-V7 were determined in the blood of patients with NMO (n=23) or MS (n=13). The effect of sera from patients on the expression of QKI in normal peripheral-bloodmononuclear-cells (PBMCs) or glial cells was explored. The mog-experimentalautoimmune-encephalomyelitis (EAE) mouse model was used to study the correlation between the changes in the expression levels of QKI in the blood to those in the brain. Results: RNA and protein expression of QKI-V5 was decreased in the peripheral blood of patients with NMO and multiple-sclerosis. Incubation of normal peripheralblood-mononuclear-cells or glial cells with sera of patients significantly reduced the expression of QKI-V5. The blood and brain of EAE mice exhibited a corresponding decrease in QKI-V5 expression.

Conclusion: The downregulation in the expression of QKI-V5 in the blood of patients with CNS-inflammatory-demyelinating-diseases and in the brain and blood of EAE mice is likely caused by a circulating factor and might promote re-myelination by regulation of myelin-associated genes.

Key words: QKI variants, Multiple sclerosis (MS), Neuromyelitis optica (NMO), Astrocytes, Demyelination

1. Introduction Neuromyelitis optica (NMO) and multiple sclerosis (MS) are inflammatory demyelinating diseases of the central nervous system (CNS). Demyelination is a pathogenic process that contributes to disability in these diseases. In MS and NMO, demyelination is caused by inflammation that damages oligodendrocytes. The damage can range from inflammatory demyelination to necrotic damage of the white and grey matter. Re-myelination is essential for restoring function and driving neuroprotection. In most animal models of MS, rapid remyelination is observed 1. In humans, the efficacy of such re-myelination is highly variable 2. Re-myelination occurs from newly

differentiated

oligodendrocytes,

not

from

pre-existing

mature

oligodendrocytes, and therefore requires ongoing oligodendrocyte differentiation 3. In a previous study, we identified 17 miRNAs that were significantly upregulated in the peripheral blood of patients with NMO compared to healthy controls (HCs) 4. The KH domain-containing RNA binding protein QKI is a putative target of 12 of the upregulated miRNAs. The myelin genes PLP1, MAG, MBP, and TF are important for oligodendrocyte differentiation and myelin production. QKI is expressed by glial progenitor cells, among other cells, and is one of the regulators of the myelin genes. QKI regulates oligodendrocyte development and differentiation, and the transition to myelinating oligodendrocytes during development is accompanied by elevated levels of QKI proteins.5, 6 There are three human QKI isoforms, which are formed by alternative splicing, namely: QKI-V5, a nuclear isoform; QKI-V6, a nuclear and cytoplasmic isoform; and the cytoplasmic isoform QKI-V7. Human QKI-V5, QKI-V6, and QKI-V7 correspond to the mouse variants QKI-V1, QKI-V2, and QKI-V3 respectively

7, 8

. The QKI

isoforms contain a KH domain and belong to the evolutionarily conserved signal

transduction and activator of RNA (STAR) family. QKI is expressed in the brain, heart, lung, testes, muscle, prostate, colon, and stomach, as well as in cells of the myeloid lineage. Although QKI knockouts are not viable, a proximal deletion breakpoint approximately 0.9 kb upstream of the QKI gene results in quaking viable mice (qkv). These mice are characterized by lower expression of QKI-V2 and hypomyelination, which causes rapid tremors (“quaking”) and seizures. The quaking mice display severely reduced levels of myelin (only 5–10% of the normal quantity). The phenotype is first observed on day 10–14, with shaking behavior often accompanied by clonic–tonic seizures, and severity increases with age. No mutations were found in any myelin structural genes. In addition to myelin impairment, qkv mice exhibit defects in mRNA processing

5, 9

; PLP and MBP levels are reduced

10, 11

and suggest

that QKI proteins are directly involved in the regulation of RNA metabolism of myelin components. A balance among the different QKI isoforms likely regulates myelination. QKI-V5 represses the expression of myelin basic protein (MBP) by binding to the 3ʹ UTR of MBP mRNAs, and retaining them in the nucleus. Consequently, during myelination, QKI-V6 and QKI-V7 are upregulated while QKI-V5 is downregulated of the central role of QKI in myelination

8, 14

12, 13

. Because

, the aim of the current study was to

investigate the involvement of QKI variants in CNS inflammatory demyelinating diseases.

2. Material and methods

2.1. Ethics Statement This study was approved by the Hadassah Medical Organization’s Ethics Committee. All of the patients included in this study provided written informed consent. 2.2. Subjects The patient cohort included 23 patients with NMO (15 females, average age: 36.7 ± 15 years; EDSS 4.2 ± 2) and 13 patients with MS (11 females, average age: 35.5 ± 8.7 years; EDSS 3.6 ± 2.1) admitted to the Hadassah MS Center. A group of 8 healthy individuals served as control. Diagnoses were conducted in accordance with 2015 diagnostic criteria. Among the NMO patient group, 100% tested positive for anti- aquaporin 4 (AQP4). 2.3. Animal experiments All animal experiments were conducted in accordance with the guidelines and supervision of the Hebrew University Ethical Committee, which approved the methods employed in this project (Permit Number: MD-13-13772-5). 2.4. Induction of EAE Myelin oligodendrocyte glycoprotein (MOG) EAE was induced as previously described 15. Briefly, 6- to 8-week-old female C57BL/6 mice were immunized with an emulsion containing 200 μg of MOG35–55 (70% purified; synthesized at Hebrew University, Jerusalem, Israel) in saline and an equal volume of complete Freund’s adjuvant containing 5 mg/mL H37RA (Difco Laboratories, Detroit, MI, USA). The inoculum (0.2 mL) was injected subcutaneously into the right and left flanks. One hundred nanograms of pertussis toxin (List Biological Labs, Campbell, CA, USA) in 0.1 mL saline was also injected intraperitoneally on day 0, and 48 hours later.

2.5. EAE scoring system Mice were observed every two days for the appearance of neurological symptoms, which were scored as follows: 0, asymptomatic; 1, partial loss of tail tonicity; 1.5, limp tail; 2, hind limb weakness (right reflex); 3, ataxia; 4, early paralysis; 5, full paralysis; and 6, moribund or dead. (Supplementary figure 3) 2.6. RNA extraction, cDNA preparation, and qPCR 2.6.1. RNA Isolation Total RNA was extracted from whole blood samples by using Tri Reagent BD (Sigma-Aldrich) and from tissue by using Tri Reagent (Sigma-Aldrich) according to manufacturers’ instructions. cDNA was prepared using a qScript cDNA synthesis kit (Quanta Biosciences). 2.6.2. cDNA cDNA was produced from 0.2 µg total RNA by using a qScript cDNA Synthesis Kit (Quanta Biosciences, Gaithersburg, MD, USA), according to the manufacturer's instructions. 2.6.3. qPCR Real-time PCR amplification and relative quantification of QKI RNA expression were performed with StepOne real time RT PCR (Life Technologies). The reaction mix included 1 μL cDNA, and 300 nmol/l concentrations of each of the following primers (Syntezza, Jerusalem, Israel): GAPDH (5ʹ-3ʹ) F-AGGGGTCATTGATGGCAACA

R-GTATTGGGCGCCTGGTCA

TUBB (5ʹ-3ʹ)

F-TCACTGATCACCTCCCAGAACTT

R-CATACATACCTTGAGGCGAGCA

QKI-V5 (5ʹ-3ʹ)

F-GGTCAGAAGGTCATAGGTTAGTTGC

R-CGGTGGCTACTAAAGTTCGAAGG

QKI-V6 (5ʹ-3ʹ)

F-TTCGTTGGGAAAGCCATACCT

R-TACACATTGGCACCAGCTACATC

QKI-V7 (5ʹ-3ʹ)

F-TGACTGGCATTTCAATCCACTCT

R-ACCCTATGAGTACCCCTACACATTG

by using 5 μL of SYBR green mix (Perfecta Sybr Green Fast Mix ROX, Quanta Biosciences) in a 10 μL total volume, according to the manufacturer’s instructions. The fold changes of target mRNAs were normalized to GAPDH and TUBB. Following normalization, the fold change of each mRNA was calculated based on the ratio to the analyzed control, as indicated. The experiment was repeated three times in triplicate and the results are presented as the mean ± SE. The statistical significance of the induction of gene expression vs the control was calculated according to student 2tailed t-test. 2.7. Western Blot analysis Western blotting was performed as previously described 16, with minor modifications. Briefly, tissue samples or blood cells were homogenized in 500 µl RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with protease inhibitors (Thermo Fisher Scientific Inc.). Protein concentration was determined using the Bradford protein assay (Bio-Rad, Richmond, CA, USA). Tissue/cells lysates containing 100 µg protein were separated on 4%-20% TrisGlycine SDS-PAGE gels (Thermo Fisher Scientific Inc) and assessed by western blot analysis, by sequential probing with polyclonal antibody targeting QKI-V5 (1:1,000, AB9904; Millipore, Amsterdam, The Netherlands) or monoclonal anti-GAPDH (0411, diluted 1:10000; Santa Cruz Biotechnologies, Santa Cruz, CA, USA,) and the corresponding secondary horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnologies).

2.8. Statistical analysis

The data were analyzed using two way Student's t-test for comparison between the control and study groups. P < 0.05 was considered statistically significant.

3. Results:

3.1. QKI is a target of most of the miRNAs upregulated in the peripheral blood of patients with NMO In a previous study, we identified 23 miRNAs that were significantly downregulated and 17 that significantly upregulated in peripheral blood of patients with NMO compared to healthy controls (HCs) 4. A search for putative target genes of the upregulated miRNAs by using the miRSystem software

17

yielded several genes. One

of these genes was QKI, which is targeted by 12/17 (70%) of the upregulated miRNAs (Table 1).

Table 1: QKI is a target of most of the miRNAs upregulated in the peripheral blood of patients with NMO

NMO is a CNS demyelination disease. Because of the known role of QKI in demyelination, we first determined whether the expression of QKI is altered in the blood of patients with NMO.

3.2. Decreased expression of QKI variant 5 in peripheral blood of patients with NMO Analysis of total QKI and individual QKI splice variant expression by real time PCR revealed significantly lower expression levels of QKI-V5 (0.45 ± 0.083; P = 0.021) in whole blood obtained from 23 patients with NMO, compared to those in 8 HCs (figure 1A ; supplementary figure 1A and 1C). The levels of total QKI and variants 6 and 7 did not differ significantly between the groups (Figure 1A). Validation of the mRNA results by western blot analysis demonstrated that the protein level of QKI-V5 is downregulated in PBMCs of patients with NMO compared to those of HCs (Figure 1B, 1C). Following this result, we analyzed the levels of the 12 QKI putative miRNAs presented in table 1 in the patients that were included in both current and previous studies. This analysis demonstrated that the average quantification levels of these miRNAs is also upregulated in this subgroup of NMO patients as compared to healthy controls (supplementary figure 2).

A.

B. C.

Figure 1: Gene expression of QKI and its variants in the blood of patients with NMO

and healthy controls. (A) RNA was isolated from whole blood of eight HCs (black bars) and 23 patients with NMO (white bars). Gene expression level of QKI and of its four human variants was analyzed using real time RT PCR. Data is expressed as fold change average ±

SE.

(B) Protein was extracted from PBMCS of 5 HCs (left panel) and five patients

with NMO (Right) and was analyzed by western blot using sequential probing with monoclonal antibody against QKI-V5 or anti-GAPDH. (C) The densitometry analysis of the immunoblot following normalization to GAPDH.

3.3. Decreased expression of QKI -V5 in peripheral blood of patients with Multiple sclerosis (MS)

Based on our observations in patients with NMO, we studied the expression of total QKI and its individual variants in whole blood of patients with MS, which is the most prevalent demyelinating disease of the CNS. Similar to our findings in patients with NMO, we found significantly lower expression levels of QKI-V5 but not of total QKI, QKI-V6, or QKI -V7 in whole blood of patients with MS compared to that of HCs (0.49 ± 0.091; P =0.02) (Figure 2; supplementary figure 1A and 1B).

Figure 2: Gene expression of QKI and its variants in the blood of patients with MS and HCs. Real time RT-PCR analysis of RNA isolated from whole blood of 8 HCs (black bars) and 13 patients with MS (white bars).

To test whether the lower expression of QKI-V5 observed in the blood of patients with NMO and MS could be identified also in the demyelinating brain, we tested the expression of this variant in the experimental autoimmune encephalomyelitis (EAE), the mouse model for MS. 3.4. Decreased expression of mice QKI –V1 (equivalent to human QKI -V5) in the brain of EAE mice.

The expression of QKI variants was studied in the brain of C57BL/6 naïve female mice (n = 18) following active induction of EAE. In this model, the mice exhibit acute neurological paralytic disease 10–14 days after the induction, followed by partial remission. Mice were scored every second day for disease signs as described in the “Materials and Methods” section. (Supplementary figure 3). Mice were sacrificed at the partial remission phase of the disease (day 51 following induction with anti-MOG EAE) which is parallel to the chronic phase of the disease in human. RNA was extracted from the mice brains and the expression of QKI was studied by quantitative RT-PCR. There was a significant decrease in the expression of m-QKI-V1 (0.54 ± 0.12; P= 0.043), but not of the other QKI variants, in the brains of mice with EAE compared to control (figure 3).

Figure 3: Gene expression of QKI variants in the in the brain of EAE mice. Real time RT-PCR analysis of RNA isolated from the brains of 18 EAE mice at the partial remission phase (pattern color fill) and from eight healthy mice (full-color fill).

3.5. Decreased expression of QKI-V1 in peripheral blood and brain of EAE mice

In order to explore whether there is a concordance between the RNA expression levels of QKI-V1 (parallel to human QKI-V5) in the blood and brains of EAE mice, another group of 6 C57BL/6 mice were subjected to EAE induction and

the

expression of QKI-V1was studied at the brains and blood of the mice at 51 days after induction. The results showed a significant decrease in QKI-V1 expression in the brains and blood of mice with EAE compared to control (0.71 ± 0.161; 0.65 ± 0.136 respectively) (p=0.042, 0.032, respectively) (figure 4A). There was a significant correlation between the expression levels of QKI-V1 in the blood and brain of EAE mice (R=0.66; p=0.03) (Figure 4B). Protein expression of QKI-V1 in the brain of five of the EAE mice and five HC mice determined by western blot further validated this result. QKI-V1 was significantly decreased (0.33 ± 0.045; P =0.0052) in the brain of EAE mice compared to that in control mice. (figure 4C).

A.

B.

C.

Figure 4: Levels of QKI –V1 in the blood and brains of EAE mice. (A) Real time RT-PCR analysis of RNA isolated from the blood and brains of six mice (age 6–8 weeks) 51 days after induction with anti-MOG EAE (pattern color fill) and from eight healthy mice (full-color fill). (B) Scatter plot showing correlation between the levels of QKI –V1 (displayed as RQ) in the blood and brains of EAE mice obtained by Real time RT-PCR (C) Western blot analysis was performed by sequential probing with monoclonal antibody against QKI-V1 or anti-GAPDH of proteins extracted from the brains of five EAE mice and five control mice. The densitometry analysis of the immunoblot following normalization to GAPDH, is demonstrated.

3.6. PBMCs incubated with NMO sera show reduced expression of QKI-V5 Following our results that there is concordance between reduced QKI-1 (parallel to human QKI-V5) in the brain and blood of EAE mice, we aimed to determine whether the reduction of QKI-V5 in the blood of NMO patients is caused by a circulating factor. For that purpose, we incubated PBMCs of HCs with serum from three NMOAQP4-positive patients and with four HCs. The expression of QKI-V5 in the PBMCs was analyzed by real time PCR following RNA extraction. The expression of QKI-V5 was downregulated (0.69 ± 0.11; P =0.011) in PBMCs incubated with serum from patients with NMO, but not in those incubated with serum of HCs (figure 5A). Expression of the other QKI variants was not changed (data not shown).

Figure 5: QKI-V5 gene expression in PBMCs after incubation with serum from patients with NMO. Real time PCR analysis of RNA isolated from PBMCs of HC, which were incubated for 24 h with serum from four healthy controls (black bar) or three patients with NMO (patterned bar). Results are shown as fold change average ± SE.

3.7. Astrocytes incubated with NMO sera show downregulated QKI expression

To determine whether QKI-V5's expression in astrocytes is altered by a circulating factor/s, as we revealed in PBMCs incubated with serum from patients with NMO, we incubated primary human astrocytes (LONZA) and two human astrocytic cell lines (A172 and U87MG) with sera from three NMO AQP4-positive patients and with four HCs. Of note is that the main target of NMO-IG in the patients' sera is AQP4 expressed by astrocytes.

As was demonstrated in PBMC's, also in primary astrocytes and in U87 and A172 cell lines, there was a significantly lower expression levels of QKI-V5 following incubation with NMO sera compared to those incubated with sera of HCs (0.55 ± 0.12; 0.54 ± 0.123; 0.55 ± 0.126 respectively) ; P = 0.033; 0.041;0.045 respectively (Figure 6).

Figure 6: QKI-V5 gene expression in glial cells after incubation with serum from patients with NMO. Real time PCR analysis of RNA isolated from human astrocytes or glioma cell lines following incubation with sera from four HCs (full-color fill) or three patients with NMO (pattern color fill) for 24 h. Results are shown as fold change average ± SE.

4. Discussion

Although QKI protein plays an important role in myelination, by governing the posttranscriptional regulation of myelin-specific genes 11, 13, 18-21, its role in demyelinating diseases is unclear. Thus, our aim was to determine its role in demyelination diseases, specifically NMO, MS, and EAE. We observed a significant reduction in QKI-V5 in the peripheral blood of patients with NMO or MS, as well as in the brain and blood of the mog-EAE mice. This result is of importance because QKI-V5 regulates myelination by affecting pre-mRNA splicing of myelin genes 8. No changes in the expression of total QKI or variants QKI-V6 and QKI-V7 were observed. This is in discrepancy with the results of Larocque D et al. 12. One explanation could be that the mechanistic role of QKI-V6/7 in Schwann cells is different from its role in blood and brain cells. A significant reduction in the expression of QKI-V5 was found in PBMCs of HCs and glial cells following incubation with sera of patients with NMO, suggesting that the reduction of QKI is caused by a circulating factor. Not much is known about the involvement of QKI in CNS diseases. In schizophrenia 14

, alterations in the balance of QKI splice variants were correlated with the

expression of several oligodendrocyte/myelin‐related genes, including myelin‐ associated glycoprotein (MAG), MBP, proteolipid protein 1 (PLP1), SOX10, and transferrin, in several human brain regions

8, 14, 22

. Defective QKI expression in

schizophrenia could lead to downstream alternative splicing defects of several myelin genes, which in turn could play a role in the etiology of schizophrenia. In addition to its role in myelination and regulation of oligodendrocyte development and differentiation

18

, QKI is also highly expressed in astrocytes

23

. Neuromyelitis

optica (NMO) is a debilitating autoimmune astrocytopathy wherein the autoantigen AQP4 is expressed only by astrocytes and not oligodendrocytes. All QKI isoforms are

expressed in astrocytes and have distinct intracellular distribution patterns

23

. We

found a decrease in QKI-V5 in the blood of patients with NMO, and a significant decrease in QKI-V5 expression in primary human astrocytes and human glial cell lines incubated with sera from patients with NMO. The role of astrocytes in the pathogenesis of MS and EAE is well-documented traditionally considered cells that form the glial scar

25

24

. Although astrocytes are

, they have been recognized as

key participants in the MS inflammatory process, with beneficial and pathogenic roles 24, 25

. The role of astrocytes in the EAE model was shown by Mayo et al

26

, who

reported that astrocyte depletion during acute EAE worsens the disease. During chronic EAE, astrocytes become inflamed and acquire a pathogenic role. One explanation for our findings is that decreased QKI-V5 affects the immune-mediated function of astrocytes in the chronic phase of demyelinating diseases. Some QKI targets are directly involved in the MS immunopathophysiological process. QKI mRNA targets include MBP, early growth factor response gene‐2 (EGR‐ 2), p27KIP1, heterogeneous nuclear ribonuclear protein A (hnRNP A1), Map1b, actin‐ interacting protein 1 (AIP‐1), and myocardin 27. EGR‐2 influences IL-17

28

, which is

essential for MS pathogenesis. Interestingly, antibodies against hnRNP A1 were recently found to influence neurodegeneration, and were detected in patients with MS 29

.

QKI targets genes as well as miRNA 30; pri‐miR‐7‐1 harbors 3 putative QKI response elements (QREs) and is tightly bound in the nucleus by QKI-V5 and QKI-V6. Interestingly, miR-7 is significantly down regulated in patients with NMO or MS compared to HCs 4, 31. Remyelination following demyelination occurs in patients with MS and patients with EAE. In some patients with MS, extensive remyelination is present in up to 90% of

the lesions. However, remyelination fails in the majority of the patients. The reason for this failure is unknown. During remyelination, QKI variants 6 and 7 are upregulated while variant 5 is downregulated

8, 12

. The decrease in QKI variant 5

levels may therefore be associated with an ongoing active remyelinating process. During active remyelination attempts, large quantities of QKI-V5 are consumed, causing the decreased QKI-V5 levels 32. Cortical lesions or subcortical white matter lesions are more frequently remyelinated than those located in periventricular areas

33

. This may explain our ability to detect

QKI variants in the brain and peripheral blood of mice with EAE as well as the peripheral blood of patients. Blood-derived QKI could act as a regulator of cortical lesion remyelination. A study in rat brains

19

demonstrated that the transition from

premyelinating to myelinating oligodendrocytes during development is accompanied by elevated levels of QKI proteins. The same study also showed that, in adult rat brains, a subset of oligodendrocytes display characteristics of actively myelinating oligodendrocytes that are normally seen during development and are characterized by increased levels of QKI proteins, indicating that QKI plays a role in adult myelination.

Almost all the therapeutic strategies for MS involve targeting the

immune system, and aim to reduce immune system attacks on the myelin sheath. However, very few therapies aim to enhance remyelination and promote the proliferation/differentiation of oligodendrocytes at the lesion sites. Although the function of QKI in adult myelination remains unclear, given the role of QKI in glial development and myelination in rodents, it likely plays a role in human myelination/remyelination 19. Our results show for the first time that QKI variants, particularly QKI-V5, might play a role in demyelinating diseases. The observation that QKI-V5 is downregulated in

the EAE mouse brain as well as in the blood of patients with MS or NMO could imply that the alteration of QKI expression in the blood plays a role in these diseases. Further study is therefore needed to elucidate the role played by QKI in demyelinating diseases.

Author Contribution Statement

Iris Lavon, PhD, corresponding author, Conceptualization, Methodology, Formal analysis, Writing - Original Draft, Supervision

Ina leykin, Investigation

Hanna Charbit, MSc, Validation, Writing - Original Draft

Orli Binyamin, MSc, Investigation Livnat Brill1, MSc, Validation

Haim Ovadia, PhD, Investigation

Adi Vaknin-Dembinsky, MD, PhD Conceptualization, Methodology, Writing Original Draft, Supervision.

Declaration of conflicting interest The authors declare that there is no conflict of interest.

Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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