Vitamin B12 as an epidrug for regulating peripheral blood biomarkers in long COVID-associated visuoconstructive deficit

doi:10.21203/rs.3.rs-3158180/v1

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Vitamin B12 as an epidrug for regulating peripheral blood biomarkers in long COVID-associated visuoconstructive deficit

https://doi.org/10.21203/rs.3.rs-3158180/v1

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Approximately four months after recovering from a mild COVID-19 infection, around 25% of individuals developed visuoconstructive deficit (VCD), which was found to be correlated with an increase in peripheral immune markers and alterations in structural and metabolic brain imaging. Recently, it has been demonstrated that supplemental vitamin B12 regulates hyperinflammation during moderate and severe COVID-19 through methyl-dependent epigenetic mechanisms. Herein, whole peripheral blood cultures were produced using samples obtained from patients with confirmed persistent VCD, and controls without impairment, between 10 and 16 months after mild COVID-19. This experimental model was used to assess the leukocyte expression patterns of 11 biomarkers previously associated with VCD in long COVID and explore the potential of pharmacological B12 in regulating these genes. The results showed that patients with persistent VCD displayed continued upregulation of CCL11 and LIF compared to controls. It is worth noting that elevated serum levels of CCL11 have been previously linked to age-related neurodegenerative diseases. Notably, the addition of 1 nM of vitamin B12 to blood cultures from individuals with VCD normalized the mRNA levels of CCL11, upregulated the neuroprotective HGF, and, to a lesser extent, downregulated CSF2 and CXCL10. There was an inverse correlation observed between CCL11 mRNA levels and methylation levels of specific cytosines in its promoter region. These findings underscore the significance of systemic inflammation in persistent VCD associated with long COVID. Moreover, the study provides evidence suggesting that B12, acting as an epidrug, shows promise as a therapeutic approach for addressing this cognitive impairment.

Long COVID

Visuoconstructive deficit

CCL11

DNA methylation

Epigenetics

Long COVID is a term used to describe the prolonged symptoms of COVID-19, including cognitive dysfunction, also known as "brain fog," which can harm memory, attention, and problem-solving abilities. These symptoms may persist for weeks or even months after the infection has resolved [1, 2]. While the exact cause of long COVID is not fully understood, it is thought to be related to the immune system's response to the virus and the systemic inflammation that it causes [reviewed in: 3].

Approximately 25% of young adults (with an average age of 38 years) who have had mild COVID-19 experience serious cognitive impairment in the copy stage of the Rey-Osterrieth Complex Figure Test (ROCF) four months after recovering from the infection [4]. The ROCF is a drawing task test used to assess visuospatial abilities, executive functions, and memory. Performance on visuoconstruction and memory tests is associated with learning, problem-solving skills, and various activities of daily living [5]. These processes integrate the perception and interpretation of visual information with memory and executive systems by involving various brain areas, including the occipito-parietal regions, the dorsal and ventral streams, and connections with the cingulate, medial temporal, and frontal cortices [6]. Notably, negative correlations were found between performance on the ROCF test and white matter volume in both the left and right genu of the corpus callosum, extending to the cingulum bundle, as well as glucose metabolism in the right dorsal anterior cingulate gyrus. Moreover, a functional interaction network was observed among ten out of eleven plasma biomarkers that were upregulated in patients with visuoconstructive deficit (VCD). Four of these biomarkers are components of the Neuroinflammation Signaling pathway, and four are components of the IL-17 Signaling pathway. Indeed, evidence suggests that peripheral innate immune system molecules can trigger a secondary neuroinflammatory response in the central nervous system (CNS) [7–9].

It is possible that epigenetic changes may contribute to the cognitive dysfunction in long COVID. For example, Lee and colleagues reported that individuals with long COVID had higher levels of DNA methylation in certain genes involved in immune response and inflammation [10]. DNA methylation is a process that involves the addition of a methyl group (-CH3) to a cytosine base in DNA. This modification can regulate gene expression by altering the interaction between the DNA molecule and the proteins that bind to it [11, 12]. It is worth noting that dysregulation of DNA methylation patterns has been linked to various brain disorders, including depression, anxiety, and cognitive decline [reviewed in: 13, 14].

Vitamin B12 is an essential cofactor for the enzyme methionine synthase, which catalyzes the conversion of homocysteine to methionine in the sulfur amino acid pathway. S-adenosylmethionine, the methyl donor utilized in all cellular methylation processes, is derived from methionine [15]. Accordingly, adjuvant vitamin B12 has been shown to hypermethylate key genes in inflammatory disorders, resulting in their downregulation. This effect has been previously demonstrated in an infant rat model of pneumococcal meningitis [16] and in whole blood cultures from human patients with acute COVID-19 [17].

The primary objective of this study was to identify peripheral blood biomarkers that are linked to long-term VCD in long COVID patients who have previously experienced a mild acute COVID-19 episode. Additionally, the study aimed to explore the methyl-dependent epigenetic mechanisms that play a role in regulating these biomarkers as well as investigate the potential of vitamin B12 in modulating the gene expression of these genes in leukocytes.

Patients

This prospective observational cohort study received approval from the Institutional Review Board (IRB) of the Federal University of Minas Gerais (UFMG) (CAAE3768820.1.0000.5149). All participants provided written informed consent. Volunteers were recruited approximately four months after being confirmed as having mild COVID-19 through RT-qPCR testing. Disease severity was categorized as 1 or 2 according to the WHO clinical ordinal scale [18]. During the recruitment appointment and subsequent follow-up appointments at six and twelve months, the participants' clinical status and mental health were evaluated, peripheral blood samples were collected, and a comprehensive investigation was conducted using neuropsychological tests, PET-CT, and magnetic resonance neuroimaging, following the previously described protocol [4]. For the present study, seven individuals diagnosed with VCD during the recruitment appointment were enrolled. Additionally, five individuals who had mild COVID-19 but did not exhibit any signs of VCD were included as controls. The research timeline can be found in Fig. 1.

Assessment of Folate, Homocysteine and Vitamin B12 basal levels

The standard chemiluminescence method was used by a commercial clinical laboratory (Geraldo Lustosa, Belo Horizonte, Brazil) to quantify the basal levels of folate, homocysteine (HCY), and vitamin B12 in plasma samples from all volunteers.

Whole Blood Cultures

Whole peripheral blood cultures were generated using samples collected in sodium heparin at 8 a.m., following the protocol outlined by Cassiano et al. [17]. In brief, the blood samples were mixed with 50% (v/v) RPMI 1640 medium (Sigma-Aldrich, Saint Louis, Missouri). Two different conditions were employed: endpoint A, where the medium was supplemented with an excipient (citrate-phosphate buffer, pH 5, Merck, Darmstadt, Germany), and endpoint B, where the medium was supplemented with cyanocobalamin (Merck) at a final concentration of 1 nM. The cultures were then incubated for 24 hours at 37°C in a humidified atmosphere with 5% CO₂.

Real-time quantitative PCR (RT-qPCR)

The total RNA was extracted from leukocytes using the QIAamp RNA Blood Kit (Qiagen, Hilden, Germany), and cDNA was produced with the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher, Waltham, MA) as previously described[17]. The mRNA levels of CCL2 (C-C Motif Chemokine Ligand 2), CCL11 (C-C Motif Chemokine Ligand 11), CSF2 (Colony Stimulating Factor 2), CXCL10 (C-X-C Motif Chemokine Ligand 10), HGF (Hepatocyte Growth Factor), IL1RA (Interleukin 1 Receptor Antagonist), IL6 (Interleukin 6), IL10 (Interleukin 10), IL31 (Interleukin 31), LIF (LIF Interleukin 6 Family Cytokine), and NGF (Nerve Growth Factor) were quantified using specific primers in SYBR Green (Thermo Fisher) or TaqMan (Thermo Fisher) platforms following the manufacturer's instructions. These genes encode for the plasma inflammatory and growth factors previously associated with VCD in long COVID [4]. Target gene expression levels were normalized by 18S ribosomal RNA (18S ribosomal N1), and relative gene expression was calculated using the 2e(-ΔCt) method [19]. The primer sequences and TaqMan assay IDs are listed in Supplementary Table 1.

Bisulfite Sequencing PCR

To assess the impact of VCD and supplemental vitamin B12 on the methylation levels of 17 CpG sites located in the promoter region and proximal portion of the first exon of CCL11, DNA libraries obtained from cultures of endpoints A and B were analyzed using Bisulfite Sequencing PCR (BSP). The individuals included in the analysis represented both the impaired and non-impaired groups.

The production of BSP libraries, sequencing, and bioinformatics analysis were performed as described in [17] with minor modifications. Following DNA isolation, unmethylated cytosine nucleotides were converted into uracil with the EZ DNA Methylation-Gold Kit (Zymo Research, Irvine, California). The DNA region spanning from − 2 569 pb to + 310 pb of CCL11 (GRCh38/hg38 chr17: 34283173–34286052) was amplified by PCR from bisulfite-converted DNA using a primer set designed to account for cytosine conversion to uracil (Bisulfite Primer Design Tool, Zymo Research) (Supplementary Table 2) and GoTaq DNA Polymerase (Promega, Madison, Wisconsin). The mapping of filtered reads to the reference sequence chr17: 34283173–34286052 of the Homo sapiens genome (Gencode, release 38), as well as the calculation of methylation percentages at CG loci, were performed using the Bismark software [20]. The functional annotations for CCL11 were obtained from the UCSC Genome Browser [21].

Statistical analysis

Statistical analyses were performed using GraphPad Prism software (version 8.0.2) from GraphPad Software Inc., Irvine, CA. The data distribution was assessed using Anderson-Darling, D'Agostino & Person, Shapiro-Wilk, and Kolmogorov-Smirnov tests. Based on the distribution, a two-tailed Student's t-test, paired Student's t-test, Mann-Whitney test, or Wilcoxon test were employed to compare the groups. Statistical significance was defined as a P-value less than 0.05. The data were presented as median ± interquartile range.

The fold change methylation levels of each CG locus were compared between groups using an unpaired t-test. Only the loci that exhibited statistically significant differences between the compared groups were chosen for further analysis. To evaluate the correlation between the fold change methylation levels of differentially methylated loci (DML) and CCL11 gene expression levels quantified by RT-qPCR in the same cultures, Pearson or Spearman tests were employed based on the data distributions.

Sample characterization

The study included female participants with an average age of 40.7 ± 8.5 years. There were no statistically significant differences in age between the groups initially diagnosed with or without VCD, as indicated in Table 1. It is worth mentioning that all participants had received the SARS-CoV-2 vaccination at the time of the follow-up appointments.

Table 1

Participant’s data
Participant ID	Age (years)	Baseline diagnosis	Follow-up appointment (months)	Diagnosis at follow-up appointment
BRA-106	34	Impaired	12	Impaired
BRA-108	51	Impaired	12	Normal
BRA-111	32	Normal	12	Normal
BRA-112	56	Normal	12	Normal
BRA-126	31	Impaired	6	Normal
BRA-138	35	Normal	6	Normal
BRA-140	33	Normal	6	Normal
BRA-143	36	Impaired	6	Impaired
BRA-187	49	Impaired	6	Impaired
BRA-196	44	Impaired	6	Impaired
BRA-200	51	Normal	6	Normal
BRA-202	37	Impaired	6	Normal

Among the seven individuals initially diagnosed with VCD, four (57%) still exhibited the condition during the follow-up appointments, while three showed no further signs of impairment. The five non-impaired control individuals maintained their initial diagnosis, as shown in Table 1. No significant statistical differences were observed in the basal levels of serum folate, HCY, and B12 among individuals with VCD, those who had recovered from the impairment, and non-impaired individuals, as detailed in Supplementary Table 3.

Supplemental B12 favorably modulates persistently dysregulated inflammatory genes in the leukocytes of patients with VCD.

After 6 to 12 months from the initial diagnosis, CCL11 (P = 0.0127) and LIF (P = 0.0485) continued to show upregulation in leukocytes from individuals with persistent VCD compared to those from non-impaired subjects (Fig. 2). Importantly, when the whole peripheral blood cultures were incubated with 1 nM of B12 for 24 hours, the expression of CCL11 in the impaired group was normalized to the levels observed in the cultures from the non-impaired group incubated with the excipient (P > 0.05). B12 also downregulated CSF2 (P = 0.0071) and CXCL10 (P = 0.0205) in leukocytes from impaired individuals, although these genes were not differentially regulated when compared to the non-impaired group. Additionally, B12 increased the mRNA levels of HGF in the impaired group compared to the non-impaired group incubated with the excipient (P = 0.0081). CCL2, IL10, IL1RA, and IL6 were not differentially expressed when impaired and non-impaired individuals were compared, and these genes did not respond to incubation with B12 (Supplementary Table 4). IL31 and NGF mRNA were not detected in leukocytes from any of the volunteers.

Impaired individuals exhibit hypomethylation of CpG sites in the CCL11 promoter region, which are then hypermethylated upon supplementation with B12

In impaired individuals, three CpG sites exhibited hypomethylation when compared to the non-impaired group. Notably, treatment of cultures with B12 significantly increased the methylation level of two of these CpG sites. Furthermore, a strong and negative correlation was observed between the methylation status of these DML and the mRNA levels of CCL11. These DML are located at predicted transcription factor binding sites (TFBS) according to the JASPAR CORE 2022 data (https://jaspar.genereg.net) (Fig. 3).

It is known that VCD, which refers to the impaired ability to visually perceive and construct complex visual-spatial designs or tasks, is a known characteristic of aging-related neurodegenerative diseases, such as Alzheimer's disease [22]. The occurrence of this symptom in the young adults enrolled in this study is noteworthy. It suggests the possibility that individuals who experience mild COVID-19 may be at a higher risk of developing severe conditions associated with neurodegeneration. Furthermore, the high prevalence (57%) of persistent VCD 6 to 12 months after the initial diagnosis is a cause for concern.

Of the 11 plasma inflammatory and growth factors previously associated with VCD in long COVID [4], only CCL11 and LIF displayed persistent upregulation in leukocytes from individuals with continued impairment. This upregulation was observed in comparison to individuals who did not develop the deficit following mild COVID-19 and those who recovered within 6 to 12 months after the initial diagnosis of VCD. CCL11, a chemokine also known as eotaxin-1, has been implicated in neuroinflammation, neurogenesis inhibition, and cognitive decline in aging, multiple sclerosis, and Alzheimer's disease [reviewed in: 23]. Recent findings have revealed CCL11 to be a critical biomarker of long-term brain damage in a mouse model of mild COVID-19 [24]. One of the primary mechanisms postulated to underlie the neurodegenerative effects of CCL11 in long COVID involves the activation of hippocampal microglia, leading to diminished neurogenesis and the deterioration of myelinated subcortical axons [23]. Additionally, LIF, a multifunctional cytokine, has been identified as a regulator of neuronal phenotype and a coordinator of glial and inflammatory cell responses in the CNS [reviewed in: 25, 26]. Moreover, the LIF protein has been shown to increase the transcription of HGF, which encodes an important neuroprotective molecule [27]. Nonetheless, the specific role of LIF in the CNS during long COVID remains to be further elucidated.

No significant increase in the levels of CCL2, CSF2, CXCL10, HGF, IL10, IL1RA, or IL6 was observed in leukocytes from individuals with persistent VCD during the follow-up. However, it is unclear whether the reduction of some of these molecules before the resolution of the impairment is beneficial. HGF is a potent neurotrophic factor that promotes neurogenesis and has been shown to have neuroprotective effects in neurodegenerative diseases [reviewed in: 28]. IL-10 is an anti-inflammatory cytokine that negatively regulates the immune response, reduces inflammation, and protects against neuroinflammatory processes associated with neurodegenerative diseases [reviewed in: 29]. IL-1RA dampens the pro-inflammatory effects of IL-1, preventing excessive inflammation and mitigating neuroinflammatory processes associated with neurological disorders [reviewed in: 30]. These findings indicate an orchestrated decline in the neuroprotective and anti-inflammatory response in individuals with persistent VCD.

Vitamin B12, recognized for its neuroprotective properties, plays a crucial role in regulating gene expression through its involvement in methyl-dependent epigenetic mechanisms, including DNA and histone methylation [31]. Incubating peripheral whole blood cultures with vitamin B12 resulted in the normalization of CCL11 expression in leukocytes from individuals with persistent VCD (Fig. 2). Notably, B12 supplementation increased the transcription of HGF and decreased the expression of CSF2 and CXCL10 in leukocytes from impaired individuals. CSF2 and CXCL10 are pro-inflammatory proteins implicated in neurodegenerative diseases [32–34]. Altogether, these findings suggest a potential beneficial effect of vitamin B12 in reducing neuroinflammation and promoting neuroprotection in patients with persistent VCD long after experiencing mild acute COVID-19.

The absence of IL31 and NGF mRNA in leukocytes from the volunteers suggests that the proteins they encode, which were previously found to be elevated in the plasma samples of patients with VCD during the recruitment appointment four months after mild acute COVID-19 [4], might be primarily produced by other cell types [35–37].

Leukocytes from cultures of individuals with persistent VCD at endpoint A showed hypomethylation of three CpG sites in the promoter region of CCL11 when compared to the non-impaired group. This VCD-associated decrease in methylation levels of each DML was negatively correlated with the CCL11 expression level (Fig. 3). Indeed, the observed DML are located at predicted TFBS [38]. The transcription factors (TF) E2F7 (JASPAR ID: MA0758.1) and E2F8 (JASPAR ID: MA0865.2) can bind to the region encompassing the DML a (chr17: 34283605), while E2F6 (JASPAR ID: MA0471.2) and TFDP1 (JASPAR ID: MA1122.1) can bind to the complementary region of this DML in the minus strand. E2F6, E2F7, and E2F8 are members of the E2F family of TF, which play important roles in cell cycle regulation, cell proliferation, and survival. and gene expression [39]. TFDP1 heterodimerizes with E2F proteins to enhance their DNA-binding activity and promote transcription from E2F target genes [39]. Unfortunately, there is limited research specifically investigating the relationship between E2F6-8 and CCL11 transcription in the CNS context. Regarding DML b (chr17: 34283701), ZNF136 (JASPAR ID: MA1588.1) can bind to the complementary region in the minus strand, but there is no evidence of its action on CCL11 expression. NPAS4 (JASPAR ID: MA2042.1) can bind to the region containing the DML c (chr17: 34285656), and RARA::RXRA (JASPAR ID: MA0159.1) and RARA::RXRG (JASPAR ID: MA1149.1) to the complementary region of this locus. NPAS4 is a neuroprotective protein and acts as a TF, regulating the transcription of a diverse set of genes involved in synaptic plasticity, neurotransmission, and neuronal survival [40]. However, there is limited research on its specific interaction with CCL11. The RARA::RXRA and RARA::RXRG complexes are heterodimeric protein complexes formed by the retinoic acid receptor alpha (RARA) in conjunction with either retinoid X receptor alpha (RXRA) or retinoid X receptor gamma (RXRG) [41]. These complexes are involved in mediating the effects of retinoic acid signaling, which plays crucial roles in development, cell differentiation, and immune response [41]. Previous studies have demonstrated that the use of RXR partial agonists can reduce eosinophilic airway inflammation, a condition mediated, in part, by the chemoattractant activity of CCL11 [42]. RXR agonists are also effective in the treatment of some neuropathies, such as Alzheimer’s disease [43] and Parkinson’s disease in animal models [44]. While direct evidence linking the RARA::RXRA and RARA::RXRG complexes to CCL11 expression in the CNS is currently not well established, it is plausible to speculate that retinoic acid signaling through these complexes could inhibit CCL11 expression in specific brain conditions. Furthermore, the DML c is located in regulatory regions regulated by EZH2, CTCF, GPS2, and TCF21 in different cell types [45]. These TF act as epigenetic regulators of chromatin structure through interactions with histone modifiers, chromatin remodeling complexes, transcriptional regulators, and/or DNA methylation machinery that modulate gene expression. It is reasonable to assume that changes in CpG methylation levels in this region may reflect on the epigenetic landscape, possibly favoring the expression of CCL11. Overall, this significant discovery highlights the potential of using CpG methylation patterns as diagnostic biomarkers for neurodegenerative conditions characterized by CCL11 upregulation.

The hypothesis that B12 could modulate CCL11 expression during persistent VCD via methyl-dependent epigenetic mechanisms was confirmed by the hypermethylation of DML a and c located in the promoter region of CCL11 in leukocytes from cultures of impaired individuals incubated with the vitamin. These two DML were initially identified as being hypomethylated in the context of the disease. The B12-induced increment in methylation levels of each of these DML negatively correlated with the CCL11 expression level (Fig. 3). These findings provide compelling evidence for the role of epigenetic regulation of CCL11 and suggest that supplemental B12 has potential as an epidrug to treat neurodegenerative conditions associated with aberrant CCL11 expression.

This study provides compelling evidence highlighting the importance of systemic inflammation in persistent VCD associated with long COVID. Notably, the study reports a persistent upregulation of CCL11 in peripheral leukocytes in patients with VCD. This upregulation has been found to strongly and negatively correlates with the methylation state of three cytosines in the promoter region of the CCL11 gene. Collectively, these findings support the potential use of CCL11 mRNA levels or promoter methylation patterns as biomarkers for VCD in long COVID. Moreover, the results suggest that supplementation with vitamin B12 holds promise as a therapeutic approach for patients with VCD in the context of long COVID.

ACKNOWLEDGEMENTS

This work was supported by INOVA FIOCRUZ (VPPCB-005-FIO-20-2-115), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—CAPES (88881.504749/2020-01. 9951), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (408817/2022-4), Instituto Nacional de Ciência e Tecnologia em Vacinas (INCT- Vacinas), and Instituto Nacional de Ciência e Tecnologia em Nanotecnologias Responsáveis (INCT-NeuroTec-R; 406935/2022). LMGC is the recipient of a CAPES doctoral scholarship.

CONFLICT OF INTEREST

The authors declare no competing interests.

CONTRIBUTIONS

Study conception and/or design: JJP, DMM, MAR-S, and RSC. Clinical assessment: JJP. Data acquisition: LMGC, JJP, DVR. Data analysis: LMGC, JJP, DVR, DMM, MAR-S, and RSC. Manuscript writing and/or revision: LMGC, JJP, DVR, DMM, MAR-S, and RSC.

AVAILABILITY OF DATA AND MATERIALS

The dataset supporting the conclusions of this study is available in the SRA repository [https://www.ncbi.nlm.nih.gov/sra/PRJNA992778].

Supplementary information is available at MP’s website

Centers for Disease Control and Prevention (CDC). Long COVID or Post-COVID Conditions. 2022. [Available in: https://www.cdc.gov/coronavirus/2019-ncov/long-term-effects/index.html].
Soriano JB, Murthy S, Marshall JC, Relan P, Diaz J V, WHO Clinical Case Definition Working Group on Post-COVID-19 Condition. A clinical case definition of post-COVID-19 condition by a Delphi consensus. Lancet Infect Dis. 2022;22:e102–e107.
Elizalde-Díaz JP, Miranda-Narváez CL, Martínez-Lazcano JC, Martínez-Martínez E. The relationship between chronic immune response and neurodegenerative damage in long COVID-19. Front Immunol. 2022;13:1039427.
de Paula JJ, Paiva RERP, Souza-Silva NG, Rosa DV, Duran FL de S, Coimbra RS, et al. Selective visuoconstructional impairment following mild COVID-19 with inflammatory and neuroimaging correlation findings. Mol Psychiatry. 2023;28:553–563.
Davies SR, Field ARJ, Andersen T, Pestell C. The ecological validity of the Rey-Osterrieth Complex Figure: predicting everyday problems in children with neuropsychological disorders. J Clin Exp Neuropsychol. 2011;33:820–831.
Kravitz DJ, Saleem KS, Baker CI, Mishkin M. A new neural framework for visuospatial processing. Nat Rev Neurosci. 2011;12:217–230.
Thomson CA, McColl A, Cavanagh J, Graham GJ. Peripheral inflammation is associated with remote global gene expression changes in the brain. J Neuroinflammation. 2014;11:73.
Tenza-Ferrer H, Magno LAV, Romano-Silva MA, da Silva JF, Gomez MV. Phα1β Spider Toxin Reverses Glial Structural Plasticity Upon Peripheral Inflammation. Front Cell Neurosci. 2019;13:306.
Riester K, Brawek B, Savitska D, Fröhlich N, Zirdum E, Mojtahedi N, et al. In vivo characterization of functional states of cortical microglia during peripheral inflammation. Brain Behav Immun. 2020;87:243–255.
Lee Y, Riskedal E, Kalleberg KT, Istre M, Lind A, Lund-Johansen F, et al. EWAS of post-COVID-19 patients shows methylation differences in the immune-response associated gene, IFI44L, three months after COVID-19 infection. Sci Rep. 2022;12:11478.
Fuks F. DNA methylation and histone modifications: teaming up to silence genes. Curr Opin Genet Dev. 2005;15:490–495.
Bird AP, Wolffe AP. Methylation-induced repression--belts, braces, and chromatin. Cell. 1999;99:451–454.
Nestler EJ, Peña CJ, Kundakovic M, Mitchell A, Akbarian S. Epigenetic Basis of Mental Illness. Neuroscientist. 2016;22:447–463.
Maity S, Farrell K, Navabpour S, Narayanan SN, Jarome TJ. Epigenetic Mechanisms in Memory and Cognitive Decline Associated with Aging and Alzheimer’s Disease. Int J Mol Sci. 2021;22.
Selhub J. Folate, vitamin B12 and vitamin B6 and one carbon metabolism. J Nutr Health Aging. 2002;6:39–42.
de Queiroz KB, Cavalcante-Silva V, Lopes FL, Rocha GA, D’Almeida V, Coimbra RS. Vitamin B12 is neuroprotective in experimental pneumococcal meningitis through modulation of hippocampal DNA methylation. J Neuroinflammation. 2020;17:96.
Cassiano LMG, Cavalcante-Silva V, Oliveira MS, Prado BVO, Cardoso CG, Salim ACM, et al. Vitamin B12 attenuates leukocyte inflammatory signature in COVID-19 via methyl-dependent changes in epigenetic markings. Front Immunol. 2023;14:1048790.
Son K-B, Lee T-J, Hwang S-S. Disease severity classification and COVID-19 outcomes, Republic of Korea. Bull World Health Organ. 2021;99:62–66.
Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc. 2008;3:1101–1108.
Krueger F, Andrews SR. Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics. 2011;27:1571–1572.
Kent WJ, Sugnet CW, Furey TS, Roskin KM, Pringle TH, Zahler AM, et al. The human genome browser at UCSC. Genome Res. 2002;12:996–1006.
Mandal PK, Joshi J, Saharan S. Visuospatial perception: an emerging biomarker for Alzheimer’s disease. J Alzheimers Dis. 2012;31 Suppl 3:S117-35.
Ivanovska M, Abdi Z, Murdjeva M, Macedo D, Maes A, Maes M. CCL-11 or Eotaxin-1: An Immune Marker for Ageing and Accelerated Ageing in Neuro-Psychiatric Disorders. Pharmaceuticals (Basel). 2020;13.
Fernández-Castañeda A, Lu P, Geraghty AC, Song E, Lee M-H, Wood J, et al. Mild respiratory COVID can cause multi-lineage neural cell and myelin dysregulation. Cell. 2022;185:2452-2468.e16.
Ostasov P, Houdek Z, Cendelin J, Kralickova M. Role of leukemia inhibitory factor in the nervous system and its pathology. Rev Neurosci. 2015;26:443–459.
Sugiura S, Lahav R, Han J, Kou SY, Banner LR, de Pablo F, et al. Leukaemia inhibitory factor is required for normal inflammatory responses to injury in the peripheral and central nervous systems in vivo and is chemotactic for macrophages in vitro. Eur J Neurosci. 2000;12:457–466.
Tomida M, Saito T. The human hepatocyte growth factor (HGF) gene is transcriptionally activated by leukemia inhibitory factor through the Stat binding element. Oncogene. 2004;23:679–686.
Desole C, Gallo S, Vitacolonna A, Montarolo F, Bertolotto A, Vivien D, et al. HGF and MET: From Brain Development to Neurological Disorders. Front Cell Dev Biol. 2021;9:683609.
Porro C, Cianciulli A, Panaro MA. The Regulatory Role of IL-10 in Neurodegenerative Diseases. Biomolecules. 2020;10.
Shaftel SS, Griffin WST, O’Banion MK. The role of interleukin-1 in neuroinflammation and Alzheimer disease: an evolving perspective. J Neuroinflammation. 2008;5:7.
Cassiano LMG, Oliveira M da S, Coimbra RS. Vitamin B12 as a neuroprotectant in neuroinflammation. In: Martin CR, Patel V, Preedy VR, editors. Vitamins and Minerals in Neurologic Disorders, . 1st ed.Academic Press; 2023. p. 399–413.
Chitu V, Biundo F, Shlager GGL, Park ES, Wang P, Gulinello ME, et al. Microglial Homeostasis Requires Balanced CSF-1/CSF-2 Receptor Signaling. Cell Rep. 2020;30:3004-3019.e5.
Sui Y, Stehno-Bittel L, Li S, Loganathan R, Dhillon NK, Pinson D, et al. CXCL10-induced cell death in neurons: role of calcium dysregulation. Eur J Neurosci. 2006;23:957–964.
Koper OM, Kamińska J, Sawicki K, Kemona H. CXCL9, CXCL10, CXCL11, and their receptor (CXCR3) in neuroinflammation and neurodegeneration. Adv Clin Exp Med. 2018;27:849–856.
GTEx Consortium. The Genotype-Tissue Expression (GTEx) project. Nat Genet. 2013;45:580–585.
Dillon SR, Sprecher C, Hammond A, Bilsborough J, Rosenfeld-Franklin M, Presnell SR, et al. Interleukin 31, a cytokine produced by activated T cells, induces dermatitis in mice. Nat Immunol. 2004;5:752–760.
Bruno F, Arcuri D, Vozzo F, Malvaso A, Montesanto A, Maletta R. Expression and Signaling Pathways of Nerve Growth Factor (NGF) and Pro-NGF in Breast Cancer: A Systematic Review. Curr Oncol. 2022;29:8103–8120.
Castro-Mondragon JA, Riudavets-Puig R, Rauluseviciute I, Lemma RB, Turchi L, Blanc-Mathieu R, et al. JASPAR 2022: the 9th release of the open-access database of transcription factor binding profiles. Nucleic Acids Res. 2022;50:D165–D173.
DeGregori J, Johnson DG. Distinct and Overlapping Roles for E2F Family Members in Transcription, Proliferation and Apoptosis. Curr Mol Med. 2006;6:739–748.
Fu J, Guo O, Zhen Z, Zhen J. Essential Functions of the Transcription Factor Npas4 in Neural Circuit Development, Plasticity, and Diseases. Front Neurosci. 2020;14:603373.
Huang P, Chandra V, Rastinejad F. Retinoic acid actions through mammalian nuclear receptors. Chem Rev. 2014;114:233–254.
Fujii U, Miyahara N, Taniguchi A, Oda N, Morichika D, Murakami E, et al. Effect of a retinoid X receptor partial agonist on airway inflammation and hyperresponsiveness in a murine model of asthma. Respir Res. 2017;18:23.
Cramer PE, Cirrito JR, Wesson DW, Lee CYD, Karlo JC, Zinn AE, et al. ApoE-directed therapeutics rapidly clear β-amyloid and reverse deficits in AD mouse models. Science. 2012;335:1503–1506.
McFarland K, Spalding TA, Hubbard D, Ma J-N, Olsson R, Burstein ES. Low dose bexarotene treatment rescues dopamine neurons and restores behavioral function in models of Parkinson’s disease. ACS Chem Neurosci. 2013;4:1430–1438.
Hammal F, de Langen P, Bergon A, Lopez F, Ballester B. ReMap 2022: a database of Human, Mouse, Drosophila and Arabidopsis regulatory regions from an integrative analysis of DNA-binding sequencing experiments. Nucleic Acids Res. 2022;50:D316–D325.

The authors have declared there is NO conflict of interest to disclose

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Vitamin B12 as an epidrug for regulating peripheral blood biomarkers in long COVID-associated visuoconstructive deficit

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Abstract

Figures

INTRODUCTION

MATERIAL AND METHODS

Patients

Assessment of Folate, Homocysteine and Vitamin B12 basal levels

Whole Blood Cultures

Real-time quantitative PCR (RT-qPCR)

Bisulfite Sequencing PCR

Statistical analysis

RESULTS

Sample characterization

DISCUSSION

CONCLUSION

Declarations

References

Additional Declarations

Supplementary Files

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