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    Topic review

    FRAb in Autism Spectrum Disorders

    Subjects: Pathology
    View times: 6
    Submitted by: Edward Quadros


    Folate deficiency and folate receptor autoimmune disorder are major contributors to infertility, pregnancy related complications and abnormal fetal development including structural and functional abnormalities of the brain. Food fortification and prenatal folic acid supplementation has reduced the incidence of neural tube defect (NTD) pregnancies but is unlikely to prevent pregnancy-related complications in the presence of folate receptor autoantibodies (FRAb). In pregnancy, these autoantibodies can block folate transport to the fetus and in young children, folate transport to the brain. These antibodies are prevalent in neural tube defect pregnancies and in developmental disorders such as cerebral folate deficiency (CFD) syndrome and autism spectrum disorder (ASD). In the latter conditions, folinic acid treatment has shown clinical improvement in some of the core ASD deficits. Early testing for folate receptor autoantibodies and intervention is likely to result in a positive outcome

    1. Background

    Folate, an umbrella term used for metabolically active forms of folic acid (B9), is an essential B-complex vitamin necessary for basic cellular metabolism including, but not limited to, essential cellular DNA synthesis, repair and methylation including regulation of synthesis and metabolism of monoamine neurotransmitters. As a nutrient found in green leafy vegetables, legumes and fruits, it is readily absorbed by the upper small intestine after breakdown from polyglutamates to monoglutamates. Folate in its active forms facilitates one-carbon transfer reactions and contributes to the synthesis of purines, pyrimidines and amino acids [1]. One of its most characterized roles is facilitating single carbon transfer to homocysteine to form methionine. This reaction is critical for maintaining intracellular S- adenosyl methionine, an essential compound for methylation reactions. Folate also has a co-dependent relationship with vitamin B12 in that both vitamins must be present in adequate amounts for conversion to the physiologic forms that participate in metabolic reactions. If folate and B12 are not adequate, cellular metabolism and replication is interrupted [2][3]. This is most critical during fetal and neonatal development because inadequate folate during this period can result in interruptions in brain development leading to structural abnormalities that produce functional deficits c of the CFD syndrome. Low cerebro-spinal fluid (CSF) folate is a characteristic feature of CFD syndrome, as first described by Ramaekers and Blau [4]. On rare occasions, CFD can also result from mutations in the FRα gene [5][6][7], but the most common cause of low CSF folate in CFD is the presence of anti-folate receptor antibodies (FRAb) that can block folate transport across the choroid plexus [8][9]. A recent report has identified mutations in the CIC transcription factor gene in children diagnosed with CFD syndrome. Mutations in the CIC gene decrease the expression of FRα to reduce folate transport across the choroid plexus [10]. No abnormalities of the FRα gene are found in ASD, but a majority of these children are positive for FRAb and have low CSF folate [11][12]. This is a priori proof that FRα is the primary transporter of folate into the brain under physiologic folate status.

    2. Folate Requirements during Pregnancy

    Since the discovery of its role in megaloblastic anemia and spina bifida, folate supplementation during pregnancy and fortification of food products have become two of the most globally accepted methods of treating and preventing folate deficiencyok. The basic folate requirement increases 75 to 100% (approximately 300–400 μg per day) in pregnancy because folate has a critical role in the growth and development of the embryo/fetus, especially during early stages of development [13]. It is, therefore, common practice to recommend that women supplement their diet with folate before conception and throughout pregnancy. The prevention of folate deficiency during pregnancy is achieved by consumption of at least 0.4 mg/day of folic acid during the first trimester of pregnancy [14][15]. In light of the recently discovered FRAb that can block folate transport, women positive for these antibodies may need additional supplementation with folinic acid to provide adequate folate to the developing fetus [16][17].

    3. Folate and Fetal Brain Development

    The importance of folate during embryonic and fetal brain development has been demonstrated in genetic animal models and dietary manipulations of folate deficiency [18][19]. If either folate transport or folate concentration in circulation is adversely manipulated, embryonic and fetal development is significantly altered. Mouse knockout models of genes such as FOLR1 that encode for folate receptor alpha (FRα) produce lethality in litters along with orbito-facial abnormalities, congenital heart defects and/or neural tube defects [20]. In FOLR1 knockout mouse, these lethalities can be prevented with adequate folinic acid (N5-formyltetrahydrofolate, a reduced form of folate) supplementation. These dramatic results occur because folate transport is lacking in the KO mouse during the early stages of neurulation and in regions where abnormalities arise [21]. In rodent models, folate deficiency causes a decrease in progenitor cells and an increase in apoptosis, and this could lead to infertility or resorption of embryos or fetal malformations [22]. Behavioral deficits are seen in rat pups born to folate-deficient mothers [23] and on methyl donor deficient diet during pregnancy [24]. In a rat model of exposure to rat folate receptor antibodies during pregnancy, resorption of embryos and malformations of the cranio-facial region and the brain were reported [25]. When the antibodies were administered at lower doses, embryos were carried to term with normal appearing pups born. However, these pups showed severe behavioral deficits [23][26]. The behavioral phenotype can be rescued by treatment with folinic acid and dexamethasone prior to antibody exposure [27]. These studies provide strong evidence in support of the pathologic consequences of exposure to FRα antibodies and the protective role of folinic acid.

    4. Folate and Neonatal Brain Development

    After birth, it is crucial for the offspring to have an adequate amount of folate in their diet. Instead of rapid cell division as embryogenesis calls for, postnatal development requires folate for neural progenitor differentiation as well as proliferation [28]. It has yet to be fully elucidated what the detailed mechanisms of folate action are, but the folate deficiency produced in animal models during early postnatal development illustrates the importance of folate in preventing developmental and cognitive deficits [23][27]. Researchers have also reported changes in neuronal excitability and maintenance that arise with a decrease in brain folate in a rat model [29]. Others have reported an increase in p53 and signs of homocysteine accumulation in the neurons and astrocytes [30]. There was a long-term effect on locomotor function and cognition in these animals. Therefore, folate is necessary for maintenance of neuronal function, as well. Based on this, further investigations into the mechanisms of folate metabolism in neurons and support cells of the brain are necessary. Thus far, folate has been linked to neuronal repair and differentiation after injury, myelin formation and maintenance and neuronal plasticity [30][31][32]. Figure 1 provides a summary of the effects of folate deficiency on fetal and post-natal brain development and the consequent sequelae that contribute to neurologic deficits.
    Figure 1. Effects of folate deficiency on the fetus and on brain development. Multiple causes lead to systemic as well as fetal folate deficiency. Folate receptor autoantibodies can block folate transport to the fetus and to the fetal as well as neonatal brain. In addition to folate deficiency, immune-mediated inflammation can contribute to the pathology. This has multipronged effects on brain development and function.

    5. Folate Receptors: Expression and Function

    In humans, there are four genes that code for folate receptors (see Table 1). The most characterized of these receptors is folate receptor alpha (FRα). As extracellular receptors, FRα, FRβ, FRγ and FRδ function as transporters of folate across different target tissues [33][34]. FRα can also act as a transcription factor [33]. Other transporters of folate include the reduced folate carrier (RFC), which requires high local concentrations (micromolar) of biologically active reduced forms of folates, and the proton-coupled folate transporter (PCFT), which can only transport folates and folic acid under acidic conditions and is the primary transporter involved in folate absorption in the gut [35].
    Table 1. Summary of folate transporters.
    Protein Gene Chromosome GPI Anchor? Localization Cofactors? Refs.
    FRα FOLR1 11q13.3 Yes Liver, kidney, uterus, placenta, choroid plexus, retinal pigment epithelium LRP2 [33][35][36][37][38]
    FRβ FOLR2 11q13.4 Yes Placenta, spleen, bone marrow, thymus, macrophages NA [33][35][36][37][38][39]
    FRγ FOLR3 11q13.4 NA Secretory granules of neutrophil granulocytes NA [33][35][36][37][38][39]
    FRδ FOLR4 11q14 Yes Oocytes NA [33][35][36][37][38][39]
    RFC SLC19A1 21q22.3 No Liver, kidney, placenta, choroid plexus, intestinal tract Vitamin D, thiamine pyrophosphate [34][40]
    PCFT SLC46A1 17q11.2 No Liver, kidney, choroid plexus, placenta, intestinal epithelium, human tumors Proton gradient [34][40]

    6. FRα Role in Maternofetal Transport of Folate

    The high demand for folate during pregnancy requires homeostatic mechanisms to ensure that sufficient folate is provided to the fetus throughout development. As the most characterized receptor in the folate transporter family of proteins, the accepted mechanism of FRα-mediated transport is translocation/endocytosis of the holo receptor subsequent to folate binding [35]. FRα is expressed on all epithelial cells including the choroid plexus. It is highly expressed in the reproductive tissues including the placenta and the fetus. To determine the mechanism of folate transport in the placenta during pregnancy, Yasuda et al. [41] manipulated osmolarity, concentrations of phosphatidylinositol-specific phospholipase C inhibition and concentrations of 3H-folic acid in vitro culture of human placental brush border membrane vesicles and determined that FRα, RFC and PCFT could transport various forms of folate, but that approximately 60% of folate was binding to FRα. They also noted that the folate requirements of Wistar rats increased across gestation, and expression of the mRNA of the transporters increased as well.

    7. FRα Role in Folate Transport to the Brain

    FRα is accepted as the main transporter of folate into the brain. However, there have been limitations to studying how FRα transports folate across the blood–brain barrier. A potential mechanism of folate transport across the choroid plexus and into the brain has been described by Grapp et al. [42]. In their experiments using immortalized Z310n rat choroid plexus cells in culture and a mouse model, they determined that transport of folate required shuttling of folates via exosomes from the basolateral side of the choroid plexus to the brain parenchyma of the apical side. Alternative transporters such as RFC and PCFT may only play a role when there is a disruption of FRα expression and transport, and adequate folate concentration is made available locally at the receptor [43]. The shuttling across the epithelial lining of the choroid plexus is a mechanism presumed to be conserved in all tissues that express FRα [44].

    8. Folate Receptor Autoantibodies: Their Role in Disrupting Folate Transport

    In some conditions, there is disruption in folate utilization that is not related to a dietary deficiency but is most likely due to a disturbance in the folate’s transport due to genetic or metabolic abnormalities. An emerging culprit of folate transport disruption is folate receptor autoimmune disorder, where autoantibodies against the FRα can interfere with folate transport to the fetus; it has been associated with subfertility, difficulty in conceiving, miscarriage and neural tube defects in the fetus [16][17][45][46].
    In infants and young children, these antibodies can block folate transport to the brain. Approximately 70% of the children diagnosed with cerebral folate deficiency syndrome or autism spectrum disorder have low CSF folate and respond to folinic acid treatment [47][48]. The majority of the autoantibodies are of the IgG class and, therefore, can readily cross the placenta and affect the fetus. Two distinct types of antibodies have been identified. One binds to FRα at the active site where folate binds and, as a consequence, blocks folate binding (blocking Ab). Another type of antibody binds to an antigenic site not involved in folate binding (binding Ab) but can trigger an immune reaction and inflammation and render the receptor nonfunctional. In most cases, one or both types of antibodies are present [49][50]. Thus, functional blocking of folate transport and inflammation are an integral part of the pathology [44].

    9. Pathologic Consequences of Folate Receptor Antibodies

    The presence of folate receptor autoantibodies can disrupt the transport of folate, and the consequences of decreased folate uptake by cells can impact development of the fetus, especially the central nervous system. There is also a correlation of folate receptor antibodies with neural tube defect pregnancy [16]. In less severe cases, a subset of children born with exposure to maternal FRα autoantibodies in utero develop low-functioning autism with or without neurological deficits after birth. Recent studies show significant association of folate receptor autoantibodies with autism spectrum disorder in children [11][51][52].

    10. Diagnosis of Folate Receptor Autoimmune Disorder

    Early indications of cerebral folate deficiency that are potentially due to maternal folate deficiency or folate receptor autoantibodies can be deduced by measuring serum folate and homocysteine and folate receptor autoantibodies in the mother during pregnancy. Other than dietary folate deficiency, folate receptor autoantibodies in the pregnant mother can contribute to fetal folate deficiency. In the latter case, blocking of folate transport across the placenta and antibody-mediated inflammation could contribute to the pathology, as shown in the rat model of exposure to rat folate receptor antibodies during pregnancy [26][27]. In infants, the presence of folate receptor autoantibodies in the blood could provide a mechanism by which folate transport to the brain via the choroid plexus could be blocked, thus leading to cerebral folate deficiency [51][52]. Therefore, determining the presence of folate receptor autoantibodies in the blood of pregnant mothers and children becomes a necessary test to prove or rule out folate receptor autoimmune disorder.
    Methodology for determination of the antibody titer in serum is well-established. Two distinct types of IgG and/or IgM antibodies have been described [50]. These antibodies can be blocking and/or binding antibodies. Both types of antibodies are capable of triggering an immune reaction due to antigen/antibody interaction, leading to local inflammation, and this could interfere with folate transport via the FR protein. Both types of assays can be performed in a laboratory setting as described below.

    11. Assay for Blocking Antibodies

    Blocking autoantibodies to FRα are determined using a functional binding radio assay. Patient’s serum (200 μL) is acidified with 300 μL 0.1 M glycine/HCl pH 2.5/0.5% Triton X-100/10 mM EDTA and added to 12.5 mg charcoal pellets in a separate tube (250 μL of 5% charcoal/1% dextran in 0.1 M Na PO4 pH 7.4/0.5% Triton X-100/10 mM EDTA, spun down and supernatant-aspirated) to remove any endogenous folate, and the pH of the supernatant fluid is neutralized with 40 μL of 1 M dibasic NaPO4 prior to using it in the assay. This assay is performed by adding purified apo human FRα protein (40 ng) to the processed serum and incubating overnight at 4 °C. The next day, 3H-folic acid (700 pg) (Moravek) is added and incubated for 20 min at room temperature. Unbound 3H folic acid is removed with dextran-coated charcoal (200 μL) and the 3H folate bound to FRα determined by counting the sample in a liquid scintillation counter. The reduction in binding of 3H-folic acid to the apo human FRα when compared to the negative control serum sample provides a measure of the blocking autoantibody present in the sample [50]. Blocking antibody can be IgG or IgM; the values are expressed as pico moles of 3HPGA blocked per ml serum, and the titer can range from >0.2 to 0.5 (low titer), >0.5 to 1.0 (medium titer) or >1.0 (high titer).

    12. Assay for Binding Antibody

    Binding of the IgG autoantibody to folate receptor alpha (FRα) is determined by an ELISA-based method. FRα (1 ug in 100 μL) purified from human milk is added to each well of an ELISA plate to covalently bind the protein to maleic anhydride-coated wells (Thermo Fisher, Waltham, MA, USA). Following blocking of additional sites by treatment with normal goat serum (200 μL) overnight to prevent non-specific binding to the wells, serum samples (4 and 8 μL) (negative control, positive control and patient samples) are added to wells along with 100 μL fresh goat serum and incubated at 4 °C overnight to facilitate binding of autoantibodies to the FRα in the wells. Following washing of the wells to remove unbound proteins, the specific IgG autoantibody bound in each well is detected by incubating with a peroxidase-conjugated, anti-human IgG secondary antibody (1:6000 dilution) (Vector Labs) for 1 h at room temperature. After washing to remove the unbound secondary antibody, the bound peroxidase-conjugated secondary antibody is determined by incubation with ultra TMB (Thermo Fisher) for 10 min. The resultant blue colored reaction is converted to yellow with 100 μL of 1.0 M HCl, and then absorbance is read at 450 nm in an ELISA plate reader. In a second set of wells, known amounts of human IgG captured in protein A-coated plates are used to construct a standard curve [50]. Values are expressed as pico moles of IgG antibody per ml serum and can range from >0.1 to 0.5 (low titer); >0.5 to 2.0 (medium titer) and >2.0 (high titer).
    Among other criteria, specific diagnosis of folate receptor autoimmune disorder is confirmed using the above tests. After correcting for background, for the blocking antibody, values of 0.2 pmoles or greater are considered positive and for the binding antibody, 0.1 pmoles or greater are considered positive. Because folate receptor alpha is a peripheral membrane protein, the antibody titer measured in the serum should be considered as excess antibody appearing in the circulation after saturating the membrane-bound antigen. Fluctuations in antibody titer have been reported in the same individual over time and can range from low to medium titer or to undetectable levels. While the reason for these changes in antibody titer are not identified, it is likely that changes in FR antigen on cells, exposure to milk FR antigen in the gut and the specific B-cell population may be contributory factors.

    The entry is from 10.3390/jpm11080710


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