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Background

Retinoic acid receptors (RARs) are a class of transcription factor proteins which are activated primarily by the ligand Retinoic acid (RA). RARs are present in all chordates and are highly conserved genes. RAR proteins are localized to the nucleus of the cell, where in the absence of a ligand particle it will form a heterodimer with Retinoic X Receptor(RXR) proteins. In this heterodiamer complex the RAR/RXR proteins are inactive and do not play a role in stimulating transcription. After the heterodimer has dissociated via the binding of the ligand to the allosteric site of the RAR/RXR complex, bound RAR proteins will act as transcription factors on target gene sequences called retinoic acid response elements or RAREs. RARs are activated by RA in both of it's naturally occurring forms all-trans retinoic acid and 9-cis retinoic acid. There are three related proteins that act as RARs, these are called RARa, RARb, and RARg. RA is a compound that, once bound to a RAR, has transcriptional control of genes which effect the patterning and development of the embryo through transcriptional control of signaling cascades. RARs are shown to be in close association with many patterning genes such as Hox genes, and Fibroblast growth factor. RARs are responsible for the proliferation and differentiation of cells that give rise to the neural crest.

RA is only beneficial to the embryo in a very small range of concentrations, cells respond to very low and very localized concentrations of RA. This complicates any issues that could arise that deal with RA concentrations.

Activation of Retinoic Acid Receptors

The RA will diffuse freely across cell membranes to target cells that need RA. Once in the cytoplasm of target cells, CRABPs will bind the RA and carry it into the nucleus. In the nucleus, retinoic acid will bind to the heterodimer that is attached to the RARE gene domain on the DNA. The heterodimer consists of RAR and RXR. The RAR binds both all-trans Retinoic acid and 9-cis Retinoic acid but RXR only binds with 9-cis Retinoic Acid. Without the RA, DNA bound RXR/RAR represses transcription by recruiting the corepressors NCOR1, NCOR2, and histone deacetylase. When bound to retinoic acid, RARs act by becoming either transcriptional repressors or activators. These complexes of receptor and ligand are able to move into the nucleus. RAR’s transcription factor activity initiates a cascade of events through hox genes (including Hox1, Hox2, Hox14) that regulate transcription of many key genes needed for proper nervous system and limb morphogenesis.

Biosynthesis & Mechanism

Retinoic Acid Receptor Pathway

Retinol (vitamin A), the precursor of RA, is known to be stored in its esterified form in the liver as complexes with Retinol binding protein or (RPB). When released from the liver into the bloodstream the RPB carries the retinol to the desired tissue. Embryos receive retinol via the placenta, therefor an embryos level of retinol depends directly upon the mothers. Retinol is oxidized into RA using a 2-step oxidation reaction with retinaldehyde as its intermediate. This synthesis pathway has high levels of functional redundancy, meaning that a single mutation will be unable to disrupt the pathway. RDH10, ADH1,5,7, and CYP1B1 are all capable of oxidizing retinol into retinaldehyde, this first step is reversible and serves as a mechanism for controlling the ratio of retinol to retinaldehyde. Retinaldehyde is then converted into RA by an irreversible reaction catalyzed by RALDH 1,2,3 and CY1B1. RA is then converted from all-trans retinoic acid to 9-cis retinoic acid. During embryonic development, retinaldehyde metabolic enzymes are expressed in selective tissues only, which establish the crucial gradients of RA in the embryo that eventually contribute to the formation of the neural crest and all of its derivatives. The control of location of this morphogen is controlled specifically by localization of the RA synthesis enzymes. These RALDH 1,2,3 proteins are localized at specific locations in the embryo over time. This spacial-temporal control of RA synthesis is what makes RA such an effective morphogenic compound.

Fetal Alcohol Syndrome and Retinoic Acid Receptor

Fetal alcohol syndrome

Fetal alcohol syndrome(FAS) is a teratogenic disorder caused by high levels of ethanol present in the developing embryo. Its main symptoms are that of microcephaly, learning disabilities, craniofacial malformations, cleft palate and a number of heart defects. Many of these symptoms are the result of deficiencies in signaling the neural crest to differentiate properly. One mechanism for the inhibition of developmental signalling by ethanol is that of effectively blocking the synthesis of RA through substrate interference. Some of the proteins capable of completing the synthesis of retinaldehyde from retinol (ADH 1,5,7) are also able to react with ethanol, converting ethanol to acetaldehyde. Other than the inherent toxic effects of ethanol on the embryo, the activity used by proteins ADH 1,5,7 to convert ethanol to acetaldehyde effectively decreases the amount of retinaldehyde available to the embryo for RA synthesis. This mechanism is supported by the fact that FAS has many symptoms in common with retinol (vitamin A) deficiency. Ethanol does not however interfere with the synthesis of RA directly from retinaldehyde, as this is carried out specifically by RALDH 1,2,3. Ethanol also has multiple degenerative effects on neural cells, acting through NMDA receptors, ethanol is capable of overstimulating the neural tissue into distress and eventually death.

Ethanol is not the only cause behind FAS symptoms; malnutrition can increase the chances of FAS diagnosis by approximately 15-fold. A study showed that in New York City, 40% of infants born to lower socioeconomic status mothers were diagnosed with FAS compared to 2.7% of infants born to middle class alcoholics. This suggests that the socioeconomic status of the mother affects the nutrition level within the placenta, which can ultimately have devastating effects depending on the well-being of the mother.

Ventricular Septal Defects (VSD) often have overlap with FAS, 29%-50% of cases of FAS also include VSD.

Treatments of FAS

Current treatment methods rely mostly on pharmacotherapy, using chemicals such as antioxidants, growth factors, nicotinamide, and various retinoids to restore the balance of developmental factors during growth in the embryo. Biopharmaceutical companies such as Tocris commercially sell agonists of the RA receptor. Postnatally, choline supplementation and cholinesterase inhibitors are used to lessen the effects of FAS. A recent study by Ballard et. al has proposed using a chemical treatment consisting of Vitamin A, folate, and choline to lessen the behavioural and physical characteristics of FAS.

However, retinoid derivative supplements can be a highly risky choice due to the fact that even the slightest deficiency of supplementation in active RA receptors can offset a sensitive balance of morphogens and gene expression needed for proper cell differentiation and growth. The cell specific expression of RALDH 1,2,3 must be maintained in order to allow for the establishment of the retinoic acid gradients. As an alternative, the concentration of alcohol and aldehyde dehydrogenases can be up-regulated through various types of gene manipulation. By having a sufficient amount of enzymes available for oxidation of alcohols, the competition between ethanol and retinol can be eliminated, allowing for proper development of the nervous system and limbs of the embryo all the while successfully metabolizing ethanol.

Therapeutic Gene Modulation

Up-regulating the production of endogenous enzymes through increasing transcription of dehydrogenase genes through injecting transcription factors (activators) into the embryo. For example, transcription factor AP-1 is involved in promoting the expression of ADH7. Increasing the concentration AP-1 in a certain tissue can increase the chances of transcription of ADH7 in that specific gene.

A potential treatment could be gene modulation of ADHs 1,5,7. Experimental determination of a specific agonist for the transcriptional activators of the ADH genes could be used to isolate an agonistic compound that is useful for modulation of ADH genes. This agonist could be introduced to both embryo and mother via injection into the placenta along with additional daily vitamin A oral supplement in non-toxic amounts (around 8,000 IU per day). This would hopefully result in the upregulation of trascription of the ADH 1,5,7 genes and result in a higher number of enzymatically active ADHs in the cytosol of the embryonic cells. This altered concentration would be potentially more able to deal with the high amount of ethanol in the cell, and could lead to levels of retinaldehyde available for the RALDHs to convert into RA. This method would not interfere with the specific localization of RA and the RAR proteins would be activated in the right place at the right time. To determine if this methodology could have positive results, a comparison of ADH 1,5,7 gene TF agonist injected embryos to those only injected with saline could be used to look at the number of ADH 1,5,7 proteins present in the cells using an antibody against the ADHs to visualize them microscopically. An antibody test could also be done on the expression of RARE proteins to determine the localization and quantity of RAR gene products. It would be expected to see more normal RA patterning and expression in those treated vs untreated.

Due to the nature of FAS, this RAR specific treatment should also be implemented with simultaneous supplementation to the mother with other prenatal vitamins and minerals such as folic acid.

If treatment was successful we would expect to see correct patterning in the developed offspring.

Gene Therapy

Gene therapy treatment for FAS

Introducing more copies of the genes that code for enzymes involved in oxidation of alcohols and aldehydes (ADH, RDH, ALDH, RADH) through the use of non-pathogenic viral vectors. These viral vectors can enter a host cell, insert the desired gene into the host’s genome, and continue to non-pathologically infect more cells under the conditions of stable viral transfection. There are two approaches to this method: in vivo and ex vivo. The in vivo approach is delivering the vector straight into the patient, whereas the ex vivo approach is transfecting an extracted tissue sample from the patient, letting it grow in vitro, and re-inserting the tissue into the patient. This treatment could eventually increase the amount of enzymes present in the body of the embryo.

A successful case of ex vivo gene therapy occurred in 2009 with two boys who were diagnosed with adrenoleukodystrophy (ALD), a brain disease. ALD is caused by the body’s lacked of ability to endogenously produce the protein ALD. The effects of the disease are fatal, resulting in paralysis, deafness, and eventually, death. Gene therapy was used as an alternative to bone marrow transplant since they couldn’t find any donors. Their innate bone marrow had to be wiped with chemotherapy, and cells treated with lentiviral vectors were implanted into their bodies. They were eventually able to produce ALD endogenously. Although they weren’t fully recovered from the effects of ALD, they showed signs of ceased degression.

In the case of treating FAS prenatally, the ex vivo gene therapy approach can be taken. A few cells from the embryo (which won’t affect the embryo’s development in their absence) can be extracted, and the cells grown in tissue culture in vitro. A modified, non-pathogenic lentivirus can be genetically modified so that it holds in its genome the gene that codes for several ADH genes along with their various enhancers. An appropriate dosage of the virus will be applied to the growing cells for transfection. Upon confirmation that the cells has been successfully infected by the virus, they can be implanted back into the embryo.

The specific mechanisms of gene therapy is not well-controlled enough at this point in time to make gene therapy an effective method of treating hereditary and somatic diseases. In the future, when more advanced technology is available and gene therapy becomes more effective, it has the potential to be a frequently-used treatment option for illnesses.

Adverse Effects

The possible side effects of gene therapy and therapeutic gene modulation are over-expression of desired proteins in undesired tissues at undesired times, which could possibly disrupt the regulation of other necessary metabolic pathways. However, in most cases of gene expression manipulation therapies, the treatments are not effective enough to show drastic change in patients.

In the case of ex vivo gene therapy, if the body cannot readily accept the non-pathogenic virus infected tissue, the side effects can be similar to the side effects of a bad organ transplant. The immune system could have difficulty recognizing the "new" tissue implanted in the body and attack its endogenous organs.

There have been several deaths that have occurred as an aftermath of gene therapy, but these deaths were not well-investigated nor well-understood to deem gene therapy failure as the cause of death.

Despite the advances in sciences and technology that allow us to be creative with preventative solutions to the disease, the most effective way to prevent FAS is to completely avoid consumption of ethanol during pregnancy.