To examine the effect of potential drugs on the splicing of the IKAP mRNA we used three FD cell lines derived from three FD patients (termed FDA, FDB and FDD). In addition, a cell line derived from a parent of an FD patient, and therefore heterozygous for the FD mutation, and four matched cell lines derived from healthy individuals were used as controls. Analysis of the splicing pattern of exon 20 was performed by RT-PCR using primers to the endogenous IKAP mRNA. reveals that exon 20 was constitutively spliced in the control cell lines (lanes 1–4); 96% inclusion was observed in the heterozygous cells (lane 5), and from 38% to 52% inclusion was observed in the three FD cell lines (lanes 6–8). also demonstrates that IKAP mRNA was either expressed at higher levels or was considerably more stable in the control compared to the FD-derived cell lines. The levels of the wild-type and mutant isoforms were further quantified by real-time quantitative PCR (QPCR) analysis (). FD cells expressed considerably lower amounts of wild-type IKAP mRNA compared to control cells (, left side). The heterozygous cell line expressed 2.5-fold less wild-type IKAP mRNA than did control cells. FD cells, FDA, FDB and FDD, expressed 5.7-, 7.2- and 3.8-fold less, respectively, wild-type IKAP mRNA than did control cells. As shown in , right side, the skipped IKAP isoform was expressed at different levels in the FD cell lines compared to the heterozygous cell line. No skipped isoform was detected in control cells (data not shown).
In order to characterize the IKAP protein levels, a western blot analysis was conducted (). The control cell lines and the heterozygous cell line expressed the wild-type IKAP protein at the expected size of 150 kDa at similar levels (1.2 less IKAP protein in heterozygous cells). However, despite the presence of both wild-type and mutant mRNA isoforms as shown by RT-PCR, in FD cells only a very faint 150-kDa band was observed. Between 4 and 5 fold less IKAP protein was observed in FD cells than in control cells. No product of the size expected for the truncated protein, 79 kDa, was detected, in agreement with previous reports 
FD is characterized by dysfunction of the autonomic and sensory nervous system resulting from incomplete neuronal development and progressive neuronal degeneration 
. We therefore sought substances that would affect neuronal function and would be safe for immediate testing in FD patients. We first examined two food supplements, one based on choline and one based on serine. The choline-based substance had little effect on the level of IKAP
mRNA. However, the serine-based substance, phosphatidylserine (produced by Enzymotec under the Sharp•PS® brand), significantly increased IKAP
mRNA and IKAP protein levels in cells derived from FD patients. PS, a nutritional supplement, is a major component of every living cell, especially neuronal cells 
, and slows cognitive degeneration in human subjects 
. We found that in FD cell lines PS significantly raises IKAP
mRNA and protein levels. PS, shown in , is used worldwide and is considered by the FDA as a safe and lawful dietary supplement. PS was added to FD cells at concentrations ranging from 0–300 µg/ml. The cells were harvested and RNA was extracted 24 and 48 hr following the addition of PS for each cell line; IKAP
mRNA was analyzed by RT-PCR analysis and QPCR. The highest effect of PS on IKAP
mRNA levels was obtained at different time points for each cell line. After 24 hr of treatment the best results were obtained for cell line FDA; we observed about a 5-fold increase in the amount of wild-type IKAP
mRNA after treatment with 5, 10 or 100 µg/ml PS compared to levels in FDA cells treated with the solvent only (). At 200 µg/ml PS, the increase was 1.5 fold, and at 300 µg/ml a 1.2-fold increase in wild-type IKAP
mRNA levels was observed. A possible explanation for lower efficacy at higher PS concentration is the toxicity of the solvent at high volumes, although the solvent itself did not affect IKAP
mRNA levels (data not shown). After 48 hr of treatment, best results were obtained for cell line FDB; IKAP
mRNA levels peaked at about 3-fold higher than levels in untreated cells at 100 and 200 µg/ml PS (). PS did not affect the ratio of the included to skipped mRNA isoforms but rather elevated the total amount of both isoforms. In FDD cells, PS had variable effects on IKAP
mRNA (Figure S1
). In the heterozygous cell line, PS treatment induced a slight increase in IKAP
mRNA (data not shown). These results indicate that the effect of PS on the level of IKAP
mRNA differed among FD cell lines.
PS raises IKAP mRNA levels in FD cell lines.
In clinical studies, PS efficacy increased as treatment was prolonged 
. We thus examined the effect of PS on the level of IKAP
mRNA in FDB cells after 3, 7 and 14 days of treatment. The level of IKAP
mRNA was evaluated by QPCR () and RT-PCR (). As shown in , PS treatment increased the level of IKAP
mRNA by 1.5 fold at 3 days, 1.7 fold at 7 days, and 2.5 fold after 14 days compared to levels in untreated cells. Proteins were harvested two weeks after the addition of the supplement and IKAP levels were analyzed by western blot. The addition of PS increased the amount of IKAP protein by 2.3 fold in FD cells treated with PS relative to untreated FD cells (). It should be noted that the antibody used in this western blot (from BD Bioscience) can detect only the wild-type IKAP protein. The antibody used in to detect the level of IKAP protein in the different cell lines (from Santa Cruz Biotechnolgy) can theoretically detect both the wild-type and truncated IKAP protein.
PS raises IKAP mRNA and protein levels following long-term treatment.
In order to test whether expression of genes in addition to IKAP was altered as a result of PS treatment, we performed a human gene expression microarray analysis (Human Gene 1.0, Affymetrix) of cDNA samples from FDB cells treated with 100 µg/ml PS. Using SAM (Significance Analysis of Microarrays) analysis, we identified 877 genes with significantly different levels of expression following PS treatment: 441 genes were up-regulated and 436 genes were down-regulated. These genes are listed in Figure S2
of the Supplementary Material. We confirmed the effect of PS on six significantly up-regulated genes (YWHAH
) and four significantly down-regulated genes (RCAN2
, and ITGB8
) using QPCR (). All values were normalized to levels of LZIC
, which were unchanged by PS treatment. A gene ontology (GO) enrichment analysis of these genes was performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) 
. A significant GO enrichment was observed for the up-regulated genes coding for proteins involved in regulation of the cell cycle and DNA metabolic processes (). Complete tables are presented in Figure S3
. As a group, the down-regulated genes did not present highly significant GO enrichment; most of them function in developmental processes (data not shown). Using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways database in DAVID we also observed a significant enrichment for genes involved in the signaling pathways for pyrmidine and purine metabolism, as well as for genes known to be involved in base excision repair ().
Validation of gene expression microarray analysis by QPCR.
GO analysis for up-regulated genes.
Signaling pathways enrichment of genes up-regulated by PS treatment.
Due to the fact that a significant number of genes up-regulated by PS treatment are involved in cell cycle regulation, we tested the effect of PS on the cell cycle distribution of FD cells using propidium iodide (PI) staining and flow cytometry. Cell cycle analysis of untreated FD cells revealed that a significantly higher fraction of the cells were in the G1 stage compared to control or heterozygous cells (). A lower fraction of the FD cells were in the S+G2 stages, 1.5-fold less than in the control cells, indicating that a low number of FD cells are in the dividing state (). Treatment of FD cells with PS significantly raised the fraction of cells in S+G2 stages by 1.5 fold compared to untreated FDA cells () and 1.7 fold compared to untreated FDB cells (). These results indicate that PS releases FD cells from cell cycle arrest.
PS alters the cell cycle distribution of FD cells.
Since PS treatment increased the amount of the wild-type IKAP
mRNA and protein present in FD cells, we obtained three additional food supplements and tested their effect on splicing. The first is produced under the name Sharp·PS GOLD4508P and is a proprietary conjugate of PS and docosahexaenoic acid (DHA), an omega-3 fatty acid. Sharp·PS GOLD resembles the functional form of natural (brain) PS, increases DHA availability in the brain, and acts to increase cognitive abilities 
. The second substance tested was L-α-glycerophosphorylcholine (GPC), a dietary supplement reported to improve mental performance that is marketed as SharpGPC 85F. GPC serves as a precursor for reconstituting a nerve cell membrane component. Clinical trials over the past two decades have demonstrated that treatment of subjects with adult-onset dementia disorders with GPC at 1,000–1,200 mg per day protects against cognitive impairment characteristics of dementia disorders 
. Third, we tested Krill oil+4225F, a proprietary complex of marine-derived DHA and eicosapentanoic acid (EPA), delivered as triglycerides or attached to phospholipids. Krill oil+4224F also contains a significant amount of astaxanthin. A similar combination improves blood-lipid markers, including LDL, HDL and triglycerides, and in clinical studies, Krill oil has greater potency than omega-3, due to its unique structure and composition 
FD cell lines were treated with these three formulations, and the effects on the splicing of IKAP mRNA were analyzed by RT-PCR and QPCR (). At 5 µg/ml, the Sharp·PS GOLD supplement increased the level of IKAP mRNA level 4.2 fold compared to levels in untreated cells (). Sharp·PS GOLD increased the level of the wild-type IKAP mRNA at one twentieth the effective concentration of PS. Treatment with GPC did not significantly increase the amount of wild-type IKAP mRNA (). At 500 µg/ml, Krill oil increased levels of the wild-type IKAP mRNA significantly ().
Other supplements also increase IKAP mRNA levels in an FD cell line.
To evaluate PS with respect to other substances already tested for FD treatment, we treated the FD cells with kinetin and tocotrienol. Kinetin is a plant cytokinin, which was reported to rescue mRNA splicing of IKAP ex vivo 
and in patients heterozygous for the FD mutation 
. A significant increase in wild-type IKAP
mRNA levels and a shift towards a higher level of exon 20 inclusion was observed with an optimal concentration of 100 µM of kinetin (), with no toxicity observed. In contrast, tocotrienol, a member of the vitamin E family that was reported to induce IKAP expression in FD cells 
, did not affect IKAP
mRNA expression in our system (); this observation is similar to that of another publication 
. These results indicate that the FD cells used in this study are sensitive to kinetin treatment but not to tocotrienol.
The effect of substances already tested in FD models or patients on IKAP mRNA levels.