Abstract

Background

   The most common gene responsible for autosomal recessive retinitis
   pigmentosa (RP) is EYS. The manner of decay of genetically defective
   EYS gene transcripts varies depending on the type of mutation using our
   cellular model, which consists of induced photoreceptor-directed
   fibroblasts from EYS-RP patients (EYS-RP cells). However,
   disease-specific profiles have not been clarified in EYS-RP cells.
   Herein we investigated comprehensive gene expression patterns and
   restoration of altered expression by low molecular weight molecules in
   EYS-RP cells.

Methods

   Using induced photoreceptor-like cells by CRX, RAX, NeuroD, and OTX2,
   we employed qRT-PCR and DNA microarray analysis to compare expression
   levels of disease-related genes in EYS-RP cells. We investigated the
   effect of antiapoptotic or anti-endoplasmic reticulum
   (ER) stress/antioxidant reagents on the restoration of altered gene
   expression.

Results

   Expression levels of phototransduction-related genes (blue opsin,
   rhodopsin, S-antigen, GNAT1, GNAT2) were lower in EYS-RP cells. CRYGD
   was extracted by global gene expression analysis, as a downregulated,
   retina-related and apoptosis-, endoplasmic reticulum (ER) stress- or
   aging-related gene. Pathway enrichment analysis suggested that
   “complement and coagulation cascades,” “ECM-receptor interaction” and
   “PI3K-Akt signaling pathway” could be involved in EYS-RP-associated
   pathogenesis. Among the matching/overlapping genes involved in those
   pathways, F2R was suggested as an EYS-RP-associated gene. The
   downregulation of CRYGD and F2R was completely restored by additional
   4-PBA, an inhibitor of ER stress, and partially restored by metformin
   or NAC. In addition, 4-PBA normalized the expression level of cleaved
   caspase-3.

Conclusions

   Our cellular model may reflect the ER stress-mediated degenerative
   retina and serve as a pathogenesis-oriented cost-effective rescue
   strategy for RP patients.

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s13287-022-02827-x.

   Keywords: Redirect differentiation, Dermal fibroblast, Photoreceptor,
   Disease modeling, Retinitis pigmentosa, EYS, Semiquantitative analysis,
   CRYGD, F2R, ER stress, 4-PBA

Background

   Retinitis pigmentosa (RP) is an inherited retinal dystrophy. Defects in
   the EYS gene on chromosome 6q12 are a major cause of autosomal
   recessive (ar) RP [[37]1–[38]4]. In Japan, defects in the EYS gene such
   as c.4957dupA (p.Ser1653Lysfs*2) and c.8805C>A (p.Tyr2935*) have been
   identified as pathogenic mutations in about 20–30% of arRP patients
   [[39]5–[40]9]. Hereafter, arRP caused by defects in the EYS gene will
   be referred to as “EYS-RP.” EYS-RP is characterized as late onset and
   progressive in severity, leading to visual loss. Therefore, it is an
   urgent global issue to develop a strategy to delay progressive retinal
   dystrophy. Although gene therapy has been reported to be safe and
   effective for one type of inherited retinal dystrophy [[41]10–[42]13],
   and several drugs are promising [[43]14–[44]17], no established
   treatments are available for EYS-RP to date.

   Because RP patients show heterogenous phenotypes due to heterogeneous
   gene defects, it may be ideal to determine inhibitory strategies
   depending on the specific pathogenesis. For this purpose, cellular
   models are available in place of human retinas. Induced photoreceptor
   cells derived from iPSCs of RP patients with defects of genes other
   than the EYS gene were reported to reproduce pathogenic phenotypes
   [[45]18, [46]19], indicating that the number of rod photoreceptor cells
   was decreased and that endoplasmic reticulum (ER) stress might be
   involved. Although methods to generate photoreceptors from iPSCs have
   been established [[47]20, [48]21], they are expensive and
   time-consuming. Another method, transduction of fate-determining
   transcription factors [[49]22–[50]25], which is called “redirect
   differentiation,” can produce photosensitive photoreceptor-like cells
   from somatic cells. Using “redirect differentiation,” we produced
   photoreceptor-like cells from dermal fibroblasts of EYS-RP patients
   with homozygous or heterozygous mutations, as a replacement for the
   degenerative retinas from EYS-RP patients [[51]26]. In that study, we
   demonstrated that the manners of decay of the EYS gene transcripts
   varied, depending on type of defect in the EYS gene. It was also
   suggested that defects in the EYS gene might be relevant for cell
   growth rates and that the cell growth rate may be determined mainly by
   donor age. Therefore, findings in the EYS-RP-derived cells should be
   compared with those in age-matched control-derived cells for accurate
   assessment of the characteristics of EYS-RP-derived photoreceptor-like
   cells.

   In the present study, we compared expression levels of
   phototransduction-related genes (blue opsin, rhodopsin, S-antigen,
   guanine nucleotide-binding protein G(t) subunit alpha-1 and 2 (GNAT1,
   GNAT2)) by qRT-PCR and gene expression profiles by microarray in
   photoreceptor-like cells from EYS-RP patients with those from
   age-matched normal volunteers. EYS-RP patients showed decreased levels
   of phototransduction-related genes, consistent with in vivo models
   reported previously [[52]27]. By global gene expression profiling,
   CRYGD and F2Y were downregulated genes in EYS-RP-derived cells. We
   further investigated whether four drugs, 4-phenyl butyric acid (4-PBA),
   rapamycin, N-acetyl-L-cysteine (NAC), and metformin, could restore
   downregulation of these genes. Rapamycin, the inhibitor of mammalian
   target of rapamycin [[53]28] (mTOR), has been suggested to inhibit
   photoreceptor degeneration in vivo [[54]29, [55]30] and in vitro
   [[56]31]. A well-known chemical chaperone, 4-PBA, is an FDA approved
   drug for urea cycle disorders owing to its effectiveness in reducing
   endoplasmic reticulum (ER) stress [[57]32] and was reported to prevent
   photoreceptor degeneration [[58]14]. The anti-inflammatory drug
   metformin had shown neuroprotective effect in rd1 mice [[59]17]. The
   antioxidant drug NAC suppressed photoreceptor death in rd10 mice
   [[60]15]. The present study showed that downregulation of
   phototransduction-related genes, CRYGD and F2R, in EYS-RP-derived cells
   was completely restored by 4-PBA, partially restored by metformin or
   NAC, but not by Rapamycin.

Methods

Isolation and culture of dermal fibroblasts

   Dermal fibroblasts were harvested from three healthy donors [N#1
   (53 years old), N#2 (48 years old), N#3 (63 years old)] and two EYS-RP
   patients (Pt#1, Pt#2, Table [61]1) under the approval of the Ethics
   Committee of the National Rehabilitation Center for Persons with
   Disabilities (NRCD). These five donors were studied in our previous
   paper [[62]26]. Signed informed consent was obtained from the donors,
   and samples were de-identified. All experiments involving human cells
   and tissues were performed in line with the Declaration of Helsinki.
   Establishment of fibroblast culture derived from those five donors was
   performed as previously reported by us [[63]26]. Seventh or
   eighth-passaged cells were used for following photoreceptor-directed
   differentiation.

Table 1.

   Defects in the EYS gene in patients for this study
   Patient # ID Sex Age (year) Allele 1 Allele 2
   Mutation Effect Mutation Effect
   1 RP38 F 67 c.4957dupA p.Ser1653Lysfs*2 c.4957dupA p.Ser1653Lysfs*2
   2 RP174 M 58 c.4957dupA p.Ser1653Lysfs*2 c.8805C>A p.Tyr2935*
   [64]Open in a new tab

Induction of photoreceptor-like cells (photoreceptor-directed fibroblasts)

   Induction experiments were performed as previously reported [[65]23,
   [66]24, [67]26]. In brief, full-length transcription factors, CRX, RAX,
   NeuroD and OTX2, were amplified from cDNAs prepared from total RNA of
   adult human retina (Clontech, CA, USA) by PCR and cloned into the
   XmnI-EcoRV sites of pENTR11 (Invitrogen). Preparation and infection
   with recombinant retrovirus were performed as previously reported
   [[68]23]. In brief, the resulting pENTR11-transcription factors were
   recombined with pMXs-DEST by use of LR recombination reactions as
   instructed by the manufacturer (Invitrogen). The retroviral DNAs were
   then transduced into 293FT cells, and 3 days later the media were
   collected and concentrated. The human dermal fibroblasts were infected
   with this media containing retroviral vector particles as a mixture of
   the four transcription factors. After the retroviral infection, the
   media were replaced with the differentiation media, DMEM/F12/B27 medium
   supplemented with 40 ng/ml bFGF, 20 ng/ml EGF, fibronectin and 1% FBS.
   The retrovirus-infected cells were cultured for one to six weeks in
   6-well or 24-well laminin-coated culture dishes. Expression of the four
   transgenes (CRX, RAX, NeuroD and OTX2 (referred to as CRNO)) was
   confirmed by endpoint RT-PCR, and CRX was confirmed by
   immunocytochemistry in all photoreceptor-directed fibroblasts tested.

Global gene expression analysis

   To compare the gene expression profiles in photoreceptor-directed
   fibroblasts derived from EYS-RP patients with those from normal
   volunteers, we analyzed the expression levels of 58,201 probes in the
   induced/non-induced photoreceptor-like cells with or without EYS genes
   defects using the SurePrint G3 Human Gene Expression Microarray
   8 × 60 K Ver.2.0 (Agilent Technologies, Palo Alto) using total RNA
   extracted from the cells. To average experimental variations, extracted
   total RNA samples were pooled into one tube from three independent
   induction experiments, and pooled samples were subjected to microarray
   analyses. To normalize the variations in staining intensity among
   chips, the 75th percentile of intensity distribution was aligned across
   arrays using GeneSpring software version 12.5 (Agilent). We first
   compared expression profiles for all the probes and then for refined
   probes based on GO terms: retina-related and apoptosis, oxidative
   stress, ER stress or aging (GO (retina) and GO (cell death),
   respectively). We extracted the intersection of two groups of genes,
   i.e., up- or downregulated and GO (retina) or GO (cell death)-related
   genes. For pathway enrichment analysis, WebGestalt2017
   ([69]http://www.webgestalt.org/) with KEGG pathway (Kyoto Encyclopedia
   of Genes and Genomes pathway) as a database was used.

Drug administration

   The dermal fibroblasts obtained from EYS-RP patients (Pt#1, Pt#2) and
   age-matched healthy individual (N#3, N#1) were seeded in laminin-coated
   (Biolamina, 521) 24-well plates. The fibroblasts were transduced with
   mixture of CRNO as described in the “Induction of photoreceptor-like
   cells (photoreceptor-directed fibroblasts)” section. After 5–6 h of
   retroviral transduction, the media in transduced fibroblast cells were
   replaced with differentiation media containing either one of the four
   drugs (rapamycin (Selleck chemicals, Cat#S1039), 4-PBA (WAKO,
   Cat#168-06471), NAC (WAKO, Cat#015-05132), metformin (Abcam,
   Cat#ab120847)), vehicle or no addition for Pt#1 and containing vehicle
   or no addition for N#3. Rapamycin was used at the final concentration
   of 10 nM from the stock solution of 1 M dissolved in 100% ethanol. The
   4-PBA was used the concentration of 5 mM from the freshly prepared
   solution of 5 M 4-PBA in 100% ethanol. A final concentration of 5 mM
   metformin was prepared from the stock solution of 100 mM metformin in
   water. Freshly prepared 1 M NAC in water was further diluted to make a
   final concentration of 5 mM NAC. The media with or without drugs or
   vehicle was replaced thrice weekly. Finally, the cells were harvested 2
   weeks post-transduction. Total RNA was isolated using a Picopure™ RNA
   isolation kit (Thermo Fisher Scientific; Cat#KIT0204) following the
   manufacturer’s protocol. cDNAs were synthesized from total RNA using
   superscript™ IV first-strand synthesis system (Thermo Fisher
   Scientific; Cat#18091050).

Reverse transcriptase (RT)-PCR

   Total RNA was isolated with RNeasy Plus mini kit® (Qiagen, Maryland,
   USA) or PicoPure™ RNA Isolation Kit (Arcturus Bioscience, CA, USA)
   according to the manufacturer's instructions. An aliquot of total RNA
   was reverse-transcribed by using an oligo (dT) primer. A cDNA template
   was amplified using the Platinum Quantitative PCR SuperMix-UDG with ROX
   (Invitrogen) and ABI7900HT Sequence Detection System (Applied
   Biosystems). Fluorescence was monitored during every PCR cycle at the
   annealing step. The authenticity and size of the PCR products were
   confirmed using a melting curve analysis (using software provided by
   Applied Biosystems) and a gel analysis. mRNA levels were normalized
   using β-actin as a housekeeping gene. The expression level in the
   photoreceptor-directed fibroblasts, HDF-a (Human Dermal
   Fibroblasts-adult, ScienCell Research Laboratories), 2 weeks
   post-transduction was used as a reference. The design of the PCR primer
   sets is shown in our previous paper [[70]23] and Table S2 (Additional
   file [71]2).

Immunocytochemistry

   The cells were seeded in laminin-coated 24-well plate (Sumilon
   MS-92132). The induction of photoreceptor-like cells was carried out
   mentioned above. The cells were fixed with 4% paraformaldehyde in
   1 × PBS 14 days after transduction. The cells were washed and
   permeabilized with 0.1% Triton in 1 × PBS. The cells were incubated in
   blocking buffer (5% normal goat serum in 1 × PBS) for an hour at room
   temperature. The cells incubated with primary antibody (anti-CRX
   (1:1000, Abnova H00001406-M02), anti-rhodopsin (1D4) (1:200, Santa Cruz
   biotechnology, sc-57432)) in blocking buffer for overnight at 4C. Then,
   cells were incubated with secondary antibody (goat anti-mouse IgG,
   alexa flour® 568 conjugate (1:500, Invitrogen, A-11004)). The nuclei
   were stained with 4',6-diamidino-2-phenylindole (DAPI). Images were
   captured with a Nikon Eclipse TE300, and contrast and brightness were
   adjusted with NIS elements AR 3.2.

Western blots

   The induced photoreceptor-directed fibroblasts were lysed with
   radioimmunoprecipitation assay (RIPA) buffer (50 mM tris pH 8.0, 150 mM
   NaCl, 0.1% sodium dodecyl sulfate, 0.5% sodium deoxycholate and 1% NP40
   substitute) containing 1X protease inhibitor cocktail (Roche). The cell
   lysates were homogenized with gentle agitation for 30 min on ice and
   centrifuged at 12,000 rpm at 4 °C for 20 min. Each supernatant was
   collected in a new microcentrifuge tube. The protein concentration of
   each sample was measured by the Pierce™ BCA Protein Assay Kit
   (ThermoScientific). The samples were denatured with 4 X Laemlli sample
   buffer (Biorad), heated at 95 °C for 5 min, resolved in 4–20% gel
   (Biorad) and transferred to PDVF membrane (Biorad). Detection of immune
   complexes formed by proteins of interest and primary antibodies
   (anti-blue opsin (1:250, Milipore, AB5407), anti-CHOP (1:1500, Cell
   Signaling Technology, #2895), anti-cleaved caspase-3 (1:1000, Cell
   signaling technology, #9664), anti-β-actin (1:1000, Cell Signaling
   Technology)) was performed by enzyme-linked color development with
   anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody
   (1:3000, Cell Signaling Technology). The signals based on chemical
   luminescence (ECL™ Prime Western Blotting system (Amersham)) were
   detected by ChemiStage (TOYOBO). The band intensity was quantified with
   ImageJ software (National Institutes of Health).

Statistical analysis

   Analysis of variance (ANOVA) with Tukey’s honest test was employed to
   compare of the gene expression level of multiple groups with the
   photoreceptor-directed fibroblasts derived from the healthy individuals
   (control group). p value less than 0.05 was considered significant.

Results

Expression levels of phototransduction-related genes were lower in induced
photoreceptor-like cells from fibroblasts of EYS-RP patients

   It was previously reported that immunostaining of blue opsin was
   decreased in EYS-deficient zebrafish [[72]33]. We compared expression
   levels of phototransduction-related genes, OPN1SW (the blue opsin
   gene), SAG (the S-antigen gene), RCVRN (the recoverin gene), RHO (the
   rhodopsin gene), GNAT1 and GNAT2. From one to six weeks after CNRO gene
   transduction, expression levels of the blue opsin gene, the recoverin
   gene and the GNAT1 gene were lower in photoreceptor-directed
   fibroblasts derived from EYS-RP patients (Fig. [73]1). In EYS-RP cells,
   the lower expression levels of the S-antigen gene initiated 2 weeks
   after transduction and expression levels were significantly lower 2, 3
   and 5 weeks after transduction. Expression levels of the GNAT2 gene
   were significantly lower one and two weeks after transduction in EYS-RP
   cells. The expression levels of the rhodopsin gene were lower in EYS-RP
   cells from one to six weeks, though not significantly (Fig. [74]1).
   Expression of blue opsin, rhodopsin and CRX were also detected by
   immunocytochemistry in photoreceptor-directed fibroblasts derived from
   both normal healthy volunteers and EYS-RP patients (Additional file
   [75]2: Fig. S1, Figure S3 in our previous paper [[76]26]); therefore,
   statistical analysis was performed on western blots for blue opsin
   (Additional file [77]2: Fig. S4).

Fig. 1.

   [78]Fig. 1
   [79]Open in a new tab

   Induction of retina-specific genes in human dermal fibroblasts derived
   from a healthy individual (N#3, open box) and an RP patient (Pt#1,
   closed box) by retroviral infection of genes for transcription factors
   CRX, RAX, NeuroD and OTX2. A RT-PCR analysis for
   phototransduction-related genes, blue opsin, S-antigen, rhodopsin,
   GNAT1, GNAT2, recoverin, in cultured human dermal fibroblasts (HDF).
   Vertical axis; relative expression, Horizontal axis; weeks after
   transduction. Columns represent mean ± SEM. All data points are
   overlaid. **p < 0.01; student’s t-test (n = 6 (blue opsin, S-antigen,
   recoverin), n = 3 (GNAT1, GNAT2). B Ratio of expression levels of rod
   photoreceptor- and cone photoreceptor-specific genes in
   photoreceptor-like cells (S-antigen vs blue opsin). Error bar: SEM.
   **p < 0.01; student’s t-test (n = 6)

NRL, a key transcription factor for rod differentiation, was downregulated
gene in the photoreceptor-directed fibroblasts derived from EYS-RP patients

   To clarify the specific gene expression profiles in induced
   photoreceptor cells derived from EYS-RP patients, we compared the
   expression profiles of 58,201 probes in the induced photoreceptor cells
   derived from EYS-RP patients (induced, RP) and healthy volunteers
   (induced, normal). We first extracted the intersection of the two
   groups of genes, i.e., up- or downregulated genes in RP versus healthy
   ([induced Pt#1] versus [induced N#3], which is an age-matched pair for
   comparison) (signal ratio ≥  + 1.5, ≤ -1.5 for “up,” “down”) and those
   in CRNO-transduced normal fibroblasts versus non-induced fibroblasts
   ([induced N#3] versus [non-induced N#3]) (signal ratio ≥  + 1.5). A
   total of 264 upregulated genes and 2677 downregulated genes were
   extracted, which suggest that there are more downregulated genes in the
   photoreceptor-directed fibroblasts derived from EYS-RP patients
   compared to those from normal controls (Fig. [80]2). Among the 2677
   downregulated genes, 50 genes were significantly decreased in another
   independently performed microarray analysis using independently
   differentiated photoreceptor-directed fibroblasts (Pt#1 vs N#2), which
   is an age-unmatched pair (Table [81]2). Among the genes whose
   expression levels were greatly decreased, NRL, a key transcription
   factor for rod differentiation, was extracted. We then compared
   non-induced EYS-RP-derived cells and non-induced normal cells. It was
   shown that there were more differentially expressed genes, EYS-RP vs
   normal, in the differentiated experimental groups (4229 upregulated
   probes, 4212 downregulated probes) (Fig. [82]2A) than in the
   non-induced groups (2552 upregulated probes, 2152 downregulated probes)
   (Fig. [83]2B).

Fig. 2.

   [84]Fig. 2
   [85]Open in a new tab

   Categorization of genes that are differentially expressed in
   photoreceptor-directed fibroblasts derived from an EYS-RP patient and
   upregulated by transduction of CRX, RAX, NeuroD and OTX2. Venn diagrams
   (A, B) and Mean-average (MA) plots (C, D) to compare EYS-RP cells vs
   normal cells. To clarify the specific gene expression profile in
   induced photoreceptor cells derived from EYS-RP patients, we compared
   the expression profiles of 58,201 probes in the induced photoreceptor
   cells or non-induced fibroblast cells derived from an EYS-RP patient
   (induced or non-induced, RP, Pt#1) and healthy volunteers (induced or
   non-induced, normal, N#3), using microarrays. We first extracted the
   intersection of the two groups of genes, i.e., up- or downregulated
   genes in RP versus healthy ([induced Pt#1] versus [induced N#3], which
   is an age-matched pair for comparison) (signal ratio ≥  + 1.5, ≤ -1.5
   for “up,” “down”) and those in CRNO-transduced normal fibroblasts
   versus non-induced fibroblasts ([induced N#3] versus [non-induced N#3])
   (signal ratio ≥  + 1.5) (A). Furthermore, we compared non-induced
   EYS-RP-derived cells and non-induced normal cells, and then extracted
   the intersection of the two groups to define up- or downregulated genes
   (non-induced Pt#1] versus [non-induced N#3]). We also determined up- or
   downregulated genes in induced RP versus induced healthy ([induced
   Pt#1] versus [induced N#3]) (signal ratio ≥  + 1.5, ≤ -1.5) (B).
   Mean-average (MA) plots indicate differential gene expression between
   induced RP and induced normal (C) and differential gene expression
   between non-induced RP and non-induced normal (D). Significantly
   differentially upregulated genes (signal ratio ≥  + 1.5) are
   highlighted in red and downregulated genes (signal ratio ≤ -1.5) are
   highlighted in n green

Table 2.

   Downregulated genes in EYS-RP-derived photoreceptor-like cells
   GeneSymbol GeneName Pt #1 non-induced (1) Pt #1 induced (1) N #3
   non-induced (1) N #3 induced (1) Pt #1 non-induced (2) Pt #1 induced
   (2) N #2 non-induced (2) N #2 induced (2)
   LZTS1 Leucine zipper, putative tumor suppressor 1 3.24 5.45 5.39 9.43
   2.81 5.24 2.98 6.85
   SST Somatostatin 1.00 4.40 1.11 8.27 2.89 4.29 2.27 8.05
   TNNC1 Troponin C type 1 (slow) 3.42 4.27 3.68 7.76 2.70 4.39 2.98 7.01
   FOXS1 Forkhead box S1 1.66 3.84 1.76 7.07 1.07 4.16 1.94 6.00
   ANO2 Anoctamin 2, calcium activated chloride channel 1.48 4.51 2.48
   7.60 1.78 4.58 1.86 6.31
   HES6 Hes family bHLH transcription factor 6 2.03 4.08 2.28 7.15 3.11
   5.29 3.23 6.47
   NLRP12 NLR family, pyrin domain containing 12 0.79 3.12 0.93 6.10 1.19
   3.17 0.29 6.42
   KRT18 Keratin 18, type I 1.63 4.02 4.34 6.90 2.40 3.67 3.05 6.57
   HES6 Hes family bHLH transcription factor 6 7.15 10.31 7.33 13.15 7.54
   10.83 8.04 12.32
   CKMT1A Creatine kinase, mitochondrial 1A 1.11 6.16 1.17 8.82 1.67 5.82
   1.42 7.10
   3.06 3.93 3.94 6.52 2.75 3.79 3.05 5.96
   DIRAS3 DIRAS family, GTP-binding RAS-like 3 4.08 7.61 5.35 10.11 4.10
   8.07 4.62 9.89
   PDLIM3 PDZ and LIM domain 3 2.93 5.31 4.28 7.78 0.59 4.16 0.50 5.70
   C8orf46 Chromosome 8 open reading frame 46 2.37 5.04 2.69 7.47 1.08
   5.38 1.02 7.36
   ASMT Acetylserotonin O-methyltransferase 3.27 3.88 3.05 6.28 1.92 4.36
   1.47 7.99
   COL4A1 Collagen, type IV, alpha 1 5.41 7.26 6.24 9.66 6.17 7.64 6.19
   9.59
   LYPD1 LY6/PLAUR domain containing 1 4.31 5.37 5.84 7.73 4.45 5.13 3.44
   7.71
   C2CD4A C2 calcium-dependent domain containing 4A 0.52 6.08 0.51 8.36
   1.21 5.85 2.08 7.60
   NRL Neural retina leucine zipper 3.15 8.05 2.60 10.31 2.86 8.76 1.91
   10.17
   COL4A2 Collagen, type IV, alpha 2 8.89 10.43 9.70 12.66 9.76 10.68 9.49
   12.02
   NRL Neural retina leucine zipper 4.04 8.84 4.12 11.06 3.83 9.51 3.46
   11.02
   LINC00463 Long intergenic non-protein coding RNA 463 0.84 4.19 0.96
   6.34 0.85 4.35 0.45 6.09
   ADRA2A Adrenoceptor alpha 2A 4.64 7.20 4.12 9.27 4.29 7.40 3.74 8.79
   NTRK3 Neurotrophic tyrosine kinase, receptor, type 3 2.36 2.14 0.59
   4.06 1.04 3.06 1.54 5.67
   NGFR Nerve growth factor receptor 0.55 4.51 0.60 6.43 0.55 4.91 0.50
   7.60
   MPPED2 Metallophosphoesterase domain containing 2 0.65 5.29 0.82 7.18
   0.37 5.64 0.33 7.84
   AMER2 APC membrane recruitment protein 2 0.78 8.01 0.80 9.76 0.68 7.97
   1.49 9.80
   CTSH Cathepsin H 6.82 7.64 7.24 9.36 6.46 7.30 6.02 9.51
   PDZD2 PDZ domain containing 2 2.19 6.21 2.38 7.92 0.39 6.33 0.99 8.45
   TCF15 Transcription factor 15 (basic helix-loop-helix) 6.12 5.76 6.57
   7.45 5.49 5.57 5.53 6.92
   F2R Coagulation factor II (thrombin) receptor 2.38 4.05 2.99 5.68 3.10
   4.80 3.53 6.61
   GRAMD1C GRAM domain containing 1C 5.88 7.06 6.06 8.62 4.88 7.00 4.46
   8.98
   GPX3 Glutathione peroxidase 3 (plasma) 4.82 8.34 5.16 9.87 4.60 8.44
   3.98 10.67
   BEGAIN Brain-enriched guanylate kinase-associated 4.33 4.39 5.47 5.92
   4.45 4.63 4.82 6.59
   TMEM176A Transmembrane protein 176A 3.66 9.80 3.69 11.31 4.13 10.15
   4.75 11.75
   A2M Alpha-2-macroglobulin 7.24 5.96 8.35 7.45 8.67 6.78 9.16 8.00
   TMSB15A Thymosin beta 15a 4.54 4.05 5.37 5.51 3.62 4.14 4.51 5.85
   GPRC5B G protein-coupled receptor, class C, group 5, member B 3.92 7.32
   3.45 8.77 4.13 7.61 3.75 10.06
   IMPG1 Interphotoreceptor matrix proteoglycan 1 0.60 4.33 0.56 5.74 0.54
   3.93 0.43 6.05
   PLEKHB1 Pleckstrin homology domain containing, family B (evectins)
   member 1 4.54 7.65 4.43 9.05 4.63 7.46 3.97 9.67
   SYPL2 Synaptophysin-like 2 7.12 7.76 6.99 9.15 6.21 7.72 5.26 9.92
   SLC16A3 Solute carrier family 16 (monocarboxylate transporter), member
   3 11.28 11.44 13.32 12.77 13.11 11.60 14.03 12.88
   CBX2 Chromobox homolog 2 2.08 5.02 1.71 6.21 3.61 5.17 3.52 6.36
   CCDC13 Coiled-coil domain containing 13 2.07 6.52 2.39 7.66 1.83 7.00
   1.92 8.75
   MECOM MDS1 and EVI1 complex locus 4.27 3.93 4.27 5.06 3.67 3.40 4.43
   5.65
   RXRG Retinoid X receptor, gamma 0.69 6.30 0.72 7.40 0.42 6.87 0.39 8.48
   CCDC181 Coiled-coil domain containing 181 3.07 8.39 3.76 9.48 3.08 8.35
   3.84 9.70
   GNB3 Guanine nucleotide binding protein (G protein), beta polypeptide 3
   3.90 6.99 3.99 8.06 3.62 7.18 4.42 8.70
   OXTR Oxytocin receptor 6.26 7.83 5.87 8.86 4.82 7.44 4.35 9.65
   4.79 4.45 5.12 5.46 3.95 4.72 4.92 6.30
   [86]Open in a new tab

CRYGD was downregulated in EYS-RP-derived cells, retina-related and
apoptosis/oxidative stress/ER stress/aging-related genes

   We then extracted intersection of the two groups of genes, i.e., up- or
   downregulated genes in RP versus healthy and retina-related genes with
   gene ontology (GO) term annotation, which was used in our previous
   study [[87]24]. A total of 119 upregulated genes and 239 downregulated
   genes were extracted, which suggests that there are downregulated genes
   in RP retinas (Fig. [88]3). CNGA1, COL11A1, SOX11, AIPL1 and CRYGD
   showing the largest decreases in expression. We then extracted the
   interaction of the two groups of genes, i.e., up- or downregulated
   genes in RP versus healthy and apoptosis/oxidative stress/ER
   stress/aging-related genes according to gene ontology (GO) term
   annotation. Those four key words were chosen based on previous studies
   of animal models of RP [[89]34–[90]37] and the fact that EYS-RP is
   late-onset and progressive disease. Ninety-six upregulated genes and
   146 downregulated genes were extracted, with CRYGD and GJB2 showed the
   largest decreases in expression. Decreased expression of CRYGD in
   EYS-RP-derived cells was confirmed by qRT-PCR.

Fig. 3.

   [91]Fig. 3
   [92]Open in a new tab

   Global gene expression analysis of EYS-RP patient-derived
   photoreceptor-like cells. A Categorization of genes differentially
   expressed in both photoreceptor-directed fibroblasts derived from an
   EYS-RP patient and retina-related genes. We extracted intersection of
   the two groups of genes, i.e., up- or downregulated genes in RP versus
   healthy and retina-related genes according to gene ontology (GO) term
   annotation, which was used in our previous study [[93]24]. A total of
   119 upregulated genes and 239 downregulated genes were extracted (left
   panel). CNGA1, COL11A1, SOX11, AIPL1 and CRYGD showed significant
   decreases in expression (Right panel). B Categorization of genes that
   are both differentially expressed in photoreceptor-directed fibroblasts
   derived from an EYS-RP patient and apoptosis/oxidative stress/ER
   stress/aging-related genes according to gene ontology (GO) term
   annotation. Ninety-six upregulated genes and 146 downregulated genes
   were extracted, with CRYGD and GJB2 displaying large decreases. C
   EYS-RP-associated pathway extracted by pathway enrichment analysis.
   Pathway enrichment analysis was performed on a gene set that is an
   intersection of up- or downregulated genes in RP versus age-matched
   healthy ([induced Pt#1] versus [induced N#3], and another intersection
   of up- or downregulated genes in RP versus age-unmatched healthy
   ([induced Pt#1] versus [induced N#2], which was served for the
   analysis. By pathway enrichment analysis, WebGestalt2017
   ([94]http://www.webgestalt.org/), three pathways, “complement and
   coagulation cascades,” “ECM-receptor interaction” and “PI3K-Akt
   signaling pathway,” were extracted from up- or downregulated genes in
   photoreceptor-directed fibroblasts derived from EYS-RP compared to
   normal volunteers. Among the matching/overlapping genes in the three
   pathways, the F2R gene was involved in both the “complement and
   coagulation cascades” and “PI3K-Akt signaling pathway.” D RT-PCR
   analysis of expression of CRYGD and F2R genes in photoreceptor-directed
   fibroblasts derived from an EYS-RP patient (Pt#1) and a normal
   volunteer (N#3). The vertical axis is relative expression, and the
   horizontal axis is weeks after gene transduction. Decreased expression
   of CRYGD and F2R in EYS-RP-derived cells was confirmed. The expression
   levels in photoreceptor-directed fibroblasts transduced with four
   transcription factor genes (CRX, RAX, NeuroD and OTX2) was calculated
   from 3 biological replicates. Columns represent mean ± SEM. All data
   points are overlaid. *p<0.05, **p<0.01; student’s t-test. Internal
   control: β-actin.

F2R may be involved in an EYS-RP-specific pathway

   We then prepared a gene set for pathway enrichment analysis, an
   intersection of up- or downregulated genes in RP versus age-matched
   healthy ([induced Pt#1] versus [induced N#3], and another intersection
   of up- or downregulated genes in RP versus age-unmatched healthy
   ([induced Pt#1] versus [induced N#2]. Using WebGestalt, we found that
   three pathways, “complement and coagulation cascades,” “ECM-receptor
   interaction” and “PI3K-Akt signaling pathway,” were extracted from up-
   or downregulated genes in photoreceptor-directed fibroblasts derived
   from EYS-RP compared to normal volunteers. Among the
   matching/overlapping genes in the three pathways, the F2R gene was
   found in both “complement and coagulation cascades” and “PI3K-Akt
   signaling pathway.” Decreased expression of F2R in EYS-RP-derived cells
   was confirmed by qRT-PCR.

Decreased expression levels of the phototransduction-related genes, OPN1SW
(blue opsin), RCVRN (recoverin), GNAT1 and GNAT2, and the genes extracted by
global gene expression analysis, GRYGD and F2R, in EYS-RP-derived cells were
increased by the chemical chaperone 4-PBA

   We tested whether drugs would increase levels of gene expression in
   photoreceptor-directed fibroblasts derived from EYS-RP to similar
   levels of those from normal volunteers. Six target genes whose
   expression levels were decreased in EYS-RP derived cells were chosen
   (blue opsin, recoverin, GNAT1, GNAT2, F2R and CRYGD), based on previous
   results in the present study. The effectiveness of four drugs was
   evaluated. 4-PBA was found to be the most effective. The treatment of
   photoreceptor-directed fibroblasts derived from Pt#1 with 5 mM 4-PBA
   restored the expression levels of target genes to levels similar to the
   control group, or higher than the control (N#3) (Fig. [95]4, Additional
   file [96]2: Fig. S2). The gene expression levels with 4-PBA treatment
   were significantly higher than those of photoreceptor-like cells with a
   vehicle, EtOH (one-way ANOVA followed by Tukey’s test). The restoration
   effect by 4-PBA treatment was confirmed by western blotting for the
   blue opsin (Additional file [97]2). Similarly, three doses of 4-PBA
   were tested to determine whether expression levels of the target genes
   in photoreceptor-directed fibroblasts derived from Pt#2 were also
   restored to levels of the control (N#1) (Additional file [98]2: Fig.
   S3). The application of metformin also increased the expression levels
   of target genes to comparable or higher levels than the control.
   However, metformin was less effective in restoring the expression of
   target genes in photoreceptor-directed fibroblasts derived from Pt#2
   (Fig. S3). This discrepancy in effectiveness of metformin might be due
   to the difference in the point mutation in EYS between Pt#1 and Pt#2;
   however, more samples are needed to clarify this discrepancy.
   Application of rapamycin and NAC did not restore the expression level
   of any target genes in photoreceptor-directed fibroblasts derived from
   Pt#1 to similar levels of the control (N#3) (Fig. [99]4), as well as
   Pt#2 versus N#1 (Fig. S3). The expression levels of these genes in
   photoreceptor-like cells derived from fibroblast of Pt#1 treated with
   vehicle (water or ethanol) were lower than in control group, however,
   not significantly (Fig. [100]4, one-way ANOVA followed by Tukey’s
   test). A similar trend in reduction in the expression level of target
   genes was also observed in photoreceptor-directed fibroblasts derived
   from Pt#2, with a heterozygous mutation in the EYS gene, and an
   aged-matched healthy individual (N#1) (Fig. S3).

Fig. 4.

   [101]Fig. 4
   [102]Open in a new tab

   Effect of drugs on gene expression in photoreceptor-directed
   fibroblasts derived from an EYS-RP patient, Pt#1. The fibroblasts of a
   healthy individual (N#3) and fibroblasts of a patient suffering from
   retinitis pigmentosa due to a homozygous mutation in the EYS gene
   (Pt#1) were transdifferentiated to photoreceptor-like cells by
   retroviral transduction of four transcription factors. Gene expression
   was compared 2 weeks post-transduction. Differentiation medium was
   supplemented with four drugs, 4-phenylbutyric acid (4-PBA), metformin
   (Metf), rapamycin (Rapa), N-acetyl-L-cysteine (NAC) or the vehicle
   (water and ethanol (EtOH)) or none (-). The gene expression with each
   pharmacological treatment was compared to the gene expression in
   photoreceptor-like cells derived from fibroblasts of healthy individual
   without any supplement (-). Columns represent mean ± SEM. All data
   points are overlaid (n = 6 (N#3 without any supplement (-)), n = 3 (N#3
   with supplement and Pt#1). a = p > 0.05, b = p > 0.01, c = p > 0.001;
   One-way ANOVA followed by Tukey’s honest test. For the sake of
   simplicity, comparison with the gene expression level in N#3 without
   any supplement (-) is shown here. Comparison among other groups is
   shown in Additional file [103]2: Fig. S2

   Based on the above results with 4-PBA, we investigated UPR-signal
   transduction in EYS-deficient cellular models. Inositol requiring
   enzyme 1α/β (IRE1) is the one of the key UPR signal activator proteins
   [[104]38–[105]40]. Thus, we examined the relative expression of ERN
   (endoplasmic reticulum to nucleus signaling), which codes for IRE1. We
   found that the relative expression of ERN was significantly lower in
   photoreceptor-directed fibroblasts derived from Pt#2 and was also lower
   in that from Pt#1, although not significantly (Additional file [106]2:
   Fig. S4). Treatment with 5 mM 4-PBA restored the relative expression
   levels of ERN in photoreceptor-directed fibroblasts derived from both
   patients to similar levels a found in healthy individuals (Fig. S4).
   CHOP (C/EBP homologous protein) is one of the target genes of the UPR
   pathway and has a role in controlling gene expression that leads to
   apoptosis [[107]38–[108]40]. We examined the expression level of CHOP
   by western blotting, using photoreceptor-directed fibroblasts derived
   from Pt#1 and N#3. CHOP was undetectable in both fibroblasts. Upon
   treatment with thapsigargin (200 mM), an ER stress inducer, the
   expression levels of CHOP were slightly higher in Pt#1-derived cells
   than N#3, albeit statistically insignificant (Fig. S4). We performed
   western blotting with a cleaved caspase-3 antibody to investigate the
   restoration effect of 4-PBA on apoptosis. The relative expression of
   cleaved caspase-3 was slightly higher in EYS-RP than photoreceptor-like
   cells derived from a healthy individual, although there was no
   significant difference. The relative expression of cleaved caspase-3
   was clearly decreased by 4-PBA treatment. By immunostaining, caspase-3
   was also detected in more photoreceptor-directed fibroblasts derived
   from Pt#1 rather than N#3 under thapsigargin supplementation
   (Additional file [109]2: Fig. S5). These results indicate that
   EYR-RP-derived cells are more sensitive to an ER stress inducer.

Discussion

   This is the first report that shows gene expression profiles of
   cellular models of EYS-RP patients. Because our previous study
   suggested that findings of patient-derived cells should be compared
   with age-matched normal volunteer to assess EYS-RP-associated
   characteristics [[110]26], we here compared gene expression profiles in
   induced photoreceptor-directed fibroblasts derived from EYS-RP patients
   with age-matched normal volunteers. As a result, expression levels of
   several phototransduction-related genes, blue opsin, recoverin, GNAT1,
   GNAT2, and an aging-related gene, CRYGD, and complement and coagulation
   cascades- and PI3K-Akt signaling pathway-related gene, F2R, were
   significantly lower in photoreceptor-directed fibroblasts derived from
   EYS-RP patients compared to age-matched normal volunteers. This
   decrease was reversed by addition of 4-PBA and metformin, but not by
   rapamycin or NAC.

   One of the downregulated genes, the γD-crystallin encoding CRYGD gene,
   which was extracted as both retina-related and apoptosis/oxidative
   stress/ER stress/aging-related genes (Fig. [111]3), is also known as a
   cataractogenesis-related gene [[112]41]. On the other hand, SLC16A10
   (LAT2), which is also a cataractogenesis-related gene [[113]42], was
   extracted by pathway enrichment analysis as involved in the “protein
   digestion and absorption pathway” (Additional file [114]1: Table S1),
   in the intersection of up- or downregulated genes in RP versus
   age-matched healthy volunteer and those in CRNO-transduced normal
   fibroblasts versus non-induced fibroblasts. RP, most likely including
   EYS-RP, is a typical late-onset retinal degenerative disease, with
   cataract formation noted at relatively younger ages [[115]43–[116]45].
   The mechanism for pathologic, not physiological, aging-related
   cataractogenesis in EYS-RP has not been elucidated; however, it may be
   explained, at least in part, by downregulation of the CRYGD gene in
   photoreceptors of EYS-RP patients. Furthermore, crystallin plays roles
   outside of the lens, with cilia-related functions in photoreceptors
   [[117]46]. Of note, defects in human γD-crystallin, known to form
   amyloid aggregates, were suggested to play a role in retinal pigment
   epithelial cells, leading to age-related macular degeneration
   [[118]47]. Our results suggest possible roles of downregulated
   γD-crystallin in EYS-associated photoreceptor degeneration as well as
   cataractogenesis. In the present study, Gap junction protein, beta 2
   (GJB2) showed decreased expression by the same analysis. The GJD2 gene,
   which encodes connexin 36, was reported to be related to myopia
   development [[119]48]. It is also known that GJD2 plays a role in
   retinal signal transduction and is expressed in gap junctions between
   cones, rods and bipolar cells, playing essential roles in rod-mediated
   visual signals [[120]49, [121]50]. Further studies are needed to
   clarify the molecular interactions of CRYGD and GJD2 with EYS in the
   retina.

   EYS, an agrin/perlecan-related extracellular matrix protein secreted by
   photoreceptors into the interrhabdomeral space [[122]51], was thought
   to be comparable to interphotoreceptor or subretinal space of the human
   retina. A recent report supports the possible function of EYS as an
   extracellular matrix protein in the retina of a zebrafish model with
   defects in the eys gene [[123]52]. From our present study using a human
   cellar model of EYS-RP, interphotoreceptor matrix proteoglycan 1
   (IMPG1) (Table [124]2) and ECM-receptor interaction pathway
   (Fig. [125]3, Table S1) were extracted as an EYS-RP-associated gene and
   pathway, suggesting the ECM-related functions of EYS throughout
   evolution.

   On the other hand, in previous reports using zebrafish, the EYS protein
   is localized near connecting cilium/transition zone in photoreceptors
   [[126]27, [127]32, [128]53] and disruption of the ciliary pocket in
   cones is observed in eys^−/− [[129]32]. The study further showed that
   expression of opsin, localized in outer segment of photoreceptors, is
   extremely lower in eys^−/−, and the authors speculate that the lower
   expression is due to disruption of transport in cilia. We here show
   that expression levels of several phototransduction-related genes, blue
   opsin, recoverin, GNAT1, GNAT2 were significantly lower in
   photoreceptor-directed fibroblasts derived from EYS-RP patients
   compared to age-matched normal volunteers. A regulatory mechanism other
   than transportation via connecting cilium may exist in EYS deficiency
   because our photoreceptor-like cells lack outer segments, although
   gathering mitochondria were observed [[130]23]. On the other hand, Nrl,
   a key molecule to control rod-photoreceptor development [[131]54,
   [132]55], was extracted as one of the significantly downregulated genes
   in EYS-RP derived cells in this study (Table [133]2). This may lead to
   the typical phenotype of RP or EYS-RP, rod-cone dystrophy [[134]56,
   [135]57], although atypical phenotypes of RP, such as cone/cone-rod
   dystrophy and sector RP, have been reported [[136]58–[137]60]. It is
   also noteworthy that expression levels of blue opsin and the ratio of
   expression levels of blue opsin vs S-antigen were significantly lower
   in the earlier days after CRNO gene transduction in EYS-RP-derived
   photoreceptor-like cells, which may be explained by the fact that
   up-regulation of cone-photoreceptor-related genes, started earlier than
   rod-photoreceptor genes [[138]23], which are consistent with previous
   reports on retinogenesis. In our redirect differentiation study, it is
   difficult to discern whether downregulation of
   phototransduction-related genes is related to delay, defects of
   retinogenesis or decrease of differentiation efficiency in EYS-RP
   cells. Correlation of gene expression profiles to phenotype should be
   helpful, as well as genotype–phenotype relation for personalized
   medicine.

   Pathway enrichment analysis revealed that three pathways were extracted
   from differentially expressed genes between EYS-RP and normal
   volunteers (Fig. [139]3). In those pathways, F2R, which encodes
   protease-activated receptor-1 (PAR-1), was matching/overlapping in both
   “complement and coagulation cascades” and “PI3K-Akt signaling pathway.”
   Decreased expression of the F2R gene in EYS-RP-derived cells was
   confirmed by qRT-PCR (Fig. [140]3). Those pathways and the F2R gene may
   be promising as therapeutic targets because there is evidence of the
   involvement of inflammation in the pathogenesis of RP
   [[141]61–[142]64]. F2R may also be involved in prevention of apoptosis
   [[143]65], morphogenesis of photoreceptors [[144]66] and survival of
   cancer cells through a Bcl-xL-dependent mechanism [[145]67]. Reduction
   of F2R expression by defects in the EYS gene may be relevant to
   multiple cascades in EYS-RP.

   It was reported that a defect in the rhodopsin gene, P23H, the most
   common cause of RP in the USA, induces rhodopsin misfolding and the
   unfolded protein response (UPR) [[146]36]. Also, it was shown that the
   endoplasmic reticulum (ER)-UPR, a known mechanism of apoptosis
   secondary to an overwhelming accumulation of misfolded protein
   [[147]68], is involved in photoreceptor degeneration caused by missense
   mutations in TULP1 [[148]69], which are associated with two forms of
   IRDs, early-onset retinitis pigmentosa (RP) and Leber congenital
   amaurosis (LCA). In this study, we demonstrated that 4-phenylbutyric
   acid (4-PBA), chemical chaperone, an ER stress inhibitor, dramatically
   restored downregulation of phototransduction-related genes, CRYGD and
   F2R, suggesting that ER stress may be a possible therapeutic target for
   photoreceptor degeneration in EYS-RP patients.

   In our previous paper, we demonstrated that the defective EYS mRNA
   containing c.4957dupA in photoreceptor-directed fibroblasts of Pt#1 and
   Pt#2 undergoes partial degradation [[149]26]. In the present study, we
   show that the expression level of ERN, which codes IRE1, was decreased
   in photoreceptor-directed fibroblasts derived from Pt#1 and Pt#2. It is
   well established that IRE1 degrades mRNA via RIDD (regulated
   Ire1-dependent decay) pathway to mitigate ER stress [[150]38–[151]40].
   The failure to degrade faulty mRNA in EYS-RP might be attributable to
   lowered expression of ERN. IRE1 has also been reported to promote
   secretion of Spacemaker/Eyes shut in the intrarhabdomeral space in
   Drosophila [[152]70]. This could be one of the reasons for depletion of
   IRE1 expression. Additionally, IRE1 is reported to play a role in
   photoreceptor differentiation via RIDD pathway [[153]70]. We speculate
   the decreased level of IRE1 might be due to the reduced protein
   translation in response to ER stress. It is plausible that the
   depletion of IRE1 might have affected the redirect differentiation in
   EYS-RP, thereby causing a decrease in expression levels of
   phototransduction-related genes. We are planning additional detailed
   studies on mRNA degradation by the RIDD pathway to clarify the
   abovementioned hypothesis. Our study also showed that the 4-PBA
   effectively restored the expression levels of ERN in EYS-RP-derived
   induced cells to levels similar of healthy individuals. Apparently,
   4-PBA may attenuate the ER stress by various mechanisms. One plausible
   mechanism of 4-PBA-mediated upregulation of several EYS-RP-associated
   genes might be due to upregulation of ERN, thereby possibly promoting
   mRNA degradation of defective EYS mRNA or facilitating the redirect
   differentiation to photoreceptors. To clarify this speculation, further
   studies are needed.

   By our pathway enrichment analysis using microarray data of
   EYS-RP-patient-derived photoreceptor-like cells, “hypertrophic
   cardiomyopathy (FDR: 0.011684)” and “dilated cardiomyopathy (FDR:
   0.014503)” were extracted (Table S1). It was suggested that EYS protein
   in Drosophila plays a role in protecting mechanoreceptor and
   chemoreceptor organs from hyperosmotic shock by providing stiffness and
   maintaining cellular integrity and tissue morphogenesis [[154]71]. A
   genetic study suggested an association between trastuzumab-induced
   cardiotoxicity and rs139944387 in exon 44 of Eyes shut homologs [EYS]
   [[155]72]. On the other hand, in Drosophila, localization and function
   of actomyosin machinery in photoreceptors were previously demonstrated
   [[156]73]. The mitochondria-related pathway may also be a common
   mechanism between photoreceptors and cardiomyocytes, considering the
   fact that both cell types include highly dense mitochondria in the
   cytosol. Creatinine kinase, mitochondrial 1A was also extracted as a
   differentially expressed gene (Table [157]2). Taken together, these
   results suggest that defects of EYS are involved in a common pathway
   between photoreceptor degeneration and dysfunction of cardiomyocytes;
   however, further studies are needed to confirm this hypothesis.

   Although iPSC lines were established from patients with defects in RHO
   [[158]31], MAK [[159]74], PDE6A [[160]75], PRPF31 [[161]76], NR2E3
   [[162]77] and EYS [[163]78], molecular mimicry of the iPSC-derived
   retina to the degenerative retina in human RP has not been confirmed. A
   high-throughput assay system for drug screening using human cellular
   models is necessary, although zebrafish model could complement such an
   assay [[164]79]. The present study suggests that our cellular model may
   be promising for such a screening system. Our system, however, has the
   limitation that our photoreceptor-like cells lack other surrounding
   cell types, such as tissue-resident microglial cells [[165]80] and
   infiltrating cells such as monocytes and lymphocytes [[166]81], which
   are relevant to pathogenesis of RP. That may be why an antioxidant,
   NAC, did not restore decreased expression of CRYGD or F2R. However, our
   photoreceptor-like cells derived from EYS-RP should be a helpful
   cellular model, considering that rod-photoreceptor cells are primarily
   injured by the genetic mutations. Another limitation is that 4-PBA,
   which was found to restore downregulation of several EYS-associated
   genes, may not restore other unknown EYS-associated changes. We are
   planning to set up single-cell analysis to overcome this limitation.
   Our cellular model can be improved by using as yet undiscovered
   exogenous factors in the differentiation media. Furthermore, we will
   have to verify the present findings using other models such as
   iPSC-derived retinas or in vivo models in the future.

Conclusions

   In summary, we here compared gene expression profiles of
   photoreceptor-like cells derived from dermal fibroblasts of EYS-RP
   patients with those of age-matched healthy donors to determine the
   molecular mechanism of photoreceptor degeneration in EYS-RP patients.
   The photoreceptor-like cells were produced by redirect differentiation
   with four transcription factors, CRX, RAX, NeuroD and OTX2, as a
   cellular model. By global gene expression analysis, several
   retina-related and apoptosis/oxidative stress/ER stress/aging-related
   genes such as opsin gene, CRYGD and F2R were differentially expressed
   in EYS-RP patient-derived cells. Decreased expression levels were
   restored by addition of a chemical chaperone, 4-PBA. These results
   suggest that retinal degeneration of EYS-RP is due, at least in part,
   to ER stress and that the redirect differentiation method could be a
   valuable tool for disease model despite some limitations as a
   replacement for the degenerative retinas.

Supplementary Information

   [167]13287_2022_2827_MOESM1_ESM.pdf^ (189.4KB, pdf)

   Additional file 1: Table S1. EYS-RP-associated pathways.
   [168]13287_2022_2827_MOESM2_ESM.pdf^ (959.5KB, pdf)

   Additional file 2 Table S2. Primer list. Figure S1. Immunostaining of
   CRX and rhodopsin in photoreceptor-like cells derived from fibroblasts
   of a healthy individual (N#3) and EYS-RP (Pt#1) after 2 weeks of
   induction. Scale bar = 50 μm. Figure S2. Comparison of the effects of
   pharmacological treatment on gene expression among groups, N#3 and
   Pt#1, with or without supplement. The same graph is shown as in
   Fig. [169]4 with other analytical results. For the sake of simplicity,
   comparison with the gene expression level in N#3 without any supplement
   (-) is shown in Fig. [170]4. Comparison among other groups is shown
   here. Column represents mean ± SEM (n = 6 (N#3 without any supplement
   (-)), n = 3 (N#3 with supplement and Pt#1). a = p > 0.05, b = p > 0.01,
   c = p > 0.001; One-way ANOVA followed by Tukey’s honest test. Figure
   S3. Effect of drugs on gene expression in photoreceptor-directed
   fibroblasts derived from an EYS-RP patient, Pt#2. The fibroblast cells
   obtained from patients suffering from retinitis pigmentosa with
   heterozygous mutations in EYS gene (Pt#2) and an age-matched healthy
   individual (N#1) were transduced with mixture of retroviral vectors
   encoding CRX, RAX, NeuroD and OTX2. Gene expression was compared
   2 weeks post-transduction. The differentiation media were supplemented
   with four drugs namely, 4-phenylbutyric acid (4-PBA; 2 mM, 5 mM and
   10 mM), metformin (METF; 5 mM), rapamycin (Rapa; 4 nM, 10 nM, 20 nM,
   50 nM and 100 nM), N-acetyl-L-cysteine (NAC; 10 mM) or the vehicle
   (EtOH) or no addition (-). The gene expression levels with different
   pharmacological treatments were compared to those of photoreceptor-like
   cells derived from a normal volunteer (N#1) without any drug or
   vehicle. a = p > 0.05, b = p > 0.01, c = p > 0.001; One-way ANOVA
   followed by Dunnett’s test. Figure S4. A. Immunoblot of blue opsin and
   β-actin in HDF-a as negative control, photoreceptor-like cells derived
   from a healthy individual (N#3), EYS-RP (Pt#1) supplemented with 5 mM
   4-PBA (Pt#1 + 4PBA) (panel a) and relative expression of the blue opsin
   (panel b). The band intensity of blue opsin was normalized by β-actin.
   Data represent mean + SEM (n = 3). *p < 0.05, **p < 0.01; Oneway ANOVA
   followed by Tukey’s test. B. Effect of 4-PBA in relative expression of
   ERN in EYS-RP. (a) Relative ERN1 expression in photoreceptor-like cells
   derived from N#3 with/out vehicle (EtOH) and RP#1 supplemented with
   5 mM 4-PBA or vehicle. (b) Relative ERN1 expression in
   photoreceptor-like cells derived from Fib#1 with/out vehicle (EtOH) and
   RP#3 supplemented with 5 mM 4-PBA or vehicle. *p > 0.05; One-way ANOVA
   followed by Tukey’s test (n = 3). C. a) Immunoblot of CHOP and β–actin
   of photoreceptor-like cells derived from fibroblasts of N#3 and RP#1
   with 0 nM, 200 nM and 2 μM thapsigargin. b) Relative expression of CHOP
   in photoreceptor-like cells derived from fibroblasts of N#3 and RP#1
   without or without thapsigargin with 0 nM, 200 nM and 2 μM
   thapsigargin. Student’s t-test (n = 3 (0), n = 5 (200 nM), n = 5
   (2 μM)). D. Immunoblot of cleaved caspase-3 and β-actin in HDF-a
   treated with 2 μM staurosporine for 4 h before harvesting
   (HDF-a + Staruo) as positive control, photoreceptor-like cells derived
   from a healthy individual (N#3) and EYS-RP patient (Pt#1) supplemented
   with 5 mM 4-PBA (Pt#1 + 4PBA) (panel a) and expression level of the
   cleaved caspase-3 (panel b). The band intensity of cleaved caspase-3
   was normalized by β-actin. Data represent mean + SEM (n = 3).
   *p < 0.05, **p < 0.01; Oneway ANOVA followed by Tukey’s test. Figure
   S5. Immunostaining of cleaved caspase-3 and numbers of positively
   stained cells in photoreceptor-directed fibroblasts of healthy
   individual (N#3) and EYS-RP (Pt#1) 2 weeks post-transduction
   supplemented by thapsigargin (1 µM). Cells were fixed in chilled
   acetone at -20 °C for 20 min followed by rinsing and incubated with 4%
   PFA in PBS for 10 min. After washing and blocking, cells were incubated
   with primary antibody (cleaved caspase-3 (Asp175) (5A1E) Rabbit mAb
   #9664, 1: 500, Cell Signaling).

Acknowledgements