Introduction
Retinoic acid (RA), synthesized from vitamin A through a series of oxidative steps, is a lipid regulator of transcription and is the mediator of the majority of vitamin A’s functions. In addition to the enzymes that synthesize RA, the binding proteins transporting RA in the cytoplasm and the nuclear retinoic acid receptors (RARs) that transduce the signal, the capacity to switch off the RA signal is vital for the action of the RA signaling pathway. This task is performed by a subset of the cytochrome P450 family: Cyp26a1, Cyp26b1 and Cyp26c1 (McCaffery and Simons
2007; Ross and Zolfaghari
2011). These microsomal enzymes oxidize RA to a range of metabolites including 4-hydroxy-RA, 18-hydroxy-RA, and 4-oxo-RA (Chithalen et al.
2002). The essential need to turn off the RA signal is illustrated by the embryonic or early postnatal lethality of
Cyp26a1 or
Cyp26b1 null mutations. Loss of
Cyp26a1 results in disruption of the posterior end of the embryo and a caudal regression phenotype, as well as disorganization of the normal patterning of the hindbrain (Abu-Abed et al.
2001). Null mutation of
Cyp26b1 leads to defects in limb development, among other abnormalities (Yashiro et al.
2004).
Cyp26b1 is also required to control timing of meiosis in germ cells of the developing mouse (Bowles et al.
2006). In contrast to its counterparts,
Cyp26c1 is expressed to a lesser extent in the embryo and its loss in the mouse has few effects, although mutation in the human results in a range of facial skin defects (Slavotinek et al.
2013).
It is generally thought that the principal role for the Cyp26 enzymes in embryonic development is to limit the diffusion of RA synthesized from vitamin A, creating regions of high and low RA concentration. This controls the patterns of gene expression that guide development (McCaffery and Simons
2007; Ross and Zolfaghari
2011). In the embryo, the enzymes that synthesize RA from vitamin A (retinol) are primarily retinol dehydrogenase 10 (Rdh10), oxidizing retinol to retinaldehyde, which is then further oxidized to RA by retinaldehyde dehydrogenases 1, 2 and 3 (Raldh1, 2, 3; also known as Aldh1a1, 1a2, 1a3) (Napoli
2012). Swindell and coworkers published a general observation that the regions of RA synthesis in the embryo do not overlap with the areas of RA degradation, which then creates the patterns of RA distribution (Swindell et al.
1999). This implies that RA predominantly acts as a paracrine factor in the developing embryo. Is this the case for its later function in the adult? This new study examined such properties in the adult brain and supports previous research indicating that RA can have such a paracrine function in regions such as the rodent hippocampus, where it performs a number of key functions (Aoto et al.
2008; Chen and Napoli
2008). Surprisingly, it was also found that RA can function in an autocrine manner in the adult human brain, with co-expression of the RA synthetic and catabolic enzymes in the same cell controlling intracellular RA concentration and signaling. This reveals an important aspect of the flexibility of RA signaling in the nervous system.
Methods
Tissues
The experiments using human tissue were approved by the Ethics Committee of the Universidade Metropolitana de Santos, SP, Brazil, and by the Brazilian Health Research Committee on April 4th 2011, under the number CONEP 16168, documents registered under the number 25000.169694/2010-18. Samples of liver, cerebellum and caudal human hippocampus from six male individuals aged 55 years or less, who did not present any neurological or psychiatric disease, were collected during post mortem procedures. Brains from individuals whose death was related to head trauma, extensive infection, or toxic, anoxic or metabolic injuries were excluded from this study. Samples from the hippocampus, measuring typically 5 mm on each side, were fixed by immersion in 10 % phosphate-buffered formalin within 24 h of death and processed into paraffin wax blocks within the following 24 h. Other samples were collected in RNAlater RNA Stabilization Reagent (Qiagen) and stored at 4 °C for qPCR or were fresh-frozen on dry ice and stored at −80 °C for analysis by western blot.
All animal procedures were carried out in accordance with UK Home Office regulations on laboratory animal use according to the Animals (Scientific Procedures) Act 1986 and local ethics committee guidelines.
Detection of Cyp26 expression by polymerase chain reaction (PCR)
Male and female adult rats were euthanized by overdose of pentobarbital and their brains removed and placed on ice. Medial cortex, lateral cortex, hippocampus, striatum, thalamus, hypothalamus, olfactory bulb, cerebellum, choroid plexus, and meninges were dissected and rapidly frozen on dry ice. Total RNA was extracted from the tissues using a Qiagen RNeasy kit. cDNA was synthesized from 500 ng of RNA using a High Capacity RNA-to-cDNA kit (Applied Biosystems) according to the manufacturer’s protocol. Primers used were
Cyp26a1 (NM_130408.2): For, TTC GGG TGG CTC TGA AGA CT, Rev, CCT CTG GAT GAA GCG ATG TAA AT.
Cyp26b1 (NM_181087.2): For, CCA GGA CTG TAT GCC CAT GA, Rev, CCA CTC ACC AAC AAA AAG ACA AG. All PCR primers used in this study were designed using PrimerBLAST (Ye et al.
2012), apart from rat
Cyp26b1 primers, which were designed and supplied by PrimerDesign Ltd. PCR products were visualized by agarose gel electrophoresis and UV transillumination.
Detection of Cyp26b1 expression by in situ hybridization
The
Cyp26b1 riboprobe template was prepared as previously described (Helfer et al.
2012) from a 925 bp cDNA fragment corresponding to 2507–3431 bp of the mouse
Cyp26b1 cDNA sequence (NM_001177713.1).
In situ hybridization was carried out as previously described using
35S-labeled riboprobe (Ross et al.
2009).
In vivo inhibition of CYP26 activity
The CYP26 inhibitor ser 2–7 (methyl 3-(1
H-imidazol-1-yl)-2,2-dimethyl-3-(4-(naphthalen-2-ylamino)phenyl)propanoate; Gomaa et al.
2011) was dissolved at 8 mg/ml in DMSO. Immediately before use, the ser 2–7 solution was mixed with an equal volume of sterile phosphate-buffered saline (PBS) to give a 4 mg/ml solution. For measurement of retinoic acid levels in tissue, female C57BL/6 mice between 8 and 12 weeks of age received a single intraperitoneal (IP) injection of 10 mg/kg ser 2–7. Control animals received an intraperitoneal (IP) injection of an equivalent volume of 1:1 PBS/DMSO. Treated animals were euthanized using pentobarbital 2, 6 or 18 h after ser 2–7/vehicle administration and the hippocampi rapidly dissected on ice and snap frozen on dry ice. Samples were stored at −80 °C before use.
For cell proliferation studies, RARE-
LacZ mice (Rossant et al.
1991) between 8 and 12 weeks of age received 10 mg/kg ser 2–7 by IP injection once every 24 h for 3 days. Injections were carried out in the morning on each day. On the third day, immediately after the last ser 2–7 treatment, all mice received three additional injections, 2 h apart, of 50 mg/kg bromodeoxyuridine (BrdU) in PBS. 2 h after the final BrdU injection, the mice were perfused transcardially first with PBS, then 4 % paraformaldehyde (PFA) in phosphate buffer. The brain was removed and placed at 4 °C in PFA overnight, then transferred to 30 % sucrose at 4 °C.
Measurement of RA levels in tissue
A reporter gene assay was used as described elsewhere (Helfer et al.
2012) to measure levels of RA activity in the hippocampus of ser 2–7-injected mice. Sil-15 cells carrying a
LacZ reporter gene driven by multiple retinoic acid response elements (Wagner et al.
1992) were maintained in DMEM/F-12 containing 10 % fetal calf serum and 0.8 mg/ml G418. Hippocampi from ser 2–7-/vehicle-injected mice were homogenized under low light conditions in an ice-cold 2:1 mix of isopropanol/ethanol containing 1 mg/ml butylated hydroxytoluene to reduce oxidation. Homogenates were centrifuged at 13,000 rpm for 5 min at 4 °C. The supernatant was diluted 1:50 in DMEM/F-12 and added to a 96-well plate containing Sil-15 cells followed by overnight incubation at 37 °C, 5 % CO
2. The cells were fixed in 1 % glutaraldehyde and β-galactosidase activity was quantified using X-gal and colorimetric analysis. The response of Sil-15 cells to tissue extracts was compared to a standard curve plotted from the response to a series of known RA concentrations from 1 μM to 1 pM.
Immunohistochemical measurement of cell proliferation using bromodeoxyuridine (BrdU)
Perfused mouse brains were cut into 40 μm coronal sections using a cryostat. Sections were washed in PBS and then incubated in 1 M hydrochloric acid for 30 min at 47 °C, followed by further washes in PBS. Sections were then blocked in PBS containing 10 % goat serum and 0.3 % Triton X-100 for 2 h at room temperature and incubated overnight at 4 °C in anti-BrdU antibody (AbD Serotec) diluted 1:200 in blocking buffer. After washing in PBS containing 0.3 % Triton X-100 (PBST), sections were incubated in fluorescent anti-rat secondary antibody (1:300 in blocking buffer; Jackson ImmunoResearch) for 2 h at room temperature. Following further washes in PBST, sections were mounted onto slides using mounting medium containing bisbenzimide. Images were obtained by fluorescence microscopy and BrdU incorporation was quantified by counting of the number of BrdU-positive cells in every 12th section from each brain (sections were approximately 480 μm apart). The ratio between the total number of BrdU-positive cells in the supra- and infrapyramidal blades was calculated. Only cells within two cell diameters of the subgranular zone (SGZ; Kuhn et al.
1997) were included in cell counts.
Quantitative PCR
RNA was extracted from samples of human hippocampus, cerebellum and liver and cDNA synthesised as described above. GAPDH was used as a reference gene. Primer sequences: CYP26A1 (NM_000783.3, NM_057157.2): For, CAC CGT ACG GGT GAT GGG CG, Rev, GCT GGC CAG TGG ACC GAC AC; CYP26B1 (NM_001277742.1, NM_019885.3): For, ACC GGC CAC TGG CTG CTG, Rev, ACG TTG ATG GCC TCG GGG TG. GAPDH (NM_002046.5): For, TCT TTT GCG TCG CCA GCC GA, Rev, AGT TAA AAG CAG CCC TGG TGA CCA.
Western blotting
Human hippocampus was homogenized in 0.01 M phosphate buffer, pH 7.0, containing a broad spectrum protease inhibitor cocktail (Roche) using mechanical homogenization and three freeze–thaw cycles. Homogenates were centrifuged for 10 min at 12,000 rpm at 4 °C. Total protein levels in each sample were quantified using the bicinchoninic acid assay (Pierce) and 50 μg protein was loaded per lane of a 12 % SDS-PAGE mini-gel. After separation, the proteins were transferred onto a Hybond-ECL nitrocellulose membrane (GE Healthcare) using a Mini Trans-Blot Cell (Bio-Rad) and equal loading was confirmed with Ponceau-S (Sigma). Membranes were blocked for 1 h at room temperature in TBS containing 0.05 % Tween-20 (TBST) and 5 % skimmed milk and then probed with mouse anti-CYP26A1 antibody (1:1000 in blocking buffer; Vertebrate Antibodies) at 4 °C overnight. Membranes were washed in TBST and then incubated in HRP-conjugated anti-rabbit secondary antibody (1:3000; Jackson ImmunoResearch) for 1 h at room temperature. The membranes were washed again in TBST and antibody binding visualized by enhanced chemiluminescence (ECL; Millipore) and exposure to X-ray film (Thermo).
Detection of CYP26 in the human brain by immunohistochemistry
Formalin-fixed samples of human hippocampus were processed into paraffin wax blocks and sectioned at 7 μm. Sections were mounted on TruBond 380 slides (Electron Microscopy Sciences) and dried overnight at 37 °C. Sections were dewaxed in xylene and rehydrated through a series of decreasing ethanol concentrations (100, 95, 80 and 70 %). The slides were washed briefly in PBS, pH 7.4, and then boiled for 10 min in sodium citrate buffer, pH 6.0. After cooling, slides were washed in PBS containing 1 % Tween-20 (Sigma) and 1 % pooled human serum (Bio-Sera), hereafter referred to as PBS(HS). Tissue sections were incubated for 1 h at room temperature in blocking solution consisting of PBS, pH 7.4, containing 0.3 % Tween-20, 5 % goat serum, 5 % bovine serum albumin and 5 % pooled human serum (Makitie et al.
2013). Sections were then incubated with primary antibodies (chicken anti-MAP2, 1:400, Abcam; goat anti-RALDH2, 1:1000, Santa Cruz; mouse anti-CYP26A1, 1:50 Vertebrate Antibodies; goat anti-Iba1, 1:300, Abcam) diluted in blocking solution and incubated overnight at 4 °C. After incubation, the slides were washed in PBS(HS). The tissue was then incubated for 1 h at room temperature in appropriate fluorescent secondary antibodies (Jackson ImmunoResearch) diluted 1:300 in PBS(HS). Slides were washed in PBS(HS) and incubated for 1 min with 10 % Sudan black (Acros Organics) in 70 % isopropanol to reduce auto-fluorescence (Schnell et al.
1999; Neumann and Gabel
2002). The slides were then thoroughly washed in distilled water and mounted with mounting medium containing bisbenzimide (Sigma). The quantitation of number of labeled cells was based on counts of between 60 and 90 cells.
SH-SY5Y treatment with vitamin A and retinoic acid
SH-SY5Y neuroblastoma cells were maintained in DMEM/F12 containing 10 % fetal calf serum and penicillin/streptomycin and Glutamax (Invitrogen). Cells were treated with either 0.35 μM retinol and 0.3 μM retinyl acetate or 1 μM retinoic acid for 8 or 24 h with serum replaced with B27 supplement (Gibco). Retinoic acid was dissolved in DMSO and so this was added at an equivalent concentration (0.1 %) to wells containing untreated control cells. Following treatment, total RNA was extracted from the cells, cDNA synthesized and qPCR performed as described above using the following primers: RALDH2 (NM_003888.3, NM_170696.2, NM_170697.2, NM_001206897.1): For, CAC TGA GCA GGG TCC CCA GAT TGA, Rev, AAC CCC TTT CGG CCC AGT CCT; RALDH3 (NM_000693.3, NM_001293815.1): For, CGC AAC CTG GAG GTC AAG TTC ACC, Rev, AGC CTT GTC CAC GTC GGG CTT A. GAPDH was used as a reference gene.
Discussion
Cyp26a1 and Cyp26b1 are the primary enzymes for RA catabolism in the body (McCaffery and Simons
2007; Ross and Zolfaghari
2011). Much initial attention was directed to their role in the developing embryo (Abu-Abed et al.
2001; Yashiro et al.
2004) where a key element to their function arose from their spatial distribution—expression in regions adjacent to areas of high RA synthesis, limiting diffusion to create precise patterns of RA concentration. The distribution of retinoic acid synthetic and catabolic enzymes in the embryo was always found to be complementary, with RA diffusing from synthetic tissue and acting as a paracrine factor on tissues distant from this source (Swindell et al.
1999). Initial findings in the adult rodent brain pointed to a similar paracrine action for RA.
In the rodent brain the predominant source of RA is the meninges surrounding the brain (Goodman et al.
2012). In situ hybridization shows the expression of the catabolic enzyme
Cyp26b1 in subregions of the brain. Expression is strong in several areas of the forebrain, presumably limiting the concentration of RA in these regions. Such regions of high
Cyp26b1 expression include the lateral and medial cortex, but it is absent from the upper layers of the medial cortex where the synthetic enzyme Raldh3 is present, potentially setting up regions of RA-regulated plasticity in the cortex (Wagner et al.
2006). There are limited regions of RA signaling in the amygdala where it is also proposed to delineate zones of plasticity (Thompson Haskell et al.
2002) and these regions would be assumed to be complementary to the intense areas of
Cyp26b1 expression in subregions of the amygdala. These patterns of
Cyp26 expression, separated from regions of RA synthesis, suggest a paracrine action of RA.
This paracrine function for RA is exemplified by its role in control of cell proliferation in neurogenic regions in the rodent hippocampus.
In situ hybridization shows strong
Cyp26b1 expression in cells of the hilus between the two blades of the dentate gyrus in the hippocampus. We previously proposed that this reduces the amount of RA that can diffuse from the meninges adjacent to the infrapyramidal blade of the dentate gyrus to reach the upper, suprapyramidal blade. We have previously shown that this asymmetry in RA between the blades of the dentate gyrus contributes to the difference in rates of cell proliferation in the subgranular zones of the two blades. Neurogenesis in these regions is crucial for several aspects of hippocampus-dependent memory, including pattern separation (Schmidt et al.
2012), the process that reduces confusion of old memories with new. Differences in the functions of the two blades are also proposed to be important in the pattern separation task of the dentate gyrus (Schmidt et al.
2012). Normally the upper, suprapyramidal blade has significantly higher levels of cell proliferation than the lower blade with a two-fold ratio between the blades. Treatment of mice with the Cyp26 inhibitor ser 2–7 resulted in equalization of the ratio of BrdU incorporation between the upper and lower blades. In the normal animal, this may have a detrimental effect on pattern separation aspects of memory.
Although there may be a detrimental effect under normal circumstances, the effectiveness of ser 2–7 to increase RA in the hippocampus may have some benefit in disorders where RA signaling is believed to decline, such as Alzheimer’s disease. Several recent articles and reviews have proposed RA treatment for this disorder because of its action to promote the non-amyloidogenic pathway of amyloid precursor protein cleavage as well as its anti-inflammatory properties, promotion of cholinergic transmission, reduction of intracellular cholesterol and cell-protective properties (Ono and Yamada
2012; Sodhi and Singh
2014). Increasing hippocampal RA may be particularly beneficial because RA levels are declining in the aging hippocampus (Etchamendy et al.
2001). However, rather than administration of RA itself, the use of Cyp26 inhibitors to raise endogenous RA is a route less likely to result in excessively high and potentially damaging amounts of RA in the brain. Further, this method avoids the problem of retinoid resistance where addition of RA induces the Cyp26 enzymes (Ray et al.
1997), limiting the efficacy of RA-based treatments over time.
The study of CYP26 in the human, versus rodent, brain has revealed a wider distribution of the catabolic enzymes in the human, suggesting a more widespread action. We have previously shown that RA signaling in the human brain differed from that of the rodent with the RA synthetic enzymes RALDH1, 2 and 3 expressed not just in the surrounding meninges but also in many neurons (Fragoso et al.
2012), suggesting a higher requirement for RA in the human brain compared to the rodent. In this study qPCR confirmed expression of both
CYP26A1 and
CYP26B1 in the human brain. The methods employed did not provide an absolute comparison of the relative amounts but previous quantitative studies (Topletz et al.
2012) found about fivefold higher levels of
CYP26B1 than
CYP26A1 in the cerebral cortex, but in the cerebellum
CYP26B1 predominated by over 100-fold.
Given the expression of
CYP26A1 and
CYP26B1 transcripts in the human brain we investigated the distribution of CYP26A1 protein, for which we selected an antibody which gave only a single band by western blotting. Immunohistochemistry indicated that, like RALDH2 (Fragoso et al.
2012), CYP26A1 was present in neurons. Comparison of CYP26A1 and RALDH2 expression indicated a high degree of colocalization in granule neurons of the hippocampus and Purkinje neurons of the cerebellum. We have previously demonstrated that these neurons express RA receptors and their co-expression of both RA synthetic and catabolic enzymes implies the capacity for autocrine RA signaling with the ability to both upregulate and downregulate the RA signal. If this were the case it would be expected that the synthetic and catabolic enzymes would respond to relative levels of RA and its retinol precursor. Expression of the genes encoding the RALDH and CYP26 enzymes was examined in the SH-SY5Y neuroblastoma cell line. As previously reported in many studies on non-neural cells [for example (Ray et al.
1997; Zhang et al.
2010)], RA rapidly induces
CYP26A1 and
CYP26B1, although this induction is attenuated by 24 h. In contrast, transcript levels of the synthetic enzymes
RALDH2 and
RALDH3 were not influenced by RA; however, when retinol levels were increased there was a significant fall in
RALDH3 transcript after 24 h. Such feedback control by the substrate for RA has not been previously described for the synthetic enzymes and suggests a mechanism that could help maintain local RA levels in the brain relatively constant by helping to prevent wide fluctuations.
Summary
In the rodent brain RA can, as a paracrine signaling factor, diffuse across tissues to control localized gene expression as it does in the embryo. The Cyp26 catabolic enzymes, expressed in cells distant from the site of synthesis, help to regulate the pattern of RA diffusion. This separation between sites of synthesis and catabolism occurs in other adult rodent tissues such as the uterus (Ma et al.
2012). However, in human neurons RA can have a different, autocrine, function that is restrained by CYP26. Neurons, but not glia, in the adult human hippocampus and cerebellum express both CYP26A1 and RALDH2 which together can modulate the balance of RA within single cells. This contrasts with the embryo and adult rodent brain in which the synthetic and catabolic enzymes control the balance of RA broadly across tissues. This would be the first time that an autocrine RA signaling system, with the capacity to switch on and off the RA signal in individual neurons, has been proposed in the human brain.