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Possible Role of Pineal Allopregnanolone in Purkinje Cell Survival

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Possible Role of Pineal Allopregnanolone in Purkinje Cell Survival Possible role of pineal allopregnanolone in Purkinje cell survival Shogo Haraguchia,b, Sakurako Haraa, Takayoshi Ubukaa, Masatoshi Mitab, and Kazuyoshi Tsutsuia,1 aLaboratory of Integrative Brain Sciences, Department of Biology and Center for Medical Life Science, Waseda University, Tokyo 162-8480, Japan; and bDepartment of Biology, Faculty of Education, Tokyo Gakugei University, Tokyo 184-8501, Japan Edited by Bruce S. McEwen, The Rockefeller University, New York, NY, and approved November 5, 2012 (received for review June 25, 2012) It is believed that neurosteroids are produced in the brain and other We thus hypothesized that pineal neurosteroid(s) may be involved nervous systems. Here, we show that allopregnanolone (ALLO), in Purkinje cell survival during development. a neurosteroid, is exceedingly produced in the pineal gland com- In the present study, we first demonstrated the biosynthetic pared with the brain and that pineal ALLO acts on the Purkinje cell, pathway of neurosteroids in the pineal gland of quail. We found a principal cerebellar neuron, to prevent apoptosis in the juvenile that the pineal gland of quail chicks exceedingly synthesizes ALLO quail. We first demonstrated that the pineal gland is a major organ compared with the brain. Px decreased ALLO concentration in the of neurosteroidogenesis. A series of experiments using molecular cerebellum and induced apoptosis of Purkinje cells, whereas ad- and biochemical techniques has further demonstrated that the ministration of ALLO to Px quail chicks prevented apoptosis of pineal gland produces a variety of neurosteroids de novo from Purkinje cells. Administration of ALLO to Px quail chicks also cholesterol in the juvenile quail. Importantly, ALLO was far more decreased the expression of caspase-3, a key protease in the apo- actively produced in the pineal gland than in the brain. Pinealectomy ptotic pathway, in Purkinje cells. Thus, ALLO rescues developing (Px) decreased ALLO concentration in the cerebellum and induced Purkinje cells in the cerebellum from apoptosis. Our results further apoptosis of Purkinje cells, whereas administration of ALLO to Px point to a role of pineal ALLO in preventing the death of de- quail chicks prevented apoptosis of Purkinje cells. We further found veloping Purkinje cells. that Px significantly increased the number of Purkinje cells that expressed active caspase-3, a key protease in apoptotic pathway, Results and daily injection of ALLO to Px quail chicks decreased the number De Novo Pregnenolone Formation from Cholesterol in the Quail Chick NEUROSCIENCE of Purkinje cells expressing active caspase-3. These results indicate Pineal Gland. Because pregnenolone (PREG) formation is the first that the neuroprotective effect of pineal ALLO is associated with the step in steroid synthesis (14, 15), we first investigated whether the decrease in caspase-3 activity during the early stage of neuronal pineal gland synthesizes PREG from cholesterol de novo. We used development. We thus provide evidence that the pineal gland is an pineal glands of male juvenile quail of posthatch day 7 (P7). Ste- important neurosteroidogenic organ and that pineal ALLO may be roidogenic acute regulatory protein (StAR; gene name StAR) involved in Purkinje cell survival during development. This is an im- delivers cholesterol to the mitochondrial cytochrome P450 side- portant function of the pineal gland in the formation of neuronal chain cleavage enzyme (P450scc; gene name Cyp11a)thatproduces circuits in the developing cerebellum. PREG. Reverse transcription PCR (RT-PCR) analyses have dem- onstrated the expressions of StAR and Cyp11a mRNAs in the pineal steroid | melatonin | HPLC | gas chromatography/mass spectrometry gland (Fig. 1 A and B and Table S1). The amplified cDNA bands from the pineal gland were sequenced, and it was verified that he cerebellar cortex has been used as an excellent model to they were authentic fragments of StAR (GenBank accession no. Tstudy synaptic formation and transmission of neural networks NM204686) and Cyp11a (GenBank accession no. NM001001756). because it forms relatively simple neuronal networks compared To investigate PREG formation from cholesterol in the quail with those of other brain regions. The Purkinje cell is a principal pineal gland, pineal glands of male chicks at P7 were incubated cerebellar neuron that integrates the process of memory and with tritiated cholesterol as a precursor, and the radioactive me- learning. It is known that in birds and mammals pinealectomy (Px) tabolite was analyzed by reversed-phase HPLC. As shown in Fig. induces cell loss in the brain including Purkinje cells during de- 1C, a single radioactive peak was detected, and it exhibited the velopment (1, 2). This observation suggests that a certain compo- same retention time as that of tritiated PREG, a reference stan- nent(s) in the pineal gland contributes to Purkinje cell survival dard, under the same chromatographic condition. The radioactive during development. peak corresponding to PREG increased in a time-dependent It is now established that steroids can be synthesized de novo manner from 0 to 24 h of incubation (Fig. 1C). In addition, 50 μM in the central and peripheral nervous systems. Such steroids are aminoglutethimide, an inhibitor of P450scc, reduced the ampli- “ ” called neurosteroids, and de novo neurosteroidogenesis from tude of this peak (Fig. 1C). cholesterol is a conserved property of the vertebrate brain (for PREG synthesis in the pineal gland was further demonstrated – reviews, see refs. 3 7). The Purkinje cell is known as a site of by gas chromatography/mass spectrometry (GC-MS) as described neurosteroidogenesis in the brain (for review, see ref. 8). This previously (16–21). Heptafluorobutyrate derivatives of the au- cerebellar neuron produces progesterone (PROG) and estradiol- thentic PREG and the metabolite of nonradioactive cholesterol 17β (E2) de novo from cholesterol during neonatal life, when cerebellar neuronal circuit formation occurs. Both PROG and E2 promote dendritic growth, spinogenesis, and synaptogenesis Author contributions: S. Haraguchi and K.T. designed research; S. Haraguchi, S. Hara, and via each cognate nuclear receptor in the developing Purkinje cell K.T. performed research; S. Haraguchi, T.U., and K.T. analyzed data; and S. Haraguchi, (9–11). Allopregnanolone (ALLO; 3α,5α-tetrahydroprogester- T.U., M.M., and K.T. wrote the paper. one), a progesterone metabolite, is also synthesized in the cer- The authors declare no conflict of interest. ebellum and facilitates Purkinje cell survival in the neonate (12). This article is a PNAS Direct Submission. Until recently, we believed that neurosteroids are produced Freely available online through the PNAS open access option. only in the brain and other nervous systems. On the basis of our 1To whom correspondence should be addressed. E-mail: [email protected]. previous study of chickens (13), however, the pineal gland may be a This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. major organ in producing neurosteroids de novo from cholesterol. 1073/pnas.1210804109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1210804109 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 quail pineal gland (Fig. 1F). No immunoreactivity was observed when anti-P450scc antibody was preadsorbed with chicken P450scc protein (Fig. 1G). Neurosteroid Formations from PREG in the Quail Chick Pineal Gland. To investigate the biosynthetic pathway of neurosteroids in the pineal gland, pineal glands of male quail chicks at P7 were used for RT-PCR analyses to demonstrate the expressions of steroidogenic enzymes, such as cytochrome P450 7α-hydroxylase (P4507α; gene name Cyp7b), 3β-hydroxysteroid dehydrogenase/Δ5-Δ4-isomerase (3β-HSD; gene name Hsd3b), 5α-reductase (gene name Srd5a), cytochrome P450 17α-hydroxylase/c17,20-lyase (P45017α,lyase; gene name Cyp17), 17β-hydroxysteroid dehydrogenase (17β-HSD; gene name Hsd17b), and cytochrome P450 aromatase (P450arom; gene name Cyp19). RT-PCR analyses showed cDNA bands of Fig. 1. De novo PREG formation from cholesterol in the pineal gland. (A and B) RT-PCR analyses of StAR (A)andCyp11a (B) mRNAs in the pineal gland of male quail chicks. Total RNA extracted from the tissue was reverse-transcribed with (+) or without (−) reverse transcriptase (RTase) followed by PCR ampli- fication. (C) HPLC analysis of PREG formation from cholesterol in the pineal gland of male quail chicks. The pineal gland was incubated with [3H]choles- terol and homogenized after different incubation times, and then each ex- tract was subjected to HPLC. The pineal gland with [3H]cholesterol was also incubated with aminoglutethimide. The arrowheads indicate the elution positions of the substrate cholesterol (open arrowhead) and its metabolite PREG (solid arrowhead). (D) GC-SIM analysis of the metabolite of non- radioactive cholesterol by the pineal gland of male quail chicks. GC-SIM was traced at m/z 298 for PREG as the metabolite of nonradioactive cholesterol. The arrowhead shows the peak corresponding to authentic PREG. (E) Western Fig. 2. Neurosteroid formation from PREG in the pineal gland. (A, D, and G) blot analysis of the pineal gland and extracts of COS-7 cells transfected with RT-PCR analyses of steroidogenic enzyme Cyp7b (A), Hsd3b (D), Srd5a (G), chicken Cyp11a cDNA with the anti-human P450scc antibody. Anti-human and Cyp17 (G) mRNAs in the pineal gland of male quail chicks. Total RNA was P450scc antibody was preadsorbed
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