Figure 1 - Arrow Legend and Network Description

Figure 1 - Arrow legend and network description.

In the description below, all ASD candidate genes/proteins that were implicated through GWASs 1-5 (Supplementary Table 1) and the other candidate genes/proteins (Supplementary Tables 3 and 4) are indicated in bold. The (genes encoding) AKAPs (A-kinase anchor proteins) (Supplementary Table 4) are underlined.

When histamine molecules bind to the histamine h2 receptor (HRH2), a signalling cascade is initiated that stimulates steroidogenesis in Leydig cells through the activation of adenylate cyclase (ADCY) 1. ADCY produces cyclic AMP (cAMP) 2 which subsequently activates protein kinase A (PKA) 2. PKA is recruited and bound by cytoplasmic AKAP10, AKAP11, AKAP13 or NBEA - all expressed in Leydig cells (http://biogps.gnf.org/) - to regulate the activity of the L-type calcium channel CACNA1C 3. This channel is highly expressed in Leydig cells and transports calcium ions into these cells 4. An increased intracellular concentration of calcium is necessary for downregulating the expression of the mitochondrial steroidogenic acute regulatory protein (STAR) 4, which plays a key role in steroid hormone synthesis by enhancing the transformation of cholesterol into pregnenolone 5.

In addition, PKA bound to AKAP10 - which is also found in the mitochondria 3 - can positively regulate STAR activity 6. TBXAS1, a cell membrane protein, downregulates the expression of STAR and hence inhibits steroidogenesis in Leydig cells 7 (not shown). The transport of cholesterol molecules into the mitochondria of Leydig cells is facilitated by vimentin (VIM) 8, a protein that itself is activated/phosphorylated by the STK33 kinase 9. The produced pregnenolone is subsequently transported from the mitochondria into the smooth endoplasmic reticulum (SER), where it undergoes a series of metabolic reactions, resulting in the production of testosterone 10. Testosterone is then either further transformed into estradiol by the CYP19A1 (or aromatase) enzyme - which is found in the (membrane) of the SER 3 - or diffuses to the cytoplasm 10. From there, testosterone is secreted into the extracellular compartment and further transported in the blood circulation and across the blood brain barrier by passive diffusion as ‘free’ testosterone or through binding to several proteins, including sex hormone-binding globuline (SHBG) and albumin 3, 11-13 (not shown). The secretion of testosterone by Leydig cells is stimulated by an increased intracellular calcium concentration - which could be e.g. achieved by CACNA1C activity (see above) 14 - and downstream of a cascade that involves the MET membrane receptor.

MET is activated by its ligand, hepatocyte growth factor (HGF) 3, which itself is activated by extracellular hepatocyte growth factor activator (HGFA) proteins 3,15. Like testosterone, the produced estradiol diffuses from the SER to the cytoplasm, where it can be degraded to toxic metabolites, such as estrogen quinones (EQ) and estradiol glucuronides (EG) 3,16,17. The NQO2 reductase enzyme is involved in the detoxification and hence degradation of EQ 16, whilst ABCC1 is a testis-expressed membrane protein that transports EG out of the cell, and is in this way involved in the detoxification of these estradiol metabolites 3,17. One of the effects of EG is the inhibition of the activity of SLCO1C1, which transports the thyroid hormones T4 (full name: thyroxine) and - to a lesser extent - T3 (full name: triiodothyronine) into Leydig cells 3,18. T4 is further metabolised into the more active T3 by the membrane-located DIO2 enzyme 3. T3 is involved in upregulating the expression of STAR 19 (see above).

The cell nucleus is an important compartment in the steroidogenesis network, as the expression of many network proteins is regulated from here. Dachshund homolog 2 (DACH2) is a transcription factor that downregulates the expression of CDKN1B 20, a cytoplasmic protein that is involved in steroidogenesis by upregulating the expression of STAR 21. Conversely, HDAC2 belongs to a repressive transcription factor complex 3 that downregulates the expression of STAR 22. The RORA transcription factor upregulates the expression of CYP19A1 23, and GATA4 is involved in activating the transcription of both STAR and CYP19A1 24. In turn, the transcriptional activity of GATA4 is repressed by the DNA-binding protein JARID2 25 and positively regulated by PKA 26 bound/anchored to nuclear AKAP8 3 . In addition, AKAP8 directly binds nuclear CASP3 27 , which in turn degrades GATA1 28, a transcription factor involved in regulating streoidogenesis in Leydig cells 29. THOC1, a component of the nuclear TREX complex, upregulates the expression of GATA1 30. Lastly, although as yet, they cannot be directly linked to the above described network, RHOXF1, RHOXF2 3,31, MAMLD1 and MAML2 32 are all nuclear proteins/transcription factors that are expressed in Leydig cells and are involved in steroidogenesis.

References

1. Mondillo C, Patrignani Z, Reche C, Rivera E, Pignataro O. Dual role of histamine in modulation of Leydig cell steroidogenesis via HRH1 and HRH2 receptor subtypes. Biol Reprod 2005; 73: 899-907.

2. Jones DC, Kuhar MJ Cocaine-amphetamine-regulated transcript expression in the rat nucleus accumbens is regulated by adenylyl cyclase and the cyclic adenosine 5'-monophosphate/protein kinase a second messenger system. J Pharmacol Exp Ther 2006; 317: 454-461.

3. Uniprot Consortium. The Universal Protein Resource (UniProt) in 2010. Nucleic Acids Res 2010; 38: D142-D148.

4. Pandey AK, Li W, Yin X, Stocco DM, Grammas P, Wang X. Blocking L-type calcium channels reduced the threshold of cAMP-induced steroidogenic acute regulatory gene expression in MA-10 mouse Leydig cells. J Endocrinol 2010; 204: 67-74.

5. Wang L, Sunahara RK, Krumins A, Perkins G, Crochiere ML, Mackey M et al. Cloning and mitochondrial localization of full-length D-AKAP2, a protein kinase A anchoring protein. Proc Natl Acad Sci USA 2001; 98: 3220-3225.

6. Dyson MT, Kowalewski MP, Manna PR, Stocco DM. The differential regulation of steroidogenic acute regulatory protein-mediated steroidogenesis by type I and type II PKA in MA-10 cells. Mol Cell Endocrinol 2009; 300: 94-103.

7. Wang X, Yin X, Schiffer RB, King SR, Stocco DM, Grammas P. Inhibition of thromboxane a synthase activity enhances steroidogenesis and steroidogenic acute regulatory gene expression in MA-10 mouse Leydig cells. Endocrinology 2008; 149: 851-857.

8. Hall PF. The roles of calmodulin, actin, and vimentin in steroid synthesis by adrenal cells. Steroids 1997; 62: 185-189.

9. Brauksiepe B, Mujica AO, Herrmann H, Schmidt ER. The Serine/threonine kinase Stk33 exhibits autophosphorylation and phosphorylates the intermediate filament protein Vimentin. BMC Biochem 2008; 9: 25.

10. Scott HM, Mason JI, Sharpe RM. Steroidogenesis in the fetal testis and its susceptibility to disruption by exogenous compounds. Endocr Rev 2009; 30: 883-925.

11. Pardridge WM. Serum bioavailability of sex steroid hormones. Clin Endocrinol Metab 1986; 15: 259-278.

12. Nanjee MN, Rajput-Williams J, Samuel L, Wootton R, Miller NE. Relationships of plasma lipoprotein concentrations to unbound, albumin-bound and sex hormone-binding globulin-bound fractions of gonadal steroids in men. Eur J Clin Invest 1989; 19: 241-245.

13. Hobbs CJ, Jones RE, Plymate SR. The effects of sex hormone binding globulin (SHBG) on testosterone transport into the cerebrospinal fluid. J Steroid Biochem Mol Biol 1992; 42: 629-635.

14. Wang SW, Hwang GS, Chen TJ, Wang PS. Effects of arecoline on testosterone release in rats. Am J Physiol Endocrinol Metab 2008; 295: E497-E504.

15. Del Bravo J, Catizone A, Ricci G, Galdieri M. Hepatocyte growth factor-modulated rat Leydig cell functions. J Androl 2007; 28: 866-874.

16. Gaikwad NW, Yang L, Rogan EG, Cavalieri EL. Evidence for NQO2-mediated reduction of the carcinogenic estrogen ortho-quinones. Free Radic Biol Med 2009; 46: 253-262.

17. Letourneau IJ, Deeley RG, Cole SP. Functional characterization of non-synonymous single nucleotide polymorphisms in the gene encoding human multidrug resistance protein 1 (MRP1/ABCC1). Pharmacogenet Genomics 2005; 15: 647-657.

18. Westholm DE, Salo DR, Viken KJ, Rumbley JN, Anderson GW. The blood-brain barrier thyroxine transporter organic anion-transporting polypeptide 1c1 displays atypical transport kinetics. Endocrinology 2009; 150: 5153-5162.

19. Manna PR, Kero J, Tena-Sempere M, Pakarinen P, Stocco DM, Huhtaniemi IT. Assessment of mechanisms of thyroid hormone action in mouse Leydig cells: regulation of the steroidogenic acute regulatory protein, steroidogenesis, and luteinizing hormone receptor function. Endocrinology 2001; 142: 319-331.

20. Li X, Perissi V, Liu F, Rose DW, Rosenfeld MG. Tissue-specific regulation of retinal and pituitary precursor cell proliferation. Science 2002; 297: 1180-1183.

21. Lin H, Hu GX, Dong L, Dong Q, Mukai M, Chen BB et al. Increased proliferation but decreased steroidogenic capacity in Leydig cells from mice lacking cyclin-dependent kinase inhibitor 1B. Biol Reprod 2009; 80: 1232-1238.

22. Clem BF, Clark BJ. Association of the mSin3A-histone deacetylase 1/2 corepressor complex with the mouse steroidogenic acute regulatory protein gene. Mol Endocrinol 2006; 20: 100-113.

23. Odawara H, Iwasaki T, Horiguchi J, Rokutanda N, Hirooka K, Miyazaki W et al. Activation of aromatase expression by retinoic acid receptor-related orphan receptor (ROR) alpha in breast cancer cells: identification of a novel ROR response element. J Biol Chem 2009; 284: 17711-17719.

24. Bouchard MF, Taniguchi H, Viger RS. The effect of human GATA4 gene mutations on the activity of target gonadal promoters. J Mol Endocrinol 2009; 42: 149-160.

25. Kim TG, Chen J, Sadoshima J, Lee Y. Jumonji represses atrial natriuretic factor gene expression by inhibiting transcriptional activities of cardiac transcription factors. Mol Cell Biol 2004; 24: 10151-10160.

26. Tremblay JJ, Viger RS. Novel roles for GATA transcription factors in the regulation of steroidogenesis. J Steroid Biochem Mol Biol 2003; 85: 291-298.

27. Kamada S, Kikkawa U, Tsujimoto Y, Hunter T. A-kinase-anchoring protein 95 functions as a potential carrier for the nuclear translocation of active caspase 3 through an enzyme-substrate-like association. Mol Cell Biol 2005; 25: 9469-9477.

28. Ribeil JA, Zermati Y, Vandekerckhove J, Cathelin S, Kersual J, Dussiot M et al. Hsp70 regulates erythropoiesis by preventing caspase-3-mediated cleavage of GATA-1. Nature 2007; 445: 102-107.

29. Qamar I, Park E, Gong EY, Lee HJ, Lee K. ARR19 (androgen receptor corepressor of 19 kDa), an antisteroidogenic factor, is regulated by GATA-1 in testicular Leydig cells. J Biol Chem 2009; 284: 18021-18032.

30. Wang X, Chinnam M, Wang J, Wang Y, Zhang X, Marcon E et al. Thoc1 deficiency compromises gene expression necessary for normal testis development in the mouse. Mol Cell Biol 2009; 29: 2794-2803.

31. Wayne CM, MacLean JA, Cornwall G, Wilkinson MF. Two novel human X-linked homeobox genes, hPEPP1 and hPEPP2, selectively expressed in the testis. Gene 2002; 301: 1-11.

32. Fukami M, Wada Y, Okada M, Kato F, Katsumata N, Baba T et al. Mastermind-like domain-containing 1 (MAMLD1 or CXorf6) transactivates the Hes3 promoter, augments testosterone production, and contains the SF1 target sequence. J Biol Chem 2008; 283: 5525-5532.

Figure 2a - Arrow legend and network description.

In the description below, all ASD candidate genes/proteins that were implicated through GWASs 1-5 (Supplementary Table 1) and the other candidate genes/proteins (Supplementary Tables 3 and 4) are indicated in bold. The (genes encoding) AKAPs (A-kinase anchor proteins) (Supplementary Table 4) are underlined.

Signalling through the network can be initiated at the neuronal cell membrane where the binding of ligands from the extracellular matrix/compartment to their respective receptors leads to the modulation of several downstream molecular cascades in the cytoplasm and nucleus that are involved in neurite outgrowth.

An important signalling cascade in the network is involved in regulating the activity of CDC42, a GTP-hydrolyzing (GTPase) protein that is an important mediator of directed neurite outgrowth 1,2. Activated CDC42 regulates a number of downstream effectors to facilitate neurite outgrowth, such as the cytoplasmic extracellular signal related kinase (ERK)1 3, which in turn phosphorylates/activates a number of cytoskeleton-associated proteins in the neuronal growth cone (see Figure 2b). Netrin axonal guidance ‘cues’ bind to the netrin receptor DCC which activates CDC42 and (eventually) results in neurite attraction towards the netrin guidance cues 4. In this respect, sonic hedgehog (SHH) molecules act as guidance cues that can mimic and supplement the chemoattractive effect of netrin on axons/neurites 5 . In addition, GDNF binding to the GFRA1 receptor activates CDC42 6, as does the membrane-located RHOG 7, which itself is activated by SGEF 8. NGF binding to NTRK1 activates the regulatory kinase PI3K 8,9, which in turn activates CDC42 10 as well as MSN 11,12. PI3K is also activated downstream of FGF1 binding to the FGFR1 membrane receptor 149,150 (not shown) and inhibited by PTEN 15, a protein that is regulated/activated by the MAST3 16 and FRK 17 kinases and degraded by cytoplasmic caspase-3 (CASP3) 18, which also degrades CDC42 itself 19. NTRK1 can also be bound by the peripheral membrane adaptor protein SH2B1, which acts as an enhancer/positive regulator of NGF-induced 20 and also FGF1-induced 21 neurite outgrowth. NEDD4L, a cytoplasmic E3 ubiquitin-protein ligase, binds and degrades NTRK1 22. Adenosine deaminase (ADA) catalyzes the hydrolysis of adenosine to inosine and is located in the cell membrane 8.

ADA binding ADORA1 promotes its activation by enhancing the binding of adenosine to this receptor 23. Activation of ADORA1 triggers a signalling cascade that leads to the inhibition of NGF-induced neurite outgrowth 24,25. When the SLIT guidance cues bind the ROBO1 receptor, the peripheral membrane proteins SRGAP1 and SRGAP3 associate with ROBO1 and inhibit CDC42 8, 26. SLITRK5 is a neuronal membrane protein that is involved in suppressing neurite outgrowth 8 and may also feed into the network because it shows a very high homology to the SLIT proteins 27 that bind ROBO1 8,28. Another important upstream regulator of CDC42 is protein kinase A (PKA), which itself has been repeatedly linked to regulating neurite outgrowth 29-31. PKA can be recruited by and bound to cytoplasmic AKAP7, AKAP10, AKAP11, AKAP13 or MSN 8,32, subsequently activating CDC42 33. MSN is positively regulated/activated by RHOA 34 which, like CDC42, is a GTP-hydrolyzing (GTPase) protein and a mediator of neurite outgrowth 35,36. RHOA itself can be activated by AKAP13 8,37 as well as ARHGEF11 8 and is inhibited by ARHGAP36 8.

A second important signalling pathway in the network leading to neurite outgrowth is CDC42-independent and centers around catenin beta (CTNNB). The N(euronal)-cadherin CDH2, which is anchored in the neuronal cell membrane and the neuronal cytoskeleton, binds to a complex consisting of CTNNB and catenin alpha 2 (CTNNA2) or catenin alpha 3 (CTNNA3) 8, 38-40. CDH2 is functionally modulated by and stably associated with cell surface N-acetylgalactosaminyltransferases such as GALNT13 and GALNTL4 8,41. In addition to inhibiting CDC42 (see above), SLIT binding to ROBO1 can inactivate CDH2, which leads to the dissolution of the CDH2-CTNNB-CTNNA2/3 complex and the CTNNB protein being released and targeted to the nucleus, where it functions as a transcription factor 38 (see below). Similar to CDH2, the neuronal cadherin CDH22 - which is mainly expressed in developing brain regions 42 - is anchored in the cell membrane and cytoskeleton and forms the same complex 8, 38-40. The nuclear translocation and hence transcriptional activity of CTNNB can be inhibited by the cytoplasmic FHIT protein 43. APCDD1 is a membrane protein that functions upstream of CTNNB and that is involved in neurogenesis during brain development by inhibiting WNT3A 44 (not shown), which itself regulates neurite outgrowth 45. Another membrane protein functioning upstream of CTNNB is LRP1B, a receptor that forms a complex with and inhibits the degradation of APP 46, a neuronal membrane protein that modulates the degradation of cytoplasmic CTNNB 47, plays an important role in both the etiology of Alzheimer's disease 46 and is involved in neurite outgrowth 48. The cytoplasmic DAB1 49 and the membrane protein COLEC12 (collectin-12) 50 bind APP and modulate its degradation and clearance. APP is also degraded by cytoplasmic CASP3 51 and it regulates the expression of NENF 52, which encodes neudesin, a secreted neurotrophic factor that is involved in the differentiation and growth of developing neurons through ERK1-, PKA- and PI3K-dependent signaling 8, 53 (not shown). Furthermore, CUL1 54 and CASP3 55 enhance the ubiquination/degradation of cytoplasmic CTNNB, whilst USP9X inhibits this process 56. Hyperphosphorylation and subsequent degradation of CTNNB can be achieved by the kinase GSK3B 57, which itself can be inhibited by ZBED3 58. Moreover, GSK3B can bind to AKAP11 together with PKA, after which the bound PKA inhibits GSK3B activity 59.