Functional genomics indicate that schizophrenia may be an adult vascular-ischemic disorder

In search for the elusive schizophrenia pathway, candidate genes for the disorder from a discovery sample were localized within the energy-delivering and ischemia protection pathway. To test the adult vascular-ischemic (AVIH) and the competing neurodevelopmental hypothesis (NDH), functional genomic analyses of practically all available schizophrenia-associated genes from candidate gene, genome-wide association and postmortem expression studies were performed. Our results indicate a significant overrepresentation of genes involved in vascular function (P<0.001), vasoregulation (that is, perivascular (P<0.001) and shear stress (P<0.01), cerebral ischemia (P<0.001), neurodevelopment (P<0.001) and postischemic repair (P<0.001) among schizophrenia-associated genes from genetic association studies. These findings support both the NDH and the AVIH. The genes from postmortem studies showed an upregulation of vascular-ischemic genes (P=0.020) combined with downregulated synaptic (P=0.005) genes, and ND/repair (P=0.003) genes. Evidence for the AVIH and the NDH is critically discussed. We conclude that schizophrenia is probably a mild adult vascular-ischemic and postischemic repair disorder. Adult postischemic repair involves ND genes for adult neurogenesis, synaptic plasticity, glutamate and increased long-term potentiation of excitatory neurotransmission (i-LTP). Schizophrenia might be caused by the cerebral analog of microvascular angina.


Table&S1.&Discovery&sample&based&on&TDT?studies&&
A list of genes extracted from the SZgene database 1 hosted by the Schizophrenia Research Forum (http://www.szgene.org) was used as discovery sample. Reproducibility of association was considered more important than a low p value. The criterion for inclusion in this sample was a p-value ≤ 0.05 obtained by at least two independent research groups in TDT studies. As of July 7, 2011, the SZgene database contained 33 genes that fulfilled this criterion. The TDT-based 33 candidate genes of schizophrenia are listed below. This sample was employed as discovery sample for the construction of the putative schizophrenia pathway depicted in Figs. 1 and 2. The TDT gene sample was updated as requested by an anonymous reviewer during the peer review process. The update increased the number of TDT genes from 33 to 41. The additional genes were (34) ACSL6, (35) DRD1, (36) ERBB4, (37) GABRB2, (38) NRG3, (39) PIP5K2A, (40) SNAP25, (41) ZNF804A. The last TDT sample of 41 candidate genes obtained in December 2014 from the SZ database was employed for the intersection analysis of the combined five samples (discovery sample and four replication sample). The results are shown in Table 1.

Table&S2.&Replication&sample&#1&based&on&case?control&studies&&
Because of the higher rate of false positive findings in case-control studies 2 , the requirement for replication was increased. The choice of three independent groups for inclusion was guided by the need to validate the findings by the highest possible number of replications and to obtain at the same time a sufficiently large number of genes for statistical analysis. On July 7, 2011, 58 candidate genes for schizophrenia were found in the SZgene database 1 , which showed positive results in case-control studies by three independent groups. The case-control based candidate genes (CC genes) were The latest update of the SZgene database obtained in December 2014 did not change the number of genes from case-control studies. Figure S1. Venn diagram of vascular, ischemia, and vascular-ischemia genes overlapping with neurodevelopmental or repair genes. The diagram was produced by using GeneVenn (available at http://genevenn.sourceforge.net). Figure S2. STRING's evidence view revealing protein-protein interactions of candidate genes for schizophrenia employed in the present study. Data from the STRING database were used for constructing the energy-supply pathway shown in Figs. 1 and 2. 9. EGLN1, PHD2, disrupted in schizophrenia 1 (DISC1) (54583). The schizophreniaassociated Leu607Phe polymorphism hinders the axonal transport of mitochondria required for energy production in presynaptic terminals 148 . Furthermore, EGLN1 is part of the HIF-VHL-prolyl hydroxylase pathway 149 , which functions as a cellular oxygen sensor and, under normoxic conditions, targets the hypoxia-inducible factor (HIF-1) alpha protein through hydroxylation for ubiquitination and proteasomal degradation via the von Hippel-Lindau (VHL) complex 150,151 . HIF-1 is a transcriptional complex that plays a central role in mammalian oxygen homeostasis and regulates, under hypoxic conditions, the transcription of numerous genes related to angiogenesis, cell survival, and glucose metabolism 152 .

Figure&S2.&Results&of&interaction&analysis&by&STRING&
10. DRD2 (1813), dopamine receptor D2 is involved in peripheral vasoconstriction 153 . With regard to the brain, central dopaminergic neurones make close contacts with the basal lamina of arterioles and with astrocytic end-feet (reviewed in 154 ). Microinjection of dopamine causes a pronounced constriction of cerebral microvessels 69 . Moreover, D2 receptor agonists produced negative changes in regional cerebral blood volume (rCBV). On the other hand, D1/D5 receptor agonists and DAT blockers induce positive hemodynamic changes 155 .
12. DTNBP1 (84062), dystrobrevin binding protein 1. Dystrobrevin and probably its binding protein is localized in the astrocytic endfeet and endothelial cells of cerebral microvessels 157,158 and is part of the signal-transduction pathway for the α1D-adrenergic receptor (α1D-AR). The latter are ubiquitously expressed on vascular smooth muscle, cause vasoconstriction when activated by noradrenaline and adrenaline 159 and are responsible for increased blood pressure during exercise, injury, and stress (reviewed in 160 ).

GABRB2
(2561), gamma-aminobutyric acid A (GABA-A) receptor beta 2. Cortical GABA interneurons provide a rich innervation to local microvessels and appear to act as local integrators for the tight coupling of neuronal activity and local perfusion, which is essential for normal brain function 161 . GABA-A receptors are present in cerebral microvessels 162,163 and respond by vasodilatation to GABA released from nerve terminals 163,164 . Muscimol, a GABA-A receptor agonist, elicites vasodilation in hippocampal microvessels 163 . Vasodilatation by cholinergic neurons is in part mediated by the local release of GABA from cholinoceptive cortical interneurons and through GABA-A receptors 165 .
The transcription of GABRB2 itself is highly sensitive to hypoxia 166 and GABA-A receptors are involved in BBB disruption during cerebral ischemia 167 . With regard to cerebral ischemia, GABA exerts neuroprotective effects (reviewed in 168,169 ) via GABA(A) and GABA(B) receptors 170 . And GABAergic interneurons survive ischemic injury for up to 30 days in all investigated brain regions 171 . Finally, the induction of ischemic tolerance by preconditioning depends on functional modifications of GABA synapses 172 . * 14* localisation and role in cortical microvessels, see GABRB2 above.
15. HTR2A (3356), 5-hydroxytryptamine (serotonin) receptor 2A mediates vasoconstrictive responses to 5-HT in many vascular smooth muscles and also potentiates the activity of growth factors (reviewed in 173 ). Intracerebrally released serotonin cause a decrease of cerebral blood flow (CBF) in several brain regions such as the neocortex suggesting a major vasoconstrictor role 174 . In the CNS, 5-HT2A receptors are abundant in the cerebral cortex and the limbic system!and are expressed in neurons as well as in astrocytes 175 . Astrocytes are involved in the regulation of cerebral blood flow (CBF) (reviewed in 176 ). In addition, the HTR2A gene appears to be associated with ischemic stroke 177 . Antagonists of the 5-HT2A receptor such as ketanserin and ritanserin increase CBF in cortical areas and reduce ischemic damage (reviewed in 174 ). Finally, atypical antipsychotic drugs (such as clozapine, aripiprazole, asenapine, iloperidone, lurasidone, olanzapine, quetiapine, risperidone, and ziprasidone) produce extensive blockade of serotonin 5-HT2A receptors and stimulation of 5-HT1A receptors at clinically effective doses 178 . Both , i.e. 5-HT1A receptor agonists and 5-HT2 receptor antagonists, have a neuroprotective effect against ischemia-induced deficits 179 .

IPO5
(3843) also known as imp5, RANBP5, IMB3, Pse1, and KPNB3 encodes the importin 5 protein, which is a member of the importin beta family, a cytoplasmic protein that binds to nuclear pore complexes (NPCs) 180 , imports ribosomal proteins in the nucleolus where they are assembled into the eukaryotic ribosomal subunits required for protein synthesis 181 and mediates the nuclear import of H2A, H2B, H3 and H4 histones 182 . These four core histones -H2A such as Hist1H2AG, H2B such as Hist1H2BJ, H3 and H4 -are localized on chromosome 6p22, a region showing a strong association with schizophrenia 183 . The import of the four core histones is essential for the S-phase of the cell cycle during which DNA is replicated and newly synthesized histones are deposited onto the DNA in order to form the chromatin structure 182 (reviewed in 184 ).
The S-phase of the cell cycle is important for the proliferation of vascular endothelial cells during angiogenesis (reviews [185][186][187]. Angiogenesis is a predictive marker of neurological outcome following hypoxia-ischemia 188,189 . Furthermore, histones H4 190 , H3, and H2A are known to play a role in ischemia protection 190-194 . 17. MTHFR (4524) methylene tetrahydrofolate reductase catalyzes the reduction of 5,10methylene tetrahydrofolate to 5-methyl tetrahydrofolate, the predominant ciruclatory form of folate and carbon donor for the re-methylation of homocysteine to methionine. Two polymorphisms are known to cause mild enzyme deficiency. A common polymorphism in the MTHFR gene (C677T, Ala --> Val) is associated with a decreased activity of the enzyme due to thermolability. In case of homozygosity for the Val allele, a relative deficiency of the enzyme leads to a mild-to-moderate hyperhomocysteinaemia 195 . Hyperhomocysteinemia is a risk factor for cerebrovascular disease 196 .
18. NOS1AP (9722) nitric oxide synthase 1 (neuronal) adaptor protein, alias CAPON, encodes a cytosolic adapter protein that activates neuronal nitric oxide synthase (nNOS/NOS1) and is involved in the synthesis of nitric oxide (NO) 197 . In humans, nNOS produces NO in nitric oxide interneurons and vascular smooth muscle cells regulating microvascular tone in humans [198][199][200] . In brain ischemia, nNOS stimulates the increase of NO from baseline nanomolar to micromolar levels NO (reviewed in 201 ). However, the activation of nNOS alone has neurotoxic effects 202 , whereas simultaneous activation of 19. NOTCH4 (4855) alias INT3, encodes for the Notch-4 protein, an endothelial cell specific homologue of Notch. The expression of NOTCH4 is restricted to endothelial cells in the embryonic and adult brain 204 . The Notch-4 protein plays a crucial role in vasculogenesis, vascular repair of injury and angiogenesis 205,206 . The latter is a key response to cerebral ischemia [185][186][187] and predicts the neurological outcome 188,189 . During angiogenesis, Notch-4 induces microvessel differentiation of brain endothelial cells 207 and the formation of new blood vessels from existing vasculature. To allow for endothelial sprouting, the extracellular matrix around existing vasculature is degraded by matrix metalloproteases (MMPs) 208 . The MMPs is induced by VEGF signaling via VEGFR-2 and the PI3K/Akt pathway 209 .

NRG1
(3084) neuregulin 1 alias glial growth factor 2 -also known as GGF; HGL; HRG; NDF; ARIA; GGF2; HRG1; HRGA; SMDF; MST131 -and two of its receptors (erbB2, erbB3) are expressed in brain microvascular endothelial cells 210 , astrocytes and oligodendrocytes 211,212 . Neuregulin 1 activates the PI3K/AKT intracellular signaling pathway by binding to erbB receptors 211,213 . Cellular survival after ischemia depends in large extent on the activation of the PI3K/Akt pathway (reviewed in 214,215 ). Like other growth factors, NRG1 activates the PI3K/Akt pathway and subsequently the mTORdependent protein synthesis 210,213,216,217 required for ischemia protection and repair. NRG1 has been shown to be a powerful neuroprotective factor in ischemia 119,[218][219][220] and to play a role in repair 119 . Following vessel hypoxia and injury, the expression of of NRG1 and erbB is upregulated whereas in uninjured vessels it is low 218,221,222 .

NTNG1
(22854) Netrin G1 belongs to a conserved family of proteins that act as axon guidance cues during vertebrate nervous system development 114,223 .
Another member of this family Netrin-1, has the ability to attract blood vessels as well as axons, and is capable of functioning as a vascular growth factor 224 . In addition, netrin-1 stimulates NO production in mature endothelial cells 225 and has been shown to protect the cerebral cortex from the effect of ischemia 226 .
Little is known about a possible vascular function of netrin G1. However, the fact that the trajectories of nerves and blood vessels are often shared, led to the hypothesis that tissues may use identical or similar factors to guide innervation and vascularization 227 . Human NTNG1 is localized at the chromosomal position 1p13.3. This region is syntenic with mouse chromosome 3, where a modifier locus for renal vascular disease lesions has been identified 228 .

PPP3CC
(5533) PPP3CC (aliases CNA3; CALNA3; PP2Bgamma) codes for calcineurin A gamma subunit 114 . Calcineurin is a serine/threonine phosphatase that is activated by calcium and calmodulin 242 . It promotes the expression of Hypoxia-inducible Factor 1 alpha (HIF-1α) via the receptor for activated C kinase 1 (RACK1) 242 . Highly localized in the brain, especially in those parts which are vulnerable to hypoxia/ischemia, it has protective as well as toxic effects and the balance may be important for the outcome of ischemia 243 .
24. PRODH (5625) encodes proline oxidase (POX), a mitochondrial inner-membrane enzyme that metabolizes l-proline. Most of the alleles associated with schizophrenia result in severely reducted POX activity and hyperprolinemia 244 . The latter impairs the activity of cytochrome c oxidase, an enzyme of the respiratory electron transport chain of mitochondria 245 . In the cerebral cortex, proline causes mitochondrial dysfunction, oxidative stress and impaired energy metabolism 246 .

RBFOX2
(23543) alias Rbm9, RNA binding protein, fox-1 homolog. The mammalian Fox genes are complex transcription units that specifically recognize the RNA element UGCAUG and generate transcripts from multiple promoters 247 . Fox-1/2 are preferentially expressed in brain, heart and muscle tissues. They target genes involved in muscle contraction and vascular regulation, such as potassium ion transport, myosin, dystrophin, calmodulin binding 92 .
Potassium (K+) channels play an important role in neurovascular coupling (reviewed in 248 ), cerebral ischemia (reviewed in 249 ) and endothelial dysfunction (reviewed in 250 ). Myosin is expressed in vascular smooth muscle and pericytes 251 suggesting a role in vasoconstriction. Dystrophin is involved in flow (shear stress)-induced endothelium-dependent dilation and its absence in mice reduces NO-dependent vascular function 252 . Calmodulin and calcium activate calcineurin, which promotes the expression of hypoxia-inducible Factor 1 alpha (HIF-1α) 242 .

RGS4
(5999), regulator of G-protein signaling 4, is selectively enriched in the heart and brain (reviewed in 253 ). RGS proteins modulate hormone and neurotransmitter signaling 254 . With regard to the former, insulin release from pancreatic beta-cells is negatively regulated by RGS4 255 . Insulin activates the PI3K/Akt pathway, which is important for ischemia protection and repair from ischemia injury by angiogenesis. The latter is inhibited by RGS4 256 . Concerning neurotransmitter signaling, RGS proteins modulate and inhibit signal transduction by G-protein-coupled receptors (GPCRs) (reviewed in 253,257,258 ). Mice deficient for RGS4 show increased concentration of serum catecholamines 259 . In addition, RGS4 is linked to regulation of cholinergic and serotonergic signaling in the brain and is expressed in most cortical layers (reviewed in 253 ). GPCRs are widely associated with the regulation of vascular smooth muscle cell contractility 260 and RGS proteins are known to play a role in the regulation of vascular tone 261,262 .

SLC6A3, DAT1
(6531), solute carrier family 6 (neurotransmitter transporter, dopamine) member 3, is situated in the plasma membrane of the dopaminergic neurons where it mediates the re-uptake of dopamine from the synaptic cleft into the presynaptic neuron 263,264 . Dopaminergic signaling in the brain is primarily modulated by dopamine transporters (DATs) (reviewed in 265 ). In rats, DAT blockers induce positive hemodynamic changes via D1/D5 dopamine receptors and smaller negative changes through D2/D3 receptors on microvessels and astrocytes 155 . In humans, the DAT blocker cocaine caused dose-dependent cerebral vasoconstriction as revealed by magnetic resonance angiography 266 . Cocaine abuse and dependence is associated with increased incidence of stroke and myocardial ischemia 267,268 . The latter has been shown to be a consequence of vascular spasms 268 .
28. SLC6A4, 5HTT (6532), solute carrier family 6 (neurotransmitter transporter, serotonin) member 4 -also known as HTT; 5HTT; OCD1; SERT; 5-HTT; SERT1; hSERT; 5-HTTLPR -encodes a membrane protein that transports the neurotransmitter serotonin from synaptic spaces into presynaptic neurons. It terminates the action of serotonin and recycles it 114 . Serotonergic perivascular nerves are involved in the regulation of cerebrovascular tone 68 . Intracerebrally released serotonin (5-HT) has a major vasoconstrictor effect resulting in a decrease of cerebral blood flow (CBF) in several brain regions including the neocortex (reviewed in 174 ). A serotoninergic pathway originating in the raphe nucleus projects to cortical microvessels endowed with several 5-HT receptors including 5-HT1B receptors that mediate their contraction (reviewed in 68,174 ). Consistent with serotonin's vasoconstrictor effect, 5-HT2 receptor antagonists such as ketanserin and ritanserin, have been shown to increase CBF in cortical areas and to exert a protective effect in ischemia 174 .

SYN2
(6854), synapsin II -is a member of the synapsin family and encodes a neuronspecific phosphoprotein that selectively binds to small synaptic vesicles in the nerve terminal 114 . Synapsin proteins have important functions in maintaining the integrity and stability of synaptic vesicles 269 and are regulators of neurotransmitter release from presynaptic nerve terminals (reviewed in 270,271 ).
SNYN2 is a negative regulator of catecholamine release. SYN2 knock-out mice showed an increase of catecholamine release 272 . Furthermore, double knock-out mice, with deletions of SYN1 and SYN2, display higher concentrations of acetylcholine in the cortex 269 . SYN2 knock-out mice also had an increase of glutamatergic and GABAergic synaptic transmission in the spinal cord after nerve injury 273 . Catecholamines and acetylcholine play a role in neurovascular regulation (reviewed in 68 ). Glutamate release following ischemia is thought to cause neuronal injury (reviewed in 274 ).

TRMT2A
(27037), HpaII tiny fragments locus 9c protein, HTF9C, TRM2 tRNA methyltransferase 2 homolog A is a protein expressed in proliferating cells. It is overexpressed in breast cancer 275 suggesting a role for TRMT2A in angiogenesis and in protection and recovery from ischemia. Angiogenesis is predictive of neurological outcome following hypoxia-ischemia 188,189 .
The transcription of TRMT2A is repressed in quiescent tissues and growth-arrested cells, activated at the G1/S transition of the cell cycle, and peaks in S phase 276,277 . The G1/S transition is the first brake-point through which the cell must pass before it can enter cell division. During S-phase of the cell cycle, DNA is replicated and de novo chromatin assembly takes place 278 .
A key response to tissue hypoxia is angiogenesis, which requires the proliferation of vascular endothelial cells (reviewed in [185][186][187] ). Prevention of endothelial cells to enter G1 phase of the cell cycle results in reduced angiogenesis 279,280 and hence in protection and 31. TNF (7124), tumor necrosis factor, is a multifunctional proinflammatory cytokine 114 which is induced within 1 hour in brain ischemia, It has oligodendrocyte cytotoxic as well as neuroprotective effects (reviewed in 281,282 ). The activation of the Akt pathway has protective effects on TNF-mediated oligodendrocyte cytotoxicity 283 . Concerning neuroprotection, TNF activates also the mammalian target of rapamycin (mTOR) which has an influence on mitochondrial energy metabolism, protein synthesis and adaptation to ischemia 284 . Moreover, TNF activates cPLA2 (reviewed in 285 ), which regulates cerebrovascular function via arachidonic acid (AA) and epoxyeicosatrienoic acids (EETs) (reviewed in 240,286 ). Finally, TNF improves ischemia repair by upregulating the erythropoietin receptor (EPOR) thereby sensitizing cerebral endothelial cells for erythropoietin-induced angiogenesis 287 .

UFD1L
(7353), ubiquitin fusion degradation 1 like. The protein encoded by this gene forms a complex with two other proteins, nuclear protein localisation-4 and valosincontaining protein. This complex is necessary for the degradation of ubiquitinated proteins 114 . Ubiquitination of proteins is the first step in the degradation of proteins by the proteasome system. The ubiquitin-proteasome system degrades hypoxia-inducible factor 1alpha (HIF-1alpha) protein under normoxic conditions, while it is stabilized and accumulated rapidly following exposure to low oxygen tensions 288,289 . HIF-1 is a master regulator of response to hypoxia by activating the transcription of many genes, including those involved in blood flow, cell survival, glucose transport, energy metabolism, i.e. genes whose protein products increase oxygen delivery or facilitate adaptation to hypoxia 114 . Ubiquitin fusion degradation protein 1 (UFD1) is a blood marker for the early diagnosis of ischemic stroke 290 .

ZDHHC8
(29801), zinc finger, DHHC-type containing 8 (DHCC8), is localised in mitochondria and presynaptic processes, mostly glutmatergic and to a lesser extent GABAergic processes. It interacts interacts with mitochondrial Complex III. ZDHHC8 dosage change is able to disrupt mitochondrial function and to influence cell survival and death 291 .

KEGG,pathway,analysis,
The Kyoto Encyclopedia of Genes and Genomes (KEGG) 292 at (http://www.kegg.jp) was used for pathway analysis and as guide for the construction of the candidate schizophrenia pathway depicted in Figs. 1 and 2.
As requested by one of the reviewers, results of these analyses are shown as Supplementary Information. The KEGG pathway analyses produced a five pages long list of pathways, which are difficult to interpret with regard to the etiology of schizophrenia. The first page of the results from the KEGG pathway analysis is shown in Table S12. Five genes from our list of candidate genes for schizophrenia were not found in the KEGG database. Yellow highlights pathways related to vascular regulation or the energydelivering pathway depicted in Figs. 1 and 2.    293,294 . Furthermore, the polymerisation status of the submembranous actin web in vascular endothelium determines the activity of eNOS and the release of NO 295 (see also Fig. 1-2). In addition, a role for calcium signaling genes in schizophrenia was reported in the 2014 GWAS by the SWGPGC 5 and has recently been emphasized by Tansey et al. (2015) 296 .

PANTHER,pathway,analysis,
The PANTHER (Protein ANalysis THrough Evolutionary Relationships) Classification System 297 at (http://www.pantherdb.org) was employed for pathway analysis of the discovery and the combined sample. The results of the latter are shown in Table S13. They involved the dopamine receptor mediated signaling pathway, adrenaline and noradrenaline biosynthesis, EGF receptor signaling pathway, 5HT2 type receptor mediated signaling pathway, nicotinic acetylcholine receptor signaling pathway. P values are Bonferroni corrected for multiple testing. Stress * 24*

Genetic,disease,association,analysis,by,DAVID,
The discovery and the combined gene samples described in this article were also analysed genetic disease associations by the Database for Annotation, Visualization and Integrated Discovery (DAVID) 298 at (http://david.abcc.ncifcrf.gov/). Table S14 shows some of the results.

Gene,set,analysis,
All genes from other species were transformed into human orthologous genes and identified by Entrez Gene IDs. The association between schizophrenia-associated genes and functional gene sets were computed by using the intersect function of the programming language R (version 3.0.2, platform: x86_64-apple-darwin10.8.0, 64-bit) 299 .

Genome,resampling,test,
Homo sapiens' complete list of genes was downloaded on April 8, 2011 from the Entrez Gene database!maintained by the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/gene). It had 45 386 entries.
To improve the power (i.e. the chances of obtaining an intersection of randomly drawn genes from the database with functional gene sets), the list was curated by deleting all genes with unknown function from the Entrez Gene database. Using the Unix stream editor SED, genes with the following key words were removed: pseudogene, hypothetical LOC, hypothetical protein, pseudo, miscRNA, readthrough, read-through, open reading frame, deletion syndrome, duplication syndrome, triplication syndrome, unknown, uncharacterised protein, putative uncharacterised protein and repeat sequence. Next, all candidate genes for schizophrenia and functional gene sets were added and duplicates removed to ensure that all genes under investigation are represented equally among the constructed representation of the Human Genome and have a chance of being drawn during genomic resampling. The final modified list of Entrez Genes was comprised of 21 012 human genes mostly of known function.
The number of intersections between schizophrenia-associated genes and functional gene sets was determined by the intersect function implemented in R 299 . A genomic resampling procedure was employed to obtain estimates of the expected numbers of intersecting genes by drawing the same number of genes as the candidate genes 1 million times at random (with replacement) from the representation of the Human Genome described above and determining the intersection of the random genes with the functional gene set. The resampling method has the avantage of being independent of the distribution assumption, but due to computational restrictions significance can only be computed up to a threshold of p ≥ 10E-6. Within this limit imposed by computational restriction, the empiciral p-values were identical to the nominal pvalues from Fisher's Exact Test.

Putative,schizophrenia,pathway,
The stress-induced increase of cortisol and desoxycorticosterone (DOC) 300 has an inhibitory influence on the expression of glucose transporters 301 , brain metabolism 302 , and serotonin uptake 303 . Furthermore, stress induces the release of adrenaline into the circulation and of noradrenaline and dopamine in the prefrontal cortex (reviewed in 304 ); these are neurotransmitters known for their vasoconstrictive effects 69,303 (reviewed in 305 ). Magnitude and duration of the signals of these neurotransmitters is primarily influenced by their plasma membrane transporters (e.g., DAT1, NAT1 and 5-HTT) (reviewed in 306 ). The expression of these transporters at the cell surface depends on the activity of the PI3K/Akt pathway and its stimulation by insulin and insulin-like growth factor 1 (IGF-1) 307 .
Numerous studies have shown that growth factors, cytokines and hormones (such as insulin, IGF-1, EGF, prolactin, estrogens and erythropoietin) have a positive effect on Akt, thereby probably reducing the vasoconstrictive impact of stress via Akt's positive influence on DAT1 264 , NAT1 308 and eNOS 309 . At low concentrations, testosterone exerts an activating effect on Akt, but an inhibitory effect at high concentrations 310 . In addition, prolactin activates Akt 311 suggesting that the hyperprolactinemia caused by typical antipsychotics is likely to have vasodilatatory and ischemia-protective effects (Fig. 2).
In summary, cerebral blood flow and energy supply depend on growth factors, hormones and genes involved in the PI3K-Akt-mTOR pathway. Activation of this pathway also protects tissue from ischemia by influencing the protein synthesis of the hypoxia-inducing factor (HIF) 312 , which subsequently induces the translation of more than 70 proteins in order to increase blood flow, cellular survival and alternative energy production from lactate.
-minus sign, overlapping genes removed; DE, differentially expressed genes; I, genes induced by cerebral ischemia; ND, neurodevelopmental genes; proximity, genes assigned by proximity to index SNP; R, post-ischemic repair genes; Repair, R and ND genes combined, because ND genes are involved in post-ischemic repair 120,127 ; SY, synaptic genes; VI, vascular-ischemia genes; VIRND, all genes involved in ischemia, i.e., V, I, R and ND genes; within range, all genes within range of index SNP; x, overlapping, i.e., interacting genes.

Quasi,experimental,study
Figure&S6.&Quasi?experimental&impairment&of&components&of&the&candidate& pathway& Figure S6. Results of quasi-experimental impairment of components of the postulated schizophrenia pathway. Disturbances of all components of the energy-supply pathway, i.e. perivascular nerves, oxygen, microvessels, oligodendrocytes, and mitochondria (see Fig. 1 in article), appear to produce a high percentage of schizophrenia-like symptoms. Multiple sclerosis (MS) is the exception, which might be due to the localized damage of myelin in MS. Very preterm birth seems to impair neurodevelopment independent of an increase in risk for ischemic disorders 314,315 . It causes high rates of neurodevelopmental disabilities from 25% to 50% such as cerebral palsy (5% to 15%) 316  Vasculitis is rare but vascular hyalinization, endothelial proliferation and perivascular gliosis are common 350 . Furthermore, small lesions in white matter (WM) (100%), diffuse WM abnormalities (43%) and cerebral infarction (29%) 351 . Cerebral hypoperfusion measured by SPECT is related to neuropsychiatric symptoms in NPSLE 352 .
Psychosis has been reported in 5% of NPSLE 353 including schizophrenia-like psychosis [354][355][356]  Cannabis-induced psychosis shares genetic predisposition and many common symptoms with schizophrenia 367,368 . The maximum proportion of psychosis attributable to cannabis in psychosis-free subjects is higher than 50 percent 369 .

Oligodendrocyte & myelin component
Metachromatic Bilateral fronto-temporal white Adult onset psychosis in 25-40% * 41* § defined as hallucinations and/or delusions with or without disorientation Experienced clinical psychiatrist will have observed, that the former, i.e., hallucinations and/or delusions with disorientation, correspond to the diagnosis of delirium and might demand an explanation for the lumping together of schizophrenia with delirium.
First, lumping and splitting of diagnostic categories are widely used in psychosis research (see Kraepelin's lumping of paranoia, hebephrenia and catatonia and his splitting of major psychoses into schizophrenia and manic depression or the current debate about the lumping of bipolar disorder and schizophrenia based on genomic findings 375,376 . Second, similarities between schizophrenia and neurodevelopmental disorders, such leukodystrophy matter is affected (reviewed in 370 ).
40% (reviewed in 370  as cerebral palsy, epilepsy, and mental retardation, have frequently been used to support the neurodevelopmental hypothesis 377,378 . See, e.g., page 401 of Weinberger's and Harrison's recent, excellent book on schizophrenia 379 : "In a sense, schizophrenia appears to be on a developmental continuum with other behavioral disorders that appear in childhood, including autism, intellectual disability, and epilepsy, arising perhaps from overlapping biological risk factors that may each have distinct covariants, but schizophrenia reflects the relatively least noise burden of this group of developmental disturbances 378 ". Third, the same reasoning may be applied to ischemia, with stroke on one end of the continuum, delirium and schizophrenia in the middle, hyperperfusion on the other end (see Fig. 4), and cerebral localization as covariant. Differences in severity or cerebral localization of ischemia might account for differences in disorientation. Consequently, an acute ischemic psychosis of known etiology would be diagnosed as delirium and an ischemic psychoses of unknown cause without disorientation as schizophrenia. For this discussion of hallucinations/delusions with disorientation, it is important to note that disorientation has also been found in some acute 380 , as well as in chronic schizophrenic patients [381][382][383][384] . In regard to cerebral localization, isolated time orientation has been observed in 4 percent of patients with thalamic ischemia 385 . * 43*

Table&S24.&Quasi?experimental&neurodevelopmental&disturbance&&
as independent variable and the production of schizophrenia-like symptoms.

Etiology of neurodevelop -mental disturbance
Evidence for neurodevelopmental disturbance Schizophrenia-like symptoms §
Since OBC is associated with the development of cardiovascular disorders 387,388 and stroke in adulthood 314,315,389,390 (reviewed in 391,392 ), OBC cannot be considered here as proving that schizophrenialike symptoms are caused by neurodevelopmental disturbance.

Perinatal brain damage
A 1966 North Finland Birth Cohort revealed that 29.9% of the children surviving perinatal brain damage developed cerebral palsy, epilepsy or mental retardation (IQ less than 71) 393 .
In the 1966 North Finland Birth Cohort, six of the 125 survivors (4.8%) of severe perinatal brain damage developed later schizophrenia 394 . However, these data cannot exclude perinatal brain damage due to OBC, which is associated with coronary heart disease and stroke (see row above).

Preterm infants
Since length of gestation and preterm birth is not associated with coronary heart disease 314 or stroke 315 , the effect of neurodevelopmental disturbances independent of vascular factors can only be investigated in preterm infants. Very preterm infants (< 32 weeks of gestational age), have shown high rates of neurodevelopmental disability in followup studies with 5% to 15% having cerebral palsy, severe neurosensory impairment and 25% to 50% having Increased rate of psychiatric hospitalization for nonaffective psychoses (i.e. schizophrenia and schizoaffective psychosis) from 0.05% (495 of 1022431 cases) for term birth to 0.1% (6 of 5125 cases) for very preterm infants (< 32 weeks of gestational age) 317  Decrease of phosphocreatine and ATP in the frontal cortex of neuroleptic-free schizophrenic patients 424 , but not in medicated or chronic patients 425 . Increase of lactate was found in the blood 426 , post-mortem brain tissue 409 , and CSF 427 of schizophrenic patients.

22
Increase in cortical conduction time 428 and reduction in the amplitude of evoked potentials (EP) 403,429 .
Increase in latency and reduction in the amplitude of EP (ERP, eventrelated potentials) is one of the most replicable biological marker in schizophrenia 430 (meta-analysis 431  Nine of 10 post-mortem studies reported focal infarctions in the brain of schizophrenic patients 442 (review 443 ). Table S25: § Values from animal studies were converted to approximate CBF values for humans by using the percentage of reductions from mean CBF. However, this approach cannot take into account the physiological and biochemical differences among species.
Increase of lactate in schizophrenic patients has been reported for blood 426 , post-mortem brain tissue 409 , and CSF 427 .

Cellular signs
Hypoxia/ischemia and chronic cerebral hypoperfusion lead to slight degeneration of astrocytic end-feet processes and BBB disruption 471,472 .
Signs of ultrastructural damage to capillaries of the neocortex in schizophrenic patients that resemble those observed in chronic hypoperfusion, oxidative stress, damaged blood brain-barrier, or cerebral ischemia 473 .
Loss of spine and dendrite structure 417 Lower density of dendritic spines, reduced dendritic arborizations 480 , and decreased presynaptic protein markers (reviewed in 481,482 ).
Gamma-oscillations are highly vulnerable to hypoxia 487,488 .
Delayed latency and decreased magnitude of gamma-oscillations 432,433
Regional cerebral hyperperfusion and hypoperfusion in unmedicated patients [397][398][399]404 . Positive symptoms correlated with either cerebral hyper-or hypoperfusion. This correlation disappeared after reduction of positive symptoms 398 . Cognitive impairment is the core of the disorder 493 and begins between the premorbid phase and first episode 497

The,foundation,of,the,neurodevelopmental,hypothesis,can,also,be,explained, by,adult,vascular,disorders,
The left column of Table S27 lists the evidence interpreted as support for the neurodevelopmental hypothesis according to reviews by Harrison 498 , Moises et al. 499 , Marenco et al. 377 , and Weinberger 378 . The right column shows findings of a literature search in PUBMED and Google SCHOLAR using the key words of the left column and "cardiovascular" or "cerebrovascular".
Summing up, the foundation of the neurodevelopmental hypothesis is not only associated with schizophrenia, but also with adult vascular disorders (depicted in supplementary Fig. S7). In conclusion, schizophrenia as adult vascular disorder is an alternative explanation for the available evidence previously interpreted as support for the neurodevelopmental hypothesis.

Alternative explanation Prenatal evidence
Broadly defined obstetric complications (OC) 377,378,498 are associated with an increased risk for schizophrenia (metaanalysis 500 ). However, overall effect of OC on the occurrence of schizophrenia is small 377 . 93% of schizophrenic patients did not experience such OC (see Table  S28).
Obstetric complications (OC) are frequently a sign of placental inefficiency 388,501 , which causes fetal undernutrition, intrauterine growth restriction (IUGR), OBC, insulin-resistance, type 2 diabetes and increased risk for cardiovascular disorders 387,388 , and stroke in adulthood 314,315,389,390 (reviewed in 391,392 ). Preeclampsia 377 : The only OC study able to adjust for mother's psychotic illness during her adult life found only preeclampsia to be significantly associated with an increased risk for schizophrenia 502 .
Preeclampsia has a strong genetic component (reviewed in 503 ) associated with an increased risk of cardiovascular or cerebrovascular disease (meta-analysis 504 ). 377 is inversely related to schizophrenia 505 .

Birth weight
Birth weight is inversely related to systolic blood pressure, ischemic heart disease, and stroke 506 .

Maternal influenza 377,378,498
Maternal influenza is associated with an 20% increase in cardiovascular disease 507 .
Prenatal famine causes increase of hypertension, raised glucose levels, increased blood pressure response to stress, * 50* and a 2-fold increase of risk for coronary heart disease (reviewed in 510 ).

Blood group incompatibilities 378,511
Thickening of the amniotic epithelium 512 and the trophoblast basement membrane 513 suggests reduced diffusion and availability of nutrients mimicking prenatal famine.
Winter birth 498 . The effect correlates with the latitude 514 (colder winter temperatures) and is not detectable in the Southern Hemisphere 515 (relatively warm winters).
Cold outdoor temperature at birth is associated with increased coronary heart disease and insulin resistance 516 .

Maternal homocysteine level elevated 378
Elevated homocysteine concentrations at pregnancy are associated with increased risk of cardiovascular disease, angina, and stroke (reviewed in 517 ).
Paternal age at conception 378 Paternal age results in reduced telomere length in his offspring 518 . Reduced telomere length is associated with premature myocardial infarction 519 .

Maternal severe stress during pregnancy 378
Maternal stress, anxiety, and glucocorticoids reduce fetal growth and birth weight, and predispose the offspring to adult cardiovascular disorder and stroke 520,521 .
Structural cerebral abnormalities (ventricular enlargement, reduced cortical volume) 498 Progressive changes in ventricular and gray matter volume challenge the neurodevelopmental hypothesis (metaanalyses 522,523 ) Neuropathology 498 : Reported cytoarchitectural abnormalities related to intrauterine development have not been replicated and are not unequivocally established 443 .
In comparison, the undisputed cytoarchitectural findings, such as alterations in neuronal size, and synaptic and dendritic organization, could well originate much later 443 .

Postnatal evidence
Delays in motor and speech development 378,498 , poor motor coordination 377 . The delays are very modest and do not cause concern to physicians or parents. Furthermore, most individuals with such a delay do not develop Maturational delay, i.e., slow growth in fetal life, infancy, and during childhood is associated with adult cerebrovascular disorder 524 . Furthermore, delays in motor development are associated with the personality dimension of neuroticism (trait anxiety) in adulthood 525 . Neuroticism seems to predispose to schizophrenia 526 .
Behavioural abnormalities as infants 498 such as hyperactivity, poor verbal abilities, nervous, withdrawn, or disruptive, aggressive, and antisocial behavior in school 377 .
These behavioural abnormalities are characteristic for the two personality dimensions frequently observed in schizophrenic patients: introversion / neuroticism (trait anxiety) 527 and impulsive/antisocial/psychopathic personality [528][529][530] . Social introversion and psychoticism/psychopathy (type A behaviour pattern) are associated with an increased risk for myocardial infarction 531,532 Soft neurological signs have been observed in up to 60% of schizophrenic patients and are interpreted as evidence for premorbid brain damage in schizophrenia 436,437 (metaanalysis 533 ).
These signs are present in up to 40.6% of normal individuals (meta-analysis 533 ). Transient ischemia increases the frequency of these signs 435 . In schizophrenic patients, soft neurological signs decrease in parallel with the remission of acute psychosis (metaanalysis 534 ).
The superior premorbid high intelligence (IQ) of a considerable number of patients{ [540][541][542] is not compatible with a premorbid fronto-cortical brain damage. However, a premorbid high IQ is not at variance with the adult ischemia hypothesis. . Figure S7. The foundation of the neurodevelopmental hypothesis of schizophrenia consists of epidemiological studies showing that prenatal factors and delayed growth are associated with an increased risk for schizophrenia (in green, for reviews 377,378,498,499 ). The same factors increase the risk for adult cardiovascular and cerebrovascular disorders (in red, references in supplementary Table S27), also known as Barker's theory 391,392,524 . Adult vascular disorder as intermediary variable between broadly defined birth complications (OC) and schizophrenia has been ignored suggesting that OC are a proxy variable for the predisposition to adult cerebrovascular disorders, and that the Barker theory might be a well-founded substitute for the neurodevelopmental hypothesis to explain prenatal risk factors and maturational delays in schizophrenia. IQ, intelligence quotient; OC, obstetric complications.

Normal,or,superior,intelligence,in,schizophrenia,
The average premorbid IQ of schizophrenic patients is on average only 0.5 SD below the population average 539 , which might be explained by their higher trait anxiety (neuroticism) 526 and not necessarily an indication of a neurodevelopmental brain damage.

Birth,complications,in,only,7%,of,schizophrenic,patients,
Data for calculating the percentage of obstetric complications in schizophrenic patients were obtained from Table 2 of the meta-analysis of prospective population-based studies by Cannon et al. (2002) 500 .
In 93% of schizophrenic patients and in 94.6% of normal controls, no evidence of (broadly defined) birth complications were found (see Table S28). In conclusion, the overall majority of schizophrenic patients were not exposed to birth complications that might have caused a defect of brain development.

Table&S28.&Percentage&of&schizophrenic&patients&with&broadly&defined&birth& complications&
Obstetric complications Schizophrenic patients Exposed  Schizophrenia,treatments,improve,CBF,and,ischemia,protection, All treatments found to improve schizophrenia also improve cerebral perfusion and/or protect against ischemia or its harmful consequences such as inflammation (see Table S29).

Treatment in schizophrenia
Effects Acetylsalicylic acid 545 prevents cerebral ischemia 546 Atypical antipsychotics 547,548 enhance CBF 397,491,549 . Clinical improvement correlates with CBF 491 . A recent meta-analysis provides a more variable picture with increased as well as decreased areas of rCBF following antipsychotic treatment 550 . Celecoxib 551-553 , nonsteroidal antiinflammatory drugs (NSAID) 554 Celecoxib is an non-steroidal anti-inflammatory drug with potent neuroprotective effect against ischemia-induced inflammatory reaction 555 Electroconvulsive therapy (ECT) 556 Epileptic seizures are accompanied by an increase in focal CBF 557,558 . ECT improves CBF and catatonia 559 Erythropoietin (EPO) 560 EPO enhances cerebral vasodilatation 561 , activates the PI3K/Akt pathway 562 and improves the consequences of cerebral ischemia 563 Exercise 564 increases cerebral vasodilatation, BDNF 565 , and cerebral blood volume in the hippocampus 566 Ginkgo Biloba Extract 567,568 increases CBF 569,570 , and protects against cerebral ischemia 571 Glucose 572,573 Glucose, the obligatory energy substrate for the brain 574 , is lacking in ischemia. Insulin Coma Therapy (ICT) 575, 576 Insulin causes cerebral vasodilatation 577 , and activates the PI3K/Akt pathway in neurons following brain ischemia 578 . Furthermore, insulininduced hypoglycemia leads to a marked increase in CBF 579 . Nicotine (alpha-7 nicotinic agonists) 580,581 enhances cholinergic vasodilation in the cerebral cortex 582 Reserpine 583 depletes dopamine and noradrenaline from the brain 584 . Its use as antihypertensive drug suggests a vascular effect 585 . Transcranial Magnetic Stimulation (rTMS) 586,587 increases CBF in some areas of the brain [588][589][590] .
Typical neuroleptics 591 Dopamine causes a dose-dependent vasoconstriction in about 50% of cortical microvessels 69 .

56*
Antipsychotic drugs block D2/3 receptors 592 and increase CBF 397,491,549,593 , mainly in paranoid patients 550 . But see also evidence for a decrease of CBF 490,550 . Clinical improvement was found in one study to correlate with CBF 491 .

Evidence 1
Adult neurogenesis and synaptic plasticity are involved in postischemic repair and in schizophrenia (reviewed in 594,595 ). 2 Adult neurogenesis and synaptic plasticity have been implicated in schizophrenia by previous pathway analyses (see supplementary information). 3 Motor endplate alterations in schizophrenic patients resemble axonal destruction followed by regeneration 596 . 4 Erythropoietin, a stimulator of adult neurogenesis, improves cognitive functions in chronic schizophrenic patients 560 . 5 Neuroleptics appear to stimulate adult neurogenesis either directly or indirectly via prolactin 597,598 . 6 Drug responding patients show signs of myelin repair in brain imaging 599 . 7 Physical exercise increases BDNF, adult neurogenesis, hippocampal volume, and improves negative symptoms in schizophrenic patients 564,565,600,601 . 8 The PI3K/Akt pathway mediates not only the effects of stress, growth factors, and hormones on metabolism, vasoconstriction, and vasodilatation, but also on synaptic plasticity and adult neurogenesis, i.e., repair 602,603 (see Fig. 2). 9 Neural stem cell proliferation required for adult neurogenesis is reduced in schizophrenia, but not in major depression 604 .

The,AVIH,seems,to,offer,a,better,explanation,for,the,evidence,compared,to, the,NDH,
Theory choice is a search for the best explanation of the evidence. The three main criteria for the evaluation of a hypothesis are consilience, simplicity, and analogy 605 . The adult vascularischemia hypothesis better fulfills the criteria of consilience, simplicity, and analogy than the neurodevelopmental hypothesis. For consilience, see Table S31.

Evidence/facts
Explanation by ND hypothesis AVI hypothesis Genetic 1 Overrepresentation of VI 606 , ND, and repair genes (Table S22, Fig. 3a)

Yes Yes
Prenatal 2 Evidence supporting the ND hypothesis (Table  S27 and Fig. S7)

Yes Yes
3 Absence of birth complications in 93% of schizophrenic patients, difference to normal population only 1.6% (meta-analysis 500 ) (see Table S28) No Yes 4 Absence of minor physical signs in 65% of patients 607 (reviewed in 608 ) No Yes 5 Absence of neuropathological evidence for neurodevelopmental brain defect 443,482 No Yes Premorbid 6 High premorbid intelligence (IQ) 540 The criterion of simplicity is met by requiring less auxiliary hypotheses, e.g., for evidence # 3-13 in Table S31 above. Finally, disorders disturbing the cerebral energy-supply (Table 23, Fig. S6) and adult vascular disorders (Fig. 7) provide useful analogies for better understanding the pathogenesis of schizophrenia, whereas the analogy of the NDH with neurodevelopmental disturbances in very preterm infants is surprisingly unconvincing (see supplementary Fig. S7, Tables S23-S24).