Table 3:

Studies Applying Systems Pharmacology Approaches to Brain Diseases

Disease / Condition	Organism/ Model	Tissue	Input	Similarity Metric	Key Findings	Validation	Measure of Reliability / Other Prioritization Methods	Public resources used	Ref
Alzheimer’s disease (AD)	Human	Hippocampus	DEGs- Top 500 up-regulated Top 500 down-regulated genes based on fold change (unadjusted P-value ≤ 0.05) Calculated 3 different ways (Limma , Limma -ChDir, and mAP-KL) for 5 independent studies (all hippocampus)	KS-like statistic Correlation coefficient	Proposed 27 candidate drugs. Highlighted potential roles of PKC, HDAC, ARG and GSK3 in mechanism of AD.	None	Negative scores CMap (permuted results P-value < 0.05) SPIEDw (results with significance value > 2) sscMap treatment set score normalized to unity with a tolerance of one false connection among all possible drugs (P < 1/1309, strictest parameterization) LINCS-L1000 (mean connectivity score across the four cell lines in which the perturbagen connected most strongly to the query (best score 4) < −0.9)) Systematically varied calculation of DEGs for input (x5 hippocampal datasets) and algorithms. Combined score across all.	GEO CMap (build 2) sscMap SPIEDw LINCS-L1000 Enrichr ChemBioServer NetworkAnalyst Mode of Action by NeTwoRk Analysis (Mantra 2.0)	(Siavelis et al. 2016)
AD	Human	Entorhinal cortex Hippocampus Middle temporal gyrus Posterior cingulate cortex Superior frontal gyrus Visual cortex	DEGs - Top 400 up-regulated Top 400 down-regulated genes based on fold change (FDR<1%)	KS-like statistic	Gene signature induced by thioridazine was similar with AD gene signatures in EC, HIP, MTG, and SFG. On the contrary, gene signatures induced by 2 histone deacetylase (HDAC) inhibitors vorinostat and trichostatin A were anticorrelated with AD gene signature in HIP and PC, which indicated that these drugs could possibly reverse the AD gene signature.	None	Positive scores (mechanistic insight) Negative scores (candidate pharmacotherapeutics)	GEO DAVID TransFind CMap KEGG	(Chen et al. 2013)
AD	Human	Hippocampus Cerebral cortex	DEGs - Hippocampus: 40 DEGs (40 genes reported by Hata, R. et al 2001; top 20 up and top 20 down) Cerebral cortex: 25 genes with FC > 5 reported by Ricciarelli, R. et al 2004; 11 up and 14 down)	KS-like statistic	No genes in common between these two query signatures, but both yielded negative connectivity scores with the two independent instances of 4,5-dianilinophthalimide (DAPH). This strengthened the candidacy of DAPH as a potential AD therapeutic.	in silico (Literature: DAPH was found to reverse the formation of fibrils (a variety of DAPH analogs have been synthesized as potential treatments for AD)	Negative scores Permutation p-value	CMap	(Lamb et al. 2006)
AD / Cognition enhancers	Human	Hippocampus Cerebral cortex	DEGs - Hippocampus: 40 DEGs (40 genes reported by Hata, R. et al 2001; top 20 up and top 20 down) Cerebral cortex: 25 genes with FC > 5 reported by Ricciarelli, R. et al 2004; 11 up and 14 down)	KS-like statistic	No genes in common between these two query signatures, but both queries resulted in a common list of negative connections that were given higher confidence. They used an integrative chemoinformatics approach combining CMap with QSAR/VS hits to emphasize connections from the CMap that one would not choose otherwise.	in vitro (validated binding to 5HT6R, also that raloxifene binds to 5HT6R) in silico (Literature: raloxifene given at a dose of 120 mg/day, but not 60 mg/day, led to reduced risk of cognitive impairment in postmenopausal women)	Negative scores (They mention statistically significant negative drugs but do not provide the threshold used.) Note: No summarization across multiple experiments for same compound (doses, timepoints, cell lines)	CMap	(Hajjo et al. 2012)
Parkinson’s disease (PD)	Human	Substantia nigra	DEGs- Top 20, 50, 100, 200 or all (535) DE genes from integrated approach. 2 GEO datasets: FC > 1.5 FDR<10% ParkDB (human): Consistent up- or down-regulation across different experiments (P ≤ 0.01)	KS-like statistic	Top 20 genes from their integrated approach for prioritizing genes outperformed the top 100, 200, 500, and all (536) DEGs for the input signature. Performance was measured by how many (out of the top 50) molecules returned by CMap from their approach were enriched with therapeutic molecules for PD. One candidate, alvespimycin (17-DMAG), was found to be neuroprotective in an in vitro rotenone model of PD.	in vitro (neuroblastoma cell line) in silico (Enrichment score)	Negative scores Integrated approach (NOTE: simply using the DEGs from the GEO datasets performed as well as or better than their integrated approach for all but the top 20 genes from their integrated approach).	CMap GEO ParkDB OMIM CTD	(Gao et al. 2014)
Huntington’s disease (HD)	Human	Caudate nucleus	DEGs - Top 100 (absolute FC) 8 up-regulated, 92 downregulated (P < 0.05)	KS-like statistic	Using a gene signature for HD, CMap identified potential therapeutic agents with multiple modes of action and validated 2 (deferoxamine and chlorzoxazone) in vivo (ameliorated neurodegeneration in flies expressing a mutant HTT fragment).	in vitro luminescent caspase-activation assay of HTT-induced apoptosis in a PC12 cell line 7 / 12 drugs with negative connectivity scores reduced HTT-induced apoptosis. Chlorzoxazone, copper sulphate, deferoxamine, felbinac, oligomycin and primidone did so in a dosedependent manner. Chemicals with positive connectivity scores had little effect on caspase activation. in vitro High-throughput recording of HTT103Q aggregation in PC12 cells using Cellomics imaging technology. Of the 7, deferoxamine and oligomycin, significantly altered the formation of HTT103Qcontaining inclusion bodies. in vivo Deferoxamine and chlorzoxazone ameliorated neurodegeneration in flies expressing a mutant HTT fragment (a widely studied fruit fly model of mutant HTT toxicity) (Steffan et al. 2001)	Negative scores	CMap ArrayExpress	(Smalley et al. 2016)
Memorymodulating compounds	Human	Saliva	Intragenic SNPs associated with aversive memory recall	Not specified	They used genomic information related to aversive memory—a trait central to posttraumatic stress disorder—to identify several potential drug targets and compounds. In a subsequent pharmacological study with one of the identified compounds, diphenhydramine, they found a druginduced reduction of aversive memory. These findings indicate that genomic information can be used as a starting point for the identification of memory-modulating compounds	in vivo A single administration of diphenhydramine (50 mg) compared with placebo significantly reduced delayed recall of aversive, but not of positive or neutral, pictures in a double-blind, placebocontrolled study in healthy volunteers	Not specified	Ingenuity Pathway Analysis (IPA) (note: not public)	(Papassoti ropoulos et al. 2013)
Motivation to exercise	Mouse (4 models of motivation to exercise and controls)	Striatum	DEGs - Top 287 genes up-regulated Top 235 genes downregulated in selected lines vs controls (FDR<5%)	KS-like statistic	Mice from 4 lines selected for wheel running (and 4 non-selected lines) were allowed full access to a running wheel for 6 days. On day 7, half of the high runners and half of the low runners were blocked from wheel access and striatum taken at the time of maximum wheel running and submitted for RNA sequencing. LINCS identified the protein kinase C δ inhibitor, rottlerin, the tyrosine kinase inhibitor, Linifanib and the delta-opioid receptor antagonist 7-benzylidenenaltrexone as potential compounds that mimic the transcriptional signature of the increased motivation to run.	None	Positive scores (to mimic the high motivational state for exercise) Considered both compounds tested across all cell lines and those only tested in neuronal cell lines No statistical p-value mentioned	DAVID LINCS-L1000	(Saul et al. 2017)
Schizophrenia Major depressive disorder Bipolar disorder Alzheimer’s disease Anxiety disorders Autistic spectrum disorders Attention deficit hyperactivity disorder	Human	10 brain areas available in GTEx: Anterior cingulate cortex (BA24) Caudate (basal ganglia) Cerebellar hemisphere Cerebellum Cortex Frontal cortex (BA9) Hippocampus Hypothalamus Nucleus accumbens (basal ganglia) Putamen (basal ganglia)	GWAS summary statistics converted to transcriptomic profiles Top K DEGs where K was varied for 50, 100, 250, 500. Results were averaged across each K.	5 methods: KS-like statistic Spearman correlation with all or with K differentially expressed genes Pearson correlation with all or with K differentially expressed genes	They imputed transcriptome profiles for 7 psychiatric conditions based on GWAS summary statistics and compared these to druginduced changes in gene expression (CMap) to find possible treatments and found that the top 15 predicted compounds were enriched with known drug-indication pairs.	in silico	Negative Scores Permutation test (shuffled the diseaseexpression z-scores and compared them to drug transcriptomic profiles. Performed 100 permutations for each drug–disease pair and combined the distribution of ranks under the null across all drug–disease pairs, such that the null distribution was derived from 347,800 ranks under H0).	CMap GTEx MetaXcan KEGG ClinicalTrials.gov MEDication Indication resource (MEDI) Psychiatric Genomics Consortium(PGC)	(So et al. 2017)
Morphine Tolerance	Rat	Whole brain	DEGs - Morphine-tolerant + saline versus morphine-tolerant + LPS Placebo-control + saline versus placebo-control + LPS Placebo-control + saline versus morphine-tolerant + saline rats (No p-value threshold reported)	KS-like statistic	Here, LINCS-L1000 gene knockdown and overexpression experiments were used to inform mechanism of action. Response to LPS was altered during morphine tolerance and indicated that VPS28 may be one of the genes responsible for the alterations associated with morphine tolerance.	None	Genetic perturbation experiments only Positive scores Negative scores	LINCS-L1000 Query App (apps.lincscloud. org/query) (now deprecated. Instead use clue.io/l1000-query)	(Chang et al. 2017)
Binge-like drinking (important risk factor for AUD)	Mouse (HDID-1 and HS/Npt controls)	Prefrontal cortex Nucleus accumbens core Nucleus accumbens shell Bed nucleus of the stria terminalis Basolateral amygdala Central nucleus of the amygdala Ventral tegmental area Ventral striatum	DEGs - Top 100 up-regulated Top 100 down-regulated genes from each brain area (based on fold change, unadjusted p < 0.05) Differentially expressed landmark genes from each brain area (unadjusted p < 0.05) (landmark genes are those whose expression is directly measured in the L1000 assay)	KS-like statistic	Many anti-inflammatory compounds had highly negative connectivity scores across brain areas, providing additional evidence for a neuroimmune component in regulating ethanol intake. The top 2 candidates, terreic acid and pergolide, were behaviorally validated to decrease ethanol intake and blood alcohol levels.	in vivo The top 2 candidates, terreic acid and pergolide, were behaviorally validated to decrease ethanol intake and blood alcohol levels.	Negative scores Integrated approach Sig_gutc tool (see text)	LINCS-L1000 GEO	Ferguson et al, 2017
Traumatic Brain Injury (TBI)	Rat	Perilesional cortex Thalamus	DEGs - Top 4964 in perilesional cortex Top 1966 in thalamus (FDR<5%)	Not reported	The study highlighted tubulins, Nfe2l2, Nfkb2, and S100a4 as target genes modulated by compounds with a high LINCS connectivity score relative to the TBI-sig. Their data suggested that desmethylclomipramine, an active metabolite of the antidepressant clomipramine, is a promising TBI treatment candidate.	in silico 2/11 top compounds had previously been investigated in epileptogenesis models in vivo.	Negative scores	LINCS-L1000 GEO IPA GSEA MsigDB	(Lipponen et al. 2016)
Ischemic stroke	Rat	Brain (whole hemisphere)	DEGs - Genes with FC1 > 1.5, FC2 > 1.2 and RR > 0 FC1: fold change between middle cerebral artery occlusion (MCAO) and Sham (FC1) FC2: fold change between MCAO and XST $RR=\frac{M_i-X_i}{M_i-C_i}$ where Ci, Mi, and Xi are the average expressions of gene i in control group, MCAO group, and XST treatment group, respectively.	KS-like statistic	This study sought to investigate the mechanism of action of a Chinese medicine called Xuesaitong injection (XST), a prescription drug made of Panax notoginseng) that is used for treating stroke in China. They looked at positive scores. Inhibition of inflammatory response and coagulation were identified as the major mechanisms involved in the protective effects of XST.	None	Positive scores Permutation p-values < 0.05	CMap	(Wang et al. 2015)
Epilepsy	Human	Cerebellar cortex Temporal cortex Frontal cortex Occipital cortex Hippocampus Thalamus White matter Medulla Putamen	Epilepsy-associated coexpression module (M30)	Fischer’s Exact Test	They used post-mortem human brain samples from healthy individuals from the UK Brain Expression Consortium (UKBEC) dataset to build gene co-expression networks (modules) and integrated modules with whole-exome-sequencing (WES) studies data of rare de novo mutations in those with epileptic encephalopathy (EE). A single module was selected: M30. The M30 genes’ functional expression for 3 epilepsies suggested downregulation of the network as a common mechanism. They used CMap to identify drugs that could up-regulate the M30 genes. Valproic acid (VPA), a widely used antiepileptic drug, was the drug most significantly predicted to up-regulate the genes in M30 (toward health).	None However, confirmed in vitro that VPA upregulates 51% of the M30 genes in neurons (FDR < 10%), replicating and strengthening the VPAsignature in the cancer cell lines from CMap	Tested the overlap of M30 genes with the list of genes upregulated by a drug using one-tail Fischer’s Exact Test (FET) (in order to prioritize drugs predicted to reverse the downregulation of M30 genes observed in epileptic hippocampi). BH corrected P values for multiple hypotheses testing Included only 152 drugs with ≥ 10 DEGs (FDR < 10%) No summarization across cell lines, doses, timepoints for each drug	UK Brain Expression Consortium Genotype-Tissue Expression (GTEx) project STRING WebGestalt GeneMANIA Hippie iRefWeb HUGO Gene Nomenclature Committee database CMap	(Delahaye-Duriez et al. 2016)
Epilepsy	Mouse (pilocarpine-induced chronic epilepsy)	Hippocampus	DEGs - FDR <0.05 & fold change ≥2 (929 up, 1,164 down)	KS-like Statistic (used query app on lincscloud.org)	The authors queried the LINCSL1000 database with an epilepsy signature consisting of the top DEGs between the hippocampus of a model of epileptic mice and control mice. They identified 123 compounds with negative scores beyond a chosen threshold (mean of best 4 ≤ −85). These 123 compounds were enriched with compounds known to have antiepileptic effects. Despite a diverse set of mechanisms of action, these compounds targeted similar biological pathways and were better at reversing pathways affected by epilepsy than common antiepileptic compounds. After filtering for practical exclusion criteria, 36 compounds remained. Of these, sitagliptin was confirmed to have antiepileptic activity in vivo.	in silico (the 123 compounds with LINCS mean connectivity score threshold of −85 or less were 6-fold more enriched with antiepileptic drugs (7/123) than the drugs in the LINCS database as a whole (203/19,767)) in vivo (Sitagliptin produced a dosedependent reduction in seizure scores) in the 6 Hz psychomotor seizure mouse model of pharmacoresistant epilepsy)	Negative scores Inclusion criterion: LINCS mean connectivity score threshold of −85 (123 compounds) Exclusion criteria: toxicity, parenteral route of administration, lack of animal or human dosage data, or BBB-impermeability (36 compounds remained). This was the only study on the table that used any practical considerations to filter compounds. No published evidence of BBBimpermeability for any of the drugs	LINCS-L1000 Drug-Set Enrichment Analysis (DSEA) Gene-Set Enrichment Analysis (GSEA)	(Mirza et al. 2017)
Nerve regeneration	Rat	Dorsal root ganglion neurons 13 separate studies: a total of 382 gene expression datasets (microarray) related to nerve injury	(1) PPI network consisting of 280 genes (2) regeneration-associated co-expression modules	KS-like statistic	The authors identified a transcriptional program observed after peripheral, but not central, nerve injury. They used the regenerationassociated modules to query CMap (2 different inputs) and tested the top 3 candidates that emerged from the intersection of the 2 queries (ambroxol, lasalocid, and disulfiram). Only ambroxol enhanced axonal outgrowth of DRG neurons in vitro and it also increased optic nerve (ON) regeneration in mice after a crush injury, albeit a modest improvement.	in vitro ambroxol, lasalocid, and disulfiram were tested, but only ambroxol enhanced axonal outgrowth of DRG neurons in vivo ambroxol increased optic nerve (ON) regeneration in mice after a crush injury, albeit a modest improvement.	Positive scores Permutation p-value	CMap GEO PubMatrix DAVID JASPAR TRANSFAC STRING ENCODE	(Chandran et al. 2016)
CNS injury	Human MCF7 breast adenocarcinoma cells	NA	DEGs - absolute fold change ≥1.5 p <0.05 (10 up and 12 down) 6 hr treatment of F05 (5 μM; a compound the group previously identified to promote neurite growth in vitro and in vivo) vs vehicle (DMSO, 0.05%)	KS-like statistic	The authors used the transcriptional signature of a compound known to induce neurite growth (F05) as a “seed” to identify other compounds with this same property. Remarkably, despite no chemical similarity to F05, a group of piperazine phenothiazine antipsychotics had similar effects on gene expression and were found to promote neurite growth in vitro at least partially through antagonism of calmodulin signaling, independent of dopamine receptor antagonism.	in vitro ¾ piperazine phenothiazine antipsychotics (but none of the other classes of antipsychotics) significantly enhanced neurite growth of dissociated hippocampal neurons and rat retinal ganglion cells on a substrate of a mixture of chondroitin sulfate proteoglycans (CSPGs) (glial scar proteins)	Positive scores	CMap	(Johnstone et al. 2012)
CNS injury / neurodegenerat ion	Mouse (Ascl1-EGFPBac transge nic reporter mouse line)	Subventricular zone (SVZ) of the dentate gyrus microdomains Regionspecific neural stem cells (NSCs) and their immediate progeny Transient amplifying cells (TAPs)	DEG’s - 1.8-fold change FDR < 5%	Pearson correlation (via SPIED webbased tool)	This study aimed to identify compounds that could direct germinal activity in the subventricular zone (SVZ), which would have therapeutic potential in nerve injury / neurodegenerative or demyelination diseases. They examined NSC lineages in the SVZ microdomains (dorsal versus ventral/lateral) and identified small molecules that direct the fate of these cells toward neurogenic as opposed to oligodendrogenic lineages. LY-294002, an inhibitor of PI3K/Akt, induces transcriptional changes that promote oligodendrogenesis. AR-A014418, an inhibitor of GSK3β induces transcriptional changes that promote rejuvenation of the adult SVZ.	in vivo Infusion of AR-A014418 or CHIR99021 in the adult (P90) dSVZ lateral ventricle dramatically stimulated the germinal activity of the adult dSVZ CHIR99021, a GSK3β inhibitor, showed regenerative potential in a neuropathological context in a model of premature injury that leads to diffuse oligodendroglial and neuronal loss throughout the cortex	Positive scores	CMap GeneGO Metacore for Process Networks SPIEDw	(Azim et al. 2017)
Down Syndrome (DS)	Human Mouse (3 models of DS: Dp16, Ts65Dn and Ts1Cje and controls)	Human: Second trimester amniocytes Induced pluripotent stem cells (iPSCs) Neurons derived from iPSCs Post-mortem human fetal cerebellum and cerebrum Mouse: Developing forebrain (E15.5)	DEGs - BH-FDR < 5% and 20% Because of the limited number of differentially regulated genes at FDR 20%, they used the top 1% up- and down-regulated genes	KS-like statistic	The authors used a human/mouse integrated approach to identify 17 high priority molecules predicted to reverse pathway changes in both human cells and mouse models. They tested the effects of apigenin, one of the molecules predicted by the CMap to treat dysregulated pathways in DS, on human amniocytes derived from fetuses with DS and on the Ts1Cje mouse model of DS. Apigenin treatment reduced oxidative stress in DS amniocytes and improved some aspects of brain morphogenesis, gene expression and postnatal behavior in the Ts1Cje mouse model (manuscript in preparation).	None – currently undergoing in vitro and in vivo testing (not yet published)	Negative scores Threshold: −0.7 or less Integrated approach (multiple inputs)	GSEA DAVID IPA GEO CMap	(Guedj et al. 2016)
Down Syndrome	Human	Second trimester amniotic fluid	DEGs - FDR<5% (414 probes individually differentially expressed between trisomy 21 and controls	KS-like statistic	This is one of the first studies to demonstrate that transcriptional profiling of RNA in uncultured amniotic fluid provides molecular insights into developmental disorders in the living human fetus. They found 4 compounds with average connectivity scores >0.7 (indicating a high correlation with the DS molecular signature), and 9 compounds with average connectivity scores less than −0.7 (indicating a high negative correlation) (NSC-5255229, celastrol, calmidazolium, NSC-5109870, dimethyloxalylglycine, NSC-5213008, verapamil, HC toxin, and felodipine). The 4 compounds that most mimic the DS phenotype were related to potassium and calcium signaling or oxidation which further supports the importance of oxidative stress and ion transport as functional classes involved in DS.	None	Positive scores (mechanistic insight) Negative scores (candidate pharmacotherapeutics)	GSEA DAVID CMap (build 1.0)	(Slonim et al. 2009)