Molecular Profiles of Pyramidal Neurons in the Superior Temporal Cortex in Schizophrenia

Figures & data

Table 1. Subjects included in this study.

Group	Sex^a	Age^b	PMI^c	pH	Smoker	Cause of death	Antipsychotic^d
Control	F	79	15.00	6.59	Yes	Cardiac arrest	N/A
Control	M	22	21.47	6.75	Yes	Myocardial infarction	N/A
Control	M	75	20.25	6.35	Yes	Unknown	N/A
Control	M	80	15.50	6.26	Yes	Myocardial infarction	N/A
Control	F	58	21.08	6.79	No	Myocardial infarction	N/A
Control	M	61	17.00	6.64	Yes	Unknown	N/A
Control	F	71	20.50	6.89	Unknown	Unknown	N/A
Control	F	90	12.66	6.10	Unknown	Lung cancer	N/A
Control	F	86	6.92	5.74	Unknown	Myocardial infarction	N/A
Mean ± SEM	4M/5F	69.11 ± 6.85	16.71 ± 1.60	6.46 ± 0.12
Schizophrenia	F	93	6.92	6.13	Unknown	Renal failure	Perphenazine (50)
Schizophrenia	M	55	21.40	6.51	Yes	Myocardial infarction	Perphenazine
Schizophrenia	F	67	21.80	5.80	Unknown	Pulmonary disease	Thioridazine, prochlorperazine (200)
Schizophrenia	F	55	22.00	5.90	Yes	Cancer	Clozapine, olanzapine
Schizophrenia	M	36	17.97	6.45	Unknown	Cardiac arrest	Clozapine (350)
Schizophrenia	M	62	10.75	6.50	Yes	Chronic obstructive pulmonary disease, lung cancer	Clozapine
Schizophrenia	F	92	17.80	6.34	Unknown	Cardiomyopathy	Unknown
Schizophrenia	M	56	21.83	6.75	No	Car accident	Olanzapine (150)
Schizophrenia	F	88	13.33	6.65	No	Unknown	Thiothixene (50)
Mean ± SEM	4M/5F	68.11 ± 6.60	16.90 ± 1.85	6.34 ± 0.11

^aF = female; M = male.

^bAge is given in years.

^cPMI = postmortem interval in hours.

^dN/A = not applicable; numbers in parentheses indicate CED.

Figure 1. Identification of pyramidal neurons and LCM procedure. Photomicrograph of a pyramidal neuron in the STG visualized with Histogene stain (A). After identification (B), pyramidal neurons are captured (C) onto the LCM cap (D). (E) Representative virtual gels showing the distribution of sizes of amplified products after two rounds of linear amplification of RNA extracted from ˜500 pyramidal neurons from a normal control (C) and a schizophrenia (S) subject. L = ladder. (F) Heatmap of 1331 differentially expressed genes identified based on the stringency criteria of fold-change > 1.1 and FDR-corrected p < 0.05. Scale bars = 25 μm.

Table 2. RNA quantity and quality.

Group	RIN of total RNA extracted from homogenized cortex^a	Microarray samples
		Number of cells captured	Total RNA RQI^b	Amount of RNA before/after amplification (μg)	A260/A280
Control	8.2	510	2.6	0.025/39.50	2.67
Control	7.9	530	N/A	0.004/69.30	2.37
Control	8.9	520	3.1	0.014/54.30	2.44
Control	8.8	500	2.8	0.004/39.49	2.51
Control	7.8	680	3.4	0.007/50.10	2.39
Control	9	600	3.7	0.009/59.92	2.54
Control	8.4	530	2.8	0.012/39.60	2.47
Control	8	520	3.6	0.007/29.83	2.75
Control	8.8	520	N/A	0.002/24.52	2.70
Mean ± SD	8.42 ± 0.15	545.56 ± 19.30	3.14 ± 0.16	0.009 ± 0.002/40.73 ± 5.15	2.54 ± 0.05
Schizophrenia	8.9	525	3.8	0.004/76.83	2.47
Schizophrenia	8	525	4.2	0.003/65.10	2.43
Schizophrenia	7.9	540	N/A	0.002/62.10	2.52
Schizophrenia	8.5	540	2.8	0.004/46.92	2.75
Schizophrenia	9.2	530	N/A	0.001/62.10	2.44
Schizophrenia	8.5	630	N/A	0.002/75.90	2.34
Schizophrenia	8.2	540	3.5	0.003/65.68	2.50
Schizophrenia	8.8	700	3.4	0.003/50.10	2.47
Schizophrenia	8.4	550	3.4	0.003/52.20	2.47
Mean ± SD	8.48 ± 0.16	564.56 ± 20.06	3.52 ± 0.19	0.002 ± 0.0003/61.88 ± 3.54	2.49 ± 0.04

^aRNA integrity number (RIN) of total RNA extracted from homogenized gray matter determined by an Agilent bioanalyzer.

^bRNA Quality Indication (RQI) of total RNA extracted from laser-captured neurons determined by a BioRad Experion Automated Electrophoresis System. N/A = not available: total RNA concentration too low to determine RQI.

Figure 2. Correlation analysis comparing fold-changes of selected genes determined by microarray and qRT-PCR. Comparison of fold-changes of differentially expressed genes within signaling pathways identified as dysregulated in schizophrenia (N = 6) and several randomly selected genes (N = 4) determined by microarray and qRT-PCR. BMP7 = bone morphogenetic protein 7; BMPR1A = bone morphogenetic protein receptor type IA; CLU = clusterin; HLA-A = major histocompatibility complex (MHC), class I, A; HPRT = hypoxanthine guanine phosphoribosyl transferase; P2RY14 = purinergic receptor P2Y, G-protein coupled, 14; MCPH1 = microcephalin 1; SMAD4 = mothers against decapentaplegic homolog 4; SMAD5 = mothers against decapentaplegic homolog 5; VCAN = versican.

Table 3a. Significantly affected genes within TGF-β/BMP signaling pathway.

Gene title	Gene symbol	p-Value	Fold-change (S vs. C)^a
bone morphogenetic protein 1	BMP1	0.04	− 1.33
bone morphogenetic protein receptor, type IA	BMPR1A	0.04	1.19
bone morphogenetic protein 5	BMP5	0.04	1.16
bone morphogenetic protein 7	BMP7	0.03	1.21
caveolin 1, caveolae protein, 22 kDa	CAV1	0.03	1.54
CREB-binding protein	CBP	0.01	1.31
growth arrest and DNA-damage-inducible, beta	GADD45B	0.03	1.30
mitogen-activated protein kinase kinase 6	MEK6	0.04	− 1.14
ribosomal protein S6 kinase, 90kDa, polypeptide 5	RPS6KA5	0.01	1.11
SHC (Src homology 2 domain containing)-transforming protein 1	SHC1	0.03	1.16
SMAD family member 4	SMAD4	0.01	1.19
SMAD family member 5	SMAD5	0.02	1.48
SKI-like oncogene	SKIL	0.02	− 1.15
transcription factor specificity protein 1	SP1	0.03	1.29
X-linked inhibitor of apoptosis	XIAP	0.02	1.10
zinc finger, FYVE domain containing 9	ZFYVE9/SARA	0.04	1.14

^aS = schizophrenia; C = control.

Table 3b. Significantly affected genes associated with extracellular matrix.

Gene title	Gene symbol	p-Value	Fold-change (S vs. C)^a
aggrecan	ACAN	0.03	− 1.26
ADAM metallopeptidase with thrombospondin type 1 motif, 1	ADAMTS1	0.03	2.56
ADAM metallopeptidase with thrombospondin type 1 motif, 6	ADAMTS6	0.05	1.15
hyaluronan and proteoglycan link protein 1	HAPLN1	0.05	− 1.14
leucine proline-enriched proteoglycan (leprecan) 1	LEPRE1	0.04	− 1.21
lumican	LUM	0.03	− 1.12
matrix metallopeptidase 16 (membrane-inserted)	MMP16	0.02	− 1.17
matrix metallopeptidase 25	MMP25	0.02	− 1.14
matrix metallopeptidase 24 (membrane-inserted)	MMP24	0.01	1.22
sperm adhesion molecule 1 (PH-20 hyaluronidase, zona pellucida binding)	SPAM1	0.04	1.15
sparc/osteonectin, cwcv and kazal-like domains proteoglycan (testican) 3	SPOCK3	0.01	1.11
spondin 1, extracellular matrix protein	SPON1	0.02	2.14
versican	VCAN	0.04	− 1.13

^aS = schizophrenia; C = control.

Table 3c. Significantly affected genes associated with apoptosis and DNA damage.

Gene title	Gene symbol	p-Value	Fold-change (S vs. C)^a
Apoptosis and survival_BAD phosphorylation
epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)	EGFR	0.02	1.59
phosphoinositide-3-kinase, regulatory subunit 2 (beta)	PIK3R2	0.04	1.23
protein kinase, cAMP-dependent, catalytic, gamma	PRKACG	0.02	1.25
protein tyrosine phosphatase, non-receptor type 11	PTPN11	0.03	− 1.11
SHC (Src homology 2 domain containing)-transforming protein 1	SHC1	0.03	1.16
DNA damage_Role of SUMO in p53 regulation
CREB-binding protein	CBP	0.01	1.31
SMT3 suppressor of mif two 3 homolog 1 (S. cervisiae)	SUMO-1	0.03	1.17
ubiquitin-like modifier activating enzyme 1	UBA1	0.04	1.14
Other genes involved in DNA damage and oxidative stress
HLA-B (major histocompatibility complex, class I, B)-associated transcript 3	BAT3/BAG6	0.01	− 1.21
calpain 9	CAPN9	0.05	1.15
calpain 10	CAPN10	0.02	1.14
clusterin	CLU	0.02	2.02

^aS = schizophrenia; C = control.

Table 3d. Significantly affected genes associated with cytoskeletal and dendritic integrity.

Gene title	Gene symbol	p-Value	Fold-change (S vs. C)^a
actin, gamma 2, smooth muscle, enteric	ACTG2	0.04	1.35
dystonin	DST	0.03	1.25
major histocompatibility complex, class I, A	HLA-A	0.02	1.86
integrin, beta 5	ITGB5	0.02	1.51
microtubule-associated protein 1 light chain 3 gamma	MAP1LC3C	0.03	1.19
myosin IIIB	MYO3B	0.01	1.13
tropomodulin 3 (ubiquitous)	TMOD3	0.02	− 1.14
tubulin polymerization promoting protein	TPPP	0.03	− 1.50
tubulin tyrosine ligase-like family, member 12	TTLL12	0.02	1.20
tubulin, alpha 4b (pseudogene)	TUBA4B	0.02	− 1.17
spectrin, beta, non-erythrocytic 1	SPTBN1	0.05	1.20

^aS = schizophrenia; C = control.

Figure 3. Representative diagram of TGF-β and BMP signaling pathways. Both TGF-β and BMP7 activate SMAD4 and CREB-binding protein, a transcription co-activator of SMAD, leading to the transcription of target genes that influence extracellular matrix composition, apoptosis, and synaptic/cytoskeletal plasticity. BMPR1A = bone morphogenetic protein receptor type IA; CBP = CREB-binding protein; SARA (ZFYVE9) = zinc finger, FYVE domain containing 9; SMAD4 = mothers against decapentaplegic homolog 4. Genes up-regulated in schizophrenia are depicted by red arrows.

Table 4. Differentially expressed miRNAs in schizophrenia.

miRNA assay name	p-Value	Log2 fold-change
hsa-miR-1243-002854	0.03	− 0.41
hsa-miR-126-4395339	0.03	1.84
hsa-miR-150-4373127	0.05	− 0.96
hsa-miR-30b-4373290	0.02	4.64
hsa-miR-328-4373049	< 0.001	2.82
hsa-miR-378-002243	0.04	− 0.31
hsa-miR-520d-3p-002743	0.01	0.12
hsa-miR-628-5p-4395544	0.03	− 1.05
hsa-miR-875-5p-002203	0.04	− 0.41
hsa-miR-99b-4373007	0.04	2.77

Table 5. Overrepressented KEGG pathways based on enrichment analysis of the predicted target genes of the differentially expressed miRNAs.

Pathway	Enrichment statistics
TGF-β signaling pathway	C = 155; O = 63; E = 8.75; R = 7.20; rawP = 1.27e-37; adjP = 1.45e-35
Focal adhesion	C = 201; O = 48; E = 11.35; R = 4.23; rawP = 9.92e-18; adjP = 1.46e-16
Regulation of actin cytoskeleton	C = 143; O = 37; E = 8.07; R = 4.58; rawP = 3.41e-15; adjP = 4.32e-14
Apoptosis	C = 82; O = 20; E = 4.63; R = 4.32; rawP = 2.12e-08; adjP = 8.06e-08
ECM-receptor interaction	C = 84; O = 16; E = 4.74; R = 3.37; rawP = 1.67e-05; adjP = 4.63e-05

Figure 4. Hypothetical model of pyramidal cell dysfunction in schizophrenia. It is hypothesized that as a result of disinhibition-induced excitotoxic insult (see text for details), TGFβ signaling within pyramidal neurons is up-regulated, which may contribute to extracellular matrix abnormalities, dysregulated apoptosis, and impaired synaptic and cytoskeletal plasticity. Shown in this highly simplified diagram are some of the hub genes that can be targeted in future experiments in the testing of this hypothesis.

Figure 5. Wisteria floribunda agglutinin (WFA) histochemical labeling of chondroitin sulfate proteoglycans (CSPGs) in the human cerebral cortex. (A) CSPG-rich perineuronal nets (arrowheads). (B) WFA also labeled intracellular CSPGs (arrowhead). Scale bars = 5 μm (A and B).