Amyotrophic lateral sclerosis, gene deregulation in the anterior horn of the spinal cord and frontal cortex area 8: implications in frontotemporal lobar degeneration.

Transcriptome arrays identifies 747 genes differentially expressed in the anterior horn of the spinal cord and 2,300 genes differentially expressed in frontal cortex area 8 in a single group of typical sALS cases without frontotemporal dementia compared with age-matched controls. Main up-regulated clusters in the anterior horn are related to inflammation and apoptosis; down-regulated clusters are linked to axoneme structures and protein synthesis. In contrast, up-regulated gene clusters in frontal cortex area 8 involve neurotransmission, synaptic proteins and vesicle trafficking, whereas main down-regulated genes cluster into oligodendrocyte function and myelin-related proteins. RT-qPCR validates the expression of 58 of 66 assessed genes from different clusters. The present results: a. reveal regional differences in de-regulated gene expression between the anterior horn of the spinal cord and frontal cortex area 8 in the same individuals suffering from sALS; b. validate and extend our knowledge about the complexity of the inflammatory response in the anterior horn of the spinal cord; and c. identify for the first time extensive gene up-regulation of neurotransmission and synaptic-related genes, together with significant down-regulation of oligodendrocyte- and myelin-related genes, as important contributors to the pathogenesis of frontal cortex alterations in the sALS/frontotemporal lobar degeneration spectrum complex at stages with no apparent cognitive impairment.

INTRODUCTION

Amyotrophic lateral sclerosis (ALS) is a progressive age-dependent neurodegenerative disease characterized by degeneration and death of upper (motor cortex) and lower (brain stem and spinal cord) motor neurons, resulting in muscle atrophy, together with variable frontotemporal lobar degeneration (FTLD). ALS may be sporadic (sALS) with unknown cause, in up to 90%-92% of cases, or inherited (fALS), accounting for about 8-10% of cases, most of them transmitted as autosomal dominant but also recessive and X-linked in some families. However, about 13% of sALS cases bear a gene mutation linked to fALS. Main pathological features in sALS are loss of myelin and axons in the pyramidal tracts and anterior spinal roots, chromatolysis of motor neurons, axonal spheroids in the anterior horn, cystatin C-containing Bunina bodies in motor neurons, ubiquitin-immunoreactive TDP-43-positive skein-like and spherical inclusions in motor neurons, and TDP-43 inclusions in oligodendroglial cells. In many cases, the frontal cortex shows cytoplasmic TDP-43-immuno-reactive intracytoplasmic inclusions in neurons and oligodendocytes, and neuropil threads. Neuron loss and spongiosis in the upper cortical layers are usually restricted to cases with severe cognitive impairment and frontotemporal dementia [1, 2].

Several mechanisms have been proposed as contributory factors in the pathogenesis of motor neuron damage in sALS including excitoxicity, mitochondrial and energy metabolism failure, oxidative stress damage, altered glial cells, inflammation, cytoskeletal ab-normalities, alterations in RNA metabolism, and altered TDP-43 metabolism, among others [3-16]. Increased understanding on the pathogenesis of sALS has emerged from the use of transcriptome analysis of the spinal cord and motor cortex [17-26]. Previous transcriptomic studies center in the spinal cord and motor cortex in separate groups of patients, cover a limited number of cases, identify and validate a few genes not coincidental among the different studies. Selection of the sample may account for these differences. Further microarray studies carried out on isolated motor neurons of the spinal cord obtained by laser micro-dissection in sALS cases have revealed up-regulation of genes associated with cell signalling and cell death and down-regulation of genes linked to transcription and composition of the cytoskeleton [27]. Curiously, similar studies performed on samples from individuals bearing mutations linked to ALS show different regulated transcripts, thus suggesting gene expression variants in the spinal cord in fALS [28, 29].

Importantly, no gene expression analyses are available in the frontal cortex area 8 in sALS in spite that frontal alterations are common in this disease. Moreover, ALS and FTLD with TDP inclusions (FTLD-TDP) are within the same disease spectrum [1].

The present study analyzes gene expression in the anterior horn of the spinal cord and frontal cortex area 8 in a series of 18 sALS cases and 23 controls. The main goals of the present study are to analyze and compare gene expression in these two regions, and more specifically to identify altered gene expression and clusters with specific functions in the anterior horn and frontal cortex area 8. Thus, the present study focuses on the pathogenesis of motor neuron damage responsible of altered motor function, and frontal cortex at preclinical stages of cognitive impairment.

RESULTS

Microarray analysis

Cofactors age and gender were not relevant for the analysis. 9,563 gene sequences were detected across all samples. Heat map indicates differences in transcripts expression levels between control and ALS cases in the anterior cord of the spinal cord and in frontal cortex area 8 (Figure 1). We identified 747 genes differentially expressed with p-value lower than or equal to 0.05 in the anterior horn of the spinal cord (up: 507 and down: 240) and 2,300 genes differentially expressed in the frontal cortex area 8 (up: 1,409 and down: 891) in sALS (Figure 1).

Figure 1

(A) Total number of significantly different expressed genes comparing transcriptomic profiles between groups and regions. (B) Hierarchical clustering heat map of expression intensities of mRNA array transcripts reflect differential gene expression profiles in the anterior horn of the spinal cord and frontal cortex area 8 in ALS compared with controls. Differences between groups are considered statistically significant at p-value ≤ 0.05. Abbreviations: ALS: amyotrophic lateral sclerosis; FC: frontal cortex area 8; mRNA: messenger RNA; SP: anterior horn of the spinal cord lumbar level

Supplementary Tables 1 and 2 identify all de-regulated genes. Post-analysis microarray data of differentially ex-pressed genes assessed with enrichment analysis against Go Ontology database are shown in Tables 1 and 2.

Table 1

Main significant clusters of altered genes in spinal cord of ALS samples

Cluster	Gene names	Size	Count	Odds Ratio	p-value	Deregulation
Activation of blood coagulation via clotting cascade	F3, ANO6	2	2	Inf	0.000574	Up
Antigen processing and presentation of exogenous peptide antigen	CTSS, FCER1G, FCGR1A, HLA-A, HLA-B, HLA-C, HLA-DMA, HLA-DMB, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DRB1, HLA-DRB5, HLA-F, HLA-G, NCF2, PSMB8, PSMB9, PSMD5, TAP1, IFI30	165	22	6.58	6.84e-11	Up
Antigen processing and presentation of exogenous peptide antigen via MHC class I	CTSS, FCER1G, FCGR1A, HLA-A, HLA-B, HLA-C, HLA-F, HLA-G, NCF2, PSMB8, PSMB9, PSMD5, TAP1, IFI30	75	14	9.66	2.45e-09	Up
Antigen processing and presentation of exogenous peptide antigen via MHC class I, TAP-independent	CTSS, HLA-A, HLA-B, HLA-C, HLA-F, HLA-G	9	6	82.7	1.45e-08	Up
Antigen processing and presentation of exogenous peptide antigen via MHC class II	CTSS, FCER1G, HLA-DMA, HLA-DMB, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DRB1, HLA-DRB5, IFI30	92	11	5.66	1.24e-05	Up
Antigen processing and presentation of peptide antigen via MHC class I	CTSS, FCER1G, FCGR1A, HLA-A, HLA-B, HLA-C, HLA-F, HLA-G, NCF2, PSMB8, PSMB9, PSMD5, TAP1, IFI30	97	14	7.09	7.58e-08	Up
Apoptotic process	AHR, APOE, FAS, BCL2A1, BCL6, BMP2, BTK, CAMK2D, CASP1, CASP4, TNFSF8, CDKN1A, CTSC, DAB2, NQO1, ECT2, EDN1, F3, FCER1G, HCK, HGF, HIF1A, HMOX1, ICAM1, IFI16, IL1A, ITGA5, JAK3, LMNB1, LYN, MNDA, MYC, NCF2, NOS3, P2RX4, PLAGL1, PLAUR, PLSCR1, PRLR, PSMB8, PSMB9, PSMD5, PTPN2, CCL2, CCL19, SNAI2, STAT1, TEK, TGFB2, TLR2, TLR3, GPR65, YBX3, NOL3, SOCS3, LY86, IKBKE, CHL1, PPP1R15A, RRM2B, SHISA5, TNFRSF12A, ACSL5, FNIP2, DNASE2B, ZMAT3, NOA1, FGD3, IL33, DEDD2, ANO6	1745	71	1.89	5.22e-06	Up
Apoptotic signaling pathway	FAS, BCL2A1, BTK, CASP4, CDKN1A, CTSC, ECT2, HGF, HIF1A, HMOX1, ICAM1, IFI16, IL1A, NOS3, P2RX4, PLAUR, PTPN2, SNAI2, TGFB2, TLR3, YBX3, NOL3, IKBKE, PPP1R15A, RRM2B, SHISA5, TNFRSF12A, ACSL5, FNIP2, FGD3, IL33, DEDD2	596	32	2.43	1.88e-05	Up
Axonemal dynein complex assembly	DNAH5, DNAI1, TEKT2, ZMYND10, ARMC4, DNAH7, CCDC114, CCDC151, DNAAF1, CCDC39	21	10	175	8.54e-18	Down
Axoneme	DNAH5, DNAH9, SPAG6, DNAI1, DCDC2, HYDIN, CFAP46, ARMC4, MNS1, DNAH7, CFAP74, CCDC114, CCDC151, DNAAF1, CFAP54, DNAH2, SPAG17, CFAP221, CCDC39, RSPH4A	89	20	52.5	1.31e-25	Down
Axoneme assembly	DNAH5, DNAI1, TEKT2, ZMYND10, HYDIN, CFAP46, ARMC4, DNAH7, CFAP74, RSPH1, CCDC114, CCDC151, DNAAF1, SPAG17, CCDC39, RSPH4A	42	16	128	5.9e-26	Down
B cell mediated immunity	FAS, BCL6, BTK, C1QB, C1QC, C7, FCER1G, HLA-DMA, HLA-DQB1, HLA-DRB1, HLA-DRB5, CFI, IL4R, CD226, TLR8	103	15	7.18	2.28e-08	Up
Cellular protein modification process	IL12RB1, INS, KCNE1, MAK, CFP, RASA4, TRAK2, MYLK3, NEK5, C17orf97, PPIAL4A	3527	11	0.473	0.00885	Down
Cellular response to interferon-gamma	CAMK2D, EDN1, FCGR1A, GBP1, HCK, HLA-A, HLA-B, HLA-C, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DRB1, HLA-DRB5, HLA-F, HLA-G, ICAM1, IRF8, OAS2, PTPN2, CCL2, CCL19, STAT1, SOCS3, IFI30, TRIM38, TRIM5	126	27	11.9	1.95e-18	Up
Clathrin-coated endocytic vesicle membrane	FCGR1A, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DRB1, HLA-DRB5	49	7	7.32	0.000108	Up
Copper ion import	ATP7B, SLC31A1, STEAP4	7	3	30.7	0.000446	Up
Cytokine production involved in immune response	BCL6, BTK, FCER1G, HLA-A, HMOX1, JAK3, SLC11A1, TEK, TGFB2, TLR2, TLR3, TREM1	69	12	8,81	7,87E-08	Up
Endolysosome membrane	TLR3, TLR7, TLR8	4	3	131	4.51e-05	Up
Fc receptor mediated stimulatory signaling pathway	FCER1G, FCGR1A, FCGR2A, FGR, HCK, ITPR3, LYN, PLSCR1, CD226, MYO1G	77	10	6.21	1.47e-05	Up
Humoral immune response mediated by circulating immunoglobulin	C1QB, C1QC, C7, HLA-DQB1, HLA-DRB1, HLA-DRB5, CFI	46	7	7.42	0.000103	Up
Igg binding	FCER1G, FCGR1A, FCGR2A, FCGR2B	10	4	28.3	5.42e-05	Up
Immunoglobulin production	FAS, BCL6, CD37, HLA-DQB1, HLA-DRB1, HLA-DRB5, IL4R, TNFSF13B, POLM, IL33	87	10	5.4	4.34e-05	Up
Inner dynein arm assembly	TEKT2, ZMYND10, DNAH7, DNAAF1, CCDC39	10	5	182	1.44e-09	Down
Integral component of lumenal side of endoplasmic reticulum membrane	HLA-A, HLA-B, HLA-C, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DRB1, HLA-DRB5, HLA-F, HLA-G	28	11	28.8	1.04e-11	Up
Interferon-alpha production	TLR3, NMI, TLR7, TLR8	18	4	11.7	0.000764	Up
Interferon-beta biosynthetic process	TLR3, NMI, TLR7, TLR8	8	4	41.1	2.12e-05	Up
Interferon-gamma biosynthetic process	TLR3, EBI3, TLR7, TLR8	16	4	13.7	0.000472	Up
Interleukin-10 production	FCER1G, HLA-DRB1, HLA-DRB5, JAK3, TLR2, PDCD1LG2	42	6	6.87	0.000463	Up
Intrinsic apoptotic signaling pathway	BCL2A1, CASP4, CDKN1A, HIF1A, HMOX1, IFI16, PLAUR, PTPN2, SNAI2, YBX3, NOL3, IKBKE, PPP1R15A, RRM2B, SHISA5, FNIP2	284	16	2.49	0.00143	Up
Macrophage activation	IL4R, SLC11A1, TLR1, SBNO2, CD93, TLR7, TLR8, IL33	48	8	8.29	1.66e-05	Up
Mast cell cytokine production	BCL6, FCER1G, HMOX1	7	3	30.7	0.000446	Up
MHC class II receptor activity	HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DRB1	11	5	35.4	2.73e-06	Up
MHC protein complex	HLA-A, HLA-B, HLA-C, HLA-DMA, HLA-DMB, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DRB1, HLA-DRB5, HLA-F, HLA-G	25	13	48.4	1.34e-15	Up
Microtubule bundle formation	DNAH5, DNAI1, TEKT2, ZMYND10, HYDIN, CFAP46, ARMC4, DNAH7, CFAP74, RSPH1, CCDC114, CCDC151, DNAAF1, SPAG17, CCDC39, RSPH4A	63	16	70.7	1.18e-22	Down
Monocyte chemotaxis	CCR1, LYN, CCL2, CCL19, PLA2G7, ANO6	49	6	5.75	0.00107	Up
Outer dynein arm assembly	DNAH5, DNAI1, ZMYND10, ARMC4, CCDC114, CCDC151, DNAAF1	11	7	325	5.6e-14	Down
Peptide antigen binding	HLA-A, HLA-B, HLA-C, HLA-DQA1, HLA-DQB1, HLA-DRB1, HLA-DRB5, HLA-F, HLA-G, TAP1	26	10	26.9	1.57e-10	Up
Platelet-derived growth factor receptor binding	TYMP, ITGA5, ITGB3, LYN	12	4	21.2	0.000123	Up
Positive regulation of Fc receptor mediated stimulatory signaling pathway	LYN, CD226	2	2	Inf	0.000574	Up
Positive regulation of interleukin-6 production	FCER1G, TLR1, TLR2, TLR3, TLR7, IL33	55	6	5.05	0.00197	Up
Positive regulation of interleukin-8 production	TLR2, TLR3, TLR5, TLR7, TLR8	42	5	5.56	0.00318	Up
Positive regulation of tumor necrosis factor production	FCER1G, CCL2, CCL19, TLR1, TLR2, TLR3	51	6	5.5	0.00133	Up
Protection from natural killer cell mediated cytotoxicity	HLA-A, HLA-B, TAP1	5	3	61.5	0.000132	Up
Regulated secretory pathway	ANXA3, FCER1G, FGR, HCK, HMOX1, IL4R, LYN, STX11, CD300A, RAB11FIP2, RAB11FIP1	73	11	7.4	1.23e-06	Up
Regulation of apoptotic process	APOE, FAS, BCL2A1, BCL6, BMP2, BTK, CAMK2D, CASP1, CASP4, CDKN1A, CTSC, DAB2, NQO1, ECT2, EDN1, F3, FCER1G, HCK, HGF, HIF1A, HMOX1, ICAM1, IL1A, ITGA5, JAK3, LYN, MNDA, MYC, NCF2, NOS3, PLAUR, PRLR, PSMB8, PSMB9, PSMD5, PTPN2, CCL2, CCL19, SNAI2, STAT1, TEK, TGFB2, TLR3, YBX3, NOL3, SOCS3, CHL1, RRM2B, TNFRSF12A, ACSL5, ZMAT3, FGD3, DEDD2, ANO6	1344	54	1.82	0.000117	Up
Regulation of B cell apoptotic process	BCL6, BTK, LYN	16	3	9.45	0.00608	Up
Regulation of coagulation	APOE, EDN1, F3, FCER1G, LYN, NOS3, PLAU, PLAUR, THBD, HPSE, ADAMTS18, ANO6	85	12	6.87	8.31e-07	Up
Regulation of cytokine biosynthetic process	CD86, HMOX1, IL1A, TLR1, TLR2, TLR3, NMI, EBI3, TLR7, TLR8	93	10	5.01	7.72e-05	Up
Regulation of extrinsic apoptotic signaling pathway	FAS, HMOX1, ICAM1, IL1A, NOS3, SNAI2, TGFB2, NOL3, TNFRSF12A, ACSL5, DEDD2	155	11	3.17	0.0013	Up
Regulation of Fc receptor mediated stimulatory signaling pathway	LYN, PLSCR1, CD226	5	3	61.5	0.000132	Up
Regulation of hemostasis	APOE, EDN1, F3, FCER1G, LYN, NOS3, PLAU, PLAUR, THBD, HPSE, ADAMTS18, ANO6	81	12	7.27	4.87e-07	Up
Regulation of leukocyte apoptotic process	BCL6, BTK, FCER1G, HIF1A, JAK3, LYN, CCL19, TGFB2	74	8	5.02	0.000386	Up
Regulation of lipid kinase activity	FGR, LYN, CCL19, TEK, NRBF2	47	5	4.89	0.0052	Up
Regulation of mast cell activation	FCER1G, FGR, HMOX1, IL4R, LYN, PLSCR1, CD226, CD300A	31	8	14.4	4.96e-07	Up
Regulation of mast cell degranulation	FCER1G, FGR, HMOX1, IL4R, LYN, CD300A	24	6	13.8	1.71e-05	Up
Regulation of microtubule movement	DNAH11, ARMC4, DNAAF1, CCDC39	18	4	51.3	3.03e-06	Down
Regulation of natural killer cell mediated immunity	HLA-A, HLA-B, PVR, TAP1, CD226	27	5	9.36	0.000405	Up
Regulation of protein metabolic process	FOXJ1, INS, CFP, RASA4, NEK5, DTHD1	2448	6	0.381	0.00803	Down
Regulation of protein modification process	INS, RASA4	1641	2	0.192	0.00288	Down
Regulation of T-helper 1 cell differentiation	HLX, IL4R, JAK3, CCL19	9	4	32.9	3.74e-05	Up
T cell costimulation	CD86, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DRB1, HLA-DRB5, LYN, CCL19, TNFSF13B, PDCD1LG2	71	11	7.65	9.25e-07	Up
TAP binding	HLA-A, HLA-B, HLA-C, HLA-F, TAP1	7	5	106	1.34e-07	Up
T-helper 2 cell differentiation	BCL6, CD86, HLX, IL4R	14	4	16.4	0.00027	Up

Table 2

Main significant clusters of altered genes in frontal cortex of ALS samples

Cluster	Gene names	Size	Count	Odds Ratio	p-value	Deregulation
Adenylate cyclase-inhibiting G-protein coupled receptor signaling pathway	ADCY1, CHRM1, CHRM3, GNAI3, MCHR1, GRM8, HTR1B, HTR1E, HTR1F, NPY1R, OPRK1, OPRM1, SSTR2	64	13	3.79	0.000164	Up
Astrocyte differentiation	ABL1, MAG, NKX2-2, NOTCH1, POU3F2, S100B, TAL1, CNTN2, SOX8	53	9	5.12	0.000184	Down
Axolemma	KCNC1, KCNC2, KCNH1, ROBO2, SLC1A2	14	5	8.26	0.00124	Up
Axon	DAGLA, CAMK2D, CCK, CHRM1, CHRM3, AP1S1, CTNNA2, DLG4, DRD1, EPHA4, PTK2B, FGF13, GAP43, GARS, GRIA1, GRIK5, GRIN2A, HTR2A, KCNB1, KCNC1, KCNC2, KCNH1, KCNK2, KCNMA1, KCNQ2, KCNQ3, MYH10, NPY1R, NRCAM, NRGN, OPRK1, PAK1, PFN2, MAP2K1, PTPRN2, ROBO2, SCN1A, SCN1B, SCN2A, SCN8A, CCL2, SLC1A2, SNCA, STXBP1, SYN1, KCNAB1, FZD3, GLRA3, PRSS12, CNTNAP1, KCNAB2, NRP1, CDK5R1, BSN, SYT7, SYNGR1, DGKI, NRXN1, HOMER1, KATNB1, SEMA3A, OLFM1, SLC9A6, CPLX1, AAK1, ADGRL1, TPX2, UNC13A, MYCBP2, NCS1, PACSIN1, STMN3, SEPT11, SLC17A7, TBC1D24, NDEL1, LMTK3, MTPN, CNTN4, LRRTM1, HCN1	358	81	4.6	1.68e-24	Up
Axon extension	BMPR2, NRCAM, PPP3CB, SLIT1, CDKL5, NRP1, CDK5R1, LHX2, SEMA3A, OLFM1, SLC9A6, BCL11A, ISLR2, NDEL1	91	14	2.7	0.00176	Up
Axon hillock	CCK, TPX2, NDEL1	7	3	11.1	0.00729	Up
Cadherin binding	CDH13, CTNNA2, TRPC4, CDK5R1, AKAP5, MMP24, PTPRT	29	7	4.81	0.00167	Up
Calcineurin complex	ITPR1, PPP3CA, PPP3CB, PPP3R1	4	4	Inf	1.59e-05	Up
Calcium channel regulator activity	CACNB2, FKBP1B, ITPR1, PRKCB, STX1A, NRXN1, TSPAN13, HPCAL4, CACNA2D3	36	9	5.05	0.000281	Up
Calcium ion-dependent exocytosis of neurotransmitter	CACNA1A, SYT1, SYT5, DOC2A, SYT7, RIMS2, RAB3GAP1, RIMS1, SYT13, SYT12	28	10	8.25	4.76e-06	Up
Calmodulin binding	ADCY1, ADD2, ATP2B1, ATP2B2, CACNA1C, CAMK4, CAMK2A, CAMK2B, CAMK2D, GAP43, ITPKA, KCNH1, KCNN1, KCNQ3, MAP2, MYH10, MYO5A, NOS2, NRGN, PDE1B, PPP3CA, PPP3CB, PPP3R1, RGS4, RIT2, RYR2, SLC8A2, SLC8A1, AKAP5, CAMKK2, ARPP21, PLCB1, KCNH5, CAMK1D, CAMK1G, CAMKV, CAMKK1, PNCK, CFAP221, RIIAD1	176	40	4.57	5.38e-13	Up
Calmodulin-dependent protein kinase activity	CAMK4, CAMK2A, CAMK2B, CAMK2D, PTK2B, ITPKA, CAMKK2, CAMK1D, CAMK1G, CAMKK1, PNCK	32	11	7.95	2.02e-06	Up
Camp binding	PDE2A, PDE4A, PRKAR1B, PRKAR2B, RAPGEF2, RAPGEF4, HCN1	24	7	6.23	0.000487	Up
Central nervous system neuron axonogenesis	EPHA4, SCN1B, NR2E1, MYCBP2, PRDM8, ARHGEF28, NDEL1	29	7	4.71	0.00187	Up
Chloride channel activity	CLIC2, GABRA1, GABRA2, GABRA3, GABRA4, GABRA5, GABRB2, GABRB3, GABRD, GABRG3, GLRB, SLC26A4, GLRA3, SLC17A7, SLC26A8, ANO5	78	16	3.93	2.05e-05	Up
Clathrin binding	SYT1, SYT5, DOC2A, SYT7, SNAP91, HMP19, SYTL2, CEMIP, SYT13, SMAP1, SYT16, SYT12	56	12	4.14	0.000141	Up
Compact myelin	MAG, SIRT2, JAM3	12	3	8.38	0.00957	Down
Cyclin-dependent protein serine/threonine kinase activity	CDK14, CDKL5, CDKL1, CDK5R1, CDKL2, CDK20	29	6	3.94	0.008	Up
Cytoskeleton of presynaptic active zone	BSN, PCLO	2	2	Inf	0.004	Up
Dendrite	BMPR2, CACNA1A, CACNA1B, CACNA1C, CCK, CHRM1, CHRM3, CRMP1, DLG3, DLG4, DRD1, EPHA4, EPHA7, PTK2B, FGF13, GABRA5, GRIA1, GRIA2, GRIA3, GRIK5, GRIN2A, GRM1, GRM5, HTR2A, ITPKA, KCNB1, KCNC1, KCNC2, KCND3, KCNH1, KCNJ4, KCNQ3, MAP2, MYH10, NELL2, NRGN, OPRK1, PAK1, PRKAR2B, PRKCG, MAP2K1, RARA, RGS7, SCN8A, CCL2, SLC8A1, CDKL5, SYN1, KCNAB1, FZD3, PRSS12, CDK5R1, BSN, NEURL1, DGKI, HOMER1, CABP1, AKAP5, ARHGAP32, FRMPD4, SEMA3A, BAIAP2, SLC9A6, ARFGEF2, CHL1, PLK2, CPLX1, LZTS1, CPEB3, NCS1, NSMF, SHANK1, IFT57, SEPT11, ANKS1B, SLC4A10, TENM2, DLGAP3, JPH4, PPP1R9B, SHANK3, LMTK3, GRIN3A, SNAP47, CNIH2, HCN1	406	86	4.24	7.25e-24	Up
Dendrite development	ADGRB3, CACNA1A, CAMK2B, CTNNA2, DLG4, EPHA4, HPRT1, ITPKA, MAP2, MEF2C, PAK1, PAK3, PPP3CA, CDKL5, NR2E1, NRP1, CDK5R1, NEURL1, AKAP5, RAPGEF2, KIAA0319, SEMA3A, BAIAP2, SLC9A6, PLK2, CIT, LZTS1, CPEB3, NEDD4L, MAPK8IP2, RBFOX2, NGEF, NSMF, SLITRK5, PACSIN1, SHANK1, DCDC2, BCL11A, FEZF2, CAMK1D, SHANK3, GRIN3A, FMN1	178	43	4.85	1.44e-14	Up
Dendrite extension	PARK2, SYT1, RIMS2, SLC9A6, RIMS1, UNC13A, NEDD4L, CPNE5	21	8	9.12	2.53e-05	Up
Dendrite morphogenesis	ADGRB3, CACNA1A, CAMK2B, CTNNA2, DLG4, EPHA4, HPRT1, ITPKA, MAP2, PAK3, PPP3CA, CDKL5, NR2E1, CDK5R1, AKAP5, RAPGEF2, SEMA3A, BAIAP2, CIT, LZTS1, NEDD4L, MAPK8IP2, RBFOX2, NGEF, NSMF, SLITRK5, SHANK1, DCDC2, SHANK3, FMN1	109	30	5.73	4E-12	Up
Dendritic shaft	CACNA1C, DLG3, DRD1, GRM5, HTR2A, MAP2, PRKAR2B, SLC8A1, HOMER1, AKAP5, LZTS1, JPH4, CNIH2	37	13	8.11	2.07e-07	Up
Dendritic spine development	CAMK2B, DLG4, EPHA4, ITPKA, MEF2C, PAK1, PAK3, CDK5R1, NEURL1, BAIAP2, SLC9A6, PLK2, CPEB3, NGEF, SHANK1, SHANK3	58	16	5.68	4.06e-07	Up
Dendritic spine membrane	ATP2B1, GRIA1, ITGA8, AKAP5, DDN	9	5	18.6	0.000102	Up
DNA metabolic process	BMPR2, CDKN2D, CIDEA, DACH1, HGF, IGF1, KCNK2, KPNA2, MAS1, KITLG, ORC4, PAK3, PIK3CA, PRKCG, CHAF1B, CDC7, NPM2, PPARGC1A, PARM1, CHD5, UBE2W, FBXW7, TSPYL2, BCL11B, SLF1, TBRG1, MAEL, XRCC6BP1, ZBED9, KLHDC3, STOX1, KIAA2022	867	32	0.549	0.000264	Up
Ensheathment of neurons	MYRF, LPAR1, KCNJ10, KEL, MAG, MAL, NGFR, CLDN11, PMP22, POU3F2, KLK6, CNTN2, QKI, ARHGEF10, OLIG2, NDRG1, SIRT2, PARD3, FA2H, SH3TC2, JAM3, NKX6-2, SERINC5	101	23	7.53	4.57e-12	Down
Excitatory postsynaptic potential	DLG4, PTK2B, GRIK5, GRIN2A, GRIN2B, MEF2C, PPP3CA, SNCA, STX1A, DGKI, NRXN1, RIMS2, RAB3GAP1, RIMS1, MAPK8IP2, SHANK1, CELF4, SLC17A7, NETO1, SHANK3	50	20	9.99	7.46e-12	Up
GABA receptor activity	GABRA1, GABRA2, GABRA3, GABRA4, GABRA5, GABRB2, GABRB3, GABRD, GABRG3, GABBR2	22	10	12.6	2.77e-07	Up
GABA receptor binding	GABRA5, AKAP5, ARFGEF2, JAKMIP1	14	4	6.03	0.0091	Up
Glial cell development	MYRF, GSN, KCNJ10, NKX2-2, POU3F2, CNTN2, ARHGEF10, NDRG1, SIRT2, PHGDH, PARD3, FA2H, SH3TC2, NKX6-2	71	14	6.19	4.84e-07	Down
Glutamate receptor activity	PTK2B, GRIA1, GRIA2, GRIA3, GRIK5, GRIN2A, GRIN2B, GRM1, GRM5, GRM8, GRIN3A	27	11	10.4	2.72e-07	Up
Innervation	GABRA5, GABRB2, GABRB3, PRKCG, NRP1, SEMA3A, UNC13A	23	7	6.47	0.000412	Up
Inositol phosphate metabolic process	PTK2B, ITPKA, MAS1, OCRL, SNCA, INPP4B, SYNJ1, PPIP5K1, PLCH1, PLCB1, NUDT11	65	11	3.02	0.00247	Up
Ionotropic glutamate receptor activity	PTK2B, GRIA1, GRIA2, GRIA3, GRIK5, GRIN2A, GRIN2B, GRIN3A	19	8	11	9.08e-06	Up
JNK cascade	ADORA2B, EPHA4, PTK2B, FGF14, MAP3K9, MAP3K10, GADD45B, PAK1, PARK2, MAPK9, CCL19, MAP2K4, MAP3K6, RB1CC1, RASGRP1, PLCB1, MAPK8IP2, KIAA1804, DUSP19, ZNF675, MAGI3	185	21	1.9	0.00716	Up
Lipid binding	ABCA1, ANXA5, APOD, AR, C3, LPAR1, HSD17B10, HIP1, HSPA2, KCNJ2, MAL, MYO1E, NPC1, P2RX7, PLD1, PTGS1, SELL, SNX1, ACOX2, IQGAP1, HIP1R, CYTH1, STARD3, FNBP1, RASGRP3, LDLRAP1, GLTP, ANKFY1, PXK, ADAP2, PARD3, PREX1, WDFY4, PLEKHF1, PRAM1, PAQR8, MVB12B, SNX29, SYTL4, ARAP1, FRMPD2, AMER2, NCF1C, C8orf44-SGK3	601	44	2.07	2.63e-05	Down
Mrna processing	LGALS3, CELF2, PPARGC1A, CELF3, CPEB3, RBFOX2, RBFOX1, MTPAP, CELF4, CELF5, SRRM4, LSM11, RBFOX3	417	13	0.466	0.00202	Up
Myelin maintenance	MYRF, NDRG1, FA2H, SH3TC2	11	4	14.2	0.000601	Down
Myelin sheath	CA2, CNP, CRYAB, GSN, HSPA2, MAG, MOBP, MOG, MYO1D, CLDN11, RDX, CNTN2, NDRG1, SIRT2, PHGDH, GJC2, ERMN, MYH14, JAM3, SERINC5	156	20	3.77	2.29e-06	Down
Myelination	MYRF, LPAR1, KCNJ10, KEL, MAG, MAL, NGFR, PMP22, POU3F2, KLK6, CNTN2, QKI, ARHGEF10, OLIG2, NDRG1, SIRT2, PARD3, FA2H, SH3TC2, JAM3, NKX6-2, SERINC5	98	22	7.38	1.81e-11	Down
Negative regulation of neuron apoptotic process	CACNA1A, PTK2B, GABRA5, GABRB2, GABRB3, MEF2C, PARK2, PIK3CA, PRKCG, CCL2, SNCB, SNCA, STAR, STXBP1, NRP1, CHL1, PPARGC1A, OXR1, AGAP2	128	19	2.59	0.000465	Up
Negative regulation of transcription, DNA-templated	ARNTL, RUNX1T1, CRYM, CYP1B1, DACH1, FGF9, FOXG1, H2AFZ, MEF2C, MAP3K10, TRIM37, PDE2A, RARA, RORB, SATB1, SNCA, SOX5, TBX15, THRB, NR2E1, WNT10B, CDK5R1, LRRFIP1, ZBTB33, BASP1, ZBTB18, KLF12, CPEB3, PLCB1, SATB2, NEDD4L, SIRT5, RBFOX2, ATAD2, TAGLN3, BCL11A, FEZF2, SMYD2, PRDM8, TENM2, MTA3, SCRT1, MAEL, PRICKLE1, EID2, ARX, ZNF675, KCTD1	1135	48	0.632	0.00083	Up
Neuron apoptotic process	CACNA1A, EPHA7, PTK2B, GABRA5, GABRB2, GABRB3, GRIK5, KCNB1, MEF2C, PAK3, PARK2, PIK3CA, PRKCG, SCN2A, CCL2, SNCB, SNCA, STAR, STXBP1, NRP1, CDK5R1, CHL1, PPARGC1A, NSMF, OXR1, FBXW7, AGAP2, SDIM1	206	28	2.35	0.000117	Up
Neuron spine	DLG4, DRD1, EPHA4, GRIA1, GRM5, ITPKA, MYH10, NRGN, PRKAR2B, SLC8A1, CDK5R1, NEURL1, DGKI, AKAP5, ARHGAP32, FRMPD4, BAIAP2, SLC9A6, ARFGEF2, LZTS1, SHANK1, SEPT11, ANKS1B, TENM2, DLGAP3, PPP1R9B, SHANK3, CNIH2	104	28	5.57	3.28e-11	Up
Neuronal postsynaptic density	ADD2, ATP1A1, BMPR2, CAMK2A, CAMK2B, CTNNA2, DLG4, DMTN, GAP43, GRIN2B, MAP2, PAK1, PRKCG, BSN, DGKI, DLGAP1, HOMER1, BAIAP2, CAP2, CNKSR2, CLSTN1, MAPK8IP2, SHANK1, CLSTN2, SHANK3	64	25	9.69	3.02e-14	Up
Neuron-neuron synaptic transmission	CA7, CACNA1A, CACNB4, CAMK4, DRD1, PTK2B, GABRA1, GABRB2, GLRB, GRIA1, GRIA2, GRIA3, GRIK5, GRIN2A, GRM1, GRM5, GRM8, HRH2, HTR1B, HTR2A, MEF2C, NPY5R, PAK1, PARK2, PRKCE, PTGS2, SNCA, STXBP1, SYT1, GLRA3, DGKI, DLGAP2, NRXN1, RAB3GAP1, UNC13A, MAPK8IP2, RASD2, TMOD2, SHC3, SLC17A7, SHANK3, GRIN3A, CNIH2	136	43	7.06	2.63e-19	Up
Neurotransmitter secretion	CACNA1A, CACNA1B, CAMK2A, GAD1, GLS, GRIK5, MEF2C, PAK1, PARK2, PFN2, SLC1A1, SLC1A2, SNCA, STX1A, STXBP1, SYN1, SYN2, SYT1, SYT5, DOC2A, PPFIA4, PPFIA2, PPFIA3, CADPS, LIN7A, SYNJ1, SYT7, DGKI, BZRAP1, NRXN1, RIMS2, RIMS3, CPLX1, HRH3, ADGRL1, RAB3GAP1, RIMS1, UNC13A, PCLO, SYTL2, SLC17A7, SYT13, SYT16, SYT12, CADPS2, SNAP47	154	46	6.52	1.93e-19	Up
Node of Ranvier	KCNQ2, KCNQ3, SCN1A, SCN1B, SCN2A, SCN8A	15	6	9.92	0.000193	Up
Nucleic acid metabolic process	ABCA2, ABL1, PARP4, AR, ATM, BMP8B, MYRF, CAPN3, CAT, CBFB, CCNA2, CDKN1C, CENPB, ELF1, EYA4, ERF, FGF1, FGFR2, GDF1, HSD17B10, HDAC1, HIP1, HOXA1, HOXA2, HOXA5, HOXB2, HOXB5, HOXD1, HOXD3, HSPA1A, FOXN2, JUP, SMAD5, SMAD9, MCM7, MEIS1, CIITA, FOXO4, NKX2-2, NOTCH1, YBX1, PBX3, PDE8A, ENPP2, POLR2L, POU3F2, PSEN1, RNH1, RPLP0, RPS5, RXRG, SALL1, SGK1, SOX10, SREBF1, STAT2, SYK, TAL1, TCF12, TRAF1, TRPS1, ZNF3, ZNF69, VEZF1, FZD5, ARHGEF5, HIST1H2AC, HIST1H3E, HIST1H4H, HIST1H4B, RNASET2, CCNE2, QKI, LITAF, ST18, ZNF536, DDX39A, OLIG2, HMG20B, SEMA4D, TXNIP, DMRT2, TCFL5, ATF7, IKZF2, ZNF652, SIRT2, SAMD4A, KANK1, HEY2, BAMBI, ZNF521, ZBTB20, GREM1, CECR2, HIPK2, KLF15, BAZ2B, SLC40A1, SOX8, ZBTB7B, RRNAD1, KLF3, DDIT4, ZNF280D, TRIM62, CHD7, SLF2, ZNF83, SLC2A4RG, OTUD7B, BBX, MAVS, SFMBT2, NCOA5, TP53INP2, ZNF462, ARHGAP22, CREB3L2, CRTC3, TRAK2, BHLHE41, DBF4B, TSC22D4, NKX6-2, ZBTB37, LOXL3, OLIG1, ZSWIM7, GABPB2, CC2D1B, ZBTB12, ZNF844, ZNF326, FRYL, C9orf142, ZNF710, GTF2IRD2B, DBX2, HIST2H4B, ZNF812, TMEM229A, GTF2H2C_2, C8orf44-SGK3	4679	144	0.718	0.000284	Up
Oligodendrocyte development	MYRF, GSN, KCNJ10, NKX2-2, CNTN2, FA2H, NKX6-2	32	7	6.99	0.000187	Down
Oligodendrocyte differentiation	BOK, MYRF, CNP, GSN, KCNJ10, NKX2-2, NOTCH1, SOX10, CNTN2, OLIG2, SOX8, FA2H, NKX6-2	75	13	5.27	5.64e-06	Down
Phosphatase activity	ALPL, ATP1A1, CDKN3, DUSP8, OCRL, PPP2R5D, PPP3CA, PPP3CB, PPP3R1, MAP2K1, PTPN3, PTPN4, PTPRN2, PTPRR, INPP4B, SYNJ1, PPIP5K1, LPPR4, PTPRT, PTP4A3, NT5DC3, PDP1, LPPR3, PTPN5, DUSP19, PPM1L, PPM1J	254	27	1.81	0.00475	Up
Phosphatidylinositol binding	HIP1, KCNJ2, MYO1E, PLD1, SNX1, IQGAP1, HIP1R, LDLRAP1, ANKFY1, PXK, ADAP2, PARD3, PLEKHF1, SNX29, ARAP1, FRMPD2, AMER2, NCF1C, C8orf44-SGK3	187	19	2.92	9.82e-05	Down
Phospholipase C-activating G-protein coupled receptor signaling pathway	ADRA1B, CCKBR, CHRM1, CHRM3, DRD1, GRM1, GRM5, HRH2, HTR2A, OPRK1, OPRM1, HOMER1, MCHR2	81	13	2.84	0.00172	Up
Phospholipid binding	ABCA1, ANXA5, LPAR1, HIP1, KCNJ2, MYO1E, PLD1, SNX1, IQGAP1, HIP1R, LDLRAP1, ANKFY1, PXK, ADAP2, PARD3, PREX1, WDFY4, PLEKHF1, SNX29, SYTL4, ARAP1, FRMPD2, AMER2, NCF1C, C8orf44-SGK3	332	25	2.1	0.000966	Down
Phospholipid translocation	ABCA1, P2RX7, ATP10B, ATP11A	20	4	6.21	0.00667	Down
Positive regulation of RNA metabolic process	ACVR1B, ARNTL, BMPR2, CAMK4, CAMK2A, CDH13, ETV1, H2AFZ, HGF, IGF1, KRAS, LUM, MEF2C, TRIM37, PPP1R12A, NEUROD2, PARK2, PLAGL1, PPP3CA, PPP3CB, PPP3R1, PRKCB, MAPK9, MAP2K1, RARA, RORB, SOX5, STAT4, THRB, NR2E1, TRAF5, WNT10B, ITGA8, LMO4, LDB2, LHX2, MICAL2, CAMKK2, TBR1, PPARGC1A, MLLT11, CELF3, KLF12, CPEB3, MAPRE3, DDN, PLCB1, SATB2, ATAD2, BCL11A, TESC, FEZF2, FBXW7, DCAF6, CELF4, ARNTL2, ATXN7L3, CAMK1D, MKL2, NEUROD6, BCL11B, CSRNP3, MED12L, RHEBL1, MTPN, SOHLH1	1455	66	0.678	0.0011	Up
Postsynapse	ADD2, ATP1A1, BMPR2, CACNA1C, CAMK2A, CAMK2B, CHRM1, CHRM3, CTNNA2, DLG3, DLG4, DRD1, DMTN, EPHA4, EPHA7, PTK2B, GABRA1, GABRA2, GABRA3, GABRA4, GABRA5, GABRB2, GABRB3, GABRD, GABRG3, GAP43, GLRB, GRIA1, GRIA2, GRIA3, GRIK5, GRIN2A, GRIN2B, GRM1, GRM5, ITPKA, ITPR1, KCNB1, KCNC2, KCNJ4, KCNMA1, MAP2, MYH10, NRGN, PAK1, PRKAR2B, PRKCG, SLC8A1, GLRA3, KCNAB2, ITGA8, LIN7A, CDK5R1, BSN, NEURL1, DGKI, DLGAP2, DLGAP1, HOMER1, CABP1, AKAP5, GABBR2, ARHGAP32, FRMPD4, LZTS3, BAIAP2, CAP2, ARFGEF2, LZTS1, CNKSR2, CLSTN1, RIMS1, SYNE1, NCS1, MAPK8IP2, NSMF, PCLO, SHANK1, SEPT11, ANKS1B, TENM2, LRFN2, KCTD16, LRRC7, DLGAP3, CACNG8, CLSTN2, LRRTM4, NETO1, PPP1R9B, SHANK3, CADPS2, GRIN3A, GRASP, CNIH2, LRRTM1, LRRTM3, IQSEC3	341	98	6.47	7.81e-39	Up
Postsynaptic membrane	CHRM1, CHRM3, DLG3, DLG4, EPHA4, EPHA7, GABRA1, GABRA2, GABRA3, GABRA4, GABRA5, GABRB2, GABRB3, GABRD, GABRG3, GLRB, GRIA1, GRIA2, GRIA3, GRIK5, GRIN2A, GRIN2B, KCNB1, KCNC2, KCNJ4, KCNMA1, GLRA3, LIN7A, NEURL1, DLGAP2, DLGAP1, HOMER1, CABP1, GABBR2, ARHGAP32, LZTS3, LZTS1, CNKSR2, CLSTN1, SYNE1, NCS1, NSMF, SHANK1, ANKS1B, TENM2, LRFN2, KCTD16, LRRC7, DLGAP3, CACNG8, CLSTN2, LRRTM4, NETO1, SHANK3, CADPS2, GRIN3A, GRASP, CNIH2, LRRTM1, LRRTM3, IQSEC3	197	61	6.98	1.99e-26	Up
Potassium channel activity	KCNB1, KCNC1, KCNC2, KCND3, KCNF1, KCNH1, KCNJ3, KCNJ4, KCNJ6, KCNJ9, KCNK2, KCNMA1, KCNN1, KCNQ2, KCNQ3, KCNS1, KCNS2, KCNAB1, KCNAB2, KCNAB3, KCNH4, KCNH3, KCNV1, KCNH5, KCNIP2, KCNQ5, KCNT1, KCNK15, KCNIP4, KCNH7, KCNG3, KCNT2, HCN1	119	33	5.93	1.53e-13	Up
Presynapse	DLG4, GABRA2, GRIA1, GRIA2, GRIN2B, ICA1, NPY1R, SNCA, STX1A, SYN1, SYN2, SYT1, SYT5, SLC30A3, FZD3, DOC2A, PPFIA4, PPFIA2, PPFIA3, BSN, SYT7, SYNGR1, DGKI, RIMS2, RIMS3, SV2B, DNM1L, RIMS1, UNC13A, DMXL2, ERC2, PCLO, SVOP, SLC17A7, SYT12, TPRG1L, SYNPR, STXBP5, SCAMP5, SLC6A17, UNC13C	142	41	6.21	5.89e-17	Up
Presynaptic active zone	SYN1, FZD3, PPFIA4, PPFIA2, PPFIA3, BSN, DGKI, RIMS2, RIMS3, RIMS1, UNC13A, ERC2, PCLO, SLC17A7, UNC13C	24	15	25	7.23e-13	Up
Protein kinase C-activating G-protein coupled receptor signaling pathway	CCK, CHRM1, DGKB, GAP43, GRM1, GRM5, HTR1B, DGKZ, DGKE, DGKI	32	10	6.74	1.85e-05	Up
Protein lipidation	ABCA1, ZDHHC9, PIGT, HHATL, ZDHHC14, ZDHHC11, MAP6D1, ATG4C, PIGM, ZDHHC20	84	10	3.38	0.00152	Down
Regulation of axon guidance	BMPR2, NRP1, SEMA3A, TBR1, FEZF2	18	5	5.68	0.00441	Up
Regulation of neuron apoptotic process	CACNA1A, EPHA7, PTK2B, GABRA5, GABRB2, GABRB3, GRIK5, KCNB1, MEF2C, PAK3, PARK2, PIK3CA, PRKCG, CCL2, SNCB, SNCA, STAR, STXBP1, NRP1, CDK5R1, CHL1, PPARGC1A, NSMF, OXR1, FBXW7, AGAP2	183	26	2.47	9.7e-05	Up
Regulation of neurotransmitter levels	DAGLA, CACNA1A, CACNA1B, CAMK2A, DRD1, GABRA2, GAD1, GLS, GRIK5, MEF2C, PAK1, PARK2, PDE1B, PFN2, SLC1A1, SLC1A2, SNCA, STX1A, STXBP1, SYN1, SYN2, SYT1, SYT5, DOC2A, PPFIA4, PPFIA2, PPFIA3, CADPS, LIN7A, SYNJ1, SYT7, DGKI, BZRAP1, NRXN1, RIMS2, RIMS3, CPLX1, HRH3, ADGRL1, RAB3GAP1, RIMS1, UNC13A, PCLO, SYTL2, SLC17A7, SYT13, SYT16, SYT12, CADPS2, SNAP47	192	50	5.4	3.37e-18	Up
Regulation of postsynaptic membrane potential	DLG4, PTK2B, FGF14, GABRB3, GRIK5, GRIN2A, GRIN2B, MEF2C, PPP3CA, SNCA, STX1A, DGKI, NRXN1, RIMS2, RAB3GAP1, RIMS1, MAPK8IP2, SHANK1, CELF4, SLC17A7, NETO1, SHANK3	59	22	8.92	3.58e-12	Up
Regulation of synaptic plasticity	ATP2B2, CAMK2A, CAMK2B, DLG4, DRD1, PTK2B, FGF14, GRIA1, GRIN2A, GRIN2B, GRM5, HRH2, ITPKA, KCNB1, MEF2C, NEUROD2, NRGN, PAK1, PPP3CB, PTGS2, PTN, SNCA, STAR, STXBP1, NR2E1, PPFIA3, SYNGAP1, SYNGR1, NEURL1, DGKI, RAPGEF2, BAIAP2, PLK2, CPEB3, RAB3GAP1, RIMS1, UNC13A, NSMF, NPTN, JPH3, NETO1, JPH4, SHANK3, SNAP47, CNTN4, LRRTM1	132	46	8.2	1.48e-22	Up
Regulatory region nucleic acid binding	ARNTL, ETV1, H2AFZ, HIVEP2, MEF2C, NEUROD2, PLAGL1, RARA, SATB1, SNCA, SOX5, STAT4, TBX15, LMO4, ZBTB33, BASP1, TBR1, KLF12, DDN, BCL11A, FEZF2, ARNTL2, PKNOX2, DMRTC1, NEUROD6, BCL11B, ZNF831, ZNF519, ARX, ZNF675, STOX1, SOHLH1, DMRTC1B	790	33	0.643	0.00634	Down
Release of cytochrome c from mitochondria	CCK, IFI6, HGF, IGF1, PARK2, MAPK9, HRK, DNM1L, MLLT11, GGCT	55	10	3.29	0.00222	Up
SNARE binding	CACNA1A, STX1A, STXBP1, SYT1, SYT5, DOC2A, NAPG, SYT7, STXBP5L, CPLX1, UNC13A, SYTL2, SYT13, NAPB, SYT16, SYT12, SNAP47, STXBP5	112	18	2.91	0.000188	Up
Sodium channel activity	SHROOM2, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SCN3B, HCN1	36	8	4.32	0.00141	Up
Synapse	ADD2, ATP1A1, ATP2B1, ATP2B2, BMPR2, CACNA1C, CACNB4, CAMK2A, CAMK2B, CAMK2D, CCK, CHRM1, CHRM3, AP1S1, CTNNA2, DLG3, DLG4, DRD1, DMTN, EPHA4, EPHA7, PTK2B, GABRA1, GABRA2, GABRA3, GABRA4, GABRA5, GABRB2, GABRB3, GABRD, GABRG3, GAP43, GLRB, GRIA1, GRIA2, GRIA3, GRIK5, GRIN2A, GRIN2B, GRM1, GRM5, GRM8, ICA1, ITPKA, ITPR1, KCNB1, KCNC2, KCNH1, KCNJ4, KCNMA1, MAP2, MYH10, NPY1R, NRCAM, NRGN, OPRK1, PAK1, PDE2A, PFN2, PRKAR2B, PRKCG, PTPRN2, CCL2, SLC8A1, SNCB, SNCA, STX1A, STXBP1, SYN1, SYN2, SYT1, SYT5, SLC30A3, FZD3, GLRA3, DOC2A, PRSS12, PPFIA4, PPFIA2, KCNAB2, ITGA8, PPFIA3, CADPS, LIN7A, CDK5R1, BSN, WASF1, SYT7, SYNGR1, NEURL1, DGKI, DLGAP2, DLGAP1, NRXN1, HOMER1, CABP1, AKAP5, GABBR2, RAPGEF2, RIMS2, ARHGAP32, FRMPD4, LZTS3, RIMS3, SV2B, DNM1L, OLFM1, BAIAP2, SLC9A6, CAP2, ARFGEF2, CPLX1, LZTS1, AAK1, CPEB3, ADGRL1, CNKSR2, CLSTN1, RIMS1, PDZRN3, UNC13A, NMNAT2, DDN, DMXL2, SYNE1, NCS1, MAPK8IP2, FRRS1L, MYRIP, NSMF, ERC2, CYFIP2, NPTN, PCLO, PACSIN1, SHANK1, NRN1, SVOP, SEPT11, SEPT3, ANKS1B, SLC17A7, TENM2, TBC1D24, LRFN2, KCTD16, LRRC7, DLGAP3, CACNG8, CLSTN2, LRRTM4, NETO1, PPP1R9B, SHANK3, SYT12, CADPS2, PRRT2, GRIN3A, OLFM3, TPRG1L, SYNPR, STXBP5, CBLN4, GRASP, SCAMP5, PHACTR1, CNIH2, LRRTM1, LRRTM3, VWC2, SLC6A17, IQSEC3, UNC13C	658	173	6.11	1.15e-62	Up
Synapse maturation	CAMK2B, NEUROD2, NEURL1, NRXN1, ADGRL1, SHANK1	18	6	7.39	0.000626	Up
Synaptic transmission	ADCY1, ATP2B2, CA7, CACNA1A, CACNA1B, CACNA1C, CACNB1, CACNB2, CACNB4, CAMK4, CAMK2A, CAMK2B, CAMK2D, CHRM1, CHRM3, DLG3, DLG4, DRD1, EGR3, PTK2B, FGF14, GABRA1, GABRA2, GABRA3, GABRA4, GABRA5, GABRB2, GABRB3, GABRD, GABRG3, GAD1, GLRB, GLS, GNAI3, GRIA1, GRIA2, GRIA3, GRIK5, GRIN2A, GRIN2B, GRM1, GRM5, GRM8, HRH2, HTR1B, HTR1E, HTR1F, HTR2A, ITPKA, KCNB1, KCNC1, KCNC2, KCND3, KCNF1, KCNH1, KCNJ3, KCNJ4, KCNJ6, KCNJ9, KCNK2, KCNMA1, KCNN1, KCNQ2, KCNQ3, KCNS1, KCNS2, KIF5A, MEF2C, MYO5A, NEUROD2, NPY5R, OPRK1, OPRM1, PAK1, PARK2, PFN2, PPP3CA, PPP3CB, PRKCB, PRKCE, PRKCG, PTGS2, PTN, RIT2, SCN1B, SCN2B, CCL2, SLC1A1, SLC1A2, SNCB, SNCA, SSTR2, SSTR4, STAR,	702	184	6.18	1.54e-66	Up
	STX1A, STXBP1, SYN1, SYN2, SYT1, SYT5, NR2E1, VIPR1, KCNAB1, GLRA3, DOC2A, PPFIA4, PPFIA2, KCNAB2, PPFIA3, CADPS, LIN7A, SYNGAP1, SYNJ1, BSN, SYT7, SYNGR1, NEURL1, DGKI, KCNAB3, DLGAP2, DLGAP1, BZRAP1, NRXN1, HOMER1, AKAP5, GABBR2, RAPGEF2, RIMS2, RIMS3, SNAP91, CACNG3, BAIAP2, CSPG5, PLK2, CPLX1, HRH3, CPEB3, ADGRL1, CLSTN1, RAB3GAP1, RIMS1, UNC13A, PLCB1, KCNH4, KCNH3, MAPK8IP2, RASD2, NSMF, SLITRK5, KCNV1, NPTN, KCNH5, PCLO, TMOD2, KCNIP2, SHANK1, SHC3, SYTL2, PCDHB13, KCNQ5, CELF4, SLC17A7, JPH3, SYT13, CACNG8, CLSTN2, NETO1, SYT16, CAMKK1, JPH4, PPP1R9B, SHANK3, KCNH7, SYT12, CADPS2, BTBD9, GRIN3A, SNAP47, CNTN4, KCNG3, CNIH2, LRRTM1, HCN1, UNC13C
Synaptic transmission, glutamatergic	CACNA1A, CACNB4, DRD1, PTK2B, GRIA1, GRIA2, GRIA3, GRIK5, GRIN2A, GRM1, GRM5, GRM8, HTR1B, HTR2A, MEF2C, PAK1, PARK2, PTGS2, SYT1, DGKI, NRXN1, RAB3GAP1, UNC13A, MAPK8IP2, SHC3, SLC17A7, SHANK3, GRIN3A, CNIH2	78	29	8.94	1.42e-15	Up
Synaptic vesicle exocytosis	GRIK5, PFN2, STX1A, STXBP1, SYN1, SYT1, SYT5, DOC2A, CADPS, SYNJ1, SYT7, RIMS3, CPLX1, ADGRL1, RIMS1, UNC13A, PCLO, SYTL2, SYT13, SYT16, SYT12, CADPS2, SNAP47	76	23	6.51	1.59e-10	Up
Synaptic vesicle localization	FGF14, GRIK5, PARK2, PFN2, SH3GL2, SNCA, STX1A, STXBP1, SYN1, SYT1, SYT5, AP3B2, DOC2A, CADPS, LIN7A, SYNJ1, SYT7, NRXN1, RIMS3, CPLX1, ADGRL1, RIMS1, UNC13A, PCLO, PACSIN1, SYTL2, SYT13, SYT16, SYT12, CADPS2, BTBD9, SNAP47	120	32	5.49	1.97e-12	Up
Synaptic vesicle membrane	ICA1, STX1A, SYN1, SYN2, SYT1, SYT5, SLC30A3, DOC2A, SYT7, SYNGR1, SV2B, DNM1L, DMXL2, SVOP, SLC17A7, SYT12, SYNPR, SCAMP5, SLC6A17	55	19	7.94	4.52e-10	Up
Synaptic vesicle priming	STX1A, STXBP1, CADPS, SYNJ1, CADPS2, SNAP47	12	6	14.8	4.34e-05	Up
Synaptic vesicle recycling	FGF14, SH3GL2, SNCA, SYT1, SYT5, SYNJ1, PACSIN1, BTBD9	29	8	5.64	0.000338	Up
Synaptic vesicle transport	FGF14, GRIK5, PARK2, PFN2, SH3GL2, SNCA, STX1A, STXBP1, SYN1, SYT1, SYT5, AP3B2, DOC2A, CADPS, LIN7A, SYNJ1, SYT7, RIMS3, CPLX1, ADGRL1, RIMS1, UNC13A, PCLO, PACSIN1, SYTL2, SYT13, SYT16, SYT12, CADPS2, BTBD9, SNAP47	116	31	5.51	4.11e-12	Up
Syntaxin binding	CACNA1A, STXBP1, SYT1, SYT5, DOC2A, NAPG, SYT7, STXBP5L, CPLX1, UNC13A, SYTL2, SYT13, NAPB, SYT16, SYT12, SNAP47, STXBP5	78	17	4.24	4.82e-06	Up
Terminal bouton	CCK, AP1S1, GRIK5, GRIN2A, KCNC2, KCNMA1, PFN2, PTPRN2, SNCA, STXBP1, SYN1, PRSS12, SYT7, SYNGR1, CPLX1, AAK1, TBC1D24	61	17	5.8	1.39e-07	Up

Up-regulated genes in ALS anterior horn of the spinal cord cluster into inflammatory responses, metal ion regu-lation and hemostasis; whereas down-regulated genes cluster into neuronal axonal cytoskeleton and apoptosis.

In contrast, clusters of up-regulated genes were involved in neurotransmission, ion channels and ion transport, synapses, maintenance of axons and dendrites, intracellular signaling and synaptic vesicle mechanisms. The majority of down-regulated genes were encoded for proteins associated with myelin and glial cell regulation (Figure 2).

Figure 2

Diagram showing de-regulated gene clusters in the anterior horn of the spinal cord (A) and frontal cortex area 8 in ALS (B) as revealed by whole transcriptome arrays.

RT-qPCR validation

Sixty-six genes from different pathways were selected for validation by RT-qPCR.

Inflammatory gene expression in the anterior horn of the spinal cord

No modifications in the expression levels of glial fibrillary acidic protein gene (GFAP) or prostaglandin-endoperoxide synthase 2 gene (PTGS2) occurred in ALS when compared with controls (p=0.31 and p=0.55, respectively). However, expression levels of AIF1 and CD68 were significantly increased in the anterior horn of the spinal cord in ALS (p=0.044 and p=0.00023, respectively). Gene expression of toll-like receptors (TLRs) TLR2, TLR and TLR7 was significantly increased in the spinal cord in ALS cases (p=2.48E-05, p=0.00011 and p=0.00074, respectively), but TLR4 was not (p=0.669). IL1B was up-regulated (p=0.005), but IL6 and IL6ST were not (p=0.26 and p=0.76, respectively). In contrast, the expression of IL10 and its corresponding receptors IL10RA and IL10RB was increased in ALS (p=0.00046, p=0.022 and p=3.23E-05, respectively). TNFA expression was significantly increased whereas a trend was found for TNFRSF1B (p=0.04 and p=0.08, respectively). The expression of CTSC and CTSS was significantly increased in spinal cord in ALS (p=5.82204E-05 and p=0.00014, respectively). Levels of SLC11A1 were also significantly increased in spinal cord of ALS (p=0.014). HLA-DRB1, a protein coding gene for the Major Histocompatibility Complex Class II (MHC-II) DR β1 protein was markedly up-regulated in ALS (p=0.004365).

PDCD1LG2, IFNγ and IL33 were significantly up-regulated in the anterior horn of the spinal cord in ALS (p=0.00153, p=0.03 and p=0.0032, respectively).

Finally, IL8 (interleukin 8) and ITGB4 (integrin subunit beta 4) expression was similar in control and ALS cases (p=0.92 and p=0.40, respectively) (Figure 3).

Figure 3

mRNA expression levels of selected deregulated genes identified by microarray analysis in the anterior horn of the spinal cord in ALS determined by TaqMan RT-qPCR assays. (A) general glial markers; (B-C) mediators of the inflammatory response; and (D) axolemal components. Up of AIGF1 and CD68, toll-like receptors, cytokines and receptors, chemokines and other mediators of the innate and adaptative inflammatory responses. Axolemal genes, excepting NEFH, which shows a non-significant trend to decrease, are significantly down-regulated. (E) glutamate transporter coding genes. The significance level is set at * p < 0.05, ** p < 0.01 and *** p < 0.001.

Axonemal gene expression in anterior horn of the spinal cord

No modifications in the expression levels of NEFH, which codes for neurofilament heavy polypeptide protein, was seen in ALS when compared with controls (p=0.30). However, DNAAF1 levels were significantly reduced (p=0.019). Expression of DNAH2, DNAH5, DNAH7 and DNAH11 mRNA was significantly reduced in ALS (p=0.029, p=0.012, p=0.005 and p=0.023, respectively), whereas DNAH9 mRNA was not altered (p=0.14). DNAI1 mRNA expression was also significantly reduced in ALS (p=0.0086) (Figure 3).

SLC1A2 and SC17A7 expression in anterior horn of the spinal cord

SLC1A2 and SLC17A7 expression levels were significantly decreased in the anterior horn of the spinal cord in ALS anterior (p=0.000115 and p=0.000125, respectively). See Figure 3.

Neurotransmission-related gene expression in frontal cortex area 8

GRIA1, which codes for the ionotropic glutamate receptor AMPA 1, and GRIN2A and GRIN2B, coding for NMDA receptors, were significantly up-regulated (p=0.018, p=0.018 and p=0.029, respectively) in frontal cortex in ALS cases. GRM5, which codes for the glutamate metabotropic receptor 5, was also up-regulated (p=0.0079). However, no significant alteration was seen in the expression of NETO1 (p=0.165).

Regarding the GABAergic system, GAD1 was up-regulated in ALS (p=0.034). Gene expression of GABA receptors GABRA1, GABRD, GABRB2 was increased (p=0.09, tendency, p=0.006 and p=0.0029, respectively). GABBR2 mRNA levels were also significantly elevated in the frontal cortex in ALS (p=0.01) (Figure 4).

Figure 4

mRNA expression levels of selected deregulated genes identified by microarray analysis in frontal cortex area 8 of ALS cases determined by TaqMan RT-qPCR assays. (A) oligodendroglial and myelin-related genes; (B) glutamatergic and GABAergic-related genes and corresponding ionotropic and metabotropic receptors; (C) genes coding for synaptic cleft proteins. Significant up of genes linked to neurotransmission and synapses, and significant down of genes linked to oligodendroglia and myelination. (D) Glutamate transporter coding genes. The significance level is set at * p < 0.05, ** p < 0.01 and *** p < 0.001, and tendencies at # < 0.1.

Synaptic cleft gene expression in frontal cortex area 8

BSN, which codes for Bassoon, a pre-synaptic cytoskeletal matrix, was up-regulated in ALS (p=0.04). mRNA levels of PCLO, coding gene for Piccolo protein, and FRMPD4 were also increased in ALS (p=0.036 and p=0.029, respectively), Finally, NRN1, which codes for neuritin 1, but not DDN, which codes for dendrin protein, was up-regulated in the frontal cortex in ALS (p=0.04 and p=0.92, respectively) (Figure 4).

Myelin- and oligodendrocyte-related gene expression in frontal cortex area 8

Significant decrease in mRNA expression of myelin transcription factor (MYRF) (p= 0.028), OLIG2 (p=0.009), SOX10 (p=0.02), NKX2-2 (p=0.032), transferring (TF) (p=0.5), proteolipid protein 1 (PLP1) (p=0.040), myelin basic protein (MBP) (p=0.061), myelin-associated oligodendrocyte basic protein (MOBP) (p=0.019), oligodendrocyte glycoprotein (MOG) (p=0.05), Mal T-cell differentiation protein (MAL) (p=0.039), myelin associated glycoprotein (MAG) (p=0.035), and 2',3'-cyclic nucleotide 3' phosphodiesterase (CNP1) (p=0.017) was seen in frontal cortex in ALS cases compared with controls (Figure 4).

SLC1A2 and SLC17A7 expression in frontal cortex area 8

SLC1A2 expression was significantly increased (p=5.25e-5) whereas SLC17A7 mRNA showed a non-significant increase (p=0.42) in frontal cortex area 8 in ALS (Figure 4).

Immunohistochemistry in spinal cord

The anterior horn of the spinal cord in ALS cases showed decreased number of neurons and altered morphology of most remaining motor neurons including loss of endoplasmic reticulum (chromatolysis) and axonal ballooning (Figure 5a) and intracytoplasmic TDP-43-immunoreactive inclusions (Figure 5b). Immunohistochemistry was carried out in the lumbar spinal cord in control and sALS cases (Figure 5a and b). VDAC was reduced in a subpopulation of neurons in the anterior horn in ALS, but not in neurons of the Clarke's column and posterior horn, when compared with controls (Figure 5c and d). Increased expression of GFAP was found in reactive astrocytes in the lateral columns and anterior horn of the spinal cord in ALS cases (Figure 5e and f). Marked differences were seen regarding microglial cell markers: IBA-1 and CD68 immunoreactivity was dramatically increased in the pyramidal tracts and anterior horn in ALS; moreover the morphology of microglia was modified in pathological cases with predominance of round, amoeboid microglia (Figure 5g-j). Similar immunoreactivity, distribution and morphology were found in reactive microglia using antibodies against HLA-DRB1 and HLA-DRB5 (Figure 5k-n). In contrast IL-10 and TNF-α immunoreactivity predominated in neurons; immunoreactivity was increased in neurons in ALS cases compared with controls (Figure 5o-r). Finally, GluT (SLC1A2), the transporter of glutamate from the extracellular space at synapses, was expressed in the membrane of neurons and in the neuropil; SLC1A2 immunoreactivity was decreased in neurons and neuropil of the anterior horn in ALS (Figure 5s, t).

Figure 5

Anterior horn of the spinal cord. Haematoxilin and eosin staining showing damaged neurons in ALS (a). Immuno-histochemistry to TDP-43 showing skein-like intracytoplasmic inclusions (b), VDAC (c, d), GFAP (e, f), IBA-1 (g, h), CD68 (i, j), HLA-DRB1 (k, l), HLA-DRB5 (m, n), IL-10 (o, p), TNF-α (q, r) and GluT (SLC1A2) (s, t) in the anterior horn of the lumbar spinal cord in control (c, e, g, I, k, m, o, q, s) and sALS (a, b, d, f, h, j, l, n, p, r, t) cases. TDP-43-immunoreactive cytoplasmic inclusions are seen in motor neurons in sALS. GFAP is increased in reactive astrocytes; microglial cells have a round, amoeboid morphology as seen with IBA-1, CD-68, HLA-DRB1, and HLA-DRB5 antibodies. VDAC immunoreactivity is decreased whereas IL-10 and TNF-α is increased in remaining motor neurons in sALS. SLC1A2 immunoreactivity is reduced in the membrane of neurons and in neuropil of the anterior horn in sALS. Paraffin sections, slightly counterstained with haematoxylin; a, c-d, o-t, bar in t = 40μm; e-n, bar in = 20μm; bar in b = 10μm

Gel electrophoresis and western blotting in frontal cortex area 8

A few tested antibodies were eventually suitable for western blotting studies. No differences in the expression levels of glutamate receptor ionotropic, NMDA 2A (NMDAR2A) and glutamate decarboxylase 1 (GAD1) were observed between control and ALS cases. However, a significant increase in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor 1 (AMPAR GluR-1) ** p < 0.01 and a tendency to increase in the expression of gamma-aminobutyric acid receptor subunit beta-2 (GABAAB2) (# p < 0.1) was found in the frontal cortex in ALS when compared to controls (Figure 6).

Figure 6

Gel electrophoresis and western blotting to glutamate receptor ionotropic, NMDA 2A (NMDAR2A), α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor 1 (GluR‐1), glutamate decarboxylase 1 (GAD1) and gamma‐aminobutyric acid receptor subunit beta‐2 (GABAAB2) in the frontal cortex area 8 of control and ALS. Significant increased levels of GluR‐1 and a tendency to increased levels of GABAAB2 are seen in ALS when compared with controls. The significance level is set at ** p < 0.01 and tendencies at # < 0.1.

DISCUSSION

Transcriptomic profiles in ALS are region-dependent when comparing the anterior horn of the lumbar spinal cord and frontal cortex area 8 in the same individuals. As an important regional difference related to excitotoxicity, the expression of glutamate transporters is markedly different in the anterior horn of the spinal cord and the frontal cortex area 8. SLC1A2 and SLC17A7 mRNA expression is significantly decreased in the anterior horn of the spinal cord, whereas SLC1A2 is significantly increased in frontal cortex area 8. SLC1A2 encodes the solute carrier family 1 member 2 or excitatory amino-acid transporter 2 (EAAT2) which clears glutamate from the extracellular space at synapses in the central nervous system. Immunohistochemistry has shown decreased SLC1A2 protein expression in the membrane of neurons and neuropil of the anterior horn in ALS. SLC17A7 encodes the vesicular glutamate transporter 1 (VGLUT1) which is a vesicle-bound, sodium-dependent phosphate glutamate transporter expressed in the synaptic vesicles. Decreased expression of these proteins is linked to increased excitotoxity which is postulated as primary factor triggering motor neuron degeneration in ALS [30, 31].

Whole transcriptome arrays show that major up-regulated clusters in the anterior horn are related with innate inflammatory and adaptative inflammatory responses. Genes involved in hemostasis and ion transport forms a small up-regulated group. The major group of down-regulated genes is linked to the neuronal cytoskeleton. The majority of significantly differentially up-regulated transcripts in sALS in frontal cortex area 8, as revealed by whole transcriptome arrays, code for proteins linked with neurotransmission, ion channels and ion transport, synapses, and axon and dendrite maintenance, whereas down-regulated genes code for proteins involved in oligodendrocyte development and function, myelin regulation and membrane lipid metabolism.

Altered gene expression as revealed by whole transcriptome arrays has been validated by RT-qPCR in 58 of 66 assessed genes. These observations increase the list of genes which are de-regulated in the anterior spinal cord and provide, for the first time, robust evidence of gene de-regulation in frontal cortex area 8 in sALS. Increased inflammatory response in the anterior horn and increased expression of selected neurotransmitter markers in frontal cortex has been further assessed using immunohistochemistry and western blotting, respectively.

Inflammation in the anterior horn of the spinal cord

AIF1 gene codes for the Allograft Inflammatory Factor 1, a protein induced by cytokines and interferon which promotes macrophage and glial activation [32, 33]. CD68 codes for the macrophage antigen CD68 glycoprotein which is expressed by microglial cells [35-37], the principal resident immune cell population in brain [38, 39]. Microglia pro-inflammatory state activation can be initiated by engagement of germline-encoded pattern-recognition receptors such as Toll-like receptors (TLRs) which are expressed in glial cells [40]. TLR activation, in turn, activates phagocytosis [41-43] and pro-inflammatory responses [44]. Up-regulated interleukins in ALS are IL1B, the coding gene for interleukin 1B an important mediator of the inflam-matory response [45], interleukin 10 (encoded by IL10) which has pleiotropic effects down-regulating the expression of Th1 cytokines, MHC class II antigens and co-stimulating the production of several molecules by macrophages through the activation of IL10 receptor subunit α and subunit β (encoded by IL10RA and IL10RB, respectively) [46]. However, IL6 mRNA, which encodes a specific pro-inflammatory cytokine with regenerative and anti-inflammatory activities in particular settings [47-50] is not modified. Tumor Necrosis Factor Receptor Superfamily Member 1A (encoded by TNFA) is involved in the regulation of a wide spectrum of biological processes including cell proliferation, cell differentiation, apoptosis, lipid metabolism and coagulation [50, 51]. CTSC gene encodes Cathepsin C which is central coordinator of activation of many serine proteinases in immune cells [52]. CTSS codes for a protein of the same family, Cathepsin S, which acts as a key protease responsible for the removal of the invariant chain from MHC class II antigens [53]. SLC11A1 encodes natural resistance-associated macrophage protein 1, which acts as a host resistance to certain pathogens [54].

Major Histocompatibility Complex Class II (MHC-II) DR β1 protein, encoded by HLA-DRB-1, plays a central role in the immune system by presenting peptides derived from extracellular proteins [55, 56] and participate in the activation of autophagosomes [57]. PDCD1LG2 codes for Programmed Cell Death 1 Ligand 2, a protein involved in co-stimulatory signals essential for T-cell proliferation and IFN-γ production [58]. IFNγ gene, which codes for the cytokine interferon-γ, is key player in antigen-specific immune responses [59]. Finally, interleukin 33, encoded by IL33, acts as a chemo-attractant for Th2 cells and functions as an ‘alarm’ that amplifies immune responses during tissue injury [60].

Increased inflammatory response in the anterior horn of the spinal cord has been further documented by immunohistochemistry showing increased expression of IBA-1, the protein encoded by AIF1, CD68, and HLA-DRB1 and HLA-DRB5 in reactive microglia. Reactive microglia has a round, amoeboid morphology and is also localized, as expected in the lateral and anterior pyramidal tracts. IL-10 and TNF-α are mainly localized in neurons of the spinal cord, and its expression is increased in remaining motor neurons of the spinal cord in ALS. These findings indicate a parallelism between gene expression and protein expression regarding inflammatory responses of assessed molecules. On the other hand the different localization of microglial markers, and IL-10 and TNF-α in neurons points to a cross-talk between microglia and neurons in the anterior horn of the spinal cord in ALS.

This is in contrast with other markers as glial fibrillary acidic protein and voltage dependent anion channel in which levels of mRNA differ from levels (or intensity) of protein expression. No modifications in the expression of GFAP mRNA are observed in the present study, but GFAP immunoreactivity is clearly increased in reactive astrocytes, as already reported in classical neuropathological studies. VDAC mRNA is not abnormally regulated in gene arrays; yet VDAC is decreased in motor neurons, but not in neurons of the Clarke's column and neurons of the posterior horn, of the spinal cord in ALS. VDAC immunohistochemistry is in line with observations in human sALS showing deficiencies in mitochondria and energy metabolism [61, 62].

Reduced expression of axolemal genes in anterior horn of the spinal cord

The expression levels of NEFH, which codes for neurofilament heavy polypeptide protein [63], are preserved in ALS. However, DNAAF1, which encodes dynein (axonemal) assembly factor 1, and mRNAs encoding several dynein axonemal heavy chains (DHC) are down-regulated thus suggesting impairment of motor ATPases involved in the transport of various cellular cargoes by ‘walking’ along cytoskeletal microtubules towards the minus-end of the microtubule [64-66].

Up-regulation of neurotransmission-related genes and synaptic cleft genes in frontal cortex

Genes involved in glutamatergic and GABAergic transmission are up-regulated in the frontal cortex in ALS. This applies to genes encoding the ionotropic glutamate receptor AMPA 1 (GRIA1), glutamate ionotropic receptor NMDA type subunit 2A (GRIN2A), the glutamate ionotropic receptor NMDA type subunit 2B (GRIN2B), and glutamate metabotropic receptor 5 (GRM5). Regarding the GABAergic system, GAD1, coding for glutamate decarboxylase 1, a rate-limiting enzyme that acts in the decarboxylation of glutamate essential for the conversion reaction of GABA from glutamate [67, 68], is up-regulated, as are GABRA1, GABRD, GABRB2, which code for different subunits of ionotropic GABA-A receptors. GABBR2, which codes for the metabotropic receptor component Gamma-Aminobutyric Acid Type B Receptor Subunit 2 and forms heterodimers with GABBR1, thus resulting in the formation of the G-protein coupled receptor for GABA [69], is also up-regulated in ALS.

In line with increased expression of neurotransmitter-related genes, several genes encoding molecules linked with the synaptic cleft are also up-regulated in ALS. BSN codes for Bassoon, a pre-synaptic cytoskeletal matrix (PCM) protein acting as a scaffolding protein and essential for the regulation of neurotransmitter release in a subset of synapses [70, 71]. PCLO codes for Piccolo protein, a component of the PCM assembled in the active zone of neurotransmitter release [72, 73]. FRMPD4 codes for PSD-95-interacting regulator of spine morphogenesis protein which regulates dendritic spine morphogenesis and is required for the maintenance of excitatory synaptic transmission [74]. DDN and NRN1 code for dendrin protein and neuritin 1 protein, respectively which are involved in the remodeling of the postsynaptic cytoskeleton and neuritic outgrowth [75-77].

De-regulation of neurotransmitters and receptors is further supported by the demonstration of significant increase in the levels of GluR-1 and a tendency in those of GABAAB2 in the frontal cortex area 8 in ALS when compared with controls. It is worth stressing that only a few antibodies of the total assessed (eight) were suitable for western blotting.

Myelin and oligodendrocyte genes in frontal cortex area 8

Myelin transcription factor (encoded by MYRF) regulates oligodendrocyte differentiation and is required for central nervous system myelination [78-81]. The basic loop- helix protein OLIG2 mediates motor neuron and oligodendrocyte differentiation [22, 82]. High mobility group protein SOX10 modulates myelin protein transcription [83, 84]. NKX2.2 homeodomain transcription factor is a key regulator of oligodendrocyte differentiation [85]. Transferrin encoded by TF participates in the early stages of myelination [86, 87]. Proteolipid protein 1 (encoded by PLP1) plays a role in the compaction, stabilization, and maintenance of myelin sheaths, as well as in oligodendrocyte development and axonal survival [88, 89]. Myelin basic protein (encoded by MBP) is the second most abundant myelin-associated protein, constituting about 30% of total myelin protein [90]. Myelin-associated oligodendrocyte basic protein (encoded by MOBP) constitutes the third most abundant protein in CNS myelin and it acts by compacting and stabilizing myelin sheaths [91]. Myelin oligodendrocyte glycoprotein (encoded by MOG) is a cell surface marker of oligodendrocyte maturation [92]. Myelin associated glycoprotein (encoded by MAG) is a type I membrane protein and member of the immunoglobulin super-family involved in the process of myelination and certain myelin-neuron cell-cell interactions [93]. Mal T-cell differentiation protein (encoded by MAL) is involved in myelin biogenesis [94]. Finally, 2',3'-cyclic nucleotide 3' phosphodiesterase (encoded by CNP1) participates in early oligodendrocyte differentiation and myelination [95-97].

Concluding comments

Results of the present study validate gene expression of individual studies performed in a limited number of samples identifying a limited number of de-regulated genes in the anterior horn of the spinal cord [17, 20, 21, 25]. Present results are more close to those carried out by using laser micro-dissection of anterior horn spinal motor neurons [27] thus reinforcing the consistence of observations in both studies. Whether some changes are related to the variable progression of the disease need further study with a larger number of cases of rapid or slow clinical course. In this line, altered mitochondria, protein degradation and axonal transport predominate in the 129Sv-SOD1(G93A) transgenic mouse with rapidly progressive motor neuron disease, whereas increased immune response is found in the C57-SOD1(G93A) transgenic mouse with more benign course [98].

The most important aspect of the present study is the description of altered gene expression and identification of altered clusters of genes in the frontal cortex area 8 in sALS cases without apparent cognitive impairment. It is worth stressing that altered clusters differ in the spinal cord and frontal cortex in sALS at terminal stages thus providing valuable information of molecular ab-normalities which can also be present within the spectrum of FTLD-TDP. Noteworthy, altered regulation of transcription related to synapses and neuro-transmission covering neurotransmitter receptors, synaptic proteins and ion channels in the frontal cortex in the absence of overt clinical symptoms of cognitive impairment are particularly important to identify early molecular alterations in frontal cortex with the spectrum of ALS/FTLD-TDP.

MATERIALS AND METHODS

Tissue collection

Post-mortem fresh-frozen lumbar spinal cord (SC) and frontal cortex (FC) (Brodmann area 8) tissue samples were from the Institute of Neuropathology HUB-ICO-IDIBELL Biobank following the guidelines of Spanish legislation on this matter and the approval of the local ethics committee. The post-mortem interval between death and tissue processing was between 2 and 17 hours. One hemisphere was immediately cut in coronal sections, 1-cm thick, and selected areas of the encephalon were rapidly dissected, frozen on metal plates over dry ice, placed in individual air-tight plastic bags, numbered with water-resistant ink and stored at −80°C until use for biochemical studies. The other hemisphere was fixed by immersion in 4% buffered formalin for 3 weeks for morphologic studies. Transversal sections of the spinal cord were alternatively frozen at −80°C or fixed by immersion in 4% buffered formalin. The whole series included 18 sALS cases and 23 controls. The anterior horn of the spinal cord was examined in 14 sALS (mean age 57 years; 6 men and 8 women) and the frontal cortex area 8 in 15 sALS (mean age 54 years; 11 men and 4 women). Spinal cord and frontal cortex were available in 11 cases. Lumbar anterior spinal cord was dissected on a dry-ice frozen plate under a binocular microscope at a magnification x4. TDP-43-immunoreactive small dystrophic neurites and/or TDP-43-positive granules and/or small cytoplasmic globules in cortical neurons in the contralateral frontal cortex area 8 were observed in 11 of 18 cases, but only abundant in three cases (cases 29, 30 and 31 in Table 3). Spongiosis in the upper cortical layers was found only in one case (case 28 in Table 3). Cases with frontotemporal dementia were not included in the present series. Patients with associated pathology including Alzheimer's disease (excepting neurofibrillary tangle pathology stages I-II of Braak and Braak), Parkinson's disease, tauopathies, vascular diseases, neoplastic diseases affecting the nervous system, metabolic syndrome, hypoxia and prolonged axonal states such as those occurring in intensive care units were excluded. Cases with infectious, inflammatory and autoimmune diseases, either systemic or limited to the nervous system were not included.

Table 3

Summary of the fifty six cases analyzed including frontal cortex area 8 of 14 controls and 15 ALS cases, and anterior horn of the spinal cord of 13 controls and 14 ALS cases

						RIN value
Case	Age	Gender	Diagnosis	PM delay	Initial symptoms	SC	FC
1	49	F	Control	07 h 00 min	-	-	7.2
2	75	F	Control	03 h 00 min	-	-	7.2
3	55	M	Control	05 h 40 min	-	-	7.7
4	59	M	Control	12 h 05 min	-	6.4	-
5	59	M	Control	07 h 05 min	-	-	7.8
6	43	M	Control	05 h 55 min	-	6.6	7.7
7	53	M	Control	07 h 25 min	-	-	5.3
8	56	M	Control	03 h 50 min	-	-	7.6
9	47	M	Control	04 h 55 min	-	5.6	7.7
10	64	F	Control	11 h 20 min	-	6.2	-
11	46	M	Control	15 h 00 min	-	5.9	7.9
12	56	M	Control	07 h 10 min	-	6.1	-
13	71	F	Control	08 h 30 min	-	5.9	-
14	64	F	Control	05 h 00 min	-	7.0	-
15	79	F	Control	06 h 25 min	-	6.7	-
16	75	M	Control	07 h 30 min	-	5.0	-
17	55	M	Control	09 h 45 min	-	5.3	-
18	52	M	Control	03 h 00 min	-	-	8.3
19	52	M	Control	04 h 40 min	-	-	6.3
20	76	M	Control	06 h 30 min	-	6.6	-
21	60	F	Control	11 h 30 min	-	-	7.5
22	51	F	Control	04 h 00 min	-	6.3	7.9
23	54	M	Control	08 h 45 min	-	-	7.0
24	56	M	ALS	10 h 50 min	NA	7.1	-
25	70	M	ALS	03 h 00 min	Respiratory	7.3	7.0
26	77	M	ALS	04 h 30 min	NA	7.4	-
27	56	F	ALS	03 h 45 min	NA	8.2	7.7
28	59	M	ALS	03 h 15 min	NA	7.5	7.7
29	63	F	ALS	13 h 50 min	Bulbar	6.8	8.2
30	59	F	ALS	14 h 15 min	NA	6.4	6.7
31	54	M	ALS	04 h 50 min	Spinal	-	7.8
32	76	M	ALS	12 h 40 min	Spinal	-	7.4
33	64	M	ALS	16 h 30 min	NA	6.3	7.3
34	57	F	ALS	04 h 00 min	Bulbar	6.2	8.6
35	75	F	ALS	04 h 05 min	Bulbar	6.8	6.8
36	79	F	ALS	02 h 10 min	NA	7.0	-
37	57	F	ALS	10 h 00 min	Bulbar	6.5	7.1
38	50	M	ALS	10 h 10 min	Spinal	-	5.9
39	59	F	ALS	02 h 30 min	Spinal	-	7.5
40	46	M	ALS	07 h 00 min	Spinal	7.0	8.0
41	69	F	ALS	17 h 00 min	Spinal	6.4	6.3

Abbreviations: ALS: amyotrophic lateral sclerosis; F: female; M: male; PM: post-mortem delay (hours, minutes); SC: anterior horn of the spinal cord lumbar level; FC: frontal cortex area 8; RIN: RNA integrity number; NA: not available.

Age-matched control cases had not suffered from neurologic or psychiatric diseases, and did not have abnormalities in the neuropathologic examination, excepting sporadic neurofibrillary tangle pathology stages I-II of Braak and Braak. No C9ORF72, SOD1, TARDBP and FUS mutations occurred in any case. Table 3 shows a summary of cases.

Whole-transcriptome array

RNA from frozen anterior horn of the lumbar spinal cord and frontal cortex area 8 was extracted following the instructions of the supplier (RNeasy Mini Kit, Qiagen® GmbH, Hilden, Germany). RNA integrity and 28S/18S ratios were determined with the Agilent Bioanalyzer (Agilent Technologies Inc, Santa Clara, CA, USA) to assess RNA quality, and the RNA concentration was evaluated using a NanoDrop™ Spectrophotometer (Thermo Fisher Scientific). Selected samples were analyzed by microarray hybridization with GeneChip® Human Gene 2.0 ST Array and WT Labeling Kit and microarray 7000G platform from Affymetrix® (Santa Clara, CA, USA). Microarray service was carried out at the High Technology Unit (UAT) at Vall d'Hebron Research Institute (VHIR), Barcelona, Spain.

Microarray data and statistical analysis

Microarray data quality control, normalization and filtering were performed using bioconductor packages in an R programming environment for genes [99] which enabled data preprocessing for differential gene expression analysis and enrichment analysis. Gene selection was based upon their values using a test for differential expression between two classes (Student's t-test). Genes differentially expressed showed an absolute fold change > 2.0 in combination with a p-value ≤ 0.05.

Complementary DNA (cDNA) preparation used High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) following the protocol provided by the supplier. Parallel reactions for each RNA sample run in the absence of MultiScribe Reverse Transcriptase to assess the lack of contamination of genomic DNA. TaqMan RT-qPCR assays were performed in duplicate for each gene on cDNA samples in 384-well optical plates using an ABI Prism 7900 Sequence Detection system (Applied Biosystems, Life Technologies, Waltham, MA, USA).

For each 10μL TaqMan reaction, 4.5μL cDNA was mixed with 0.5μL 20x TaqMan Gene Expression Assays and 5μL of 2x TaqMan Universal PCR Master Mix (Applied Biosystems). Table 4 shows identification numbers and names of TaqMan probes. The mean value of one house-keeping gene, hypoxanthine-guanine phosphoribosyltransferase (HPRT1), was used as internal control for normalization of spinal cord samples, whereas the mean values of the three house-keeping genes, alanyl-transfer RNA synthase (AARS), glucuronidase Beta (GUS-β) and X-prolyl amino-peptidase (aminopeptidase P) 1 (XPNPEP1) were used as internal controls for normalization of frontal cortex samples [100, 101]. The parameters of the reactions were 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 sec and 60°C for 1 min. Finally, capture of all TaqMan PCR data used the Sequence Detection Software (SDS version 2.2.2, Applied Biosystems). The double-delta cycle threshold (ΔΔCT) method was used to analyze the data; results with T-student test. The significance level was set at * p < 0.05, ** p < 0.01 and *** p < 0.001, and tendencies at # < 0.1. Pearson's correlation method assessed a possible linear association between TDP-43 pathology in frontal cortex area 8 and gene deregulation in the same region; significant correlations were not found.

Table 4

Genes, gene symbols and TaqMan probes used for the study of gene expression in the anterior horn of the spinal cord and frontal cortex area 8 in ALS cases and controls including probes for normalization (AARS, GUS-β, HPRT-1 and XPNPEP-1)

Gene	Gene symbol	Reference
2',3'-Cyclic Nucleotide 3' Phosphodiesterase	CNP	Hs00263981_m1
Alanyl-TRNA Synthetase	AARS	Hs00609836_m1
Allograft Inflammatory Factor 1	AIF1	Hs00741549_g1
Bassoon Presynaptic Cytomatrix Protein	BSN	Hs01109152_m1
Cathepsin C	CTSC	Hs00175188_m1
Cathepsin S	CTSS	Hs00356423_m1
C-X-C Motif Chemokine Ligand 8	IL8	Hs00174103_m1
Dendrin	DDN	Hs00391784_m1
Dynein (Axonemal) Assembly Factor 1	DNAAF1	Hs00698399_m1
Dynein Axonemal Heavy Chain 11	DNAH11	Hs00361951_m1
Dynein Axonemal Heavy Chain 2	DNAH2	Hs00325838_m1
Dynein Axonemal Heavy Chain 5	DNAH5	Hs00292485_m1
Dynein Axonemal Heavy Chain 7	DNAH7	Hs00324265_m1
Dynein Axonemal Heavy Chain 9	DNAH9	Hs00242096_m1
Dynein Axonemal Intermediate Chain 1	DNAI1	Hs00201755_m1
Gamma-Aminobutyric Acid Type A Receptor Alpha 1 Subunit	GABRA1	Hs00971228_m1
Gamma-Aminobutyric Acid Type A Receptor Beta 2 Subunit	GABRB2	Hs00241451_m1
Gamma-Aminobutyric Acid Type A Receptor Delta Subunit	GABRD	Hs00181309_m1
Gamma-Aminobutyric Acid Type B Receptor Subunit 2	GABBR2	Hs01554996_m1
Glial Fibrillary Acidic Protein	GFAP	Hs00909240_m1
Glutamate Decarboxylase 1	GAD1	Hs01065893_m1
Glutamate Ionotropic Receptor AMPA Type Subunit 1	GRIA1	Hs00181348_m1
Glutamate Ionotropic Receptor NMDA Type Subunit 2A	GRIN2A	Hs00168219_m1
Glutamate Ionotropic Receptor NMDA Type Subunit 2B	GRIN2B	Hs01002012_m1
Glutamate Metabotropic Receptor 5	GRM5	Hs00168275_m1
Hypoxanthine Phosphoribosyltransferase 1	HPRT1	Hs02800695_m1
Integrin Subunit Beta 4	ITGB4	Hs00173995_m1
Interferon, Gamma	IFNG	Hs00989291_m1
Interleukin 1 Beta	IL1B	Hs01555410_m1
Interleukin 10	IL10	Hs00961622_m1
Interleukin 10 Receptor Subunit Alpha	IL10RA	Hs00155485_m1
Interleukin 10 Receptor Subunit Beta	IL10RB	Hs00988697_m1
Interleukin 33	IL33	Hs00369211_m1
Interleukin 6	IL6	Hs00985639_m1
Interleukin 6 Signal Transducer	IL6ST	Hs00174360_m1
Macrophage Antigen CD68	CD68	Hs02836816_g1
Major Histocompatibility Complex, Class II, DR Beta 1/4/5	HLA-DRB	Hs04192463_mH
Mal T-Cell Differentiation Protein	MAL	Hs00360838_m1
Myelin Associated Glycoprotein	MAG	Hs01114387_m1
Myelin Basic Protein	MBP	Hs00921945_m1
Myelin Oligodendrocyte Glycoprotein	MOG	Hs01555268_m1
Myelin Regulatory Factor	MYRF	Hs00973739_m1
Myelin-Associated Oligodendrocyte Basic Protein	MOBP	Hs01094434_m1
Neuritin 1	NRN1	Hs00213192_m1
Neurofilament, Heavy Polypeptide	NEFH	Hs00606024_m1
Neuropilin And Tolloid Like 1	NETO1	Hs00371151_m1
NK2 Homeobox 2	NKX2-2	Hs00159616_m1
Oligodendrocyte Lineage Transcription Factor 2	OLIG2	Hs00377820_m1
Piccolo Presynaptic Cytomatrix Protein	PCLO	Hs00382694_m1
Programmed Cell Death 1 Ligand 2	PDCD1LG2	Hs01057777_m1
Prostaglandin-Endoperoxide Synthase 2	PTGS2	Hs00153133_m1
Proteolipid Protein 1	PLP1	Hs00166914_m1
PSD-95-Interacting Regulator Of Spine Morphogenesis	FRMPD4	Hs01568794_m1
Solute Carrier Family 1 (Glial High Affinity Glutamate Transporter), Member 2 (EAAT-2)	SLC1A2	Hs01102423_m1
Solute Carrier Family 11 Member 1	SLC11A1	Hs01105516_m1
Solute Carrier Family 17 (Vesicular Glutamate Transporter), Member 7 (VGLUT-1)	SLC17A7	Hs00220404_m1
SRY (Sex Determining Region Y)-Box 10	SOX10	Hs00366918_m1
Toll Like Receptor 2	TLR2	Hs00610101_m1
Toll Like Receptor 3	TLR3	Hs01551078_m1
Toll Like Receptor 4	TLR4	Hs01060206_m1
Toll Like Receptor 7	TLR7	Hs00152971_m1
Transferrin	TF	Hs01067777_m1
Tumor Necrosis Factor Receptor Superfamily Member 1A	TNFRSF1	Hs01042313_m1
Tumor Necrosis Factor-Alpha	TNFa	Hs01113624_g1
X-prolyl aminopepidase P1	XPNPEP1	Hs00958026_m1
β-glucuronidase	GUS-β	Hs00939627_m1

Immunohistochemistry

De-waxed sections, 4μm thick, of the lumbar spinal cord from control and ALS cases were processed in parallel for immunohistochemistry. Endogenous peroxidases were blocked by incubation in 10% methanol-1% H2O2 for 15 min followed by 3% normal horse serum. Then the sections were incubated at 4°C overnight with one of the primary antibodies: rabbit polyclonal antibodies to IBA-1 (019-19749, Wako Chemicals Gmbh, Neuss, GE) were used at a dilution of 1:1,000; VDAC (voltage dependent anion channel, ab15895, Abcam, Cambridge, UK) at 1:100; HLA-DRB1 (GTX104919, GeneTex, Barcelona, Spain) at 1:100; HLA-DRB5 (NBP2, Novusbio, Littleton, Colorado, USA) at 1:100; IL-10 (AP52181PU, ACRIS, ProAlt, Madrid, Spain) at 1:100; and GFAP (glial fibrillary acidic protein, RP014-S, Diagnostic Biosystem, Palex Medica, Sant Cugat, Spain) at 1:400. Mouse monoclonal antibodies to CD68 (ab955, Abcam, Cambridge, UK) and TNF-α (ab1793, Abcam, Cambridge, UK), were used at dilutions of 1:200 and 1:150, respectively. Antibodies to GluT: SLC1A2 (ab1783, Millipore, Billerica, MA, USA) were used at a dilution of 1:100. Following incubation with the primary antibody, the sections were incubated with EnVision + system peroxidase (Dako, Agilent, Santa Clara, CA, USA) for 30 min at room temperature. The peroxidase reaction was visualized with diamino-benzidine and H2O2. Control of the immunostaining included omission of the primary antibody; no signal was obtained following incubation with only the secondary antibody. Sections were slightly stained with haematoxylin.

Gel electrophoresis and western blotting

Frozen samples of the somatosensory cortex were homogenized in RIPA lysis buffer composed of 50mM Tris/HCl buffer, pH 7.4 containing 2mM EDTA, 0.2% Nonidet P-40, 1mM PMSF, protease and phosphatase inhibitor cocktail (Roche Molecular Systems, USA). The homogenates were centrifuged for 20 min at 12,000 rpm. Protein concentration was determined with the BCA method (Thermo Scientific). Equal amounts of protein (20μg) for each sample were loaded and separated by electrophoresis on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred onto nitrocellulose membranes (Amersham, Freiburg, GE). Non-specific bindings were blocked by incubation in 3% albumin in PBS containing 0.2% Tween for 1 h at room temperature. After washing, membranes were incubated overnight at 4°C with antibodies against glutamate receptor ionotropic, NMDA 2A (NMDAR2A, 130 kDa, rabbit, 1:200, Abcam, Cambridge, UK), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor 1 (AMPAR GluR-1, 100 kDa, rabbit, 1:200, Cell Signaling Technology, Danvers, MA, USA), glutamate decarbo-xylase 1 (GAD1, 67 kDa, rabbit, 1:200, Cell Signaling Technology, Danvers, MA, USA) and gamma-aminobutyric acid receptor subunit beta-2 (GABAAB2, 59 kDa, mouse, 1:1000, Abcam, Cambridge, UK). Protein loading was monitored using an antibody against β-actin (42 kDa, 1:30,000, Sigma). Membranes were incubated for 1 h with appropriate HRP-conjugated secondary antibodies (1:2,000, Dako); the immunoreaction was revealed with a chemilumines-cence reagent (ECL, Amersham). Densitometric quantification was carried out with the ImageLab v4.5.2 software (BioRad), using β-actin for normalization. Seven samples of FC area 8 per group were analyzed.

These antibodies were selected on the basis of a larger screening which included antibodies against proteins whose RNA levels were de-regulated as revealed by RT-qPCR. Only antibodies working for western blotting were eventually assessed. The significance level was set at ** p < 0.01 and tendencies at # < 0.1.