Perturbation of Cell-subtype specific Active Kinome Networks in Schizophrenia

Perturbation of Cell-Subtype Specific Active Kinome Networks in Schizophrenia Ali Imami1,, Khaled Alganem1, Nicholas Henkel1, Alex Joyce1, Emily Devine3, Jessica Jiron1, Elizabeth Sheddroff1, Abdul Hamoud1, Jarek Meller3, Robert E. McCullumsmith1, 2 1University of Toledo Department of Neurosciences and Psychiatry, Toledo, OH, 2Promedica, Toledo, OH; 3Cincinnati Children’s Hospital Medical Center, Cincinnati, OH; University of Cincinnati, OH DEPARTMENT OF NEUROSCIENCES AND PSYCHIATRY UNIVERSITY OF TOLEDO STE CK1 AGC CAMK CMGC TKL STE CK1 AGC CAMK CMGC TKL STE CK1 AGC CAMK CMGC TKL A B STE CK1 AGC CAMK CMGC TKL C D Pyramidal Neurons SCZ vs CTL DLPFC SCZ vs CTL Rat PFC HLD vs CTL hiPSC Neurons DISC1 vs CTL Schizophrenia is a serious illness with significant effects on patients and their families. Because the disease impacts executive function, the Dorsolateral Prefrontal Cortex (DLPFC) has been a focus of study. We previously reported altered protein kinase activity, including AKT, in postmortem brain samples from patients with schizophrenia. Now, we extend this research to the cellular level, concentrating on frontal cortical pyramidal neurons. Using laser capture microdissection, we isolated DLPFC pyramidal neurons from matched pairs of schizophrenia and control postmortem brain samples (n = 20 per group). We then used the PamChip STK kinome array assay for high-throughput analysis of kinase activity. With well-established bioinformatics methods, we identified upstream kinases involved in schizophrenia. Several kinases of interest emerged, such as c-Jun N-terminal kinases (JNK), extracellular signal-regulated kinases (ERK), and p38 mitogen-activated protein kinases (P38). Of these, the P38 kinases were of particular importance as they are involved in the inflammation cascade and immune function. Next, we applied a new technique to identify kinase interaction networks within high-throughput kinase activity data. This modeling enabled us to detect network-level changes in pyramidal neurons in schizophrenia. This is the first study to analyze the subkinome at the cellular level in schizophrenia and to reveal active kinome network changes in this often-devastating illness. Prevailing hypotheses regarding the etiology of schizophrenia include aberrant synapse turnover, which is mediated by microglia and immune function pathways. Our findings offer a crucial new starting point for understanding how disrupted signaling networks may mediate the pathophysiology of this often devastating severe mental illness. Background Methods Pairwise SCZ vs CTL Conclusions • The CAMK Family, and MAP Kinases in particular, appear to have an intracellular role in excitatory neurons the pathogenesis of schizophrenia. • The individual MAP Kinase members, JNK, ERK and P38, show differential activity in schizophrenia subjects. • JNK and P38 activity was increased, while ERK was decreased, with the kinome array in SCZ. • We confirmed decreased ERK activity using a highly selective individual kinase activity assay. ns 2 4 6 8 10 CTL SCZ Pair01 ns 2 4 6 8 10 CTL SCZ Pair02 ns 2 4 6 8 10 CTL SCZ Pair03 ns 2 4 6 8 10 CTL SCZ Pair04 ns 2 4 6 8 10 CTL SCZ Pair05 ns 2 4 6 8 10 CTL SCZ Pair06 ns 2 4 6 8 10 CTL SCZ Pair07 ns 2 4 6 8 10 CTL SCZ Pair08 * 2 4 6 8 10 CTL SCZ Pair09 ns 2 4 6 8 10 CTL SCZ Pair11 ns 2 4 6 8 10 CTL SCZ Pair12 ns 2 4 6 8 10 CTL SCZ Pair13 ns 2 4 6 8 10 CTL SCZ Pair14 ns 2 4 6 8 10 CTL SCZ Pair15 ns 2 4 6 8 10 CTL SCZ Pair16 ns 2 4 6 8 10 CTL SCZ Pair17 ns 2 4 6 8 10 SCZ CTL Pair18 ns 2 4 6 8 10 SCZ CTL Pair19 CTL SCZ Pair01 CTL SCZ Pair02 CTL SCZ Pair03 CTL SCZ Pair04 CTL SCZ Pair05 CTL SCZ Pair06 CTL SCZ Pair07 CTL SCZ Pair08 CTL SCZ Pair09 CTL SCZ Pair11 CTL SCZ Pair12 CTL SCZ Pair13 CTL SCZ Pair14 CTL SCZ Pair15 CTL SCZ Pair16 CTL SCZ Pair17 CTL SCZ Pair18 CTL SCZ Pair19 Legend. Phylogenetic trees of kinome array activity profiles in (A) Schizophrenia (SCZ) vs Control (CTL) dorsolateral prefrontal cortex (DLPFC) pyramidal neurons, (B) region-level, (C) human iPSC-derived frontocortical neurons with DISC1 mutation and (D) haloperidol-treated (HDL) rat prefrontal cortex (PFC) substrates. (E) Quartile-ranked kinase activity comparison across all pairs. (F) MAP Kinase Family-Specific (P38, JNK and ERK) activity across all pairs. Red color means statistically significant result (p < 0.1) and larger circles represent bigger effect sizes. (G) AssayQuant targeted ERK activity assay for SCZ vs CTL showing a decrease in activity between groups About Me DAPK AMPK SGK PRKDC ATR MLCK PKN CK2 CAMK2 QIK ATM PKCA COT IRAK SLK MTOR GSK ULK TTK MST VRK1 NIK RAF MLK CLK CAMKK PEK CAMK1 LKB NEK NUAK MELK BUD32 KHS TAO MAPKAPK STE11 RIPK PDHK RAD53 MARK WNK MOS PDK1 PAKB BARK2 PIM IKK MSN HAL PHK CHK1 BRSK PKD NDR RSK PASK TLK GRK FRAY PKG AKT NAK PKCH PKCI PAKA PKA DMPK AUR PLK NMO CK1 STE7 DYRK PKCD STKR P38 CDK ERK JNK P01 P02 P03 P04 P05 P06 P07 P08 P09 P10 P11 P12 P13 P14 P15 P16 P17 P18 P19 -1.6 -1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 P10 P12 P11 P15 P18 P04 P19 P09 P05 P16 P08 P14 P17 P13 P02 P06 P07 P03 P01 o g 2 F o l d C h a n g e P10 P11 P12 P02 P18 P16 P19 P04 P15 P05 P08 P14 P09 P01 P13 P06 P17 P03 P07 P10 P12 P18 P15 P11 P14 P19 P16 P02 P01 P04 P03 P06 P09 P17 P07 P05 P08 P13 Subject Pair Effect Size 0.0 0.2 0.4 0.6 0.8 1.0 JNK ERK P38 Kinase Family P38 Core Function JNK ERK Change in SCZ Impacted processes Clinical RelevanceMembers • P38α • P38β • P38γ • P38δ • JNK1 • JNK2 • JNK3 • ERK1 • ERK2 • ERK3/4 • ERK5 • ERK7 • Inflammation • Cytokine Signaling •Microglia Activation • Stress response • Apoptosis • Synaptic Pruning • Cell Growth • Cell Differentiation • Synaptic Plasticity • Neuroinflammation • Immune Response • Neuro-Glial Communication • Neuronal stress signaling • Dendritic remodeling • Long-term potentiation • Learning and Memory circuits Links SCZ to immune dysfunction; candidate for anti-inflammatory therapy Drives excessive synapse turnover; potential stress pathway modulation target Reduced plasticity may explain cognitive deficits; confirmed by in-vitro assay F E G CTRL SCZ 0 100 200 300 400 ERK Activity slope p=0.0319