Elevated neuregulin-1 and ErbB4 protein in the prefrontal cortex of schizophrenic patients

Abstract

Neuregulin-1 (NRG1) and its receptor, ErbB4, have been implicated in schizophrenia at both gene and transcript levels. The present investigation compared NRG1 and ErbB4 protein levels in prefrontal cortical (PFC) cytoplasmic and nuclear fractions among normal, schizophrenic, bipolar and major depressed subjects from the Stanley Consortium. We used immunoblotting procedures to examine potential NRG1 and ErbB4 immunoreactive bands, but specifically quantified NRG1 immunoreactive signals at 42, 48 and 53 kDa and ErbB4 immunoreactive signals at 21, 55, 60 and 180 kDa. PFC cytoplasmic 53 kDa NRG1 protein levels were significantly increased (approximately 20%) in schizophrenic patients relative to each of the other subject groups. We also detected diagnostic effects on PFC cytoplasmic full-length (180 kDa) ErbB4 protein levels, and post hoc tests revealed that these quantities were significantly increased (approximately 30%) in schizophrenic patients relative to normal and to depressed subjects. In addition, we examined the levels of potential ErbB4 cleavage products at 21, 55 and 60 kDa relative to those of full-length ErbB4 in the PFC fractions. We detected trends for diagnostic effects on PFC cytoplasmic 21 kDa/180 kDa and 55 kDa/180 kDa ratios, and post hoc tests revealed that these ratios were significantly reduced in schizophrenic patients relative to normal individuals. Our investigation suggests that schizophrenia-associated NRG1 and ErbB4 mRNA elevations also occur at the protein level and may be specific to schizophrenia. We hypothesize that ErbB4 proteolytic processing may also be altered in schizophrenia, yielding altered ratios of functionally distinct forms of ErbB4.

Figure 1

Representative sections of immunoblots showing NeuN detection in PFC nuclear but not cytoplasmic fractions of Stanley cohort subjects. Three predominant NeuN immunoreactive bands were detected in PFC nuclear fractions at 48, 62 and 66kDa, as labeled. N, Normal; S, Schizophrenia; B, Bipolar disorder; D, Depressed.

Figure 2

Representive immunoblots showing immunoreactive bands detected (a) by the NRG1 antibody and (b) by the ErbB4 antibody in the internal control. Internal control consists of a pool of all PFC protein samples. In each figure, the first lane displays the resulting immunoreactive bands following no preabsorption (None), the second lane displays the resulting immunoreactive bands following preabsorption with the NRG1 peptide (N) or the ErbB4 peptide (E) and the third lane displays the resulting immunoreactive bands following preabsorption with an unrelated oxytocin peptide (O) used as a negative control. A molecular weight ladder is shown on the left of each figure, and arrowed bands are fragments further analyzed in the investigation.

Figure 3

Relative PFC cytoplasmic NRG1-53kDa protein quantities among diagnostic groups. Representative sections of immunoblots are shown below the graph. Data are expressed relative to normal (N) subjects, and error bars represent standard errors. S, Schizophrenia; B, Bipolar disorder; D, Depressed (***p ≤ 0.01; ****p ≤ 0.001).

Figure 4

Relative PFC (a) cytoplasmic and (b) nuclear ErbB4-180kDa protein quantities among diagnostic groups. Representative sections of immunoblots are shown below the graphs. Data are expressed relative to normal (N) subjects, and error bars represent standard errors. S, Schizophrenia; B, Bipolar disorder; D, Depressed (*p = 0.058; **p ≤ 0.03).

Figure 5

Relative PFC cytoplasmic (a) ErbB4 55kDa/180kDa and (b) ErbB4 21kDa/180kDa ratios among diagnostic groups. Data are expressed relative to normal (N) subjects, and error bars represent standard errors. S, Schizophrenia; B, Bipolar disorder; D, Depressed (***p ≤ 0.01).

Figure 6

Correlation between ErbB4-180kDa and NRG1-53kDa protein quantities in PFC cytoplasmic fractions of schizophrenic patients. A correlation was observed specifically between these fragments exclusively in schizophrenic subjects. Equation of best fit line (solid line), along with correlation coefficient (R) and p-value, is inset. Dots represent data points; dotted line encloses data within 95% confidence.