PAK1 Protein Expression in the Auditory Cortex of Schizophrenia Subjects

Deficits in auditory processing are among the best documented endophenotypes in schizophrenia, possibly due to loss of excitatory synaptic connections. Dendritic spines, the principal post-synaptic target of excitatory projections, are reduced in schizophrenia. p21-activated kinase 1 (PAK1) regulates both the actin cytoskeleton and dendritic spine density, and is a downstream effector of both kalirin and CDC42, both of which have altered expression in schizophrenia. This study sought to determine if there is decreased auditory cortex PAK1 protein expression in schizophrenia through the use of quantitative western blots of 25 schizophrenia subjects and matched controls. There was no significant change in PAK1 level detected in the schizophrenia subjects in our cohort. PAK1 protein levels within subject pairs correlated positively with prior measures of total kalirin protein in the same pairs. PAK1 level also correlated with levels of a marker of dendritic spines, spinophilin. These latter two findings suggest that the lack of change in PAK1 level in schizophrenia is not due to limited sensitivity of our assay to detect meaningful differences in PAK1 protein expression. Future studies are needed to evaluate whether alterations in PAK1 phosphorylation states, or alterations in protein expression of other members of the PAK family, are present in schizophrenia

Characteristics of control subjects and subjects with schizophrenia.

<p>665: Min (12.9) Max (43.2) Avg (28.1) **One Pair Not Shown: HU 1201 (C) and 1189 (S) 802: Min (19.9) Max (37.9) Avg (29) 1222: Min (18.6) Max (43) Avg (30.8)</p><p>All subject pairs shown were included in analyses of PAK1 and kalirin protein levels. Subject pairs also analyzed for spinophilin protein levels (Observed PMI≤24 hours) are indicated by an asterisk. Control and schizophrenia subjects significantly differed in pH and handedness, but not in age, sex, PMI, and tissue storage time. S/R/A, Sex/Race/Age; PMI, postmortem interval (calculated as lag from midpoint of time last seen alive – time discovered dead to time of brain fixation. [Observed] is calculated as time discovered dead to time of brain fixation); COD, cause of death; Hand, handedness; Meds ATOD, medications used at time of death; ADC, Alcohol Dependence, current at time of death; ADR, Alcohol Dependence, in remission at time of death; AAC, Alcohol Abuse, current at time of death; AAR, Alcohol Abuse, in remission at time of death; ODC, Other Substance Dependence, current at time of death; ODR, Other Substance Dependence, in remission at time of death; OAC, Other Substance Abuse, current at time of death; OAR, Other Substance Abuse, in remission at time of death; L, Left; M, Mixed; R, Right; U, Unknown; B, Benzodiazepines; C, Anticonvulsants; D, Antidepressants; L, Lithium; N, No medications; O, Other medication(s); P,Antipsychotic; U, Unknown.</p

Processing of human tissue for PAK1.

<p><b>A</b>–<b>C</b>) show an example of a frozen coronal slab through the right temporal lobe from which a block containing the STG and Heschl's gyrus (located between the arrows in <b>C</b>) has been excised and mounted for cryostat sectioning. The boundary between the gray and white matter of Heschl's gyrus (dotted line) was undercut so that only gray matter was collected for protein extraction. <b>D</b>) Demonstrates our approach to quantification of PAK1 and tubulin. Lanes from all blots were simultaneously imported into the analysis software, and for quantification of each protein the peaks were aligned, and then a single band definition applied to all blots concurrently, indicated by the black horizontal lines. <b>E</b>) PAK1 optical density as a function of micrograms of protein loaded per lane. The mean (SD) of 3 human subjects, assayed together in duplicate runs is shown. It can be seen that the protein loading used in the comparison of schizophrenia and control subjects (20 µg) sits within the linear detection range. <b>F</b>) Western blot demonstrating isoform specificity of anti-PAK1 antibody. Lanes were loaded with 0.5 µg of each full length GST-tagged recombinant protein (ProQinase # 0357-0000-1, 0304-0000-1, 0422-0000-1, Freiburg, Germany), and detected with Coomassie Blue (top) or with anti-PAK1 antibody (bottom) as described for human and mouse studies.</p

PAK1 Expression in schizophrenia.

<p><b>A</b>) Detection of PAK1 in human gray matter extracts. Bands corresponding to the predicted molecular weights of PAK1 and Tubulin are readily detected in human gray matter extracts from subjects with schizophrenia (S) and matched control subjects (C). MW, Molecular Weight Markers. <b>B</b>) Comparison of PAK1 expression (normalized to tubulin expression) for the 25 pairs of subjects. Each point represents a pair of subjects. The diagonal reference line represents a control: schizophrenia ratio of one. Points above the line indicate increased expression in schizophrenia subjects, points below the line indicate decreased expression in schizophrenia subjects. <b>C</b>) Mean (SD) expression PAK1:tubulin in schizophrenia and control subjects. <b>D</b>) Correlation of within pair differences in log PAK1 expression with the corresponding measure of log total kalirin protein expression (r = 0.55, p = 0.004). <b>E</b>) Correlation of within pair differences in PAK1 expression with the corresponding measure of spinophilin protein expression (r = 0.61, p = 0.004).</p

Effect of subject variables on PAK1 expression in schizophrenia.

<p>Each point represents the mean within pair difference in log PAK1 expression. Horizontal bars are group means. No differences were significant.</p

PAK1 Signaling Pathway.

<p>PAK1 resides in inactive homo- or heterodimers. Binding of the Rho-GTPase, RAC1 (or CDC42) causes dissociation of the dimers and activation of PAK1. PAK1 activates LIMK1, which inhibits cofilin-mediated f-actin depolymerization. PAK1 may be subject to activation by non-GTPase mechanisms, and can activate other effector pathways, some of which (e.g. MLCK and MLC) may also impact spine dynamics. Blue indicates promotion of dendritic spine persistence, red indicates promotion of spine elimination.</p

Functional Interplay between the 53BP1-Ortholog Rad9 and the Mre11 Complex Regulates Resection, End-Tethering and Repair of a Double-Strand Break

<div><p>The Mre11-Rad50-Xrs2 nuclease complex, together with Sae2, initiates the 5′-to-3′ resection of Double-Strand DNA Breaks (DSBs). Extended 3′ single stranded DNA filaments can be exposed from a DSB through the redundant activities of the Exo1 nuclease and the Dna2 nuclease with the Sgs1 helicase. In the absence of Sae2, Mre11 binding to a DSB is prolonged, the two DNA ends cannot be kept tethered, and the DSB is not efficiently repaired. Here we show that deletion of the yeast 53BP1-ortholog <i>RAD9</i> reduces Mre11 binding to a DSB, leading to Rad52 recruitment and efficient DSB end-tethering, through an Sgs1-dependent mechanism. As a consequence, deletion of <i>RAD9</i> restores DSB repair either in absence of Sae2 or in presence of a nuclease defective MRX complex. We propose that, in cells lacking Sae2, Rad9/53BP1 contributes to keep Mre11 bound to a persistent DSB, protecting it from extensive DNA end resection, which may lead to potentially deleterious DNA deletions and genome rearrangements.</p></div

Effects of postmortem interval on PAK1 expression.

<p><b>A</b>) Example western blot of PAK1 and Tubulin in mice in which the interval from sacrifice to brain harvesting was experimentally varied between 0 and 48 hours. MW, Molecular Weight Markers. <b>B</b>) Optical densities for PAK1 and Tubulin in mice in which the interval from sacrifice to brain harvesting was experimentally varied between 0 and 48 hours. Two sets of mice (each set containing one mouse for each PMI) were tested. Sets were run concurrently on separate gels, and the run was repeated. Mean (SEM) values for the four observations at each time point are shown. Time points sharing the same superscript letter differ significantly.</p

Rad9 limits an Sgs1- and Sae2- dependent initial step of DSB resection.

<p>(A) Scheme of the <i>MAT</i> locus. The figure shows the positions of the HO-cut site, and the probe used in experiments shown in (B and C) and in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004928#pgen.1004928.s003" target="_blank">S3</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004928#pgen.1004928.s004" target="_blank">S4 Figs</a>. (B, C) Exponentially growing YEP+raf cell cultures of the wild type JKM139 strain and the indicated derivatives, carrying a unique HO cut site at <i>MAT</i> locus and expressing the HO nuclease under GAL1 promoter, were synchronized and kept in G2/M phases by nocodazole treatment. Galactose was added at time 0 to induce HO. SspI-digested genomic DNA, extracted from samples taken at the indicated times, was analysed by Southern blotting to test 3′ filament formation. (C) The mean values ± SEM corresponding to the resection products of two independent experiments were determined by densitometry. (D) Schematic representation of the quantitative PCR method used to monitor HO-induced DSB resection. (E–F) Plots showing the ratio of resected DNA among HO cut DNAs at each time points by qPCR analysis. The mean values from three independent experiments are shown with SEM. Significance was calculated by one-tailed paired Student's <i>t</i> test (* for P<0.05; ** for P<0.01; where not indicated, the P value was higher than 0.05) (G) JKM139 derivatives were nocodazole-arrested in G2/M and 2% galactose was added to induce HO cut. After 2 hours of HO induction, cells were plated on YEP+raf and YEP+raf+gal, and incubated at 28°C for three days. Viability results were obtained from the ratio between number of colonies on YEP+raf+gal and YEP+raf. The mean values from three independent experiments are shown with SD.</p

Deletion of <i>RAD9</i> rescues DSB repair defects of <i>sae2</i>Δ cells through <i>SGS1</i> and <i>DNA2</i>.

<p>(A) Map of the YMV80 Chr III region, containing the HO-cut site. The indicated vertical bars show KpnI restriction sites. The short thick lines indicate the position where the probe hybridizes. After the HO mediated cleavage, DNA ends are resected. Once the indicated <i>leu2</i> cassettes have been exposed as ssDNA, repair through SSA can occur and be monitored by the appearance of an SSA product fragment by Southern blot. (B and D) Exponentially growing YEP+raf cell cultures of the wild type YMV80 strain and the indicated derivatives were synchronized and kept blocked in G2/M phase with nocodazole treatment; galactose was added at time zero to induce HO-cut. <i>KpnI</i>-digested DNA was analysed by Southern blotting with a <i>LEU2</i> probe. An <i>ATG5</i> (uncut locus on chromosome XVI) probe was also used to normalize the signals. In (D) <i>LEU2</i> and <i>ATG5</i> probes were added contemporarily to the filter. (C and E) Densitometric analysis of the product band signals of the experiments shown in (B) and (D). The intensity of each band was normalized respect to unprocessed <i>ATG5</i> locus (*).</p