Comparison of the adolescent and adult mouse prefrontal cortex proteome

<div><p>Adolescence is a developmental period characterized by unique behavioral phenotypes (increased novelty seeking, risk taking, sociability and impulsivity) and increased risk for destructive behaviors, impaired decision making and psychiatric illness. Adaptive and maladaptive adolescent traits have been associated with development of the medial prefrontal cortex (mPFC), a brain region that mediates regulatory control of behavior. However, the molecular changes that underlie brain development and behavioral vulnerability have not been fully characterized. Using high-throughput 2D DIGE spot profiling with identification by MALDI-TOF mass spectrometry, we identified 62 spots in the PFC that exhibited age-dependent differences in expression. Identified proteins were associated with diverse cellular functions, including intracellular signaling, synaptic plasticity, cellular organization and metabolism. Separate Western blot analyses confirmed age-related changes in DPYSL2, DNM1, STXBP1 and CFL1 in the mPFC and expanded these findings to the dorsal striatum, nucleus accumbens, motor cortex, amygdala and ventral tegmental area. Ingenuity Pathway Analysis (IPA) identified functional interaction networks enriched with proteins identified in the proteomics screen, linking age-related alterations in protein expression to cellular assembly and development, cell signaling and behavior, and psychiatric illness. These results provide insight into potential molecular components of adolescent cortical development, implicating structural processes that begin during embryonic development as well as plastic adaptations in signaling that may work in concert to bring the cortex, and other brain regions, into maturity.</p></div

62 Differentially expressed proteins in adult versus adolescent mPFC Identified in proteomics analysis.

<p>62 Differentially expressed proteins in adult versus adolescent mPFC Identified in proteomics analysis.</p

Adolescent development impacts a functional protein network involved with neurological disease, skeletal and muscular disorders, and psychological disorders.

<p>Visualization of a protein interaction network identified by Ingenuity Pathway Analysis as being altered by adolescent brain development (<i>p</i>-score = 22). Proteins shown in red were up-regulated in the adult mPFC, proteins in green were up-regulated in the adolescent mPFC, and proteins in yellow indicate statistically significant interaction proteins identified by IPA network analysis. Solid lines indicate a direct interaction, and dashed lines indicate an indirect interaction mediated by additional, non-significant proteins. Asterisks denote proteins that were identified multiple times in the proteomic analysis that have been consolidated into a single point in the functional network. Focus molecule cofilin-1 (CFL1) is indicated in bold.</p

Proteomic analysis of the adolescent and adult medial prefrontal cortex (mPFC).

<p><b>(A)</b> Timeline of experimental procedure. <b>(B)</b> Adult body weight was significantly greater than adolescent body weight at tissue collection (left), but brain weight did not differ between the two ages (right). <b>(C)</b> Photomicrograph (left) and schematic (right) of mouse brain section showing location of mPFC tissue punch for two-dimensional differential in-gel electrophoresis (2D-DIGE) and immunoblot studies. <b>(D)</b> Schematic describing proteomics workflow. Adult tissue is combined with Cy3 (red) and adolescent with Cy2 (green) dye and run in 2D-DIGE, with protein separating in the <i>y</i> plane via molecular weight and the <i>x</i> plane via isoelectrical focusing (IEF). DeCyder software identifies protein spots with significantly different florescent signals. Selected spots are subjected to tryptan digestion and identified via tandem MALDI TOF/TOF mass spectrometry. <b>(E)</b> Representative 2D-DIGE gel run in the proteomics analysis of mPFC. Adult samples were combined with red Cydye (left top); adolescent samples were combined with green CyDye (left bottom). Overlay of adult and adolescent samples (right). IEF is indicated on the <i>x</i> axis with pH values and molecular weight is indicated on the <i>y</i> axis in kDa. Circles indicate location of differentially expressed spots on the gel, with numeric markers prior to identification. Focus proteins are indicated in bold, with red representing increased expression in adults and green representing increased expression in adolescents. (**** indicates p≤0.0001).</p

Adult and adolescent expression of dynamin-1 (DNM1).

<p><b>(A)</b> Representative 3D plot of DNM1 expression in adult (left) and adolescent (right) mice for Spot #4. <b>(B)</b> Standardized abundance (log) of DNM1 (Spot 4) demonstrating higher expression in adults versus adolescents <b>(C)</b> Representative 3D plot of DNM1 expression in adult (left) and adolescent (right) mice for Spot #6. <b>(D)</b> Standardized abundance (log) of DNM1 (Spot 6) demonstrating higher expression in adults versus adolescents. <b>(E)</b> Representative gel image of a Western blot for DNM1 expression to confirm 2D-DIGE changes. <b>(F)</b> Quantification of Western blot results, confirming reduced expression of DNM1 (normalized to actin) in adolescents as compared to adults. <b>(G)</b> Top, representative gel images for each brain region; bottom, quantification of Western blots for each brain region. DNM1 expression was higher in adults in dStr, M1, Amy, and VTA. There was no significant change in DNM1 expression in NAc (p>0.05). Data were expressed as percent change from mean adult within the same blot and graphed as mean ± SEM. (* indicates <i>p</i>≤0.05, ** indicates <i>p</i>≤0.01, *** indicates <i>p</i>≤0.001, **** indicates <i>p</i>≤0.0001).</p

Adult and adolescent expression of cofilin-1 (CFL1).

<p><b>(A)</b> Representative 3D plot of CFL1 expression in adult (left) and adolescent (right) mice for Spot #54. <b>(B)</b> Standardized abundance (log) of CFL1 demonstrating higher expression in adults versus adolescents. <b>(C)</b> Representative gel image of a Western blot for CFL1 expression to confirm 2D-DIGE changes. <b>(D)</b> Quantification of Western blot results, confirming reduced expression of CFL1 (normalized to actin) in adolescents as compared to adults. <b>(E)</b> Top, representative gel images for each brain region; bottom, quantification of Western blots for each brain region. Adolescents show higher expression of CFL1 in VTA. There were no significant changes in CFL1 expression in dSTR, NAc, MC or Amy (<i>p</i>>0.05). (* indicates <i>p</i>≤0.05, *** indicates <i>p</i>≤0.001).</p

Adult and adolescent expression of dihydropyrimidinase-like-2 (DPYSL2).

<p><b>(A)</b> Representative 3D plot of DPYSL2 expression in adult (left) and adolescent (right) mice for Spot #20. <b>(B)</b> Standardized abundance (log) of DPYSL2 demonstrating higher expression in adults versus adolescents. <b>(C)</b> Representative gel image of a Western blot for DPYSL2 expression to confirm 2D-DIGE changes. Both resulting bands were quantified. <b>(D)</b> Quantification of Western blot results, confirming reduced expression of DPYSL2 (normalized to actin) in adolescents as compared to adults. <b>(E)</b> Top, representative gel images for each brain region; bottom, quantification of Western blots for each brain region. Adults show increased expression of DPYSL2 in dStr, NAc, MC and Amy. No significant age differences were observed in the VTA (<i>p</i>>0.05). Data were expressed as percent change from mean adult within the same blot and graphed as mean ± SEM. (* indicates <i>p</i>≤0.05, *** indicates <i>p</i>≤0.001).</p

Adolescent development impacts a functional protein network involved in behavior, cell-to-cell signaling and interaction, and nervous system development and function.

<p>Visualization of a protein interaction network identified by Ingenuity Pathway Analysis as being altered by adolescent brain development (<i>p</i> score = 31). Proteins shown in red were up-regulated in the adult mPFC, proteins in green were up-regulated in the adolescent mPFC, and proteins in yellow indicate statistically significant interaction proteins identified by IPA network analysis. Solid lines indicate a direct interaction, and dashed lines indicate an indirect interaction mediated by additional, non-significant proteins. Asterisks denote proteins that were identified multiple times in the proteomic analysis that have been consolidated into a single point in the functional network. Focus molecule syntaxin binding protein 1 (STXBP1) is indicated in bold.</p

Adult and adolescent expression of syntaxin binding protein 1 (STXBP1).

<p><b>(A)</b> Representative 3D plot of STXBP expression in adult (left) and adolescent (right) mice for Spot #20. <b>(B)</b> Standardized abundance (log) of STXBP1 (Spot 20) demonstrating higher expression in adults versus adolescents. <b>(C)</b> Representative 3D plot of STXBP1 expression in adult (left) and adolescent (right) mice for Spot #79. <b>(D)</b> Standardized abundance (log) of STXBP1 (Spot 79) demonstrating higher expression in adults versus adolescents. <b>(E)</b> Representative gel image of a Western blot for STXBP1 expression to confirm 2D-DIGE changes. <b>(F)</b> Quantification of Western blot results, confirming reduced expression of STXBP1 (normalized to actin) in adolescents as compared to adults. <b>(G)</b> STXBP1 expression was greater in adults in dStr, NAc, M1, Amy, and VTA. Top, representative gel images for each brain region; bottom, quantification of Western blots for each brain region. (*** indicates <i>p</i>≤0.001, **** indicates <i>p</i>≤0.0001.)</p