The Ubiquitin Ligase Praja1 Reduces NRAGE Expression and Inhibits Neuronal Differentiation of PC12 Cells

<div><p>Evidence suggests that regulated ubiquitination of proteins plays a critical role in the development and plasticity of the central nervous system. We have previously identified the ubiquitin ligase Praja1 as a gene product induced during fear memory consolidation. However, the neuronal function of this enzyme still needs to be clarified. Here, we investigate its involvement in the nerve growth factor (NGF)-induced differentiation of rat pheochromocytoma (PC12) cells. Praja1 co-localizes with cytoskeleton components and the neurotrophin receptor interacting MAGE homologue (NRAGE). We observed an enhanced expression of Praja1 after 3 days of NGF treatment and a suppression of neurite formation upon Praja1 overexpression in stably transfected PC12 cell lines, which was associated with a proteasome-dependent reduction of NRAGE levels. Our data suggest that Praja1, through ubiquitination and degradation of NRAGE, inhibits neuronal differentiation. The two murine isoforms, Praja1.1 and Praja1.2, appear to be functionally homologous in this respect.</p></div

Unaltered auditory cued fear memory in SST deficient mice.

<p>(A) Freezing towards the conditioned auditory stimulus (CS+) did not differ between genotypes or time points of testing. (B) Likewise, treezing towards the context also was not influenced by the time point of training. No deficits in could be observed in SST^-/- mice. Values are mean ± SEM. </p

Lack of circadian fluctuation of anxiety-like behavior in SST deficient mice.

<p>(A) SST^-/- mice displayed hyperactivity during the second half of their active phase as indicated by total distance walked in the open field, compared to their SST+/+ littermates. (B) However, time spent in center did not differ between genotypes are time of testing. (C,D) In the light/dark- avoidance test, SST^-/- mice showed reduced exploration of the light compartment, indicating increased anxiety-like behavior during the second half of the active phase. In fact the mutants failed to show a reduction of anxiety as seen in SST^+/+ mice, thus indicating a deficit in the circadian modulation of anxiety- like behavior. Values are mean ± SEM. * significant differences between time point of testing, p<0.05; #, significant differences between SST^-/- and SST^+/+ mice, p < 0.05; # # p < 0.01.</p

Long-term changes in the CA3 associative network of fear-conditioned mice

<div><p></p><p>The CA3 associative network plays a critical role in the generation of network activity patterns related to emotional state and fear memory. We investigated long-term changes in the corticosterone (CORT)-sensitive function of this network following fear conditioning and fear memory reactivation. In acute slice preparations from mice trained in either condition, the ratio of orthodromic population spike (PS) to antidromic PS was reduced compared to unconditioned animals, indicating a decrease in efficacy of neuronal coupling within the associative CA3 network. However, spontaneous sharp wave–ripples (SW-R), which are thought to arise from this network, remained unaltered. Following CORT application, we observed an increase in orthodromic PS and a normalization to control levels of their ratio to antidromic PS, while SW-R increased in slices of fear conditioned and fear reactivated mice, but not in slices of unconditioned controls. Together with our previous observations of altered hippocampal gamma activity under these learning paradigms, these data suggest that fear conditioning and fear reactivation lastingly alters the CORT-sensitive configuration of different network activity patterns generated by the CA3 associational network. Observed changes in the mRNA expression of receptors for glutamate, GABA and cannabinoids in the stratum pyramidale of area CA3 may provide a molecular mechanism for these adaptive changes.</p></div

Home cage activity of SST deficient mice.

<p>(A) The general activity profile of SST^-/- mice in their home cages did not differ from that of SST^+/+ mice over the 24h cycle (lights off indicated by gray shaded area on the x-axis). (B) Planned comparison of the average activity over the daytime corresponding to the behavioral test periods showed a reduced activity of SST^-/- mice during the first half of the active phase (D1^st) but no difference between genotype in the second half (D2^nd). Values are mean ± SEM. #, significant differences between SST^-/- and SST^+/+ mice, p < 0.05. </p

Circadian modulation of SST expression in the BLA.

<p>(A) Per2 mRNA levels display pronounced circadian fluctuation over a 24h period, confirming previous reports. Expression of SST is well correlated (Pearson’s correlation: r=0.439) with expression levels of Per2 in the BLA over the different time points. Significant correlation with Per2 expression is also observed for NPY (r=0.455) and GAD65 expression levels (r=0.514). Shaded area illustrates 12h lights off period and brackets indicate time of behavioral testing. Planned comparison revealed a significant increase of Per2 and SST, but also NPY mRNA levels towards the second half of the active phase (D2^nd). Values are relative expression after normalization to house keeping gene GAPDH (dCT) ± SEM for each time point. (B) Accordingly, the peptide concentration of SST in the BLA is increased in the second half of the dark phase compared to the first half.. Values are mean ± SEM. * significant difference T1 vs. T7, p<0.05 .</p

Long-term changes in the CA3 associative network of fear-conditioned mice

<div><p></p><p>The CA3 associative network plays a critical role in the generation of network activity patterns related to emotional state and fear memory. We investigated long-term changes in the corticosterone (CORT)-sensitive function of this network following fear conditioning and fear memory reactivation. In acute slice preparations from mice trained in either condition, the ratio of orthodromic population spike (PS) to antidromic PS was reduced compared to unconditioned animals, indicating a decrease in efficacy of neuronal coupling within the associative CA3 network. However, spontaneous sharp wave–ripples (SW-R), which are thought to arise from this network, remained unaltered. Following CORT application, we observed an increase in orthodromic PS and a normalization to control levels of their ratio to antidromic PS, while SW-R increased in slices of fear conditioned and fear reactivated mice, but not in slices of unconditioned controls. Together with our previous observations of altered hippocampal gamma activity under these learning paradigms, these data suggest that fear conditioning and fear reactivation lastingly alters the CORT-sensitive configuration of different network activity patterns generated by the CA3 associational network. Observed changes in the mRNA expression of receptors for glutamate, GABA and cannabinoids in the stratum pyramidale of area CA3 may provide a molecular mechanism for these adaptive changes.</p></div