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

Probe location in the EPN.

<p>(A) Injection cannula were stereotactically implanted at a 10° angle, aiming at the dorsal portion of the EPN (B) Histological verification of probe location in a cresyl violet stained coronal section from a representative animal. A small lesion indicates the injection site, which is located in the dorsal EPN. LA, lateral amygdala; BLA, basolateral amygdala; EPN, endopiriform nucleus; PC, piriform cortex; III, third ventricle (C) Summary of injection sites in individual animals of all three test groups. Illustrations modified from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002695#pone.0002695-Paxinos1" target="_blank">[37]</a>.</p

Behavioral NPS effects.

<p>A selective reduction of contextual fear responding was observed upon NPS administration. (A) Injection of 0.1 nmole NPS to the endopiriform nucleus (n = 14), resulted in significantly reduced freezing behavior during contextual fear memory retrieval, as compared to vehicle injected controls (n = 12). At a dose of 0.01 nmole, NPS had no such effect (n = 12). Time course analysis of conditioned freezing behavior revealed a continuous reduction during the contextual retrieval session. For better comparison the 2-min context exposure was dissected into eigth intervals of either 10 s 1,3,5,7) or 20 s (2,4,6,8) length, analogous to those in cued retrieval. (B) In cued fear memory retrieval, a somewhat reduced freezing response was observed in the 0.1 nmole group, but this changes failed to reach significance level. The reduction was mostly observed during inter-stimulus-intervals and hence may have resulted from the animals' reduced fear response to the background context, whereas the responses during tone exposure were almost identical between groups. 1,3,5,7, conditioned stimluli (CS+, 10 s), 2,4,6,8 inter-stimulus-intervals (20 s). (C, D) A similar specific reduction during contextual versus cued memory retrieval was observed for a second defensive behavior, risk-assessment. (E, F) No change of anxiety-related behavior was apparent in an elevated plus maze. All values are means±SEM; *, p<0.05; compared to vehicle, **, p<0.01.</p

NPS stimulates GABAergic input of BLA projection neurons.

<p>In BLA projection neurons, NPS stimulates GABA-mediated synaptic activity dependent on action potential propagation. Examples of GABAergic sIPSCs recorded in a BLA projection neuron before (A) and during action of NPS (B). (C) Cumulative amplitude and (D) inter-event interval histograms obtained from the same neuron shown in (A, B) before addition of NPS and after a steady-state effect had been reached. (E) sIPSC amplitude and (F) frequency pooled during control conditions and after addition of NPS demonstrates a significant increase in sIPSC amplitude as well as frequency. (G) mIPSCs recorded in the presence of TTX were unchanged in amplitude as well as (H) frequency by addition of NPS. (I) Representative traces of IPSCs recorded before and after addition of 200 nM NPS. (J) Time course of mean paired-pulse ratio of eIPSCs showing lack of NPS effects on paired-pulse facilitation in BLA projection neurons. **, p<0.01. Bar, time of NPS addition.</p

NPS stimulates glutamatergic input of BLA interneurons.

<p>NPS stimulates glutamatergic synaptic activity dependent on action potential propagation in BLA interneurons. (A) Examples of glutamatergic sEPSCs recorded in a BLA interneuron before and (B) during action of NPS. (C) Cumulative amplitude and (D) inter-event interval histograms obtained from the same neuron shown in (A, B) before addition of NPS and after a steady-state effect had been reached. (E) sEPSC amplitude and (F) frequency pooled during control conditions and after addition of NPS demonstrates a significant increase in sEPSC frequency. (G) mEPSCs recorded in the presence of TTX were unchanged in amplitude as well as (H) frequency by addition of NPS. (I) Representative traces of EPSCs recorded before and after addition of 200 nM NPS. (J) Time course of mean paired-pulse ratio of eEPSCs showing lack of NPS effects on paired-pulse facilitation in BLA interneurons. **, p<0.01. Bar, time of NPS addition.</p

Effects of mechanical circuit dissection.

<p>(A) Cuts separating medial/cortical (Me/Co) and BLA do not interfere with the significant increase in amplitude as well as frequency of sIPSCs recorded from BLA projection neurons after addition of NPS. (B) Cuts detaching endopiriform nucleus from BLA eliminate NPS action upon sIPSCs in BLA projection neurons. (C, D) Only dissection in between EPN and BLA diminish (C) amplitude or (D) frequency ratio: NPS/control significantly. (E, F) Examples of GABAergic sIPSCs recorded in a BLA projection neuron before (E, top traces) and after puff application of NPS (F, top traces) into the ventral EPN. Mean normalized amplitude (E) and frequency (F) of sIPSCs during control conditions and after puff application of NPS. *, p<0.05; **, p<0.01. Illustrations modified from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002695#pone.0002695-Paxinos1" target="_blank">[37]</a>.</p

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

NPS stimulates glutamatergic input of BLA projection neurons.

<p>NPS stimulates glutamatergic synaptic activity dependent on action potential propagation in BLA projection neurons. (A) sEPSC amplitude and (B) frequency pooled during control conditions and after addition of NPS demonstrates a significant increase in sEPSC amplitude as well as frequency. (C) mEPSCs recorded in the presence of TTX were unchanged in amplitude as well as (D) frequency by addition of NPS. (E) Under current-clamp conditions, NPS application induces a depolarizing response associated with increased spike activity triggered upon depolarizing current injections in a subset of BLA projection neurons. *, p<0.05.</p