Nucleolar Integrity Is Required for the Maintenance of Long-Term Synaptic Plasticity

<div><p>Long-term memory (LTM) formation requires new protein synthesis and new gene expression. Based on our work in <i>Aplysia</i>, we hypothesized that the rRNA genes, stimulation-dependent targets of the enzyme Poly(ADP-ribose) polymerase-1 (PARP-1), are primary effectors of the activity-dependent changes in synaptic function that maintain synaptic plasticity and memory. Using electrophysiology, immunohistochemistry, pharmacology and molecular biology techniques, we show here, for the first time, that the maintenance of forskolin-induced late-phase long-term potentiation (L-LTP) in mouse hippocampal slices requires nucleolar integrity and the expression of new rRNAs. The activity-dependent upregulation of rRNA, as well as L-LTP expression, are poly(ADP-ribosyl)ation (PAR) dependent and accompanied by an increase in nuclear PARP-1 and Poly(ADP) ribose molecules (pADPr) after forskolin stimulation. The upregulation of PARP-1 and pADPr is regulated by Protein kinase A (PKA) and extracellular signal-regulated kinase (ERK)—two kinases strongly associated with long-term plasticity and learning and memory. Selective inhibition of RNA Polymerase I (Pol I), responsible for the synthesis of precursor rRNA, results in the segmentation of nucleoli, the exclusion of PARP-1 from functional nucleolar compartments and disrupted L-LTP maintenance. Taken as a whole, these results suggest that new rRNAs (28S, 18S, and 5.8S ribosomal components)—hence, new ribosomes and nucleoli integrity—are required for the maintenance of long-term synaptic plasticity. This provides a mechanistic link between stimulation-dependent gene expression and the new protein synthesis known to be required for memory consolidation.</p></div

Forskolin-induced increase of PARP-1 and pADPr requires the ERK pathway.

<p>Left column (A, C, E): Representative Western blots used for quantification shown in right column (B, D, F). Phosphorylation of the ERK pathway substrate S6K was used as a stimulation-dependent positive control quantified as the ratio of p-S6K to total S6K (A, B). Histone 2B was used as a loading control (C, E). Fsk treatment (black bar) produced an increase of p-S6K, PARP-1, and pADPr (B, D, and F, respectively). The Fsk induced increase was prevented by the ERK inhibitor U0126 (grey bar; B, D, F). The inhibitor alone had no effect on PARP-1 or pADPr levels (striped bar; D, F respectively) compared with basal controls (white bar; D, F). Student's t-test (B,D,F); * = p<0.05; ** = p<0.01; NS = not significant.</p

PAR is required for Fsk-induced synthesis of new rRNAs.

<p>(A) Fsk treatment upregulates unedited precursor rRNA (ht rRNA) (black bar) 1.8 fold in hippocampal slices compared with untreated controls (white bar). This increase is prevented when PAR is blocked by 3-AB (grey bar). (B) The PARP-1 dependent immediate-early gene <i>c-jun</i>, was used as a positive control. Student's t-test; * = p<0.05; ** = p<0.01.</p

Forskolin-induced PARP-1 and pADPr increase requires PKA activity.

<p>Left column (A, C, E): Representative Western blots used for quantification shown in right column (B, D, F). Phosphorylation of the PKA pathway substrate S6K was used as a stimulation-dependent positive control quantified as the ratio of p-S6K to total S6K (A, B). Histone 2B was used as a loading control (C, E). Fsk treatment (black bar) produced an increase of p-S6K, PARP-1, and pADPr (B, D, and F, respectively). The Fsk induced upregulation of both PARP-1 and pADPr was prevented by the PKA inhibitor KT5720 (grey bar; B, D, F). The inhibitor alone had no effect on PARP-1 or pADPr levels (striped bar; D, F respectively) compared with basal controls (white bar; D, F). Student's t-test (B,D,F); * = p<0.05; ** = p<0.01; NS = not significant.</p

PAR inhibitor 3-AB produces changes in nucleolar fibrillarin intensity in cultured hippocampal neurons.

<p>DAPI staining (blue; A, B, G and H) shows the area of nuclei. Nomarski images (grey; E and F) show the area of the nuclei and nucleoli (arrowheads). (I) 3-AB treatment (100 µM, 3h) has no effect on the average area of the nucleolar marker fibrillarin (green). (J) In contrast, there was an increase in the average intensity of fibrillarin that was ∼1.6 fold stronger than control; compare arrowheads A,C,G to B, D,H. Bar = 10 µm (A–F) and 2 µm (G, H). Student's t-test ** = p<0.01; NS = not significant.</p

The specific Pol I inhibitor CX-5461 causes nucleolar disruption, blocks LTP maintenance and Fsk-induced synthesis of new rRNA.

<p>DAPI staining (blue; A, B,C) shows the area of the nucleus. Nomarski images (grey; G, H,I) show the area of the nuclei and nucleoli (arrowheads). Application of Pol I specific inhibitor CX-5461 (200 nM) causes nucleolar disruption as indicated by the distribution of fibrillarin (green); compare A, D to B, E and C, F. (J) After 1 h CX-5461 treatment, the average area of fibrillarin staining became smaller than control (5.5 µm² and 7.1 µm² respectively). After 3 h of CX-5461, fibrillarin formed small punctate domains indicative of nucleolar disruption (average area 4.7 µm²). (K) No changes in the average intensity of fibrillarin staining were observed after CX-5461 application. (L) The maintenance of LTP is blocked by application of CX-5461 (200 nM) (45 min). (M) Quantification of the changes in LTP amplitude between Fsk and Fsk+CX-5461 groups show significant changes 4 and 5 h after initiating Fsk treatment. CX-5461 alone had no effect on baseline stimulation (left inset). Representative fEPSP traces for times designated a, b and c in (L) are shown in middle and right inset. N) Fsk treatment upregulates the immediate-early gene <i>c-jun</i> (black bar) in hippocampal slices compared with untreated controls (white bar). This increase is not affected by Pol I inhibitor CX-5461 (grey bar). O) Fsk treatment upregulates unedited precursor rRNA (ht rRNA) (black bar) in hippocampal slices compared with untreated controls (white bar). This increase is prevented when Pol I inhibitor CX-5461 is applied during Fsk treatment (grey bar). Student's t-test (J,K,N,O). Student-Newman-Keuls test (M). * = p<0.05; ** = p<0.01; Bar = 10 µm; NS = not significant.</p

Low-dose Actinomycin D (Act-D) treatment dramatically disrupts nucleolar localization of PARP-1 in cultured hippocampal neurons.

<p>DAPI staining (blue; A, B) shows the area of the nucleus. Nomarski images (grey; G and H) show the area of the nuclei and nucleoli (shown by arrowheads). Low-dose Act-D treatment (100 nM, 45 min) causes nucleolar disruption as indicated by the distribution of fibrillarin (green). Fibrillarin forms punctate domains of increased intensity under Act-D treatment (compare large arrowheads A,C to small arrowheads B,D). Under the same treatment PARP-1 (red) exits the nucleolus (compare large arrowheads in E to small arrowheads in F). Bar = 10 µm.</p

Model for the Role of the Nucleolus in Synaptic Activation leading to Long-Term Plasticity.

<p>Synaptic stimulation activates adenylate cyclase (AC) resulting in the rapid release of cAMP and the activation of the cAMP→PKA→ERK pathway (kinases are indicated in yellow). Stimuli leading to long-term plasticity activate mTOR dependent protein synthesis of preexisting RNA granules (red ovals) allowing the transition into late-phase synaptic plasticity. Simultaneously, the cAMP→PKA→ERK pathway induces the synthesis and activation of chromatin remodeling proteins (e.g. PARP-1) and new gene expression. It is not yet known whether the mTOR pathway directly contributes to the activation and up-regulation of PARP-1 (dashed line). PARP-1 opens the chromatin allowing activity-dependent transcription to take place. Among the new genes expressed are activity-dependent Pol I transcripts required for the consolidation of late-phase synaptic plasticity. In this model, the new stimulation-evoked rRNAs give rise to new ribosomes. The new plasticity-dependent ribosomes are assembled into new RNA granules (green ovals) and shipped to activated synapses to maintain, through local protein synthesis, the long-lasting changes required for long-term synaptic plasticity and memory.</p

Low-dose Act-D treatment blocks LTP maintenance.

<p>(A) The maintenance of LTP is blocked by low-dose (45 min, 100 nM) Act-D treatment. (B) Quantification of the change in LTP amplitude 1 and 2 h after initiating Fsk treatment. Representative fEPSP traces for times a, b, and c (from A) appear above bars. Student-Newman-Keuls test ** = p<0.01.</p