Learning-induced ribosomal RNA is required for memory consolidation in mice-Evidence of differentially expressed rRNA variants in learning and memory.

The transition from short-term to long-term forms of synaptic plasticity requires protein synthesis and new gene expression. Most efforts to understand experience-induced changes in neuronal gene expression have focused on the transcription products of RNA polymerase II-primarily mRNAs and the proteins they encode. We recently showed that nucleolar integrity and activity-dependent ribosomal RNA (rRNA) synthesis are essential for the maintenance of hippocampal long-term potentiation (LTP). Consequently, the synaptic plasticity and memory hypothesis predicts that nucleolar integrity and activity dependent rRNA synthesis would be required for Long-term memory (LTM). We tested this prediction using the hippocampus-dependent, Active Place Avoidance (APA) spatial memory task and found that training induces de novo rRNA synthesis in mouse dorsal hippocampus. This learning-induced increase in nucleolar activity and rRNA synthesis persists at least 24 h after training. In addition, intra-hippocampal injection of the Pol I specific inhibitor, CX-5461 prior to training, revealed that de novo rRNA synthesis is required for 24 h memory, but not for learning. Using qPCR to assess activity-dependent changes in gene expression, we found that of seven known rRNA expression variants (v-rRNAs), only one, v-rRNA IV, is significantly upregulated right after training. These data indicate that learning induced v-rRNAs are crucial for LTM, and constitute the first evidence that differential rRNA gene expression plays a role in memory.

New ribosomes for new memories?

Widely thought to be a housekeeping process, the regulation and synthesis of rRNA emerges as a potentially central mechanism for the maintenance of synaptic plasticity and memory. We have recently shown that an essential component of late-phase synaptic plasticity is rRNA biosynthesis - the rate-limiting step in the production of new ribosomes. We hypothesize that a particular population of ribosomes is generated upon learning-associated neural activity to alter the rate of synthesis of plasticity factors at tagged synapses that will support the maintenance of synaptic plasticity and memory.

Nucleolar integrity is required for the maintenance of long-term synaptic plasticity.

Long-term memory (LTM) formation requires new protein synthesis and new gene expression. Based on our work in Aplysia, 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.

Polyglutamine toxicity is controlled by prion composition and gene dosage in yeast.

Polyglutamine expansion causes diseases in humans and other mammals. One example is Huntington's disease. Fragments of human huntingtin protein having an expanded polyglutamine stretch form aggregates and cause cytotoxicity in yeast cells bearing endogenous QN-rich proteins in the aggregated (prion) form. Attachment of the proline(P)-rich region targets polyglutamines to the large perinuclear deposit (aggresome). Aggresome formation ameliorates polyglutamine cytotoxicity in cells containing only the prion form of Rnq1 protein. Here we show that expanded polyglutamines both with (poly-QP) or without (poly-Q) a P-rich stretch remain toxic in the presence of the prion form of translation termination (release) factor Sup35 (eRF3). A Sup35 derivative that lacks the QN-rich domain and is unable to be incorporated into aggregates counteracts cytotoxicity, suggesting that toxicity is due to Sup35 sequestration. Increase in the levels of another release factor, Sup45 (eRF1), due to either disomy by chromosome II containing the SUP45 gene or to introduction of the SUP45-bearing plasmid counteracts poly-Q or poly-QP toxicity in the presence of the Sup35 prion. Protein analysis confirms that polyglutamines alter aggregation patterns of Sup35 and promote aggregation of Sup45, while excess Sup45 counteracts these effects. Our data show that one and the same mode of polyglutamine aggregation could be cytoprotective or cytotoxic, depending on the composition of other aggregates in a eukaryotic cell, and demonstrate that other aggregates expand the range of proteins that are susceptible to sequestration by polyglutamines.

Effects of ubiquitin system alterations on the formation and loss of a yeast prion.

The yeast prion [PSI+] is a self-propagating amyloidogenic isoform of the translation termination factor Sup35. Overproduction of the chaperone protein Hsp104 results in loss of [PSI+]. Here we demonstrate that this effect is decreased by deletion of either the gene coding for one of the major yeast ubiquitin-conjugating enzymes, Ubc4, or the gene coding for the ubiquitin-recycling enzyme, Ubp6. The effect of ubc4Delta on [PSI+] loss was increased by depletion of the Hsp70 chaperone Ssb but was not influenced by depletion of Ubp6. This indicates that Ubc4 affects [PSI+] loss via a pathway that is the same as the one affected by Ubp6 but not by Ssb. In the presence of Rnq1 protein, ubc4Delta also facilitates spontaneous de novo formation of [PSI+]. This stimulation is independent of [PIN+], the prion isoform of Rnq1. Numerous attempts failed to detect ubiquitinated Sup35 in the yeast extracts. While ubc4Delta and other alterations of ubiquitin system used in this work cause slight induction of some Hsps, these changes are insufficient to explain their effect on [PSI+]. However, ubc4Delta increases the proportion of the Hsp70 chaperone Ssa bound to Sup35, suggesting that misfolded Sup35 is either more abundant or more accessible to the chaperones in the absence of Ubc4. The proportion of [PSI+] cells containing large aggregated Sup35 structures is also increased by ubc4Delta. We propose that UPS alterations induce an adaptive response, resulting in accumulation of the large "aggresome"-like aggregates that promote de novo prion generation and prion recovery from the chaperone treatment.

Hsp70 chaperones as modulators of prion life cycle: novel effects of Ssa and Ssb on the Saccharomyces cerevisiae prion [PSI+].

[PSI(+)] is a prion isoform of the yeast release factor Sup35. In some assays, the cytosolic chaperones Ssa1 and Ssb1/2 of the Hsp70 family were previously shown to exhibit "pro-[PSI(+)]" and "anti-[PSI(+)]" effects, respectively. Here, it is demonstrated for the first time that excess Ssa1 increases de novo formation of [PSI(+)] and that pro-[PSI(+)] effects of Ssa1 are shared by all other Ssa proteins. Experiments with chimeric constructs show that the peptide-binding domain is a major determinant of differences in the effects of Ssa and Ssb proteins on [PSI(+)]. Surprisingly, overproduction of either chaperone increases loss of [PSI(+)] when Sup35 is simultaneously overproduced. Excess Ssa increases both the average size of prion polymers and the proportion of monomeric Sup35 protein. Both in vivo and in vitro experiments uncover direct physical interactions between Sup35 and Hsp70 proteins. The proposed model postulates that Ssa stimulates prion formation and polymer growth by stabilizing misfolded proteins, which serve as substrates for prion conversion. In the case of very large prion aggregates, further increase in size may lead to the loss of prion activity. In contrast, Ssb either stimulates refolding into nonprion conformation or targets misfolded proteins for degradation, in this way counteracting prion formation and propagation.

Pleiotropic effects of Ubp6 loss on drug sensitivities and yeast prion are due to depletion of the free ubiquitin pool.

Mutation of the mouse Usp14 gene, encoding the homolog of yeast deubiquitinating enzyme Ubp6, causes ataxia. Here we show that deletion of the UBP6 gene in Saccharomyces cerevisiae causes sensitivity to a broad range of toxic compounds and antagonizes phenotypic expression and de novo induction of the yeast prion [PSI+], a functionally defective self-perpetuating isoform of the translation termination factor Sup35. Conversely, overexpression of ubiquitin (Ub) increases phenotypic expression and induction of [PSI+] in the wild type cells and suppresses all tested ubp6Delta defects, indicating that they are primarily due to depletion of cellular Ub levels. Several lines of evidence suggest that Ubp6 functions on the proteasome. First, Ub levels in the ubp6Delta cells can be partly restored by proteasome inhibitors, suggesting that deletion of Ubp6 decreases Ub levels by increasing proteasome-dependent degradation of Ub. Second, fluorescence microscopy analysis shows that Ubp6-GFP fusion protein is localized to the nucleus of yeast cell, as are most proteasomes. Third, the N-terminal Ub-like domain, although it is not required for nuclear localization of Ubp6, targets Ubp6 to the proteasome and cannot be functionally replaced by Ub. The human ortholog of Ubp6, USP14, probably plays a similar role in higher eukaryotes, since it fully compensates for ubp6Delta defects and binds to the yeast proteasome. These data link the Ub system to prion expression and propagation and have broad implications for other neuronal inclusion body diseases.