Nitric oxide (NO) signaling was inhibited via N(omega)-nitro-L-arginine methyl ester (L-NAME) during and after training Aplysia that a food is inedible. Treating animals with L-NAME 10 min before the start of training blocked the formation of three separable memory processes: (1) short-term, (2) intermediate-term, and (3) long-term memory. The treatment also attenuated, but did not block, a fourth memory process, very short-term memory. L-NAME had little or no effect on feeding behavior per se or on most aspects of the animals' behavior while they were being trained, indicating that the substance did not cause a pervasive modulation or poisoning of many aspects of feeding and other behaviors. Application of L-NAME within 1 min after the training had no effect on short- or long-term memory, indicating that NO signaling was not needed during memory consolidation. Treating animals with the NO scavenger 2-phenyl-4,4,5,5-tetramethyl-imidazdine-1-oxy-3-oxide before training also blocked long-term memory. Memory was not blocked by D-NAME, or by the simultaneous treatment with L-NAME and the NO donor S-nitroso-N-acetyl-penicillamine, confirming that the effect of L-NAME is attributable to its effect as a competitive inhibitor of L-arginine for NO synthase in the production of NO rather than to possible effects at other sites. These data indicate that NO signaling during training plays a critical role in the formation of multiple memory processes.

To gain insight into the function of NO transmission during learning we tested whether blocking NO synthesis affects aspects of feeding that are expressed both in a nonlearning context and during learning. Inhibiting NO synthesis with L-NAME and blocking guanylyl cyclase with methylene blue decreased the efficacy of ad libitum feeding. D-NAME had no effect. L-NAME also decreased rejection responses frequency, but did not affect rejection amplitude. The effect of L-NAME was explained by a decreased signaling that efforts to swallow are not successful, leading to a decreased rejection rate, and a decreased ability to reposition and subsequently consume food in ad libitum feeding. Signaling that animals have made an effort to swallow is a critical component of learning that food is inedible. Stimulation of the lips with food alone did not produce memory, but stimulation combined with the NO donor SNAP did produce memory. Exogenous NO at a concentration causing memory also excited a key neuron responding to NO, the MCC. Block of the cGMP secondmessenger cascade during training by methylene blue also blocked memory formation after learning. Our data indicate that memory arises from the contingency of three events during learning that food is inedible. One of the events is efforts to swallow, which are signaled by NO by cGMP.

Maintaining functional protein homeostasis ( proteostasis) is a constant challenge in the face of limited protein-folding capacity, environmental threats, and aging. Cells have developed several quality-control mechanisms that assist nascent polypeptides to fold properly, clear misfolded molecules, respond to the accumulation of protein aggregates, and deposit potentially toxic conformers in designated sites. Proteostasis collapse can lead to the development of diseases known as proteinopathies. Here we delineate the current knowledge on the different layers of protein quality-control mechanisms at the organelle and cellular levels with an emphasis on the prion protein (PrP). We also describe how protein quality control is integrated at the organismal level and discuss future perspectives on utilizing proteostasis maintenance as a strategy to develop novel therapies for the treatment of proteinopathies.

SummaryDespite the activity of cellular quality-control mechanisms, subsets of mature and newly synthesized polypeptides fail to fold properly and form insoluble aggregates. In some cases, protein aggregation leads to the development of human neurodegenerative maladies, including Alzheimer's and prion diseases. Aggregates of misfolded prion protein (PrP), which appear in cells after exposure to the drug cyclosporin A (CsA), and disease-linked PrP mutants have been found to accumulate in juxtanuclear deposition sites termed 'aggresomes'. Recently, it was shown that cells can contain at least two types of deposition sites for misfolded proteins: a dynamic quality-control compartment, which was termed 'JUNQ', and a site for terminally aggregated proteins called 'IPOD'. Here, we show that CsA-induced PrP aggresomes are dynamic structures that form despite intact proteasome activity, recruit chaperones and dynamically exchange PrP molecules with the cytosol. These findings define the CsA-PrP aggresome as a JUNQ-like dynamic qualitycontrol compartment that mediates the refolding or degradation of misfolded proteins. Together, our data suggest that the formation of PrP aggresomes protects cells from proteotoxic stress.

Do different neurodegenerative maladies emanate from the failure of a mutual protein folding mechanism? We have addressed this question by comparing mutational patterns that are linked to the manifestation of distinct neurodegenerative disorders and identified similar neurodegeneration-linked proline substitutions in the prion protein and in presenilin 1 that underlie the development of a prion disorder and of familial Alzheimer's disease (fAD), respectively. These substitutions were found to prevent the endoplasmic reticulum (ER)-resident chaperone, cyclophilin B, from assisting presenilin 1 to fold properly, leading to its aggregation, deposition in the ER, reduction of c-secretase activity, and impaired mitochondrial distribution and function. Similarly, reduced quantities of the processed, active presenilin 1 were observed in brains of cyclophilin B knockout mice. These discoveries imply that reduced cyclophilin activity contributes to the development of distinct neurodegenerative disorders, propose a novel mechanism for the development of certain fAD cases, and support the emerging theme that this disorder can stem from aberrant presenilin 1 function. This study also points at ER chaperones as targets for the development of counter-neurodegeneration therapies.