Ubiquitination acutely regulates presynaptic neurotransmitter release in mammalian neurons.

The ubiquitin proteasome system (UPS) plays a crucial role in modulating synaptic physiology both presynaptically and postsynaptically, but the regulatory mechanisms remain obscure. To determine acute effects of proteasome inhibition on neurotransmission, we performed whole-cell voltage-clamp recordings from cultured rodent hippocampal neurons. We find that proteasome inhibitors induce a strikingly fast, severalfold increase in the frequency of both miniature (mini) and spontaneous synaptic currents at excitatory and inhibitory synapses. The lack of change in mini amplitude and kinetics indicates a presynaptic site of action. This effect does not depend on increased levels of presynaptic proteins, previously suggested as proteasomal targets. Furthermore, blockade of the UPS using E1-activating enzyme inhibitors also increases mini frequency, demonstrating that accumulation of ubiquitinated proteins is not required. Overall, these data suggest that the UPS not only orchestrates protein turnover, but also dynamically regulates the activity state of presynaptic proteins, thus crucially shaping synaptic transmission.

Functional effects of spinocerebellar ataxia type 13 mutations are conserved in zebrafish Kv3.3 channels.

BACKGROUND: The zebrafish has been suggested as a model system for studying human diseases that affect nervous system function and motor output. However, few of the ion channels that control neuronal activity in zebrafish have been characterized. Here, we have identified zebrafish orthologs of voltage-dependent Kv3 (KCNC) K+ channels. Kv3 channels have specialized gating properties that facilitate high-frequency, repetitive firing in fast-spiking neurons. Mutations in human Kv3.3 cause spinocerebellar ataxia type 13 (SCA13), an autosomal dominant genetic disease that exists in distinct neurodevelopmental and neurodegenerative forms. To assess the potential usefulness of the zebrafish as a model system for SCA13, we have characterized the functional properties of zebrafish Kv3.3 channels with and without mutations analogous to those that cause SCA13. RESULTS: The zebrafish genome (release Zv8) contains six Kv3 family members including two Kv3.1 genes (kcnc1a and kcnc1b), one Kv3.2 gene (kcnc2), two Kv3.3 genes (kcnc3a and kcnc3b), and one Kv3.4 gene (kcnc4). Both Kv3.3 genes are expressed during early development. Zebrafish Kv3.3 channels exhibit strong functional and structural homology with mammalian Kv3.3 channels. Zebrafish Kv3.3 activates over a depolarized voltage range and deactivates rapidly. An amino-terminal extension mediates fast, N-type inactivation. The kcnc3a gene is alternatively spliced, generating variant carboxyl-terminal sequences. The R335H mutation in the S4 transmembrane segment, analogous to the SCA13 mutation R420H, eliminates functional expression. When co-expressed with wild type, R335H subunits suppress Kv3.3 activity by a dominant negative mechanism. The F363L mutation in the S5 transmembrane segment, analogous to the SCA13 mutation F448L, alters channel gating. F363L shifts the voltage range for activation in the hyperpolarized direction and dramatically slows deactivation. CONCLUSIONS: The functional properties of zebrafish Kv3.3 channels are consistent with a role in facilitating fast, repetitive firing of action potentials in neurons. The functional effects of SCA13 mutations are well conserved between human and zebrafish Kv3.3 channels. The high degree of homology between human and zebrafish Kv3.3 channels suggests that the zebrafish will be a useful model system for studying pathogenic mechanisms in SCA13.

Disordered sleep, nocturnal cytokines, and immunity: interactions between alcohol dependence and African-American ethnicity.

Sleep disturbance is one of the most prominent complaints of alcohol-dependent patients. In view of recent evidence that the immune system is integrated with other homeostatic processes ultimately regulated by the brain, the influence of sleep on host defense mechanisms and the expression of proinflammatory and T helper cell cytokines deserves attention in alcohol dependence. Although not all immune alterations found in alcohol-dependent persons are related to disordered sleep, it is exceedingly important to know whether sleep influences immunity in alcoholism because of the recognized impact of disordered sleep on infectious disease risk. Conversely, feedback systems are also operating between the brain and the immune system, and abnormalities in the expression of cytokines might contribute to sleep disturbances in alcohol-dependent persons. In this review, we identify the immune alterations found in association with alcohol dependence and discuss the implications of these findings for infectious disease risk, with particular attention to the interaction between African-American ethnicity and alcoholism in contributing to this risk. We provide evidence that sleep disruption occurs in association with alcohol dependence and that African-American alcohol-dependent persons show greater abnormalities in sleep and sleep regulatory processes than shown by Euro-American alcohol-dependent persons. The relations among alcoholism, sleep, and immunity are discussed, with an emphasis on understanding how the cytokine network is altered during sleep in the African-American alcohol-dependent populations. The potential is to use cytokine agonists or antagonists to determine whether physiologic changes in cytokines have a role in the homeostatic regulation of sleep in human beings, which has tremendous implications for the development of novel treatments of alcohol-related sleep disorders.

Nocturnal proinflammatory cytokine-associated sleep disturbances in abstinent African American alcoholics.

Animal studies reveal that cytokines play a key role in the regulation of sleep. Alcoholic patients show profound alterations of sleep and a defect in the homeostatic recovery of sleep following sleep loss. In this study, we investigated whether nocturnal plasma levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF) were associated with disordered sleep in alcohol dependence by testing the temporal relationships between these inflammatory cytokines and sleep, before and after sleep deprivation. All-night polysomnography and serial blood sampling at 23:00, 03:00, and 06:30 h were conducted across baseline, partial sleep deprivation, and recovery nights in abstinent African American alcoholics (n=16) and matched controls (n=15). Coupled with prolonged sleep latency and increased rapid eye movement sleep, alcoholics showed nocturnal elevations of IL-6 and TNF as compared to controls after adjustment for alcohol consumption and body mass index. Following sleep deprivation, alcoholics showed greater nocturnal levels of IL-6 and greater nocturnal increases of TNF as compared to controls. Pre-sleep IL-6 levels at 23:00 h correlated with prolonged sleep latency after adjustment for potential confounders whereas IL-6 levels at 03:00 h correlated with rapid eye movement sleep in the second half of the night. Taken together, these findings indicate that circulating levels of proinflammatory cytokines may have a negative influence on sleep initiation. These findings have implications for determining why sleep is disordered in alcoholics and may aid in the development of novel treatments to optimize sleep in this population.