Research ArticleAlcohol and IL-6 Alter Expression of Synaptic Proteins in Cerebellum of Transgenic Mice with Increased Astrocyte Expression of IL-6
Graphical abstract
Introduction
The cerebellum is a CNS region that shows high sensitivity to alcohol both during development and in the adult (Alfonso-Loeches and Guerri, 2011, Kumar et al., 2013, Oscar-Berman and Marinkovic, 2007). Both cerebellar neurons and glial cells are targets of alcohol actions, and show altered cellular or synaptic function following acute or chronic alcohol exposure, effects that underlie alcohol-induced changes in cerebellar function (Dar, 2015, Luo, 2015, Riikonen et al., 2002, Stowell and Majewska, 2020, Valenzuela and Jotty, 2015, Worst and Vrana, 2005, Zhang et al., 2015). Well-known behavioral consequences of excessive alcohol use include ataxia and motor dysfunction, among other cerebellar symptoms (Dar, 2015, Luo, 2015). In addition to the well-known role of the cerebellum in motor coordination and learning, recent studies indicate that the cerebellum participates in cognitive and emotional processes, and psychiatric disorders (Carta et al., 2019, Fitzpatrick and Crowe, 2013, Phillips et al., 2015, Rossi and Richardson, 2018). For example, connections occur between the cerebellum and CNS regions involved in fear and anxiety, and studies indicate a role of the cerebellum in these higher-order processes (Lange et al., 2015, Moreno-Rius, 2018, Timmann et al., 2010). The cerebellum also communicates with CNS regions involved with alcohol reward and alcohol drinking (Rossi and Richardson, 2018). Thus, effects of alcohol on the cerebellum could impact the function of several CNS regions and a variety of behaviors.
The actions of alcohol on the cerebellum and other CNS regions depend on a variety of factors such as alcohol dose, route of alcohol administration, age of the subjects, and local environmental factors. Recent studies indicate that alcohol induces the production of neuroimmune factors, including the cytokine IL-6, in the cerebellum and other CNS regions, thus affecting the local cellular environment (Kane et al., 2014, Lippai et al., 2013). IL-6 is produced primarily by glial cells of the CNS, the astrocytes, and microglia, and is an important signaling molecule in the CNS. IL-6 has been shown to alter the physiology of cerebellar cells (Gruol, 2015, Gruol et al., 2014, Peng et al., 2005). Thus, indirect effects of alcohol on the cerebellum could occur as a consequence of alcohol-induced production of IL-6.
Given that little is known about the actions of IL-6 and interactions between IL-6 and alcohol in the CNS, and that emerging research indicates that neuroimmune activation is an important aspect of the effects of alcohol on the CNS (Crews et al., 2017, Robinson et al., 2014), our studies have focused on the issue of alcohol/IL-6 interactions. In recent studies we assessed potential interactions between effects of IL-6 and chronic alcohol on CNS function using transgenic mice that express elevated levels of IL-6 in the CNS through increased astrocyte expression (Gruol et al., 2018). The IL-6 tg mice were used as a model for subjects that have experienced tonically elevated levels of IL-6 in the CNS, such as occurs with long-term alcohol abuse. This model can inform on structural and functional consequences of CNS produced IL-6 and how these consequences affect the actions of alcohol, information that is important to an understanding of the mechanisms underlying the actions of alcohol on the CNS. Results showed that IL-6 produces neuroadaptive changes in the CNS and that interactions occurred between the neuroadaptive changes and actions of chronic intermittent alcohol exposure/withdrawal (CIE/withdrawal). These changes included altered brain activity as measured in EEG recordings and altered expression/activation of proteins involved in IL-6 signal transduction and inhibitory GABAergic synaptic transmission in the hippocampus. Results also showed that IL-6 and alcohol have similar cellular and synaptic targets in the hippocampus. Actions of IL-6 and alcohol at these targets could underlie the IL-6/alcohol interactions affecting EEG activity in these studies and could be a contributing factor to the effects of alcohol on the CNS under conditions of alcohol-induced IL-6 production.
In the current studies we have extended our investigation into the cellular and synaptic actions of IL-6 and IL-6/alcohol interactions in the CNS of IL-6 tg mice to the cerebellum, which is the brain region that expresses the highest level of IL-6 mRNA in the CNS of the IL-6 tg mice (Campbell et al., 1993). Interestingly, the IL-6 tg mice express ataxia, a process that is altered by alcohol, suggesting a potential role for IL-6 in cerebellar control of movement. Both acute and chronic alcohol have been reported to produce elevated levels of IL-6 mRNA in the cerebellum. For example, in adult rats an acute high dose of alcohol (4 g/kg, i.p.) increased cerebellar levels of IL-6 mRNA, but reduced cerebellar levels of IL-1 and TNFα mRNA (Doremus-Fitzwater et al., 2014). Chronic exposure to alcohol (6 g/kg by gavage for 10 days) also increased expression of IL-6 mRNA in the cerebellum with no effect on TNFα mRNA levels (Kane et al., 2014).
Little is known about the consequences of the alcohol-induced IL-6 production in the cerebellum. To address this issue, we have analyzed effects of IL-6 and alcohol on synaptic proteins in cerebella from alcohol naïve and CIE/withdrawn IL-6 tg and non-tg littermate control mice. Based on results from our studies of the hippocampus, we focused on proteins involved in inhibitory synaptic transmission mediated by the GABAA subtype of GABA receptors (GABAAR), which are an important and highly sensitive target of alcohol in cerebellar circuits and have been implicated in cerebellar dysfunction associated with excessive alcohol use (Belmeguenai et al., 2008, Botta et al., 2007, Carta et al., 2004, Carta et al., 2006, He et al., 2013, Hirono et al., 2009, Kaplan et al., 2016, Li et al., 2018, Mapelli et al., 2009, Rossi and Richardson, 2018, Saeed Dar, 2006, Simonyi et al., 1996, Su et al., 2010, Valenzuela and Jotty, 2015, Wadleigh and Valenzuela, 2012, Zamudio-Bulcock et al., 2018). Alcohol-induced effects on GABAAR function in the cerebellum impacts both motor and non-motor processes associated with cerebellar circuitry (e.g., cognitive/emotive/reward) (Blednov et al., 2017, Rossi and Richardson, 2018, Valenzuela and Jotty, 2015, Wu et al., 2014). The GABAA subtype of GABA receptors (GABAAR) are multi-subunit (pentameric) ligand-gated ionotropic channels. The pentamers are comprised of different subunits (e.g., α1-α6, β1-β3, γ1-γ3, δ, ɛ, π, θ, and ρ1-ρ3) that form channels permeable to chloride ions. Depending on the subunit composition, the GABAARs mediate phasic (i.e., synaptic) or tonic (extrasynaptic) inhibition. In the cerebellum, receptors mediating phasic inhibition typically contain the α1 subunit, whereas receptors mediating tonic inhibition typically contain the α5 or α6 subunit. For example, in cerebellar granule neurons from adult rodents, tonic inhibition is mediated by GABAARs containing the α6 subunit (Santhakumar et al., 2006). Tonic inhibition is the primary form of synaptic inhibition in the granule neurons and is enhanced by low doses of acute alcohol (5–30 mM), although the exact mechanism mediating this action of alcohol is controversial (Botta et al., 2007, Hamann et al., 2002, Valenzuela and Jotty, 2015). The increased tonic inhibition produced by acute alcohol has been proposed to play a role in alcohol-induced motor effects (Hanchar et al., 2005).
In the current studies a number of synaptic proteins were examined for effects of IL-6, alcohol and IL-6/alcohol interactions included the GABAAR subunits alpha-1, alpha-5 and alpha-6, the postsynaptic scaffolding protein gephyrin, the GABA synthesizing enzymes GAD65/67, and the GABA vesicular transporter VGAT. In addition, three proteins involved in excitatory synaptic transmission were examined, the AMPA receptor subunit GluR1, the glutamate vesicular transporter VGLUT1, and the astrocyte transporter GLAST. Glutamine synthetase, the astrocyte protein involved in both GABA and glutamate cycles, was also examined. Results show that prolonged exposure to IL-6 in the IL-6 tg cerebellum alters the expression of proteins involved in both inhibitory and excitatory synaptic function. Both increases and decreases in protein levels were observed. CIE/withdrawal reversed effects of IL-6 for some proteins but enhanced effects of IL-6 for others. Taken together, these results provide support for the hypotheses that alcohol-induced production of IL-6 can influence the effects of alcohol on synaptic pathways. Presumably behaviors mediated by those pathways would also be affected.
Section snippets
Transgenic mice
Heterozygous IL-6-tg mice (n = 21) with elevated expression of astrocyte produced IL-6 in the CNS and littermate controls (n = 23) were used in these experiments. Male and female mice of both genotypes (similar numbers) were used, although sex differences were not a focus of the studies. Construction of the transgenic mice was described previously (Campbell et al., 1993). The elevated expression of IL-6 was targeted to astrocytes using an expression vector derived from the murine glial
GABARs
Postsynaptic GABAARs containing the alpha-1 subunit are widely expressed in the cerebellum, with abundance in the granule cell layer and mainly localized to the synapse where they participate in phasic synaptic inhibition (Hortnagl et al., 2013, Pirker et al., 2000, Poltl et al., 2003). In alcohol naïve mice, GABAAR alpha-1 levels were significantly lower in cerebellum from IL-6 tg mice compared to cerebellum from non-tg mice (U = 4, n1 = 9, n2 = 7, p = 0.004, Mann–Whitney) (Fig. 1A). No
Discussion
In the current studies, we identify neuroadaptive changes in the expression of synaptic associated proteins in the cerebellum as a consequence of persistently expressed elevated levels of IL-6, a condition that occurs with excessive alcohol consumption. The proteins examined included neuronal and glial proteins associated with inhibitory (i.e., GABAAR alpha-1, alpha-5 and alpha-6, gephyrin, GAD65/67, VGAT, Glu Syn), and excitatory (i.e., GluR1, VGLUT1, Glu Syn and GLAST) synaptic transmission.
Overview
Comparison of effects of IL-6 and alcohol, alone and together, on cerebella from non-transgenic (littermate controls) and IL-6 transgenic mice revealed that proteins involved in inhibitory GABAergic synaptic transmission are important targets of both IL-6 and alcohol.
Acknowledgements
Author contributions: DLG and AJR conceived of the project and designed the experiments. CM, KL, JS, and YT performed the research. DLG analyzed and interpreted the data with support from AJR, CM, KL, JS, and YT. All authors contributed to manuscript preparation and approved the final version.
Funding: This work was supported by National Institutes of Health Grants AA024484 and the Integrated Neuroscience Initiative on Alcoholism (INIA)-West grant AA020893, and The Scripps Research Institute’s
Ethics declarations
Conflict of interest: The authors have no conflict of interest to declare.
Research Involving Animals: All animal procedures were performed in accordance with the National Institutes of Health Guideline for the Care and Use of Laboratory Animals. Animal facilities and experimental protocols were in accordance with the Association for the Assessment and Accreditation of Laboratory Animal Care.
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