Our analysis revealed that alcoholism is causing changes to the synaptic proteome in both the SFG and the OC, with a small subset of proteins altered in both regions. This group of proteins is quite different to those changes that occur in whole tissue proteomes of similar brain regions [14
], a result most likely due to the focus of this study specifically on synaptosomal proteins. Additionally, we have identified that the visual association area represented by the OC region of the brain is reacting to alcoholism in a very significant way, with our finding that almost twice as many proteins are altered between alcoholics and controls in this region than in the SFG.
This result suggests that the OC is not as spared as previously thought. Considering that the OC receives input from areas of higher brain function such as the frontal cortex and thalamus, these changes could be, in part, a response to the alterations taking place in other brain regions. The brain-wide neuropathological effects of alcoholism may also be causing direct protein changes, although this is not necessarily to the detriment of the OC. The greater response of the OC could be an indication that those changes are induced to increase protection from the effects of alcoholism in this region, resulting in no changes to the functionality of visual processes.
As expected, many proteins altered by alcoholism are involved in energy metabolism or glycolytic pathways. Alcoholism affects various metabolic pathways throughout the body, including the brain, and alcoholism-induced alterations could lead to disruption of essential energy-producing processes, resulting in reduced cellular functionality and possibly cell death. Some altered proteins are involved in aerobic glycolysis and mitochondrial oxidative phosphorylation which supply the brain's high energy requirements through ATP production [25
]. Without appropriate control of ATP production by glycolytic and other mitochondrial pathways in the brain, certain highly-used, and thus energy-hungry, areas are bound to be adversely affected – possibly to the point of death due to energy starvation.
The decrease of creatine kinase B (CKB) in the alcoholic SFG (this study), OC (this study), hippocampus [26
], and cerebellar vermis [26
] shows that energy metabolism is altered in many parts of the alcoholic brain. The corpus callosum is notable in its differences in that this protein is not altered in the splenium [28
], and is increased in the genu [29
], suggesting region specific regulation.
CKB is an essential enzyme in energy-hungry tissues such as the brain where it catalyses the phosphorylation of creatine to phosphocreatine. This is not only an energy storage molecule required for rapid ATP synthesis, but can act as a ‘shuttle’ to move the phosphate from the site of consumption to the site of generation [25
]. Those brain regions showing reduced CKB are likely to have a reduced capacity for energy storage and production which would severely compromise synapse function.
Changes to the glycolysis proteins fructose-bisphosphate aldolase A and C are specific to the OC, and are interconnected with changes to the cytoskeleton. Aldolases catalyze the cleavage of fructose 1,6-bisphosphate (FBP) to glyceraldehyde 3-phosphate and dihydroxyacetone in glycolysis, but they also interact with several other proteins, including filamentous actin [30
], possibly for co-localization of glycolytic proteins [31
] such as triosephosphate isomerase [32
], another protein altered by alcoholism (this study). In addition to direct interaction with actin, aldolase sequesters the actin filament nucleation protein Wiskott-Aldrich syndrome protein (WASP), which inhibits both WASP and aldolase activity until enough substrate (FBP) is present to dislodge it [33
]. Actin itself is also decreased specifically in the OC of alcoholics (this study), suggesting that alcoholism may affect the actin dynamics of OC cells directly, through γ-actin, and indirectly, through disruption of aldolase's metabolic function and through WASP.
Altered regulation of cytoskeletal components and associated signaling pathways can cause significant changes to synaptic and axonal function. Dihydropyrimidinase-related protein (DRP
2), involved in neuronal repair in the adult brain through control of axonal outgrowth of regenerated neurons, is down-regulated in Alzheimer's disease brain [35
], but has increased oxidation [36
]. In this disease, loss of non-oxidized DRP
2 may increase neurodegeneration by preventing neuronal repair. The increase in DRP
2 levels in the OC found in this study may be a compensatory mechanism in response to alcoholism-induced damage as a means to increase axonal growth, repair and regeneration.
Several proteins involved in synaptic transmission were altered by alcoholism. The brain-specific protein, dynamin-1, controls synaptic-vesicle recycling via endocytosis where it is involved in scission of clathrin-coated vesicles from a parent membrane in the pre-synaptic cell [38
]. It is essential only during the application of a strong or sustained stimulus when exocytosis of neurotransmitter-containing vesicles is extreme and thus requires rapid retrieval of clathrin-coated vesicles via endocytosis to maintain the pool of synaptic vesicles [39
]. There were 12 dynamin-1 isoforms identified in both brain regions, but more were significantly lowered in response to alcoholism in the SFG (5 isoforms) than in the OC (1 isoform). Three of these isoforms show region specific expression in non-alcoholic controls indicating that the different isoforms may be responsible for different functions in each region of normal brain. For two isoforms, these expression differences are lost in alcoholics due to significant decrease of these isoforms in the alcoholic SFG region. Alcoholics exhibit defects in cognitive processes controlled by the SFG such as learning and decision-making, thus reduced or impaired synaptic-vesicle recycling, and thus neuronal signaling, due to loss of dynamin-1 in this area may underpin alcoholism's neurodegenerative effects and its general disruption of cognitive function.
The modification responsible for multiple expression forms of dynamin-1 is not known, however each is likely to have a distinct function since those altered by alcoholism do so independent of the modification. In this manner, chronic alcohol misuse may be disrupting multiple synaptic functions through a single protein.
Other studies of the alcoholic SFG either did not report a change in dynamin-1 [15
], or found an increase [14
]. No other study has shown that multiple isoforms of dynamin-1 are regulated in alcoholics. This disparity is likely due to differences in protein preparation: this study focused on an analysis of enriched synaptic proteins, while the other studies utilized total protein extracts [14
]. By fractionating the synaptosomal proteins we have enriched those proteins which are normally of low abundance within the total protein extract, such as the different isoforms of dynamin-1, and have thus been able to identify differences in the levels of these low-abundant proteins in alcoholics.
Other proteins, such as several HSP70s, showed an alcoholism-induced shift from one isoform to another. Most HSP70 expression is stress-induced [40
], and different HSP70s are induced by short-term [41
] and long-term [14
] alcohol consumption; however, some are also expressed at a basal level (e.g., in human brain HSP70-2 [46
], HSP70-5 [47
], and HSP70-8 [48
]). The chaperone-related housekeeping roles of constitutively expressed HSP70s work to prevent aberrant protein folding and targeting [40
]. These functions are subsumed into protective roles during stress, along with the inducible HSP70s, to combat the effects of excitotoxicity (particularly HSP70-1 [51
]) and oxidative damage (HSP70-1 [41
]; HSP70-8 [52
]). Excessive consumption of alcohol causes both of these stresses: ethanol metabolism pathways produce free radicals that cause oxidative damage in the liver [54
] and brain [56
], and chronic alcohol abuse enhances excitotoxicity through altered regulation of the neurotransmitters γ-aminobutyric acid (GABA) and glutamate, and their receptors [57
Changes to the levels of different HSP70 isoforms may be elicited as a protective response against these stresses. Differently modified isoforms are likely to have slightly varying functions, thus this shift in the predominance of particular isoforms suggests an altered requirement for specific functional isoforms. When compared to the same region in controls, the alcoholic SFG showed fewer changes to the HSP70 proteins than the OC, potentially leaving it more susceptible to oxidative damage or excitotoxicity. Additionally, the basal level of some HSP70 isoforms in non-alcoholic brain differs between the SFG and OC (). Many of these differences are lost in alcoholics further suggesting region-specific changes resulting in loss of HSP70 functionality.
These results give an interesting glimpse into the synaptic-specific changes induced by alcoholism and indicate pathways involved in alcoholism's effect. The differences between the changes that occur in the two brain regions demonstrate that the synapses of the SFG and OC have both been affected by alcoholism to different degrees. Changes to vesicle transport and cytoskeleton proteins are indicative of alcoholism-induced changes to synaptic transmission pathways and could potentially explain alcoholism's neurodegenerative effects and disruptions to cognitive function. Evidence of enhanced protective functions is present in the changes to chaperone levels and isoforms, particularly in the functionally operational OC. Further study of these proteins will further our knowledge of alcoholism's effect on the brain, and also help us to gain insight into mechanisms of neurodegeneration and synaptic loss.