Immunochemical Characterization of the Deficiency of the α‐Ketoglutarate Dehydrogenase Complex in Thiamine‐Deficient Rat Brain

Abstract

Abstract:Mitochondrial dysfunction is a common feature of many neurodegenerative disorders. The metabolic encephalopathy caused by thiamine deficiency (TD) is a classic example in which an impairment of cerebral oxidative metabolism leads to selective cell death. In experimental TD in rodents, a reduction in the activity of the thiamine diphosphate-dependent, mitochondrial enzyme a-ketoglutarate dehydrogenase complex (KGDHC) occurs before the onset of pathologic lesions and is among the earliest biochemical deficits found. To understand the molecular basis and the significance of the deficiency of KGDHC in TD-induced brain damage, the enzyme activity and protein levels of KGDHC were analyzed. The effect of TD on the subregional/cellular distribution of KGDHC and the anatomic relation of KGDHC with selective cell death were also tested by immunocytochemistry. Consistent with several previous studies, TD dramatically reduced KGDHC activity in both anatomically damaged (thalamus and inferior colliculus) and spared (cerebral cortex) regions. Immunocytochemistry revealed no apparent correlation of regional KGDHC immunoreactivity or its response to TD with affected regions in TD. The basis of the enzymatic and immunocytochemical behavior of KGDHC was further assessed by quantitative immunoblots, using antibodies specific for each of the three KGDHC components. Despite the marked decrease of KGDHC activity in TD, no reduction of any of the three KGDHC protein levels was found. Thus, TD impairs the efficacy of the KGDHC catalytic machinery, whereas the concentration of protein molecules persists. The generalized decline of KGDHC activity with no apparent anatomic selectivity is consistent with the notion that the compromised mitochondrial oxidation sensitizes the brain cells to various other insults that precipitate the cell death. The current TD model provides a relevant experimental system to understand the molecular basis of many neurodegenerative conditions in which mitochondrial dysfunction and KGDHC deficiency are prominent features. Key Words: Alzheimer's disease-et-Ketoglutarate dehydrogenase-2-Oxoglutarate dehydrogenaseOxidative stress-Thiamine deficiency-Thiamine diphosphate-dependent enzymes-Wernicke-Korsakoff's syndrome.

Responses of the mitochondrial alpha-ketoglutarate dehydrogenase complex to thiamine deficiency may contribute to regional selective vulnerability

“…Immunoblotting analysis of whole thalamus and cortex from TD11 and TD13 rats reveals no changes in the immunoreactivity in any of the subunits of KGDHC compared to controls (Sheu et al, 1998). Immunocytochemical staining of a typical coronal section of TD13 and control rats brains with an anti-KGDHC antibody also reveals a similar staining pattern in both unaffected and vulnerable regions (Sheu et al, 1998). In the current experiments, a novel micropunch technique was utilized to precisely sample brain samples from small brain regions such as SmTN, which is one third the size of the Substantia nigra (Karuppagounder et al, 2007) and to quantitatively measure changes of gene expression and immunoreactivity of KGDHC in SmTN and nonvulnerable regions such as cortex.…”

“…For these brain regions, the changes in in vitro glucose flux reflected selective vulnerability better than KGDHC activity (Gibson et al, 1989). Immunoblotting analysis of whole thalamus and cortex from TD11 and TD13 rats reveals no changes in the immunoreactivity in any of the subunits of KGDHC compared to controls (Sheu et al, 1998). Immunocytochemical staining of a typical coronal section of TD13 and control rats brains with an anti-KGDHC antibody also reveals a similar staining pattern in both unaffected and vulnerable regions (Sheu et al, 1998).…”

“…Diminished activities of thiamine dependent-enzymes such as the α-ketoglutarate dehydrogenase complex (KGDHC) have been consistently observed in brains of TD animal models and in post mortem brains of humans with age-related diseases (Sheu et al, 1998;Gibson et al, 1999;Mastrogiacoma et. al, 1996;Bubber et al, 2004).…”

“…Although the temporal and regional response of various cell types to TD has been characterized in SmTN, the corresponding biochemical changes of KGDHC have not been assessed in the same region. Previous studies that tested the relation of KGDHC to neurodegeneration did not compare changes in the SmTN to non-damaged regions of brain because the prior studies were completed before the sensitivity of the SmTN was identified (Gibson et al, 1984;Butterworth et al, 1986;Sheu et al, 1998).…”

“…TD diminishes KGDHC activity in whole brain homogenates (Freeman et al, 1987;Bubber et al, 2004) or dissected brain regions such as cerebral cortex, entire thalamus or inferior colliculus (Butterworth et al, 1986;Sheu et al, 1998). For these brain regions, the changes in in vitro glucose flux reflected selective vulnerability better than KGDHC activity (Gibson et al, 1989).…”