Reduction of brain mitochondrial β-oxidation impairs complex I and V in chronic alcohol intake: the underlying mechanism for neurodegeneration.

Neuropathy and neurocognitive deficits are common among chronic alcohol users, which are believed to be associated with mitochondrial dysfunction in the brain. The specific type of brain mitochondrial respiratory chain complexes (mRCC) that are adversely affected by alcohol abuse has not been studied. Thus, we examined the alterations of mRCC in freshly isolated mitochondria from mice brain that were pair-fed the ethanol (4% v/v) and control liquid diets for 7-8 weeks. We observed that alcohol intake severely reduced the levels of complex I and V. A reduction in complex I was associated with a decrease in carnitine palmitoyltransferase 1 (cPT1) and cPT2 levels. The mitochondrial outer (cPT1) and inner (cPT2) membrane transporter enzymes are specialized in acylation of fatty acid from outer to inner membrane of mitochondria for ATP production. Thus, our results showed that alterations of cPT1 and cPT2 paralleled a decrease β-oxidation of palmitate and ATP production, suggesting that impairment of substrate entry step (complex I function) can cause a negative impact on ATP production (complex V function). Disruption of cPT1/cPT2 was accompanied by an increase in cytochrome C leakage, while reduction of complex I and V paralleled a decrease in depolarization of mitochondrial membrane potential (ΔΨ, monitored by JC-1 fluorescence) and ATP production in alcohol intake. We noted that acetyl-L-carnitine (ALC, a cofactor of cPT1 and cPT2) prevented the adverse effects of alcohol while coenzyme Q10 (CoQ10) was not very effective against alcohol insults. These results suggest that understanding the molecular, biochemical, and signaling mechanisms of the CNS mitochondrial β-oxidation such as ALC can mitigate alcohol related neurological disorders

Freshly isolated mitochondria were assessed for changes in membrane potential by JC-1 red and green fluorescence assay and Cytochrome C release by titerzyme enzyme immuno-metric assay kit.

<p>(A) Changes in mitochondria membrane potential by JC-1 in which the ratio of red to green determines the rate of membrane potential decay and cells dead. (B) Cytochrome c release. Results are expressed as mean values ± SD; (n = 6). Statistical significance (p<0.01) is indicated by asterisk compared with controls or compared with EtOH (double stars).</p

ALC protects the inner membrane protein cPT2 integrity from chronic alcohol ingestion.

<p>(A) Expression of mitochondria inner membrane specific cPT2 protein in frontal cortex tissue sections, in which cPT2 protein (red) is colocalized with neurofilament (green). Scale bar indicated 10 μm and original magnification ×20. (B) Representative immunoreactive bands and (C) quantitative immunoreactive intensity of cPT2 in purified mitochondria. Results are expressed as mean ± SD; n = 6. Statistical significance (p<0.01) is indicated by asterisk compared with controls or compared with EtOH (double stars).</p

Mitochondria isolated from 7–8 weeks pair-fed the control/EtOH liquid-diets with/without ALC or CoQ10 mice brain tissues were assayed for ATP level by luminescence SpectraMax M5 microplate reader.

<p>(A) Steady-state level of ATP in isolated mitochondria from respective groups. (B) ATP production in freshly isolated mitochondria in respective groups following supplementation of ALC or CoQ10 (0.1 mM each) to purified mitochondrial fractions. (C) Utilization of palmitic acid (PA) in isolated mitochondria in respective experimental conditions after supplementation of ALC or CoQ10 during PA oxidation. The levels of ATP were calculated from ATP standard curve run in parallel. Results are expressed as mean nmoles/mg protein ± SD; (n = 6). Statistical significance (p<0.01) is indicated by asterisk compared with controls.</p

Summarizes the significance of the present findings in a schematic pathways.

<p>Summarizes the significance of the present findings in a schematic pathways.</p

Acetyl-L-carnitine preserves the integrity of mitochondrial outer membrane protein cPT1 from chronic alcohol intake in mice.

<p>(A) cPT1 protein colocalization with neurons in brain tissue sections. Neuronal marker protein neurofilament (green) and cPT1 (red). Scale bar indicated 10 μm and original magnification is ×20. (B) Representative immunoreactive bands and (C) quantitative immunoreactive intensity of cPT1 in purified mitochondria. Results are expressed as mean ± SD; n = 6. Statistical significance (p<0.01) is indicated by asterisk compared with controls or double asterisks compared with EtOH condition.</p

ALC prevents the alcohol-induced reduction of LTP and LFS synaptic transmission in the CA1 region of mice hippocampal slices.

<p><i>(A)</i> shows time course and LTP synaptic responses induced by a constant current stimulation (twin pulses, 150–300 μA, 40 μs, 0.05 Hz) of Schafer collateral fibers in three respective conditions. The graph plots the initial slope of the first evoked excitatory postsynaptic potentials (EPSPs) recorded from the CA1 dendrite field (stratum radium) in response to constant current stimuli. <i>(B)</i> Illustrates the LFS synaptic transmission in the CA1 region of mice hippocampal slices. <u>Control (<b>Black</b>), EtOH (Red), EtOH+ALC (Green),</u> and results were expressed as mean values (± SD; n = 3). <b>Right panels:</b> Alcohol impairs mitochondria outer and inner membrane proteins. The marker proteins were probed with antibody to monoamine oxidase A+B (outer membrane marker, Green) and antibody to H6/C12 (inner membrane marker, Red) from Abcam (Cambridge, MA). Conjugated primary antibody was detected by fluorescence Alexa Fluor 488 anti-mouse or Fluor 555 goat-anti chicken secondary antibody. Scale bar indicated 10 μm and original magnification ×40.</p

Freshly isolated mitochondria from frontal cortex of mice pair-fed the control or ethanol liquid-diets for 7–8 weeks were examined for alterations of mitochondrial complexes.

<p>(A) Representative immunoreactive bands and (B) relative intensity of immunoreactive bands of complex I. (C) Representative immunoreactive bands and (D) relative intensity of complex V immunoreactive bands. Results are expressed as mean values ± SD; n = 6. Statistical significance (p<0.01) is indicated by asterisk compared with controls or double asterisks compared with EtOH condition.</p

Acetyl-L-carnitine protects neuronal function from alcohol-induced oxidative damage in the brain

The studies presented here demonstrate the protective effect of acetyl-L-carnitine (ALC) against alcoholinduced oxidative neuroinflammation, neuronal degeneration, and impaired neurotransmission. Our findings reveal the cellular and biochemical mechanisms of alcohol-induced oxidative damage in various types of brain cells. Chronic ethanol administration to mice caused an increase in inducible nitric oxide synthase (iNOS) and 3-nitrotyrosine adduct formation in frontal cortical neurons but not in astrocytes from brains of these animals. Interestingly, alcohol administration caused a rather selective activation of NADPH oxidase (NOX), which, in turn, enhanced levels of reactive oxygen species (ROS) and 4-hydroxynonenal, but these were predominantly localized in astrocytes and microglia. Oxidative damage in glial cells was accompanied by their pronounced activation (astrogliosis) and coincident neuronal loss, suggesting that inflammation in glial cells caused neuronal degeneration. Immunohistochemistry studies indicated that alcohol consumption induced different oxidative mediators in different brain cell types. Thus, nitric oxide was mostly detected in iNOS-expressing neurons, whereas ROS were predominantly generated in NOXexpressing glial cells after alcohol ingestion. Assessment of neuronal activity in ex vivo frontal cortical brain tissue slices from ethanol-fed mice showed a reduction in long-term potentiation synaptic transmission compared with slices from controls. Coadministration of ALC with alcohol showed a significant reduction in oxidative damage and neuronal loss and a restoration of synaptic neurotransmission in this brain region, suggesting that ALC protects brain cells from ethanol-induced oxidative injury. These findings suggest the potential clinical utility of ALC as a neuroprotective agent that prevents alcohol-induced brain damage and development of neurological disorders