Differential sensitivity of prefrontal cortex and hippocampus to alcohol-induced toxicity.

The prefrontal cortex (PFC) is a brain region responsible for executive functions including working memory, impulse control and decision making. The loss of these functions may ultimately lead to addiction. Using histological analysis combined with stereological technique, we demonstrated that the PFC is more vulnerable to chronic alcohol-induced oxidative stress and neuronal cell death than the hippocampus. This increased vulnerability is evidenced by elevated oxidative stress-induced DNA damage and enhanced expression of apoptotic markers in PFC neurons. We also found that one-carbon metabolism (OCM) impairment plays a significant role in alcohol toxicity to the PFC seen from the difference in the effects of acute and chronic alcohol exposure on DNA repair and from exaggeration of the damaging effects upon additional OCM impairment in mice deficient in a key OCM enzyme, methylenetetrahydrofolate reductase (MTHFR). Given that damage to the PFC leads to loss of executive function and addiction, our study may shed light on the mechanism of alcohol addiction

Templates for qPCR-based BER activity assay.

<p>Whole cell extracts were isolated from brain tissue and exposed to a template DNA containing a single nucleotide lesion (A) and control template (B). BER activity was calculated by comparing the ΔΔCt values (Ct is the number of cycles required for the fluorescent signal to cross the threshold) of the repaired and control templates.</p

OCM impairment is involved in ethanol impact on DNA repair in the PFC.

<p>(A) Blood Hcy levels in WT and <i>Mthfr+/−</i> mice following acute or chronic 3- week (3 w) or 5-week (5 w) ethanol exposure. Values are means ± SEM; *p<0.01; **p<0.001. Note chronic alcohol-induced increase in blood Hcy levels, compared with the acute exposure and the heightening this increase by MTHFR deficiency. (B) DNA repair activity in the PFC of WT and <i>Mthfr+/−</i> mice exposed to acute and chronic ethanol. Values are means ± SEM; *p<0.01; **p<0.005; ***p<0.002. Note a decrease in DNA repair activity in the PFC following 3-week (3 w) and even more so following 5 week (5 w) exposure, compared with acute alcohol exposure and a significant exaggeration of this decrease by MTHFR deficiency.</p

PFC is more vulnerable to ethanol-induced oxidative stress than hippocampus.

<p>(A) oxo-8dG expression in neurons (neuronal marker MAP-2) was quantified in PFC and hippocampus (Hip) by stereological counting. Neither the number of neurons nor volumes of the brain structures were affected. Values are mean ± SEM; *p<0.01. (B) Oxo-8dG expression in neurons normalized to corresponding controls (Δ). Note significantly higher density of oxo-8dG -labeled neurons and Δ in PFC, compared with hippocampus of ethanol-exposed mice. (C) DNA repair activity in response to oxidative DNA damage (oxo-8dG) assessed by qPCR in whole cell extracts obtained from PFC and hippocampus (Hip) of control mice and mice exposed to acute or chronic ethanol. Values are means ± SEM; *p<0.05, **p<0.01. Note the response to oxidative DNA damage by PFC lysate is significantly stronger than those in the hippocampus.</p

PFC is more vulnerable to ethanol-induced neuronal apoptosis than hippocampus.

<p>Brain sections double-labeled with TUNEL and MAP-2 in the PFC and hippocampus (Hip) and quantified by stereological counting; Values are means ± SEM; *p<0.01. Neither the number of MAP-2-positive cells (neurons) nor volumes of the brain structures were affected by chronic 3-week ethanol exposure. Note significantly higher density of TUNEL- positive neurons in PFC than in hippocampus of ethanol-exposed mice.</p

OCM impairment is involved in ethanol-induced oxidative DNA damage and neuronal apoptosis effects in the PFC.

<p>The brain sections (PFC) of WT and <i>Mthfr+/−</i> mice exposed for 3 weeks or 4 days (acute) to the Lieber-DeCarli liquid diet with- or without ethanol (5%) were triple-labeled with NeuN (purple), TUNEL (green) and cleaved caspase-3 (red). Hoechst 33342 (blue) was used to identify all cell nuclei. Fluorescence was visualized by confocal microscopy. Scale bar = 20 µm. Note increased number of TUNEL/caspase-3-positive neurons in PFC of <i>Mthfr+/−</i> mice chronically exposed to ethanol, compared with corresponding WT mice (arrows). Also, note a higher density of TUNEL/caspase-3-positive neurons in PFC of chronically, compared with acutely exposed to ethanol WT mice (arrows).</p

Integrin α7 Mutations Are Associated With Adult-Onset Cardiac Dysfunction in Humans and Mice

BACKGROUND: Integrin α7β1 is a major laminin receptor in skeletal and cardiac muscle. In skeletal muscle, integrin α7β1 plays an important role during muscle development and has been described as an important modifier of skeletal muscle diseases. The integrin α7β1 is also highly expressed in the heart, but its precise role in cardiac function is unknown. Mutations in the integrin α7 gene (ITGA7) have been reported in children with congenital myopathy. METHODS AND RESULTS: In this study, we described skeletal and cardiac muscle pathology in Itga7−/− mice and 5 patients from 2 unrelated families with ITGA7 mutations. Proband in family 1 presented a homozygous c.806_818del [p.S269fs] variant, and proband in family 2 was identified with 2 intron variants in the ITGA7 gene. The complete absence of the integrin α7 protein in muscle supports the ITGA7 mutations are pathogenic. We performed electrocardiography, echocardiography, or cardiac magnetic resonance imaging, and histological biopsy analyses in patients with ITGA7 deficiency and Itga7−/− mice. The patients exhibited cardiac dysrhythmia and dysfunction from the third decade of life and late-onset respiratory insufficiency, but with relatively mild limb muscle involvement. Mice demonstrated corresponding abnormalities in cardiac conduction and contraction as well as diaphragm muscle fibrosis. CONCLUSIONS: Our data suggest that loss of integrin α7 causes a novel form of adult-onset cardiac dysfunction indicating a critical role for the integrin α7β1 in normal cardiac function and highlights the need for long-term cardiac monitoring in patients with ITGA7-related congenital myopathy