Gap junctions are made up of connexin proteins, which comprise a multigene family in mammals. Targeted mutagenesis of connexin43 (Cx43), one of the most prevalent connexin proteins, showed that its absence was compatible with survival of mouse embryos to term, even though mutant cell lines showed reduced dye coupling in vitro. However, mutant embryos died at birth, as a result of a failure in pulmonary gas exchange caused by a swelling and blockage of the right ventricular outflow tract from the heart. This finding suggests that Cx43 plays an essential role in heart development but that there is functional compensation among connexins in other parts of the developing fetus.

Analysis of transgenic mice expressing familial amyotrophic lateral sclerosis (ALS)-linked mutations in the enzyme superoxide dismutase (SOD1) have shown that motor neuron death arises from a mutant-mediated toxic property or properties. In testing the disease mechanism, both elimination and elevation of wild-type SOD1 were found to have no effect on mutant-mediated disease, which demonstrates that the use of SOD mimetics is unlikely to be an effective therapy and raises the question of whether toxicity arises from superoxide-mediated oxidative stress. Aggregates containing SOD1 were common to disease caused by different mutants, implying that coaggregation of an unidentified essential component or components or aberrant catalysis by misfolded mutants underlies a portion of mutant-mediated toxicity.

The discovery that some cases of familial amyotrophic lateral sclerosis (FALS) are associated with mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1) has focused much attention on the function of SOD1 as related to motor neuron survival. Here we describe the creation and characterization of mice completely deficient for this enzyme. These animals develop normally and show no overt motor deficits by 6 months in age. Histological examination of the spinal cord reveals no signs of pathology in animals 4 months in age. However Cu/Zn SOD-deficient mice exhibit marked vulnerability to motor neuron loss after axonal injury. These results indicate that Cu/Zn SOD is not necessary for normal motor neuron development and function but is required under physiologically stressful conditions following injury.

Apoptotic neuronal cell death has recently been associated with the development of infarction after cerebral ischemia. In a variety of studies, CuZn-superoxide dismutase (CuZn-SOD) has been shown to protect the brain from ischemic injury. A possible role for CuZn-SOD-related modulation of neuronal viability is suggested by the finding that CuZn-SOD inhibits apoptotic neuronal cell death in response to some forms of cellular damage. We evaluated this possibility in the model of transient focal cerebral ischemia in mice bearing a disruption of the CuZn-SOD gene (Sod1). Homozygous mutant (Sod1 Ϫ/Ϫ) mice had no detectable CuZn-SOD activity, and heterozygous mutants (Sod1 ϩ/Ϫ) showed a 50% decrease compared with wild-type mice. Sod1 Ϫ/Ϫ mice showed a high level of bloodbrain barrier disruption soon after 1 hr of middle cerebral artery occlusion and 100% mortality at 24 hr after ischemia. Sod1 ϩ/Ϫ mice showed 30% mortality at 24 hr after ischemia, and neurological deficits were exacerbated compared with wildtype controls. The Sod1 ϩ/Ϫ animals also had increased infarct volume and brain swelling, accompanied by increased apoptotic neuronal cell death as indicated by the in situ nick-end labeling technique to detect DNA fragmentation and morphological criteria. These results suggest that oxygen-free radicals, especially superoxide anions, are an important factor for the development of infarction by brain edema formation and apoptotic neuronal cell death after focal cerebral ischemia and reperfusion. Key words: CuZn-superoxide dismutase; focal cerebral ischemia; blood-brain barrier; Evans blue extravasation; neuronal apoptosis; TUNEL; oxidative stressOxygen-free radicals are believed to be involved in the pathogenesis after cerebral ischemia and reperfusion. During cerebral ischemia, a number of events that predispose the brain to the formation of oxygen-free radicals may occur (Siesjö, 1984;McCord, 1985). After reperfusion, these events can set off a cascade of other biochemical and molecular sequelae such as the xanthine-xanthine oxidase reaction and phospholipase activation, leading to free-radical production (Gaudet and Levine, 1979;Chan et al., 1984;Siesjö, 1984;McCord, 1985). Among these oxygen-free radicals, superoxide anion (O 2 Ϫ ), being directly toxic to neurons (Fridovich, 1986;Patel et al., 1996), may initiate a free radical-mediated chain reaction causing additional CNS damage (Saugstad and Aasen, 1980;Chan, 1994).One of the manifestations of CNS damage after cerebral ischemia is the formation of brain edema caused by the breakdown of the blood-brain barrier (BBB). CuZn-superoxide dismutase (CuZn-SOD), a cytosolic protein, prevents vasogenic brain edema after several kinds of injuries Kinouchi et al., 1991;Shukla et al., 1993), suggesting that O 2 Ϫ is an important factor for BBB disruption. Another manifestation of CNS damage is the direct injury of neuronal cells, including excitatory events that are induced by glutamate release after cerebral ischemia. Glutamate elevates cytosolic free calcium (Ca 2ϩ ) (...

Fibrillar amyloid deposits are defining pathological lesions in Alzheimer's disease brain and are thought to mediate neuronal death. Amyloid is composed primarily of a 39-42 amino acid protein fragment of the amyloid precursor protein (APP), called amyloid beta-protein (Abeta). Because deposition of fibrillar amyloid in vitro has been shown to be highly dependent on Abeta concentration, reducing the proteolytic release of Abeta is an attractive, potentially therapeutic target. Here, the turnover rate of brain Abeta has been determined to define treatment intervals over which a change in steady-state concentration of Abeta could be measured. Mice producing elevated levels of human Abeta were used to determine approximate turnover rates for Abeta and two of its precursors, C99 and APP. The t1/2 for brain Abeta was between 1.0 and 2.5 hr, whereas for C99, immature, and fully glycosylated forms of APP695 the approximate t1/2 values were 3, 3, and 7 hr, respectively. Given the rapid Abeta turnover rate, acute studies were designed using phorbol 12-myristate 13-acetate (PMA), which had been demonstrated previously to reduce Abeta secretion from cells in vitro via induction of protein kinase C (PKC) activity. Six hours after intracortical injection of PMA, Abeta levels were significantly reduced, as measured by both Abeta40- and Abeta42-selective ELISAs, returning to normal by 12 hr. An inactive structural analog of PMA, 4alpha-PMA, had no effect on brain Abeta levels. Among the secreted N-terminal APP fragments, APPbeta levels were significantly reduced by PMA treatment, whereas APPalpha levels were unchanged, in contrast to most cell culture studies. These results indicate that Abeta is rapidly turned over under normal conditions and support the therapeutic potential of elevating PKC activity for reduction of brain Abeta.

The effects of dietary cholesterol on brain amyloid precursor protein (APP) processing were examined using an APP gene-targeted mouse, genetically humanized in the amyloid ␤-peptide (A␤) domain and expressing the Swedish familial Alzheimer's disease mutations. These mice express endogenous levels of APP holoprotein and abundant human A␤. Increased dietary cholesterol led to significant reductions in brain levels of secreted APP derivatives, including sAPP␣, sAPP␤, A␤1-40, and A␤1-42, while having little to no effect on cellassociated species, including full-length APP and the COOH-terminal APP processing derivatives. The changes in levels of sAPP and A␤ in brain all were negatively correlated with serum cholesterol levels and levels of serum and brain apoE. These results demonstrate that secreted APP processing derivatives and A␤ can be modulated in the brain of an animal by diet and provide evidence that cholesterol plays a role in the modulation of APP processing in vivo. APP gene-targeted mice lacking apoE, also have high serum cholesterol levels but do not show alterations in APP processing, suggesting that effects of cholesterol on APP processing require the presence of apoE.Alzheimer's disease (AD) 1 pathology includes extracellular amyloid deposits, intracellular neurofibrillary tangles, synaptic loss, and neuronal death (for a review, see Ref. 1). Alterations in the production or processing of APP have been implicated in the etiology of at least some forms of AD (2, 3). Multiple pathways for APP processing have been described, including a nonamyloidogenic pathway in which a putative ␣-secretase cleaves within the A␤ domain (4, 5), resulting in the formation of a secreted NH 2 -terminal fragment, sAPP␣, and a cell-associated 9-kDa COOH-terminal derivative. Another fraction of APP is processed along an amyloidogenic pathway in which cleavage by a putative ␤-secretase at the NH 2 terminus of the A␤ domain results in the formation of a secreted NH 2 -terminal fragment, sAPP␤ (6), and a cell-associated 12-kDa COOH-terminal derivative that may be the immediate precursor of A␤ (7,8). Cleavage of APP by both ␤-secretase and ␥-secretase results in formation of A␤, 40 or 42 amino acids in length (9, 10), that is found deposited in extracellular amyloid plaques in the AD brain (1, 11).In order to elucidate mechanisms of APP processing and A␤ generation in vivo, an animal model was developed by gene targeting that converted the mouse A␤ sequence to human and incorporated the Swedish familial Alzheimer's disease mutations (12). Enhanced amyloidogenic APP processing by the Swedish mutations, resulting in higher level A␤ production has been well documented in cell culture systems (13-15) and in the APP gene-targeted mice (12). These mice are well suited for investigating modulation of APP processing in vivo, because brain A␤ levels are nearly 10-fold above normal endogenous levels, thereby reducing the stringency for assays to detect A␤, particularly for the less abundant but more amyloidogenic 42-residue form. Further...

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