Deborah A. Shackelford

Is DNA repair compromised in Alzheimer’s disease?

Mammalian cells utilize multiple mechanisms to repair DNA damage that occurs during normal cellular respiration and in response to genotoxic stress. This study sought to determine if chronic oxidative stress proposed to occur during Alzheimers disease alters the expression or activity of DNA double-strand break repair or base excision repair proteins. Double-strand break repair requires DNA-dependent protein kinase, composed of a catalytic subunit, DNA-PKcs, and a regulatory component, Ku. Ku DNA binding activity was reduced in extracts of postmortem AD midfrontal cortex, but was not significantly different from the age-matched controls. Decreased Ku DNA binding correlated with reduced protein levels of Ku subunits, DNA-PKcs, and poly(ADP-ribose) polymerase-1. Expression of the base excision repair enzyme Ref-1, however, was significantly increased in AD extracts compared to controls. Ku DNA binding and DNA-PK protein levels in the AD cases correlated significantly with synaptophysin immunoreactivity, which is a measure of synaptic loss, a major correlate of cognitive deficits in AD. Immunohistochemical analysis suggested that DNA-PK protein levels reflected both number of neurons and regulation of cellular expression.

DNA end joining activity is reduced in Alzheimer's disease

Evidence indicates that oxidative stress-induced damage to DNA, protein, and other cellular components contributes to the progression of Alzheimers disease (AD). Several studies indicate that postmitotic neurons have a reduced capacity for some types of DNA repair, which is further compromised by aging. Thus in AD, the cellular response to increased oxidative DNA damage may be inadequate to protect the genome. Mammalian cells use several mechanisms to repair DNA damage generated during normal oxidative metabolism or by genotoxic insults. The predominant mechanism to repair double strand breaks is non-homologous end joining (NHEJ) which utilizes the DNA-dependent protein kinase (DNA-PK) complex. A cell-free DNA end joining assay was employed to determine if NHEJ was reduced in nuclear cortical extracts from brains of AD versus normal subjects. This report demonstrates that end joining activity and protein levels of DNA-PK catalytic subunit are significantly lower in AD brains compared to normal controls. The amount of end joining activity correlates with the expression of DNA-PK and is dependent on DNA-PK catalytic activity. This indicates that repair of DNA double-strand breaks by the DNA-PK-dependent NHEJ pathway may be deficient in AD.

Differences in a dinucleotide repeat polymorphism in the tau gene between Caucasian and Japanese populations: implication for progressive supranuclear palsy

Previous studies of a tau polymorphism in Caucasian subjects with progressive supranuclear palsy (PSP) showed an over-representation of one genotype, A0/A0, versus normal control subjects. This result suggested that tau may be playing a genetic role in the progression of PSP. This study examines whether the over-representation of A0/A0 is Caucasian-specific or universal to PSP. Unfortunately, we found this dinucleotide repeat was relatively non-polymorphic in Japanese subjects. As a result, the genotypes were virtually the same, A0/A0, between Japanese PSP and control subjects. However, this outcome, albeit negative, does suggest two possible roles of the tau gene in PSP pathogenesis: (1) the role of this dinucleotide repeat in PSP may be different between Caucasian and Japanese populations or (2) this repeat may not be causal for PSP but represents a marker for other molecular genetic risk factors within or close to the tau gene on chromosome 17.

Changes in Phosphorylation of τ During Ischemia and Reperfusion in the Rabbit Spinal Cord

Abstract: The microtubule‐associated protein τ plays an important role in the dynamics of microtubule assembly necessary for axonal growth and neurite plasticity. Ischemia disrupts the neuronal cytoskeleton both by promoting proteolysis of its components and by affecting kinase and phosphatase activities that alter its assembly. In this study the effect of ischemia and reperfusion on the expression and phosphorylation of τ was examined in a reversible model of spinal cord ischemia in rabbits. τ was found to be dephosphorylated in response to ischemia with a time course that closely matched the production of permanent paraplegia. Dephosphorylation of τ was limited to the caudal lumbar spinal cord. In a similar manner, Ca2+/calmodulin‐dependent kinase II activity was reduced only in the ischemic region. Thus, dephosphorylation of τ is an early marker of ischemia as is the rapid loss of Ca2+/calmodulin‐dependent kinase II activity, τ, however, was rephosphorylated rapidly during reperfusion at site(s) that cause a reduction in its electrophoretic mobility regardless of the neurological outcome. Alterations in phosphorylation or degradation of τ may affect microtubule stability, possibly contributing to disruption of axonal transport but also facilitating neurite plasticity in a regenerative response.

Activation of nuclear factor-κB in the rabbit spinal cord following ischemia and reperfusion

Abstract The transcription factor NF-κB is a ubiquitously expressed inducible regulator of a broad range of genes. Recent studies have shown that activation of NF-κB predominantly is associated with protecting cells from apoptosis, but in some cell models, it is associated with promoting cell death. We used a rabbit spinal cord model of reversible ischemia to determine whether NF-κB was activated by ischemic and reperfusion injury. DNA binding activity of NF-κB was analyzed by an electrophoretic mobility shift assay in animals subjected to varying durations of ischemia and reperfusion. A low level of constitutive NF-κB DNA binding was detected in normal lumbar spinal cord extracts. Animals subjected to a short ischemic insult of 15 min, from which they usually recover neurologic function, had a significant increase in the amount of active NF-κB in nuclear extracts after 18 h reperfusion. There was no change in nuclear NF-κB DNA binding in animals occluded for 60 min that are permanently paraplegic and exhibit extensive neuropathological damage. The amount of deoxycholate-releasable NF-κB sequestered in the cytosol, however, decreased after 18 h reperfusion in rabbits occluded for 60 min. This correlated with a decrease in the amount of RelA(p65) NF-κB subunit. The results suggest that activation of NF-κB after a limited ischemic injury may participate in a neuroprotective response and not in cell death.

Modulation of ERK and JNK activity by transient forebrain ischemia in rats.

The mitogen‐activated protein (MAP) kinase families of ERK and JNK participate in numerous intracellular signaling pathways and are abundantly expressed in the CNS. Activation of ERK and JNK during reperfusion of ischemic tissue is implicated in promoting cell death, insofar as inhibition of either pathway reduces neuronal cell death. However, ERK or JNK activation provides protection in other neuronal injury models. In this study, we monitored the concurrent modulation of ERK and JNK activity in the hippocampus, neocortex, and striatum during ischemia and immediately upon reperfusion in a rat model of transient global ischemia. All three regions incur a similar reduction in blood flow during occlusion but show different extents and temporal patterns of injury following reperfusion. ERK and JNK were active in the normal rat forebrain, and phosphorylation was reduced by ischemia. Upon reperfusion, ERK was rapidly activated in the hippocampus, neocortex, and striatum, whereas JNK phosphorylation increased in the hippocampus and striatum but not in the neocortex. The response of JNK vs. ERK more closely reflects the susceptibility of these regions. JNK1 was the predominant phosphorylated isoform. A minor pool of phosphorylated JNK3 increased above the control level after reperfusion in hippocampal but not in neocortical particulate fractions. In addition, a novel 32–35‐kDa c‐Jun kinase activity was detected in the hippocampus, neocortex, and striatum. The results show that ERK and JNK activities are rapidly, but not identically, modulated by ischemia and reperfusion and indicate that the MAP kinase pathways contribute to regulating the response to acute CNS injury.

Inactivation and subcellular redistribution of Ca2+/calmodulin-dependent protein kinase II following spinal cord ischemia.

Abstract: Reversible spinal cord ischemia in rabbits induced a rapid loss of Ca2+/calmodulin‐dependent protein kinase II (CaM kinase II) activity measured as incorporation of phosphate into exogenous substrates. About 70% of the activity was lost from the cytosolic fraction of spinal cord homogenates after 15 min of ischemia preceding irreversible paraplegia, which takes 25 min in this model. The loss of enzyme activity correlated with a loss of in situ renaturable autophosphorylation activity and a loss of CaM kinase II α and β subunits in the cytosol detected by immunoblotting. CaM kinase II activity in the particulate fraction also decreased but the protein levels of the a and β subunits increased. Thus ischemia resulted in an inactivation of CaM kinase II and a sequential or concurrent subcellular redistribution of the enzyme. However, denaturation and renaturation in situ of the CaM kinase subunits immobilized on membranes partly reversed the apparent inactivation of the enzyme in the particulate fraction. CaM kinase II activity was restored after reperfusion following short (≤25 min) durations of ischemia but not after longer durations (60 min) that result in irreversible paraplegia. The ischemia‐induced inactivation of CaM kinase II, which phosphorylates proteins regulating many cellular processes, may be important in the cascade of events leading to delayed neuronal cell death.

Differential effects of ischemia and reperfusion on c-Jun N-terminal kinase isoform protein and activity

Activation of the c-Jun N-terminal (JNK) or stress-activated protein kinases (SAPK) is associated with a wide range of disparate cellular responses to extracellular stimuli, including either induction of or protection from apoptosis. This study investigates the effect of ischemia and reperfusion on JNK isoform activities using a reversible rabbit spinal cord ischemia model. High basal JNK activity, attributed to the p46 JNK1 isoform, was expressed in the CNS of untreated rabbits. JNK activity decreased in the lumbar spinal cord of rabbits occluded for 15-60 min. During reperfusion animals occluded for 15 min recovered neurological function and JNK activity returned to normal levels. In contrast animals occluded for 60 min remained permanently paraplegic and JNK activity was half the control activity after 18 h of reperfusion. In these animals proteolytic fragments of JNK1 and JNK3 were observed and protein levels, but not activity, of JNK isoforms increased in a detergent-insoluble fraction. Two novel c-Jun (and ATF-2) kinase activities increased during reperfusion of animals occluded for 60 min. An activity designated p46(slow) was similar in M(r) to a JNK2 isoform induced in these animals. A second 30-kDa activity associated with the detergent-insoluble fraction co-migrated with a JNK3 N-terminal fragment. The results show that JNK1 is active in the normal CNS and increased activity is not associated with durations of ischemia and reperfusion that induce cell death. However, specific JNK isoform activation may participate in the cell death pathways as increased activity of novel c-Jun (ATF-2) kinase activities was observed in paraplegic animals.

Activation of extracellular signal-regulated kinases (ERK) during reperfusion of ischemic spinal cord

The extracellular signal-regulated kinases (ERK) participate in numerous signaling pathways and are abundantly expressed in the CNS. It has been proposed that ERK activation promotes survival in models of neuronal injury. Inhibition of MEK, the upstream kinase that activates ERK, however, leads to neuroprotection in models of cerebral ischemia and trauma, suggesting that in this context ERK activation contributes to cellular damage. The effect of ischemia and reperfusion on activity and expression of ERK was investigated using a reversible model of rabbit spinal cord ischemia. Active ERK was observed in nai;ve animals, which decreased during 15 to 60 min of ischemia. Upon reperfusion, a robust activation of ERK was observed in animals occluded for 60 min that remained permanently paraplegic. Immunohistochemical analyses revealed increased staining of phosphorylated ERK (pERK) in glial cells and faint nuclear staining in motor neurons of animals occluded for 60 min and reperfused for 18 h. In contrast ERK activity did not increase in animals occluded for 15 min that regained motor function. No evidence of increased pERK immunoreactivity in motor neurons or nuclear translocation was noted in these animals. ERK1 was demonstrated to be identical to a p46 c-Jun/ATF-2 kinase previously shown to be activated by reperfusion after a 60-min occlusion. The results suggest that activation of ERK during reperfusion of ischemic spinal cord participates in the cellular pathways leading to neuronal damage.

Reversible ischemia increases levels of Alzheimer amyloid protein precursor without increasing levels of mRNA in the rabbit spinal cord

In a rabbit spinal cord ischemia model (RSCIM), the time courses of neuropathological damage of the spinal cord and neurological impairment of the motor functions are well established, demonstrating that the extent of neuropathological damage and the severity of neurological impairment are closely correlated. We used the RSCIM to elucidate the effects of reversible (15 min) and irreversible (60 min) ischemia on the endogenous levels of amyloid protein precursors (APPs) at both the mRNA and protein levels in the caudolumbar/sacral region of the spinal cord. We speculate that endogenous APPs are induced by ischemia as either trophic factors or stress-induced proteins in the RSCIM. A 15-min occlusion transiently increased the APP protein levels in neurons, which returned to the original levels by the end of 60 min occlusion. The increase in APP protein levels during 15-min ischemic insult does not appear to involve regulation at the mRNA level. The increased level of APPs, particularly of the soluble form, could support the possibility that APPs play a neuroprotective role in the RSCIM as stress-induced proteins. In contrast, failure to maintain the increased APP protein levels or to increase the mRNA, as seen in the 60-min ischemia samples, may be one of the causal factors that induce necrosis and neuronal cell death leading to irreversible neurological impairment.