Aileen J. Anderson

A potential role for apoptosis in neurodegeneration and Alzheimer's disease

Previous studies have shown that β-amyloid (Aβ) peptides are neurotoxic. Recent data suggest that neurons undergoing Aβ-induced cell death exhibit characteristics that correspond to the classical features of apoptosis, suggesting that these cells may initiate a program of cell death. This chapter explores the criteria and precautions that must be applied to evaluate mechanisms of cell death in vitro and in vivo, discusses the evidence supporting an apoptotic mechanism of cell death in response to Aβ in cultured neurons, and describes potential correlations for these findings in the Alzheimers disease brain. In addition, cellular signaling pathways that may be associated with apoptosis in response to Aβ are examined, and support for apoptosis as a mechanism of cell death for other neurodegeneration-inducing stimuli (e.g., oxidative injury) is described. The connection of multiple stimuli that induce neuronal cell death to an apoptotic mechanism suggests that apoptosis could play a central role in neurodegeneration in the brain.

Quantitative analysis of cellular inflammation after traumatic spinal cord injury: evidence for a multiphasic inflammatory response in the acute to chronic environment

Traumatic injury to the central nervous system results in the disruption of the blood brain/spinal barrier, followed by the invasion of cells and other components of the immune system that can aggravate injury and affect subsequent repair and regeneration. Although studies of chronic neuroinflammation in the injured spinal cord of animals are clinically relevant to most patients living with traumatic injury to the brain or spinal cord, very little is known about chronic neuroinflammation, though several studies have tested the role of neuroinflammation in the acute period after injury. The present study characterizes a novel cell preparation method that assesses, quickly and effectively, the changes in the principal immune cell types by flow cytometry in the injured spinal cord, daily for the first 10 days and periodically up to 180 days after spinal cord injury. These data quantitatively demonstrate a novel time-dependent multiphasic response of cellular inflammation in the spinal cord after spinal cord injury and are verified by quantitative stereology of immunolabelled spinal cord sections at selected time points. The early phase of cellular inflammation is comprised principally of neutrophils (peaking 1 day post-injury), macrophages/microglia (peaking 7 days post-injury) and T cells (peaking 9 days post-injury). The late phase of cellular inflammation was detected after 14 days post-injury, peaked after 60 days post-injury and remained detectable throughout 180 days post-injury for all three cell types. Furthermore, the late phase of cellular inflammation (14-180 days post-injury) did not coincide with either further improvements, or new decrements, in open-field locomotor function after spinal cord injury. However, blockade of chemoattractant C5a-mediated inflammation after 14 days post-injury reduced locomotor recovery and myelination in the injured spinal cord, suggesting that the late inflammatory response serves a reparative function. Together, these data provide new insight into cellular inflammation of spinal cord injury and identify a surprising and extended multiphasic response of cellular inflammation. Understanding the role of this multiphasic response in the pathophysiology of spinal cord injury could be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions.

Activated caspase-3 expression in Alzheimer’s and aged control brain: correlation with Alzheimer pathology

Several studies have suggested that activated caspase-3 has properties of a cell death executioner protease. In this study, we examined the expression of activated caspase-3 in AD and aged control brains. Activated caspase-3 immunoreactivity was seen in neurons, astrocytes, and blood vessels, was elevated in AD, and exhibited a high degree of colocalization with neurofibrillary tangles and senile plaques. These data suggest that activated caspase-3 may be a factor in functional decline and may have an important role in neuronal cell death and plaque formation in AD brain.

Correlation between Caspase Activation and Neurofibrillary Tangle Formation in Alzheimer’s Disease

Although evidence suggests that neurofibrillary tangles (NFTs) and neuronal cell loss are prominent features of Alzheimers disease (AD), the relationship between the two remains unknown. In the present study, the relationship between the activation of apoptotic mechanisms and NFT formation in AD was investigated using a caspase-cleavage site-directed antibody to fodrin, an abundant neuronal cytoskeleton protein. This antibody recognized cleavage products of fodrin after digestion by caspase-3, but did not recognize full-length fodrin. In vitro analysis of this fodrin caspase-cleavage product (CCP) antibody demonstrates that it is a specific probe for the detection of apoptotic but not necrotic pathways in cultured neurons. To determine whether caspases cleave fodrin in vivo, tissue sections from controls and AD were immunostained for fodrin (CCPs). Although no staining was observed in control cases, labeling of neurons was observed in the hippocampus of all AD cases, which increased as a function of disease progression. To determine a possible relationship between caspase activation and NFT formation, double-labeling experiments with fodrin CCP and PHF-1 were performed. Co-localization of these markers was observed in many neurons, and quantitative analysis showed that as the extent of NFT formation increased, there was a significant corresponding increase in fodrin CCP immunolabeling (r = 0.84). Taken together, these results provide evidence for the activation of apoptotic mechanisms in neurons in the AD brain and suggest that there is an association between NFT formation and the activation of apoptotic pathways in AD.

Increased Immunoreactivity for Jun- and Fos-Related Proteins in Alzheimer's Disease: Association with Pathology

The protein products of the Jun and Fos immediate early gene (IEG) families are cooperative transcriptional regulatory factors implicated in regulating the expression of many genes. The levels of a variety of proteins such as the amyloid precursor protein and basic fibroblast growth factor are altered in Alzheimers disease (AD), thus the events regulating these changes are of interest. Both of these genes contain an activator protein-1 consensus sequence which may be responsive to regulation by immediate early genes. In order to evaluate the potential involvement of IEGs in AD pathology, we have examined Jun- and Fos-related protein immunoreactivity in control and AD brain. Specifically, we investigated the correspondence of immunoreactivity for Jun and Fos proteins with immunoreactivity for paired helical filament-1 (PHF-1), a marker for neurofibrillary tangles which recognizes abnormally phosphorylated tau, glial fibrillary acidic protein (GFAP), and thioflavine staining in double-labeling experiments. An intensification of both Jun and Fos immunoreactivity was observed in AD cases; in addition, both Jun and Fos immunoreactivity were colocalized with PHF-1 in some neurons in AD brain. Jun and Fos immunoreactivity were also colocalized with GFAP-positive astrocytes distributed in the cortex of AD and control cases, and surrounding thioflavine-stained plaques in AD brain. These observations suggest that members of the Jun and Fos IEG families may play a role in AD pathology.

Human neural stem cells differentiate and promote locomotor recovery in an early chronic spinal cord injury NOD-scid mouse model.

Background Traumatic spinal cord injury (SCI) results in partial or complete paralysis and is characterized by a loss of neurons and oligodendrocytes, axonal injury, and demyelination/dysmyelination of spared axons. Approximately 1,250,000 individuals have chronic SCI in the U.S.; therefore treatment in the chronic stages is highly clinically relevant. Human neural stem cells (hCNS-SCns) were prospectively isolated based on fluorescence-activated cell sorting for a CD133+ and CD24−/lo population from fetal brain, grown as neurospheres, and lineage restricted to generate neurons, oligodendrocytes and astrocytes. hCNS-SCns have recently been transplanted sub-acutely following spinal cord injury and found to promote improved locomotor recovery. We tested the ability of hCNS-SCns transplanted 30 days post SCI to survive, differentiate, migrate, and promote improved locomotor recovery. Methods and Findings hCNS-SCns were transplanted into immunodeficient NOD-scid mice 30 days post spinal cord contusion injury. hCNS-SCns transplanted mice demonstrated significantly improved locomotor recovery compared to vehicle controls using open field locomotor testing and CatWalk gait analysis. Transplanted hCNS-SCns exhibited long-term engraftment, migration, limited proliferation, and differentiation predominantly to oligodendrocytes and neurons. Astrocytic differentiation was rare and mice did not exhibit mechanical allodynia. Furthermore, differentiated hCNS-SCns integrated with the host as demonstrated by co-localization of human cytoplasm with discrete staining for the paranodal marker contactin-associated protein. Conclusions The results suggest that hCNS-SCns are capable of surviving, differentiating, and promoting improved locomotor recovery when transplanted into an early chronic injury microenvironment. These data suggest that hCNS-SCns transplantation has efficacy in an early chronic SCI setting and thus expands the “window of opportunity” for intervention.

Differential Induction of Immediate Early Gene Proteins in Cultured Neurons by β-Amyloid (Aβ): Association of c-Jun with Aβ-Induced Apoptosis

Abstract: β‐Amyloid (Aβ) is a 39–42 amino acid that is the primary component of plaques in Alzheimers disease (AD). Previous studies from our laboratory and others have shown that Aβ induces neurodegeneration via apoptosis in vitro, suggesting that Aβ may also initiate an apoptotic pathway of cell death in AD. Apoptosis has been suggested to proceed by a gene‐directed program in several systems. Accordingly, we have investigated whether Aβ‐mediated apoptosis is associated with the induction of genes that may regulate or play a role in cell death in vitro. Immediate early genes (IEGs) respond to cellular stimuli and participate in cellular signaling pathways. The protein products of some IEGs, e.g., c‐jun, are capable of forming dimers and acting as transcriptional regulatory proteins, and have been implicated in apoptosis in both nonneuronal and neuronal cells. In this study, we report a selective and abnormally sustained induction of c‐Jun in cultured hippocampal neurons treated with Aβ. In addition, we describe the lack of induction of c‐Jun in neurons that are relatively resistant to Aβ‐mediated toxicity, and a correspondence between immunoreactivity for c‐Jun and changes in nuclear morphology that are indicative of apoptosis. These data demonstrate that c‐Jun is induced in cultured neurons that undergo Aβ‐mediated apoptosis and suggest that c‐Jun may participate in a cell death program in these neurons.

Polymorphonuclear leukocytes promote neurotoxicity through release of matrix metalloproteinases, reactive oxygen species, and TNF-α

As the first immune cells to infiltrate the nervous system after traumatic PNS and CNS injury, neutrophils (polymorphonuclear leukocytes, PMNs) may promote injury by releasing toxic soluble factors that may affect neuronal survival. Direct neurotoxicity of matrix metalloproteinases (MMPs), reactive oxygen species (ROS), and cytokines released by PMNs was investigated by culturing dorsal root ganglion (DRG) cells with PMN‐conditioned media containing MMP inhibitor (GM6001), ROS scavengers, or tumor necrosis factor αR (TNF‐αR) neutralizing antibody. Although DRGs exposed to PMN‐conditioned media had 53% fewer surviving neurons than controls, neuronal cell loss was prevented by GM6001 (20 μmol/L), catalase (1000 U/mL), or TNF‐αR neutralizing antibody (1.5 μg/mL), elevating survival to 77%, 94%, and 95%, respectively. In accordance with protection by GM6001, conditioned media collected from MMP‐9 null PMNs was less neurotoxic than that collected from wild‐type PMNs. Additionally, MMP inhibition reduced PMN‐derived ROS; removal of ROS reduced PMN‐derived MMP‐9 activity; and TNF‐α inhibition reduced both PMN‐derived MMP‐9 activity and ROS in PMN cultures. Our data provide the first direct evidence that PMN‐driven neurotoxicity is dependent on MMPs, ROS, and TNF‐α, and that these factors may regulate PMN release of these soluble factors or interact with one another to mediate PMN‐driven neurotoxicity.

The complement cascade: Yin–Yang in neuroinflammation – neuro–protection and –degeneration

The complement cascade has long been recognized to play a key role in inflammatory and degenerative diseases. It is a ‘double edged’ sword as it is necessary to maintain health, yet can have adverse effects when unregulated, often exacerbating disease. The contrasting effects of complement, depending on whether in a setting of health or disease, is the price paid to achieve flexibility in scope and degree of a protective response for the host from infection and injury. Loss or even decreased efficiency of critical regulatory control mechanisms can result in aggravated inflammation and destruction of self‐tissue. The role of the complement cascade is poorly understood in the nervous system and neurological disorders. Novel studies have demonstrated that the expression of complement proteins in brain varies in different cell types and the effects of complement activation in various disease settings appear to differ. Understanding the functioning of this cascade is essential, as it has therapeutic implications. In this review, we will attempt to provide insight into how this complex cascade functions and to identify potential strategic targets for therapeutic intervention in chronic diseases as well as acute injury in the CNS.

Up-regulation of Bcl-2 is associated with neuronal DNA damage in Alzheimer's disease

Cell death and neurofibrillary tangle formation are prominent features of Alzheimers disease (AD). It has been suggested that DNA damage may reflect neuronal vulnerability. In this context, the Ced homologue Bcl-2 is able to repress a number of cell death programs. Recently we found both numerous nuclei exhibiting DNA damage within neurons in the AD brain and increases in Bcl-2 immunoreactivity. In this study, we examined the relationship between Bcl-2 expression and nuclear DNA damage or tangle formation. Nuclei exhibiting DNA damage were associated with an up-regulation of Bcl-2 expression, whereas tangle-bearing neurons were associated with a down-regulation of Bcl-2 expression.