Restorative Neurology and Neuroscience

Injury to the nervous system initiates a cascade of signal transduction events that mobilize survival-promoting gene products and post-translational modifications of existing proteins involved in neuronal injury responses. These ‘cell life programs’ appear to converge on gene products involved in maintenance of calcium homeostasis and suppression of free radical accumulation. Central to the hypothesis of ‘programmed cell life’ is that neurons die (either by apoptosis or necrosis) when the severity or duration of the insult overcomes the ability of the cell life programs to protect the cell. Whether cell death manifests as apoptosis or necrosis depends upon the severity and duration of the insult, the cell type encountering the insult, and the state of the cell rather than the type of insult. For example, activation of glutamate receptors and oxidative insults can kill neurons by a rapid necrotic mode or a delayed apoptotic mode. In either case, calcium and free radicals mediate the cell injury. Several categories of anti-apoptotic signaling molecules (AASMs) are released from neurons and/or glia in response to brain injury including: classic neurotrophic factors such as nerve growth factor, brain-derived neurotrophic factor and basic fibroblast growth factor; cytokines such as tumor necrosis factor and interleukin-1; protease inhibitors such as protease nexin-1; and novel AASMs such as secreted forms of the β-amyloid precursor protein. The specific ways in which AASMs promote cell survival range from induction of antioxidant enzymes to regulation of glutamate receptor expression to stimulation of calcium-binding protein expression to activation of K⁺ channels. The intracellular messengers mediating programmed cell life pathways include intermediate kinases. cyclic nucleotides and transcription factors such as NFκB. As details of AASM signal transduction pathways emerge so do novel therapeutic approaches to reducing neuronal degeneration. Because neuronal degeneration in many, if not all, neurodegenerative conditions results from excessive accumulation of free radicals and disruption of calcium homeostasis, activiation of AASM signaling pathways has broad applicability to both acute and chronic neurodegenerative disorders.

Although there is not yet any in vivo evidence of the neurotoxic action of β-amyloid in humans, it is well established that the insoluble form of full length β-amyloid 1–40, and the fragment comprised of amino acids 25–35, are both toxic in vitro to neurons in tissue culture. β-amyloid 25–35 increases cytosolic calcium in rat PC12 cells and in rat cortical neurons in primary culture by facilitating the entry of extracellular calcium into the cell. This effect is not altered by calcium channel blocking drugs but is prevented by U-83836E, one of the lazaroid anti-oxidant drugs, and by vitamin E. Similarly, the neurotoxic actions of β-amyloid 25–35 are also prevented by U-83836E and by vitamin E. These observations indicate that the actions of β-amyloid 25–35 are mediated by free radicals. In vivo, β-amyloid 1–40 is cleaved from a precursor protein that appears to be synthesized and inserted into cellular membranes following damage to cells. To form neurotoxic β -amyloid, the precursor protein must be cleaved within the transmembrane portion of its structure. In spite of extensive world-wide effort, an enzyme capable of doing this has not been found. However, a peroxidation cascade propagated through the lipid bilayer of the cellular membrane would cleave the precursor protein at a site needed to form β-amyloid. If this is the case, then free radicals would play a role both in the formation of β-amyloid and in its neurotoxic actions.

This study demonstrates elastase immunoreactivity in the neurofibrillary pathology of Alzheimer disease. Using an antiserum against elastase, we show that elastase immunoreactivity is restricted to neurons and is markedly elevated in a proportion of neurofibrillary tangle-bearing neurons. Elastase is a proteolytic enzyme that might be a candidate protease for the generation of amyloid-β from β-protein precursor. These findings support the hypothesis that proteases play an important role in Alzheimer disease and furthers the notion that an imbalance in proteolytic regulation contributes towards the pathogenic presentation of the disease. Moreover, since α₁-antitrypsin, the principal inhibitor of elastase, is highly susceptible to oxidative stress, our findings suggest a link between proteolytic imbalance and oxidative stress in the pathogenesis of Alzheimer disease.

Many toxic factors are generated during stroke that contribute directly to the death of neurons. Several recent studies suggest that a suicide-like phenomena similar to apoptosis or programmed cell death also contributes to the loss of neurons in stroke. The evidence implicating apoptosis in stroke can be divided into three categories; biochemical, molecular and pharmacological. Biochemical evidence: One hallmark of apoptosis is the early activation of destructive enzymes, including endonucleases and proteases. Endonuclease-mediated DNA fragmentation can be observed within 4 h after focal cerebral ischemia and precedes morphological evidence of cell death. Cells with damaged DNA appear to concentrate in the salvageable tissue of the penumbra while necrosis predominates in areas where the sustained lack of blood flow may make tissue salvage impossible. Molecular evidence: Bcl-2 is an anti-apoptotic gene that confers the ability to block apoptosis from a wide variety of stimuli. The levels of bcl-2 can be enhanced by viral gene delivery or transgenic methodology. In cortical tissue where bcl-2 was elevated, neurons were protected from a subsequent ischemic attack. In contrast to bcl-2, p53 is a pro-apoptotic protein. Levels of p53 are elevated after cerebral ischemia and transgenic p53 knockouts exhibit smaller infarcts than wild type control mice. Pharmacological evidence: The process of apoptosis typically involves the activation of enzymes and genes, leading to an irreversible committment to die. Inhibition of new protein synthesis by cycloheximide reduces brain damage after a stroke, suggesting that newly synthesized proteins are contributing to the death of neurons. In addition, inhibition of calpain (an enzyme implicated in certain forms of apoptosis) protects neurons in models of global ischemia, focal ischemia, and hypoxia. In conclusion, the observation that an apoptotic-like process contributes to stroke may have important therapeutic implications since therapies that inhibit apoptosis improve outcome in experimental stroke.

Numerous studies have demonstrated evidence of DNA nick end-labeling and DNA laddering following cerebral ischemia. To determine whether genes directly implicated in apoptosis were induced by ischemia, the expression of bcl-2, bcl-x and ICE mRNAs were examined using oligonucleotide probes. Northern blots demonstrated induction of bcl-2 mRNA and bcl-x mRNAs in hippocampus 24 and 72 h following 5 min of global ischemia. In situ hybridization demonstrated induction of bcl-2 and bcl-x mRNAs in CAl pyramidal neurons of hippocampus at 24 h following ischemia which decreased by 72 h. ICE-like mRNA was induced in non-neuronal cells in the CAl region at 72 h following global ischemia. The data show that genes implicated in either protecting against or promoting programmed cell death in other systems are induced following cerebral ischemia. It is hypochesized that CAl neuronal cell death could be accounted for by the failure of the ischemic cells to make protective proteins that protect the cells from an ischemic induced apoptotic-like cell death.

Secondary brain injury is a well-demonstrated contributor to the morbidity and mortality of severe head injury. At least ten new compounds which antagonize the effects of glutamate in the brain are currently undergoing clinical evaluation as putative protectants against this secondary injury. None have yet shown clear benefit in humans. It is accepted that excitatory amino acids, glutamate in particular, have ‘neurotoxic’ effects on the brain, especially when present in excessive amounts. Whether or not this excitatory amino acid toxicity represents the major pathway for secondary damage is disputed. In the laboratory, over 300 studies have now demonstrated the ability of glutamate antagonist drugs of various types to prevent ischemic and post-traumatic acute brain damage. The magnitude and consistency of protection afforded by this group of compounds exceeds that which has ever been shown with any other mechanisms. Laboratory studies using in vitro neuronal models have implicated glutamate as a promoter of ionic flux and calcium entry across the cell membrane, which may then initiate astrocytic swelling and neuronal necrosis. In vivo animal models of brain trauma and ischemia have demonstrated glutamate release and potassium efflux into the extracellular fluid (ECF). Outcome in these models is improved, as assessed by both histopathology and behavioral studies, when glutamate antagonists are used. Additionally, presynaptic glutamate blockade in animal models such as middle cerebral artery (MCA) occlusion and subdural hematoma, creates reduction in lesion size which is paralleled by reduced glutamate production. In bridging the gap between the laboratory and the patient care setting, human microdialysis studies have shown massive release of excitatory amino acids into the ECF after severe head injury. Early studies with TV-methyl-D-aspartate (NMDA) antagonists in head injured humans have demonstrated a reduction of intracranial pressure and an improvement in cerebral perfusion. Future studies are needed to examine further the value of protection from excitatory amino acid induced injury.

The proto-oncogene bcl-2 is an important suppressor of apoptotic cell death in development and of both apoptotic and necrotic cell death in mature neurons. We studied expression of bcl-2 and the related gene, bax, which may promote cell death, after seizures induced by systemic kainate injection in rats. Expression of bcl-2 mRNA was studied by in situ hybridization. Bax and bcl-2 protein expression was studied by immunocytochemistry. Histologic analysis of cresyl violet-stained paraffin sections was performed at 72 h. bcl-2 protein was expressed in CA1 neurons, a region that is injured, yet survives after seizures. Bcl-2 mRNA was expressed in CA3, a region where there is extensive neuronal death at 72 h, but the bcl-2 protein was not translated. However, bax protein expression in CA3 was increased at 24 h. These results support a possible role for bcl-2 in promoting survival of CA3 after seizures.

We recently reported that the pineal hormone melatonin protected neuronal cultures from excitotoxicity mediated via kainate-sensitive glutamate receptors and from oxidative stress-induced apoptosis. It has been shown that in rats, a systemic administration of kainate induces apoptotic cell death in various brain regions. In this study, we assayed the extent of brain injury after intraperitoneal (i.p.) administration of 10 mg/kg kainate to rats, using the quantitative TUNEL technique and Nissl staining. We examined the role of melatonin on kainate-induced brain injury by (a) injecting melatonin (4 × 2.5 mg/kg i.p.) prior to and after kainate injection and (b) injecting kainate at the time of low circulating melatonin levels (day/light), and high melatonin levels (night/dark). The extent of kainate-triggered DNA damage and the loss of Nissl staining were lower in animals treated with melatonin, or when kainate was injected at night, i.e. in the presence of high endogenous levels of melatonin. Our results suggest that both the pharmacological use of melatonin and the circadian secretion of endogenous melatonin during the night may reduce the extent of excitotoxic brain injury. Further studies are needed to fully characterize the relevance of our findings for the treatment of progressive neurodegenerative processes which involve excitotoxicity and apoptotic neuronal death.