The Neurobiology of Aging: Free Radical Stress and Metabolic Pathways

Environmental stressors and several genetic pathways play complex and crucial roles in the neurobiology and control of aging. This chapter will summarize current knowledge on these two specific research areas divided into two sections, one on free radical stressors and the other on genetic control of metabolic pathways.

Nitrosative and Oxidative Stress in the Neurobiology of Aging

Tomohiro Nakamura
Stuart A. Lipton

Aging represents a major risk factor for neurodegenerative diseases, such as Parkinson disease (PD), Alzheimer disease (AD), amyotrophic lateral sclerosis (ALS), polyglutamine (polyQ) diseases such as Huntington disease (HD), glaucoma, human immunodeficiency virus (HIV)–associated neurocognitive disorder (HAND), multiple sclerosis, and ischemic brain injury, to name but a few. Although many intra- and extracellular molecules may participate in neuronal injury and loss, the accumulation of nitrosative and oxidative stress, due to excessive generation of reactive nitrogen species (RNS) such as nitric oxide (NO) and of reactive oxygen species (ROS), appears to be a potential factor contributing to neuronal cell damage and death. A well-established model for NO production entails a central role of the N -methyl- d -aspartate (NMDA)–type glutamate receptors in the nervous system. Excessive activation of NMDA receptors drives Ca ²⁺ influx, which in turn activates neuronal NO synthase (nNOS) as well as the generation of ROS ( Fig. 11-1 ). Accumulating evidence suggests that NO can mediate protective and neurotoxic effects by reacting with cysteine residues of target proteins to form S-nitrosothiols (SNOs), a process termed S-nitrosylation because of its effects on the chemical biology of protein function. Importantly, normal mitochondrial respiration also generates free radicals, principally ROS, and one such molecule, superoxide anion (O ₂ ⁻ ), reacts rapidly with free radical NO under nitrosative stress conditions to form the very toxic product peroxynitrite (ONOO ⁻ ) ( Fig. 11-2 ).

Figure 11-1, Activation of the NMDA receptor (NMDAR) by glutamate (Glu) and glycine (Gly) induces Ca 2+ influx and consequent ROS and RNS production. NMDAR hyperactivation triggers the generation of ROS and RNS and cytochrome C release from mitochondria associated with subsequent activation of caspases, causing neuronal cell death and damage. SNO-PARK, S-nitrosylated parkin; SNO-PDI, S-nitrosylated PDI.

Figure 11-2, Pathways of ROSand RNS neurotoxicity. NO activates soluble guanylate cyclase (sGC) to produce cyclic guanosine monophosphate (cGMP), which in turn activates cGMP-dependent protein kinase. Excessive NMDA receptor activity, leading to the overproduction of NO, can be neurotoxic. For example, S-nitrosylation of parkin and PDI can contribute to neuronal cell damage and death, in part by triggering accumulation of misfolded proteins. Other neurotoxic effects of NO are mediated by peroxynitrite (ONOO − ), a reaction product of NO and superoxide anion (O 2 − ). In contrast, S-nitrosylation can also mediate neuroprotective effects—for example, by inhibiting caspase activity and preventing overactivation of NMDA receptors.

An additional feature of most neurodegenerative diseases is accumulation of misfolded and/or aggregated proteins. These protein aggregates can be cytosolic, nuclear, or extracellular. Importantly, protein aggregation can result from a mutation in the disease-related gene encoding the protein, or posttranslational changes to the protein engendered by nitrosative and oxidative stress. A key theme of this chapter, therefore, is the hypothesis that age-related nitrosative or oxidative stress contributes to protein misfolding in the brains of neurodegenerative patients. In the first section of this chapter, we discuss specific examples showing that S-nitrosylation of ubiquitin E3 ligases such as parkin or endoplasmic reticulum (ER) chaperones such as protein disulfide isomerase (PDI) are critical factors for the accumulation of misfolded proteins in neurodegenerative diseases such as PD and other conditions.

Protein Misfolding in Neurodegenerative Diseases

A shared histologic feature of many neurodegenerative diseases is the accumulation of misfolded proteins that adversely affect neuronal connectivity and plasticity and trigger cell death signaling pathways. For example, degenerating brains contain aberrant accumulations of misfolded aggregated proteins, such as α-synuclein and synphilin-1 in PD, and amyloid-β (Aβ) and tau in AD. The inclusions observed in PD are called Lewy bodies (LBs) and are mostly found in the cytoplasm. AD brains show intracellular neurofibrillary tangles, which contain tau, and extracellular plaques, which contain Aβ. Other disorders manifesting protein aggregation include HD (polyQ), ALS, and prion disease. These aggregates may consist of oligomeric complexes of non-native secondary structures and demonstrate poor solubility, even in the presence of detergents.

In general, protein aggregates do not accumulate in unstressed healthy neurons due in part to the existence of cellular quality control mechanisms. For example, molecular chaperones are believed to provide a defense mechanism against the toxicity of misfolded proteins because chaperones can prevent inappropriate interactions within and between polypeptides and can promote refolding of proteins that have been misfolded because of cell stress. In addition to the quality control of proteins provided by molecular chaperones, the ubiquitin-proteasome system (UPS) and autophagy-lysosomal degradation are involved in the clearance of abnormal or aberrant proteins. When chaperones cannot repair misfolded proteins, they may be tagged via the addition of polyubiquitin chains for degradation by the proteasome. In neurodegenerative conditions, intra- or extracellular protein aggregates are thought to accumulate in the brain as a result of a decrease in molecular chaperone or proteasome activities. In fact, several mutations that disturb the activity of molecular chaperones or UPS-associated enzymes can cause neurodegeneration. Along these lines, postmortem samples from the substantia nigra of PD patients manifest a significant reduction in proteasome activity compared to non-PD controls.

Historically, lesions that contain aggregated proteins were considered to be pathogenic. Several lines of evidence have suggested that aggregates are formed through a complex multistep process whereby misfolded proteins assemble into inclusion bodies; soluble oligomers of these aberrant proteins are thought to be the most toxic forms via interference with normal cell activities, whereas large insoluble aggregates may be an attempt by the cell to wall off potentially toxic material.

Generation of Reactive Oxygen and Reactive Nitrogen Species

Induction of Ca ²⁺ Influx by NMDA Receptor–Mediated Glutamatergic Signaling Pathways

It is well known that the amino acid glutamate is the major excitatory neurotransmitter in the brain. Glutamate is present in high concentrations in the adult central nervous system and is released for milliseconds from nerve terminals in a Ca ²⁺ -dependent manner. After glutamate enters the synaptic cleft, it diffuses across the cleft to interact with its corresponding receptors on the postsynaptic face of an adjacent neuron. Excitatory neurotransmission is necessary for the normal development and plasticity of synapses and for some forms of learning and memory; however, excessive activation of glutamate receptors is implicated in neuronal damage in many neurologic disorders, ranging from acute hypoxic-ischemic brain injury to chronic neurodegenerative diseases. It is currently thought that overstimulation of extrasynaptic NMDA receptors mediates this neuronal damage, whereas, in contrast, synaptic activity may activate survival pathways. Intense hyperstimulation of excitatory receptors leads to necrotic cell death, but milder or chronic overstimulation can result in apoptotic cell death.

NMDA receptor–coupled channels are highly permeable to Ca ²⁺ , thus permitting Ca ²⁺ entry after ligand binding if the cell is depolarized to relieve block of the receptor-associated ion channel by Mg ²⁺ . Subsequent binding of Ca ²⁺ to various intracellular molecules can lead to many significant consequences. In particular, excessive activation of NMDA receptors leads to the production of damaging free radicals (e.g., NO and ROS) and other enzymatic processes, contributing to cell death. *

* References .

Ca ²⁺ Influx and Generation of Reactive Oxygen and Reactive Nitrogen Species

Excessive activation of glutamate receptors is implicated in neuronal damage in many neurologic disorders. Olney coined the term excitotoxicity to describe this phenomenon. This form of toxicity is mediated at least in part by excessive activation of NMDA-type receptors, resulting in excessive Ca ²⁺ influx through a receptor's associated ion channel.

Increased levels of neuronal Ca ²⁺ , in conjunction with the Ca ²⁺ -binding protein Ca ²⁺ calmodulin (CaM), trigger the activation of nNOS and subsequent generation of NO from the amino acid l -arginine. NO is a gaseous free radical (thus highly diffusible) and a key molecule that plays a vital role in normal signal transduction but, in excess, can lead to neuronal cell damage and death. This discrepancy in NO effects on neuronal survival can also be caused by the formation of different NO species or intermediates—NO radical (NO⋅), nitrosonium cation (NO ⁺ ), nitroxyl anion (NO ⁻ , with a high-energy singlet and lower energy triplet forms).

Studies have further pointed out the potential connection between ROS-RNS and mitochondrial dysfunction in neurodegenerative diseases, especially in PD. Pesticide and other environmental toxins that inhibit mitochondrial complex I result in oxidative and nitrosative stress, with consequent aberrant protein accumulation. Administration to animals of complex I inhibitors, such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 6-hydroxydopamine, rotenone, and paraquat, which result in the overproduction of ROS-RNS, reproduces many of the features of sporadic PD, such as dopaminergic neuron degeneration, upregulation and aggregation of α-synuclein, LB-like intraneuronal inclusions, and behavioral impairment.

Increased nitrosative and oxidative stress are associated with chaperone and proteasomal dysfunction, resulting in the accumulation of misfolded aggregates. However, until recently, little was known regarding the molecular and pathogenic mechanisms underlying the contribution of NO to the formation of inclusion bodies, such as amyloid plaques in AD or LBs in PD.

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