Current Alzheimer Research (www.bentham.org/car),
2004, 1, 11-25
Bentham Science Publishers Ltd.(www.bentham.org)
The Identification and Characterization of Excitotoxic Nerve-endings in Alzheimer Disease
Rudi K. Tannenberg, Heather L. Scott, Robert I. Westphalen† and Peter R. Dodd*
Department of Biochemistry, University of Queensland, Australia
*Address correspondence to this author at the Department of Biochemistry, University of Queensland, Brisbane 4072, Australia; Tel: +61-7-3365 3364; Fax: +61-7-3365 4699; E-mail: p.dodd@uq.edu.au
†Present address: Department of Anesthesiology, Cornell
University Medical School, New York NY, USA
Abstract: Regionally
specific neuronal loss is a distinguishing feature of Alzheimer disease (AD).
Excitotoxicity is a mechanism commonly invoked to explain this. We review the
accumulating evidence for such a hypothesis, particularly the altered
expression and pharmacology of glutamate receptors and transporters in
pathologically susceptible regions of the AD brain. Loss of neurons would be
expected to lead to the retrograde degeneration of their afferents, which
should be reflected in a loss of presynaptic markers such as
synaptophysin. We discuss the
possibility that neurons may be
destroyed locally, but that
glutamatergic presynaptic terminals may remain, or even re-proliferate. The reduced glutamate uptake site density in AD brain may signify a loss of the transporters on otherwise intact terminals, rather than the loss of glutamatergic afferents. Neuronal death may follow if cells are exposed to excessive amounts of glutamate; the loss of transporters from functioning, but defective, glutamate terminals would mean they could continue to release glutamate to exacerbate excitotoxicity. We discuss experimental methods to quantitate synapses, which are crucial for deciding between the various possibilities.
Keywords: Excitatory Amino Acids; Neurotoxicity; Cerebral Cortex; Glutamate — Receptors; Glutamate — Transporters; Synaptic Terminals; Neurodegenerative Diseases.
Introduction
Alzheimer disease (AD) is characterized by progressive deterioration of memory, cognition, behavior, speech, and visual-spatial perception. Differential diagnosis is difficult [1], and AD can only be confirmed when characteristic pathological changes in brain morphology and histology are evaluated at autopsy [2]. These changes include excessive b-amyloid deposition forming senile plaques, intraneuronal paired helical filaments forming neurofibrillary tangles, and localized neuronal loss [2].
In common with many neurological diseases, the pattern of cellular damage in AD is not distributed uniformly through the brain. There are gross changes that are mainly confined to certain cortical regions, where the shrinkage of gyri and widening of the sulci in the frontal and temporal lobes contrasts with a relatively normal morphology in motor and occipital regions [3]. Other brain areas such as the brainstem, basal ganglia, and cerebellum are little affected [3]. This cerebral atrophy is associated with nerve cell loss. The pyramidal neurons in the superior, middle and inferior temporal gyri, and the superior and middle frontal gyri, are the major cellular populations affected [4]. There is no significant neuronal loss in motor or occipital cortical regions [4]. Counts of pyramidal cells give, on average, a 60% loss in temporal cortex, hippocampus, nucleus basalis of Meynert, and locus ceruleus [3]. The area-specificity is further exemplified by the pattern of cell loss within the hippocampus. Specific pathological changes occur in the subiculum and CA1 regions, and in the neighboring entorhinal cortex [5]. Pyramidal neurons from the CA3 region and granule cells from the dentate gyrus are relatively spared [6].
In contrast to many neurological diseases, AD exhibits destruction of cells belonging to most neurotransmitter systems [7, 8]. Markers of cholinergic neurons such as choline acetyltransferase activity, high-affinity choline uptake, and acetlylcholine synthesis and release, are lost [7, 8]. Radioligand binding to the dopamine uptake site is markedly reduced in autopsy samples of putamen [9]. Reduced concentrations of norepinephrine and reduced dopamine b-hydroxylase activity demonstrate the loss of noradrenergic neurons [7]. The markers of serotonergic neurons 5-HT and 5-hydroxyindole acetic acid are also reduced in autopsy samples [7]. The uptake of g‑aminobutyric acid (GABA) and the density of GABA uptake binding sites are reduced in the temporal cortex [10, 11], suggesting a loss of GABAergic neurons. Both Na+‑dependent D‑[3H]aspartate binding to glutamate uptake sites, and the initial rate of D‑[3H]aspartate uptake into synaptosomes, are reduced in affected areas [12]. Since the glutamate uptake site is thought of as a marker for glutamatergic nerve terminals, these measures have been taken to indicate that glutamate neurons are lost.
A large portion of cortical cell loss in AD comprises pyramidal neurons [3, 4]. Neurofibrillary tangles are concentrated in the pyramidal neurons of layers III and V, which provide the major cortico-cortical and projection pathways [13]. The loss of pyramidal neurons correlates with the clinical severity of the disease [14]. Pyramidal neurons of the cerebral cortex use glutamate as their neurotransmitter [15].
The key problem is to explain how a pathogenic process that is indiscriminate in targeting cells of all transmitter systems can nevertheless produce a generally consistent regional pattern of neuronal loss throughout the AD brain. The amyloid cascade hypothesis [16] alone seems inadequate, since plaque deposition does not conform to the distribution of neuronal loss (v.i.). To obtain a synthesis, we suggest that any simplistic general mechanism (deposition of a toxic protein; formation of reactive oxygen species; excitotoxicity; etc) is of itself insufficient, but requires refinement. We propose that modified excitotoxicity would suffice, given the hypotheses that
1) Glutamate is an intrinsic neurotoxin capable of mediating the death of all neuronal classes.
2) Specific variations in receptors and/or transporters underlie regional differences in susceptibility within those classes.
Neurons of a particular class can be targeted by a selective neurotoxin. For example, the general mitochondrial toxin MPP+ is preferentially taken into dopaminergic neurons by the dopamine transporter, for which it is a selective substrate [17]. Because the dopamine transporter is located almost exclusively on dopaminergic neurons alone, the toxin kills only those neurons. Loss of nigrostriatal dopamine neurons precipitates a Parkinsonian syndrome [18]. In contrast, the predominance of glutamate neurotransmission (v.i.) means that neurons of all transmitter classes carry glutamate receptors and are subject to glutamate-mediated excitation. In the event that such excitation should become excessive, and give rise to excitotoxicity, neurons of virtually every transmitter class would be vulnerable. Hence, glutamate fulfills the requirement of an intrinsic toxin with the potential to kill all (or most) classes of neurons, if it achieves toxic concentrations in the extracellular milieu. The pathophysiology of the potentially excitotoxigenic nerve-ending needs to be explored; experimental approaches are proposed that aim to delineate these entities, should they exist.
If glutamate-mediated excitotoxicity is to circumvent global damage and produce the regional pattern observed in AD, local modulation of cellular vulnerability is required. One approach to this question is to determine how the brain responds to generalized, non-specific insults. We recall that global hypoxia/ischemia insult leads to neural damage in areas that are susceptible to AD pathology [5,6]. We suggest that neurons in these areas may be more prone to excitotoxicity. We review the accumulating literature showing that the components that mediate glutamate transmission, especially receptors and transporters, exhibit highly specific variations that conform to the AD-vulnerability status of the region in which they are expressed. We discuss the experimental approaches that are required to elucidate excitotoxic mechanisms in a tissue in which glutamate is both the most abundant and ubiquitous excitatory transmitter, but also the most abundant and active intermediary metabolite. In particular, we note that approaches that are appropriate to study neuronal toxicity in specialized, locally discrete transmitter systems (e.g., MPP+ in dopamine neurons), are inappropriate and misleading in this context. A concerted approach to delineate the excitotoxigenic synapse should help clarify the rôle of abnormal glutamate-mediated transmission in the mechanism of neuronal death in Alzheimer disease.
Intrinsic Vulnerability
If brain regions vary in their propensity for damage, the patterns of pathology elicited by general insults should provide a clue to such inherent susceptibility. Hypoxia/ischemia produces a regional pattern of transmitter-nonspecific damage in experimental animals that mimics that in AD [6, 19–21]. Peripheral administration of plant (Lathyrism) and shellfish (domoic acid) neurotoxins also produces patterns of pathology that match those in AD. Lathyrism, domoate poisoning, and hypoxia may be linked to a common intrinsic neurotoxin.
Intrinsic Neurotoxins
Three agents endogenous to the CNS have been suggested as mediators of neuronal damage in AD: free radicals [22], b-amyloid [23], and excitatory amino acids (EAAs) [24, 25]. Their concentration would have to be either locally increased or redistributed to cause neurotoxicity. Each will be discussed in terms of its potential to mimic AD neuropathology.
Free Radicals
Free radicals are cytotoxic [22, 26]. CNS neurons are at risk because of their high rate of oxygen consumption and high content of polyunsaturated fatty acids that are susceptible to peroxidation [22]. Markers of oxidative injury (e.g. lipid peroxidation) are associated with the neuropathological lesions of AD [27]. Oxygen free radicals are involved in neuronal death induced by b-amyloid [26] and EAAs [28, 29].
Many pieces of evidence suggest the involvement of excitotoxicity in hypoxic brain damage. Increased glutamate release [30] and reduced glutamate uptake [31] follow hypoxic-ischemic events. EAA receptor antagonists protect against neuronal loss in global ischemia [19]. When EAA innervation to a brain region is cut, that area is protected against subsequent ischemia [32]. Hypoxic-ischemic brain damage is concentrated in regions that have high densities of EAA receptors [6]. These regions are also susceptible to AD pathology. They include the CA1 region of the hippocampus, cerebellar Purkinje cells, and superficial cortical layers [5, 6].
The maintenance of the membrane potential is energy-dependent, so bioenergetic insults like hypoxia/ischemia could initiate excitotoxicity. Dysfunctional membrane ion pumps would impair the maintenance of a membrane potential [21]. Activation of non-NMDA glutamate receptors depolarizes neurons [21] and removes the NMDA-site voltage block (v.i.). No defect either in membrane potentials, or in the generation of action potentials, has been reported in AD. This suggests that, if glutamate terminals are operating near-normally, receptor-mediated excitation will be maintained, and probably exacerbated, in pathologically susceptible areas.
b-Amyloid
The clinical severity of Alzheimer dementia has been correlated with counts of senile plaques at autopsy [33]. A causative rôle for b-amyloid in AD underpins the amyloid cascade hypothesis [16]. Support for this derives from several findings: a set of mutations in the amyloid precursor protein gene give rise to some familial forms of AD [16]; b-amyloid deposition precedes the development of neurofibrillary changes [3]; and b-amyloid is neurotoxic [23]. b‑Amyloid accumulation may render neurons vulnerable to excitotoxicity [34, 35], but support for its central rôle is somewhat diminished by reports that plaque counts do not distinguish between demented and non-demented individuals [33, 36]. Moreover, the distribution of plaques does not correlate with the distribution of neuronal death: both temporal and occipital cortices usually carry a heavy plaque load in AD, but the latter commonly shows much less cell loss [37].
Domoic Acid
An outbreak of neurological illness resulted from the consumption of mussels contaminated with domoic acid [38]. Autopsy studies showed hippocampal damage in a pattern resembling that caused by excitotoxicity [38]. The excitotoxic nature of domoate poisoning is most likely due to the compound’s structural relationship to glutamate [39].
Lathyrism
Chronic ingestion of the chickling pea, Lathyrus sativus, causes a spastic paraparesis known as neurolathyrism [39]. A non-protein-amino-acid constituent, b-N-oxalylamino-L-alanine (BOAA), which causes excitotoxic damage, is thought to be the causative agent [40, 41].
EAAs
Glutamate, aspartate, homocysteate, and cysteine sulfonate are putative EAA neurotransmitters. The notion that glutamate may induce neurotoxicity was first raised by Lucas and Newhouse [42]. The ability of EAAs to destroy neurons in culture was later demonstrated [43], and the concept that over-excitation could be toxic was labeled “excitotoxicity”. Selective glutamate antagonists prevent excitation and subsequent neuronal death, demonstrating the receptor-mediated nature of excitotoxicity [44]. It has been inferred that dysfunctional EAA pathways play rôles in the clinical and pathological manifestations of AD [6]. The neurotoxic insults mentioned above, domoate poisoning, Lathyrism and hypoxia, are associated with excitotoxic mechanisms.
Excitotoxicity
The activation of all ionotropic glutamate receptor subtypes (NMDA, quisqualate, and kainate) can induce excitotoxicity experimentally [21]. Two mechanisms are involved: passive Cl– influx and Ca2+ influx [45], such that excessive intracellular Ca2+ is accumulated [21]. Cytoplasmic Ca2+ can activate a variety of Ca2+-dependent enzymes, including protein kinase C, phospholipase A2, phospholipase C, Ca2+/calmodulin-dependent protein kinase II, nitric oxide synthase, and some protease and endonucleases, all of which have the potential to destroy neurons [21, 46]