Matthew R. Hynd

Glutamate-mediated excitotoxicity and neurodegeneration in Alzheimer's disease.

Alzheimers disease (AD) is the most common form of dementia, accounting for 60-70% of cases in subjects over 65 years of age. Several postulates have been put forward that relate AD neuropathology to intellectual and functional impairment. These range from free-radical-induced damage, through cholinergic dysfunction, to beta-amyloid-induced toxicity. However, therapeutic strategies aimed at improving the cognitive symptoms of patients via choline supplementation, cholinergic stimulation or beta-amyloid vaccination, have largely failed. A growing body of evidence suggests that perturbations in systems using the excitatory amino acid L-glutamate (L-Glu) may underlie the pathogenic mechanisms of (e.g.) hypoxia-ischemia, epilepsy, and chronic neurodegenerative disorders such as Huntingtons disease and AD. Almost all neurons in the CNS carry the N-methyl-D-aspartate (NMDA) subtype of ionotropic L-glutamate receptors, which can mediate post-synaptic Ca2+ influx. Excitotoxicity resulting from excessive activation of NMDA receptors may enhance the localized vulnerability of neurons in a manner consistent with AD neuropathology, as a consequence of an altered regional distribution of NMDA receptor subtypes. This review discusses mechanisms for the involvement of the NMDA receptor complex and its interaction with polyamines in the pathogenesis of AD. NMDA receptor antagonists have potential for the therapeutic amelioration of AD.

Biochemical and molecular studies using human autopsy brain tissue

The use of human brain tissue obtained at autopsy for neurochemical, pharmacological and physiological analyses is reviewed. RNA and protein samples have been found suitable for expression profiling by techniques that include RT‐PCR, cDNA microarrays, western blotting, immunohistochemistry and proteomics. The rapid development of molecular biological techniques has increased the impetus for this work to be applied to studies of brain disease. It has been shown that most nucleic acids and proteins are reasonably stable post‐mortem. However, their abundance and integrity can exhibit marked intra‐ and intercase variability, making comparisons between case‐groups difficult. Variability can reveal important functional and biochemical information. The correct interpretation of neurochemical data must take into account such factors as age, gender, ethnicity, medicative history, immediate ante‐mortem status, agonal state and post‐mortem and post‐autopsy intervals. Here we consider issues associated with the sampling of DNA, RNA and proteins using human autopsy brain tissue in relation to various ante‐ and post‐mortem factors. We conclude that valid and practical measures of a variety of parameters may be made in human brain tissue, provided that specific factors are controlled.

Differential expression of N‐methyl‐d‐aspartate receptor NR2 isoforms in Alzheimer's disease

We have previously shown that the expression of NMDA receptor NR1 subunit mRNA splice variants in Alzheimers disease (AD) brain varies according to regional susceptibility to pathological damage. Here we investigated the expression of the modulatory NR2 subunits of the NMDA receptor using quantitative RT–PCR to assay all NR2 isoforms. Significantly lower expression of NR2A and NR2B transcripts was found in susceptible regions of AD brain, whereas expression of NR2C and NR2D transcripts did not differ from that in controls. Western blot analysis confirmed a lower expression of the NR2A and NR2B isoforms at the protein level. The results suggest that NR2 subunit composition may modulate NMDA receptor‐mediated excitotoxicity. NMDA receptor dysfunction might give rise to the regionally selective pattern of neuronal loss that is characteristic of AD.

Glutamate NMDA receptor NR1 subunit mRNA expression in Alzheimer's disease

We analyzed the expression profile of two NMDAR1 mRNA isoform subsets, NR10XX and NR11XX, in discrete regions of human cerebral cortex. The subsets are characterized by the absence or presence of a 21‐amino acid N‐terminal cassette. Reverse transcription polymerase chain reaction for NR1 isoforms was performed on total RNA preparations from spared and susceptible regions from 10 pathologically confirmed Alzheimers disease (AD) cases and 10 matched controls. Primers spanning the splice insert yielded two bands, 342 bp (NR10XX) and 405 bp (NR11XX), on agarose gel electrophoresis. The bands were visualized with ethidium and quantified by densitometry. NR11XX transcript expression was calculated as a proportion of the NR11XX + NR10XX total. Values were significantly lower in AD cases than in controls in mid‐cingulate cortex, p < 0.01, superior temporal cortex, p < 0.01 and hippocampus, p ∼ 0.05. Cortical proportionate NR11XX transcript expression was invariant over the range of ages and areas of controls tested, at ∼50%. This was also true for AD motor and occipital cortex. Proportionate NR11XX expression in AD cingulate and temporal cortex was lower at younger ages and increased with age: this regression was significantly different from that in the homotropic areas of controls. Variations in NR1 N‐terminal cassette expression may underlie the local vulnerability to excitotoxic damage of some areas in the AD brain. Alternatively, changes in NR1 mRNA expression may arise as a consequence of the AD disease process.

Selective loss of NMDA receptor NR1 subunit isoforms in Alzheimer's disease

Previous work had shown that the ratio of NMDA receptor NR1 subunit mRNA transcripts containing an N‐terminal splice cassette to those that do not is markedly lower in regions of the Alzheimers disease (AD) brain that are susceptible to pathological damage, compared with spared regions in the same cases or homotropic regions in controls. To elucidate the origins of this difference in proportionate expression, we measured the absolute levels of each of the eight NR1 transcripts by quantitative internally standardized RT‐PCR assay. Expression of transcripts with the cassette was strongly attenuated in susceptible regions of Alzheimers brain, whereas expression of non‐cassette transcripts differed little from that in controls. The expression of other NR1 splice variants was not associated with pathology relevant to disease status, although some combinations of splice cassettes were well maintained in AD cases. The population profile of NR1 transcripts in occipital cortex differed from the profiles in other brain regions studied. Western analysis confirmed that the expression of protein isoforms containing the N‐terminal peptide was very low in susceptible areas of the Alzheimers brain. Cells that express NR1 subunits with the N‐terminal cassette may be selectively vulnerable to toxicity in AD.

Quantitation of alternatively spliced NMDA receptor NR1 isoform mRNA transcripts in human brain by competitive RT-PCR

The N-methyl-D-aspartate (NMDA)-selective subtype of ionotropic glutamate receptor is of importance in neuronal differentiation and synapse consolidation, activity-dependent forms of synaptic plasticity, and excitatory amino acid-mediated neuronal toxicity [Neurosci. Res. Program Bull. 19 (1981) 1; Lab. Invest. 68 (1993) 372]. NMDA receptors exist in vivo as tetrameric or pentameric complexes comprising proteins from two families of homologous subunits, designated NR1 and NR2(A-D) [Biochem. Biophys. Res. Commun. 185 (1992) 826]. The gene coding for the human NR1 subunit (hNR1) is composed of 21 exons, three of which (4, 20 and 21) can be differentially spliced to generate a total of eight distinct subunit variants. We detail here a competitive RT-PCR (cRT-PCR) protocol to quantify endogenous levels of hNR1 splice variants in autopsied human brain. Quantitation of each hNR1 splice variant is performed using standard curve methodology in which a known amount of synthetic ribonucleic acid competitor (internal standard) is co-amplified against total RNA. This method can be used for the quantitation of hNR1 mRNA levels in response to acute or chronic disease states, in particular in the glutamatergic-associated neuronal loss observed in Alzheimers disease [J. Neurochem. 78 (2001) 175]. Furthermore, alterations in hNR1 mRNA expression may be reflected at the translational level, resulting in functional changes in the NMDA receptor.

Quantitation of NMDA receptor NR2 mRNA transcripts in human brain by competitive RT-PCR

The NMDA-selective ionotropic receptor constitutes one of the three principal classes of L-glutamate receptors within the mammalian brain. It plays key roles in neuronal differentiation and synapse consolidation, activity-dependent forms of synaptic plasticity, and excitatory amino acid-mediated neuronal toxicity [Lab. Invest., 68 (1993) 372-387]. NMDA receptors exist as multimeric complexes comprising proteins from two families, NR1 and NR2(A-D) [J. Biol. Chem., 271 (1996) 15669-15674]. Studies on recombinant receptors have revealed that while homomeric NR2 receptors are non-functional, co-expression of an NR1 with an NR2 subunit modulates the efficacy of the resulting channel [Nature, 357 (1992) 70-74]. The RT-PCR assay we describe here was developed to allow quantitation of all hNR2 transcripts in a single-tube PCR assay. Each hNR2 isoform is quantified on the basis of standard curves in which a known amount of synthetic ribonucleic acid competitor is co-amplified against total RNA. The protocol has been applied to the quantitation of hNR2 mRNA levels in autopsy brain. Used in conjunction with a method for the quantitation of hNR1 transcripts [Brain Res. Protoc., in press], a complete analysis of NMDA receptor mRNA expression can be obtained.

Role of ionotropic glutamate receptors in neurodegenerative and other disorders

Disorders of the central nervous system (CNS) are amongst the most complex disease states and lead to some of the most devastating conditions found in medicine today. It is well established that both genetic and epigenetic factors influence the pathogenesis of neurodegenerative disorders. More recently a growing body of evidence suggests that changes in neurotransmitter systems may underlie the pathogenic mechanisms of neurodegenerative disorders, such as Alzheimer’s disease and Parkinson’s disease. Excitatory amino acids (EAA) are known to play key roles in neurotransmission, neuromodulation, and neurotoxicity. EAA receptors are classified into two major subdivisions, ionotropic glutamate receptors (iGluRs) and metabotropic glutamate receptors (mGluRs). L-Glu-gated ion channels, more commonly known as (iGluRs), are the primary mediators of fast excitatory synaptic transmission between neurons in the mammalian CNS. Evidence suggests that the iGluR receptor systems are involved in a number of pathophysiology conditions. This chapter will detail the current state of knowledge regarding the involvement of iGluRs in neurodegenerative and other disorders.