Neuronal loss is a major pathological hallmark observed in the Alzheimer (AD) brain. The mechanism to explain this neuronal loss is yet to be fully elucidated. Excitotoxicity is thought to occur in some neurological diseases, including AD. Glutamate is the major excitatory neurotransmitter in the brain and is regulated by specific transporter proteins located on glia and neurones.
The focus of this project was to examine the rOle excitotoxicity plays in the loss of postsynaptic cells in AD. Excitotoxicity could arise non-specifically - for example, by a global failure of glutamate transport leading to an increase of extracellular glutamate; or specifically - for example, by an increased activity in trans-synaptic excitatory pathways. The latter mechanism was explored in this project. To study this, a range of synaptic markers was chosen such that general synaptic proteins and proteins specific to excitatory synapses were quantified and compared.
A sandwich ELISA was designed and optimised using a novel antibody raised against the amino acid sequence of the first vesicular loop of synaptophysin. The assay proved to be a sensitive and accurate method of determining the relative amount of synaptophysin levels in human brain.
Synaptophysin and dynamin I were chosen as representative general presynaptic markers. Synaptophysin is localised to synaptic vesicles and dynamin I is involved in synaptic vesicle recycling. Sandwich ELISA analysis showed no significant difference between AD cases and controls in the levels of synaptophysin. An immunohistochemical stain for synaptophysin showed that the cortical thickness of AD brain tissue was not significantly thinner compared to controls. Western blot analysis of dynamin I also showed no significant difference between AD cases and controls.
Complexin I and II regulate neurotransmitter exocytosis in inhibitory and excitatory presynaptic terminals respectively. Quantification of complexin I by indirect ELISA revealed a significant reduction in AD cases compared with controls (P = 0.005). Analysis of the levels of complexin II by indirect ELISA also showed significantly lower levels in AD cases compared with controls (P = 0.007).
An excitatory synaptic junction protein, N-cadberin, and the most abundant protein of the postsynaptic density, aCaMKII, were chosen as markers of excitatory postsynaptic terminals. The levels of these proteins were examined by Western blot. There was no significant difference between AD and controls for N-cadherin or aCaMKII. N-cadberin levels in AD cases were non-significantly higher compared to controls, whereas the levels of the other five proteins were lower in AD cases than in controls.
All the proteins showed significantly different levels in the three areas studied. Moreover, the distribution of the complexins suggested there might be varied patterns of neurotransmitter exocytosis regulation in the three areas. The ratio of complex in II to complexin I was highest in hippocampus and lowest in occipital cortex, reflecting not only the heterogeneity of synaptic contacts, but also the heterogeneity of excitatory and inhibitory networks. With in the human cortex.
Genetic risk factors may accelerate the progression of Alzheimer disease. By categorising the cases according to risk factor, the effect of severity of the disease on the levels of synaptic markers was assessed. Across all presynaptic proteins, there was an association of the presence of the APOE £4 allele with lower levels of presynaptic proteins in AD cases. This reduction was independent of the number of £4 alleles.
The principal finding of this project was that the levels of synaptic proteins did not conform to the observed loss of neurones in AD brain tissue. Although the AD cases showed lower levels for synaptophysin, dynamin l, and aCaMKII, and higher levels of N -cadherin, these differences were not significant. The levels of the complexins were significantly different in AD cases as compared with controls. This may signify a disordered regulation of excitatory and inhibitory neurotransmitter exocytosis in AD, which has implications for the excitotoxicity theory of neuronal cell death in AD. For the AD case group, the presence of at least one copy of the £4 allele of APOE was associated with greater differences in the levels of synaptic proteins compared with AD cases without any e4 alleles, although the presence of two copies had no further effect.