Molecular changes of acetylcholinesterase and butyrylcholinesterase in Alzheimer patients during the natural couse of the disease and treatment with cholinesterase inhibitors : Insight into neurochemical mechanisms affecting the progression of the disease

Sammanfattning: Deficits in central cholinergic neurotransmission in the brain correlate with cognitive impairment in Alzheimer disease (AD) patients. This fact has resulted in the introduction of cholinesterase inhibitors (ChEls) e.g. tacrine, donepezil, rivastigmine and galantamine, which are so far the most successful therapeutic agents for symptomatic treatment of AD patients. The ChEls principally act by inhibiting acetylcholinesterase (AChE), which is primarily associated with cells involved in cholinergic neurotransmission and is responsible for inactivating acetylcholine at the synapses in the central and peripheral nervous systems. The neurobiological role of butyrylcholinesterase (BuChE) is not yet fully understood. Long-term clinical observations indicate that the ChEls may, in addition to cognitive, functional and global improvement of AD patients, have stabilizing effect on the progression of AD. This in turn implies that the ChEls may induce some changes in the brain that gradually affect the pathological processes of the disease. The overall aim of this thesis was to characterize changes, in AChE and BuChE, at the molecular, protein expression and activity levels in AD patients during the natural course of the disease as well as treatment with ChEls, in conjunction with changes in cognitive function of the AD patients. Comparative analysis of the neurochemical findings in brain and cerebrospinal fluid (CSF) in this thesis suggested that the expression of the globular (G) catalytic subunit of AChE variants and their monomeric (G1), dimeric (G2), and tetrameric (G4) molecular isoforms in CNS neurons were most likely the major source of the AChE protein in CSF. The protein expression of AChE variants was found to decrease during the time course of AD in untreated patients, whereas expression of the G2 isoform of the synaptic variant (AChE-S) was unexpectedly increased. These changes were reversed after treatment with the ChEIs. These bi-directional changes in the expression and composition of the AChE isoforms were shown to have an impact on the cognitive function of the patients, suggesting different mechanisms adapted by neurons in response to the ChEls and controlling the intrinsic stimulatory signals in untreated AD patients. In particular, preferential and distinct changes in the expression and activity levels of the "readthrough" variant (AChER) were observed in both untreated and treated patients with a consistent strong positive correlation with cognitive improvement in the AD patients. These observations support experimental data, which ascribe a putative neuroprotective role of AChE-R variant in CNS. These findings may in addition offer insight into the neurochemical processes underlying the possible stabilizing effect of ChEls in AD patients. Long-term treatment with the pseudoirreversible ChEI, rivastigmine was shown in this thesis to cause a sustained inhibition of both AChE and BuChE in CSF of AD patients, whereas the reversible ChEls, tacrine, donepezil and galantamine upregulated the AChE activity. A crucial and clinically relevant question is whether such an increase in response to the reversible ChEls reflects a tolerance against long-term treatment with the ChEls. This question was approached by a combination of different methodologies, which showed that the elevated CSF AChE activity following donepezil and galantamine treatment reflects the in vivo brain AChE inhibition and is not a tolerance phenomenon. Attention was shown to be the main cognitive domain improved by long-term AChE inhibition and an optimal AChE inhibition appeared to be a prerequisite to prevent cognitive deterioration in the AD patients. Positive correlations were also observed between inhibition of BuChE activity and several cognitive measures of attention and episodic memory in the AD patients. These findings suggest that BuChE may be involved in neuronal function and that simultaneous inhibition of both enzymes in AD patients may clinically be advantageous. Possession of certain genetic variants of BuChE and apolipoprotein E epsilon4 allele (ApoE epsilon4) increases the risk of developing late-onset AD, pointing at a pathological role of BuChE in AD. In this thesis, the majority of AD patients with high BuChE levels in CSF were found to be epsilon4 non-carriers, whereas most of those with moderate-low CSF BuChE levels were carriers of one or two epsilon4 alleles. The AD patients with the high CSF BuChE level had also a high score in cognitive tests. In addition, some measures of attention and episodic memory as well as cerebral glucose utilization, assessed by PET, positively correlated with the BuChE level in CSF. A reduced BuChE level in CSF, in particular in the epsilon4 carriers, may hence reflect an inverse relationship to its deposition in the brain regions, where much of the increased BuChE in AD brain may be ascribed to amyloid plaques. In conclusion, these studies show that selective and distinct changes occur in the transcription, alternative splicing and inter-protein interactions of AChE variants during the natural course of AD or during the ChEls treatment, which may influence the long-term efficacy of the therapeutics and progression of the AD. Different levels of CSF BuChE might clinically be useful to differentiate responders from non-responders of ChEl therapy. This thesis may provide a further understanding of pharmacological features of the ChEls in conjunction with the clinical responses in AD patients and optimizing the efficiency of the therapy.