Apolipoprotein E and cholinergic enzymes : the interface in dementia

The main confirmed genetic risk factor for sporadic form of Alzheimer’s disease (AD) is apolipoprotein E (ApoE) ε4 allele (APOE4). ApoE protein aids in receptor ligand mediated transport, distribution and metabolism of fatty acids and cholesterol. However, the molecular mechanism of its contribution to the pathological events in AD is still highly obscure. Some reports implicate ApoE in amyloid-beta (Aβ) deposition in the brain, one of the main hallmarks of AD. But there are no consensuses about how ApoE causes the accumulation of Aβ deposits. The main neuronal network that becomes severely affected in the course of AD and Lewy body dementia disorders (LBD) is the central cholinergic system, and the main available therapeutic options are cholinesterase inhibitors which prevents the degradation of cholinergic neurotransmitter, acetylcholine (ACh). However, it is unclear, why these particular neuronal populations are selectively affected in AD and LBD. In addition to the classical view of the cholinergic neurotransmission, extrasynaptic cholinergic signaling also plays a crucial role in the regulation of immune system. Although, it is not clear how ACh can reach and act on such non-neuronal cell population in the brain or in the peripheral organs.

This thesis aimed to provide some insights in these unresolved issues. We found that APOE4 gene dose-dependently was associated with the highest ApoE protein expression in cerebrospinal fluid (CSF) of dementia patients (AD and DLB). This finding was observed in two independent study cohorts. Correlation analyses between the CSF ApoE protein and clinical measures (various cognitive tests), paraclinical measures (cerebral glucose metabolism and Aβ load by positron emission tomography) and AD biomarkers (CSF Aβ and tau, etc) suggested that high levels of ApoE protein may act as one of the driving forces of the pathological events in AD and DLB. This thesis also examined a postulated genetic interaction between APOE4 and a certain genetic variant of the ACh-degrading enzyme, butyrylcholinesterase (BuChE), namely BCHE-K allele. This directed genetic analysis was then to some extent scrutinized at their protein expression levels. The findings indicated that in the absence of APOE4, BCHE-K was associated with the lowest CSF ApoE protein, and the slowest annual cognitive decline, suggestive of some protective effects by BCHE-K. In the presence of APOE4, this protective effect of BCHE-K was either insufficient or rather increased the negative impact of APOE4 on both CSF ApoE levels and the rate of annual cognitive decline in patients. Given that astrocytes, one of the key players of the immune system in the central nervous system are the main sources of ApoE and BuChE, this thesis also aimed at providing a 7 mechanistic understanding of the interplays between extrasynaptic cholinergic immune-regulatory signaling, Aβ, ApoE and BuChE.

In this context, our previous studies have provided evidence for APOE4 dependent molecular interaction between ApoE protein, Aβ and BuChE leading to formation of hyperactive BuChE-Aβ-ApoE complexes (BAβACs), which then accumulates in the brain, causing abnormal extrasynaptic ACh levels, astroglial dysfunction, altered Aβ clearance and aggregation in AD and DLB patients. Nonetheless, this hypothesis and the well-established cholinergic anti-inflammatory hypothesis faced a major dilemma, since ACh is a highly instable signaling molecule to be able to diffuse and act on cholinoceptive cells located far from the cholinergic synapses. This thesis provided a solution to this dilemma by providing for the first time, compelling evidence for the presence of ACh-synthesizing enzyme, choline acetyltransferase (ChAT) in human extracellular fluids, such as CSF and plasma. We showed that both lymphocytes and astrocytes might be the sources of soluble ChAT, as they were capable of acquiring cholinergic phenotype (expressing ChAT) on demand, and releasing ChAT into extracellular fluids. We showed that soluble ChAT was fully functional, and could maintain steady state ACh equilibrium in the presence of highly active ACh-degrading enzymes in CSF. Thus this thesis also provided for the first time evidence that astrocytes are cholinergic cells and hence might be an active part of the non-neuronal cholinergic network in the brain. In addition, the undertaking of this thesis led to development of a novel, sensitive integrated assay for sequential quantitative measurements of ChAT activity and protein levels in practically any biological fluid deemed to contain this enzyme.

Conclusions and future perspectives: This thesis provided important insights into the complex interplays that are ongoing in vivo in the course of two major dementia disorders (AD and DLB) between several key genetic and/or molecular factors and cellular networks. This thesis also provided a novel research tool, facilitating future studies for reviewing the above hypothesis in the field of dementia as well as other neuroinflammatory disease such as multiple sclerosis, which this thesis has pointed out.

List of scientific papers

I. S. Vijayaraghavan, W. Maetzler, M. Reimold, C. U. Lithner, I. Liepelt-Scarfone, D. Berg and T. Darreh-Shori. (2014). High apolipoprotein E in cerebrospinal fluid of patients with Lewy body disorders is associated with dementia. Alzheimer's & Dementia. 10(5): 530-540.e1.
https://doi.org/10.1016/j.jalz.2013.03.010

II. S. Vijayaraghavan, A. Karami, S. Aeinehband, H. Behbahani, A. Grandien, B. Nilsson, K. N. Ekdahl, R. Lindblom, F. Piehl and T. Darreh-Shori. (2013). Regulated extracellular choline acetyltransferase activity-the plausible missing link of the distant action of acetylcholine in the cholinergic anti-inflammatory pathway. PloS one. 8(6): e65936.
https://doi.org/10.1371/journal.pone.0065936

III. S. Vijayaraghavan, T. Darreh-Shori, A. Rongve, G. Berge, S. B. Sando, L. R. White, B. H. Auestad, A. Witoelar, O. A. Andreassen, I. D. Ulstein and D. Aarsland. (2016). Association of Butyrylcholinesterase-K Allele and Apolipoprotein E varepsilon4 Allele with Cognitive Decline in Dementia with Lewy Bodies and Alzheimer's Disease. J Alzheimers Dis. 50(2): 567-576.
https://doi.org/10.3233/JAD-150750

IV. S. Vijayaraghavan, D. Aarsland, T. Darreh-Shori. Interplay of genetic and phenotypic variations of Apolipoprotein E and Butyrylcholinesterase in patients with Alzheimer’s disease and Lewy body dementia. [Manuscript]