Alzheimer’s disease (AD) is accompanied by increased brain levels of soluble amyloid-β (Aβ) oligomers (McLean et al., 1999). It has been suggested that Aβ oligomers directly impair synaptic function (Haass and Selkoe, 2007) and modulate high voltage-activated calcium channels (Sberna et al., 1997; Bobich et al., 2004; Nimmrich et al., 2008a). State-dependent drugs are hypothesized to target pathologically overactivated channels without altering physiological activity, which may reduce adverse side effects (Winquist et al., 2005). Here, the effect of Aβ globulomer, a synthetic, stable, and pathologically relevant oligomeric Aβ preparation (Barghorn et al., 2005; Gellermann et al., 2008), on high voltage-activated calcium channels was further elucidated. Furthermore, it was tested whether novel state-dependent calcium channel blockers can ameliorate oligomeric Aβ-induced deficits in synaptic transmission. First, the effect of oligomeric Aβ on excitatory synaptic transmission was investigated in rat organotypic hippocampal slice cultures. Specific block of P/Q-type and N-type calcium channels by ω-agatoxin IVA and ω-conotoxin MVIIA, respectively, completely reversed Aβ oligomer-induced deficits. By contrast, (additional) L-type calcium channel block by nimodipine (Diochot et al., 1995; Furakawa et al., 1999), a potential antidementia medicine (Birks and López-Arrieta, 2002), was ineffective. As assessed by whole cell patch clamp analysis, oligomeric Aβ shifted the activation of P/Q-type and N-type calcium channels, recombinantly expressed in HEK293 cells, to more hyperpolarized values. Application of non-aggregated Aβ peptide had no effect. These findings suggest that overactivation of presynaptic calcium channels by oligomeric Aβ may lead to functional synaptic deficits, which can be prevented with presynaptic calcium channel blockers. In a second part of this work, novel state-dependent calcium channel blockers were identified (published in Mezler et al., 2012a) and their potential to protect from Aβ-induced functional deficits investigated. Compounds were initially detected by a fluorescence imaging plate reader-based primary high throughput screen (previously performed at Abbott) using HEK293 cells recombinantly expressing P/Q-type calcium channels. For subsequent compound validation using a more direct measure of channel function, an automated patch clamp secondary screening assay was established and incorporated into the hit-to-lead cycle of a drug discovery process. Representative compounds out of this screen were characterized for state-dependent P/Q-type calcium channel block by manual patch clamp recordings. Finally, these blockers were able to protect from Aβ-induced functional decline in synaptic transmission similarly as the state-independent peptide toxins.
Findings from this work hint towards the therapeutic potential of state-dependent presynaptic calcium channel block, which needs to be further elucidated in an in vivo model of AD. As P/Q-type calcium channel gain-of-function is also associated with migraine and epilepsy, novel specific channel blockers may also alleviate symptoms of other neurological diseases, beyond AD.
|Supervisor:||Draguhn, Prof. Dr. Andreas|
|Date of thesis defense:||20 February 2013|
|Date Deposited:||27 Feb 2013 12:50|
|Date:||20 February 2013|
|Faculties / Institutes:||The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences|
|Subjects:||570 Life sciences|
|Controlled Keywords:||Alzheimer-Krankheit, Amyloid <beta->, Patch-Clamp-Methode, Calciumkanal|