Elektrophysiologische Charakterisierung der Locus coeruleus Neuronen von Parkin­-Knockout Mäusen mit Hilfe der Patch­-Clamp­-Technik

Parkinson’s Disease (PD) is the second most common neurodegenerative disorder worldwide following Alzheimer’s Disease. Loss-of-function mutations in the PARK2 gene, which encodes the enzyme Parkin, have been implicated in nearly 50 % of inherited autosomal recessive forms of PD. Typically, this hereditary PD variant manifests itself before the age of 40 and is therefore referred to as early-onset PD. Parkin is an E3 ubiquitin ligase that plays a curcial role in labelling a range of proteins in central neurons via ubiquitin conjugation. Different types of ubiquitin conjugation cause misfolded or damaged proteins to be directed towards degradation in proteasomes or lysosomes. Moreover, Parkin-dependent ubiquitylation contributes to cellular signaling pathways, which are involved in neuroprotective cellular processes. Functional loss of Parkin triggers mitochondrial dysfunction and inflammation, which leads to neurodegeneration in vulnerable neurons. A clear explanation at the molecular level for selective vulnerability of certain neuronal populations for PD-dependent cell loss has not yet been found. A variety of factors that predispose to high mitochondrial oxidative stress levels have already been discussed. Previous studies show a multifactorial triggered disturbed calcium homeostasis, which increases susceptibility for oxidative stress in the context of pathologic conditions. In the literature Parkin is described to play a key in the context of cellular calcium homeostasis. This hypothesis was supported by ubiquitylome profiling of murine Parkin knockout (KO) brains. These studies have revealed dysregulation of the calcium homeostasis factors ATP1A2, Hippocalcin and GNA11. The listed neuron-specific Parkin substrates are known to modulate the generation and the shape of action potentials of central neurons. Therefore, the question of functional consequences arises, such as whether neuronal excitability is altered in vulnerable neurons, due to loss of Parkin function. Due to the specific vulnerability of Locus coeruleus (LC) neurons for PD specific pathological changes it can be assumed that phenotypical alterations most likely occur early in that nucleus. Therefore, in the current study patch clamp experiments of LC neurons in acute brain slices of Parkin-KO or wildtype mice were performed time-dependently. My objective was to reveal the role of Parkin in the context of PD pathophysiology especially in electrophysiological alterations. Patch clamp experiments in acute Parkin-KO brain slices of 12 month old mice indeed revealed progressive alterations in the intrinsic pacemaker activity of noradrenergic LC neurons, which can be correlated to an impaired calcium homeostasis. LC neurons of aged Parkin-KO brain showed an acceleration of the spontaneous pacemaker frequency, a reduction in slow afterhyperpolarization and a shortening of action potential duration. Inconspicuous results from the Whole-Cell Voltage-Clamp measurements of potassium and calcium currents suggest that the changed pacemaker activity in Parkin-KO LC neurons does not result from a disturbed expression or dysfunction of specific potassium or calcium channels. Taking into account the interaction between hippocalcin accumulated in Parkin-KO tissue and voltage-dependent KCNQ channels, the pharmacological isolation of KCNQ mediated potassium currents was carried out by applying the selective blocker HMR1556. KCNQ-borne potassium currents also remained unchanged in Parkin-KO LC neurons. The results of the current study indicate that the altered pacemaker activity of murine Parkin-KO LC neurons is most likely due to an altered calcium-dependent excitability of the plasma membrane, caused by diminished turnover of potential Parkin substrates such as ATP1A2 and Hippocalcin. The results of this work should contribute to a better understanding of the increased vulnerability of monaminergic neurons due to an altered calcium homeostasis and the selective neuronal loss in context of PD.