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AbstractSelective degeneration of substantia nigra dopaminergic (SN DA) neurons presents the underlying neuropathological hallmark of Parkinson’s disease (PD). However, the reason, why especially SN DA neurons are highly vulnerable to PD stressors, while other DA neurons within the ventral tegmental area remain much more resistant, is still unclear. Activity-related Ca2+ load and resulting metabolic stress have emerged as important factors that contribute to PD pathology. SN DA neurons are autonomously active to secure a basal level of striatal dopamine release and voluntary movement control. However, this pacemaker activity and related activation of voltage-gated Ca2+ channels generate oscillatory elevated Ca2+ levels in SN DA neurons that induce elevated levels of mitochondrial oxidant stress, high vulnerability to PD stressors and are supposed to trigger neurodegeneration. Cav2.3 channels have been recently identified as significant contributors to somatic Ca2+ oscillations and hypothesized as degenerative stressors in SN DA neurons. In contrast, Ca2+-dependent neuronal Ca2+ sensor NCS-1 function can reduce SN DA neuron activity and thereby prevent overexcitability by sensitized dopamine D2 autoreceptor (D2-AR)-mediated inhibition. This activity reducing feedback mechanism has been suggested as neuroprotective mechanism to counteract activity-related stress. In this thesis, I aimed to study the role of Cav2.3 R-type Ca2+ channels and the neuronal Ca2+ sensor NCS-1 in defining differential vulnerability of DA midbrain neurons in PD paradigms, also in the context of altered Ca2+ homeostasis. First, I quantified expression levels of distinct voltage-gated Ca2+ channels, i.e. Cav1.2, Cav1.3, Cav2.3 and Cav3.1 that are associated with SN DA viability, as well as NCS 1 and its related D2-AR signaling complex in individual SN DA neurons by combining cell-specific UV-laser microdissection and reverse transcription qPCR. I focused on expression changes in mice during maturation and in response to general loss of Cav2.3 or NCS 1, respectively. I identified Cav2.3e as the major splice variant expressed in SN DA neurons. I found 2-fold higher expression levels of Cav2.3 and both existing D2 AR splice variants in SN DA neurons of adult compared to juvenile mice, while Cav1.2 and Cav1.3 expression levels were significantly downregulated in SN DA neurons during maturation. In line with aging being the highest risk factor to develop PD, these findings might point towards a higher relevance of stressful Cav2.3 activity in comparison to L-type Ca2+ channels and of the NCS-1 dependent D2-AR feedback mechanism with aging. This might as well explain the more stressful Ca2+ homeostasis of mature SN DA neurons and their higher vulnerability during aging. I also detected 2-fold lower expression levels of Cav2.3 in SN DA neurons of juvenile and aged NCS-1 KO mice, but 1.5-fold higher expression levels of NCS 1 in SN DA neurons of adult Cav2.3 KO mice. These data indicate a transcriptional link of Cav2.3 and NCS-1 in SN DA neurons. As I detected no transcriptional change of Cav1.2, Cav1.3 and Cav3.1 in SN DA neurons of adult Cav2.3 KO mice, this argues against a possible compensation of the loss of Cav2.3 by the here analyzed other Ca2+ channels. Next, I addressed the role of Cav2.3 and NCS-1 on the vulnerability of DA midbrain neurons to degeneration in PD paradigms by using the chronic low dose MPTP/probenecid (MPTP/p) model. I quantified pattern and loss of DA midbrain neurons and their respective projection fibers within the striatum in NCS-1 KO and Cav2.3 KO mice compared to control mice that were chronically treated with MPTP/p or saline, as control, via densitometry, unbiased stereology and automated counting. As prerequisite for the automated counting approach, I successfully established and optimized a suitable convolutional neural network (CNN) algorithm for automated counting of neurons by correlating CNN algorithm total counts with stereology estimates. Using densitometry, I found a lower vulnerability of tyrosine hydroxylase (TH)-positive fibers in the dorsal striatum of Cav2.3 KO mice compared to Cav2.3 WT mice after MPTP/p treatment. SN DA neurons of Cav2.3 KO mice were surprisingly fully protected in the chronic MPTP/p PD model, analyzed via stereology. These findings were confirmed by automated counting of all neurons within the SN, excluding downregulation of TH expression as possible confounder. On the other hand, using the same approach, nigrostriatal fibers and SN DA neurons of NCS-1 KO mice were significantly more susceptible to MPTP/p-induced degeneration compared to WT mice. This neuroprotection in Cav2.3 KO mice and higher susceptibility in NCS-1 KO mice in response to MPTP/p were only present in the nigrostriatal but not in the mesocorticolimbic pathway. These data provide substantial evidence, that indeed Cav2.3 and NCS-1 are crucial players in defining the vulnerability of SN DA neurons to PD degeneration. Cav2.3 channels trigger SN DA loss and NCS-1 function seems to protect SN DA neurons in PD paradigms. These implicated antagonistic functions of Cav2.3 and NCS-1 in SN DA neurons are believed to keep SN DA function and activity within a physiological bandwidth, ensuring a certain Ca2+-based activity level on the one hand, but protecting them from overexcitability on the other hand. In this scenario, PD-trigger factors could disturb this delicate balance, a situation, where blocking Cav2.3 channels or stimulating NCS-1 function should have neuroprotective effects. In conclusion and in line with the here presented hypothesis, the findings of this thesis open novel therapeutic avenues for PD by providing two novel targets, Cav2.3 and NCS-1, which were both formerly not linked to PD. Given the ubiquitous expression and function of NCS 1, particularly selective inhibitors or genetic inactivation of Cav2.3 are highly recommended to pursue. In this context, additional studies are desired to further develop and evaluate targeting of Cav2.3 channels as neuroprotective PD therapy.dc.description.abstract
PublisherUniversität Ulmdc.publisher
LicenseCC BY 4.0 Internationaldc.rights
Link to license text
KeywordVoltage-gated calcium channelsdc.subject
KeywordNeuronal calcium sensordc.subject
KeywordSelective vulnerabilitydc.subject
Dewey Decimal GroupDDC 610 / Medicine & healthdc.subject.ddc
MeSHParkinson diseasedc.subject.mesh
MeSHNeuronal calcium-sensor proteinsdc.subject.mesh
TitleRole of Cav2.3 channels and NCS-1 in defining differential vulnerability of dopaminergic neurons in Parkinson's diseasedc.title
Resource typeDissertationdc.type
Date of acceptance2020-01-24dcterms.dateAccepted
RefereeLiss, Birgitdc.contributor.referee
RefereeDanzer, Karindc.contributor.referee
FacultyMedizinische Fakultätuulm.affiliationGeneral
InstitutionInstitut für Angewandte Physiologieuulm.affiliationSpecific
InstitutionUKU. Klinik für Neurologieuulm.affiliationSpecific
Grantor of degreeMedizinische Fakultätuulm.thesisGrantor
DCMI TypeTextuulm.typeDCMI
University Bibliographyjauulm.unibibliographie

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