Abstract:
Neuroimaging is a valuable tool to investigate the brain in a healthy and diseased state. Imaging techniques such as positron emission tomography (PET) and magnetic reso-nance imaging (MRI) make it possible to structurally visualize the brain and display the brain in action on a molecular and functional level. Functional MRI (fMRI) detects neu-ronal activity via the hemodynamic response at rest, during a task, or after a pharmaco-logical stimulation. The picomolar sensitivity of PET enables imaging of the distribution of various molecular targets and related biological processes in the brain. The combina-tion of pharmacological MRI and PET offers excellent potential to investigate brain network modulations of different neurotransmitter systems and provides information on molecular neurotransmitter-receptor interactions in vivo. Both imaging modalities may provide a deeper insight into the coupling of molecular and macroscopic neural mecha-nisms underlying specific processes in the healthy and the diseased brain.
This work focuses on imaging synucleinopathies, a group of heterogeneous neurodegen-erative diseases characterized by the pathological aggregation of the protein α-synuclein (αSYN) in different cells and regions in the brain. Relevant biomarkers of αSYN aggre-gation are still missing in the field of neuroimaging. Therefore, there is an unmet need to develop a novel PET tracer to detect αSYN aggregates for diagnosis and therapy control and to develop a multimodal imaging approach to link protein aggregation to functional neuronal changes.
The first part of this work aimed to investigate different chemical derivatives of anle138b, a compound that has been shown to have therapeutic activity in animal mod-els of neurodegenerative diseases, towards their binding affinities to recombinant αSYN fibrils to develop a specific αSYN PET tracer. One compound, MODAG-001, showed a very high affinity towards human recombinant αSYN fibrils. Furthermore, binding se-lectivity over Aβ in human and mouse Alzheimer’s disease (AD) tissue and the binding specificity in human synucleinopathies with αSYN pathology were determined. [3H]MODAG-001 showed specific binding to Aβ-pathology. However, this was be-tween 5- and 14-fold lower compared to the gold standard AD marker, Pittsburgh Compound B. Using [3H]MODAG-001 in autoradiography experiments on human brain sections of different proteinopathies revealed binding of [3H]MODAG-001 in cortical areas of PD and AD brain tissue, with a weak signal to noise ratio. In vivo evaluation of [11C]MODAG-001 revealed good pharmacokinetics of the PET tracer with a fast wash-out of the brain and one metabolite with 90% of [11C]MODAG-001 present in the brain at 15 minutes after tracer injection. In vivo, evaluation of [11C]MODAG-001 in a mouse model of Parkinson’s disease showed no specific binding in brain areas with high αSYN pathology. The metabolite observed in the [11C]MODAG-001 metabolite analysis was the demethylated precursor MODAG-005, which was then tritiated for further specifici-ty experiments in human brain sections of different proteinopathies. Autoradiography experiments revealed binding of [3H]MODAG-005 in cortical areas of PD and AD brain tissue.
The second part of this thesis aimed to develop a simultaneous PET/fMRI approach in rats to correlate changes in dopamine availability and presynaptic dysfunction to whole-brain functional changes. For this purpose [11C]raclopride, a widely used PET tracer to measure striatal changes in dopamine release, in combination with BOLD-fMRI, was used in a rat model overexpressing the protein αSYN in the caudate putamen of one brain hemisphere. The reproducibility and reliability of both measures were first evaluat-ed using test-retest experiments in healthy rats. The variability within and between [11C]raclopride-PET scans and the reliability within and between fMRI scans were de-termined. Between scan variability of [11C]raclopride was 20.8 ± 14.2% at end of the acquisition (time interval T5 (86-95 minutes)) compared to the baseline, while within scan variability was 11.4 ± 17.1 %. Reliability between fMRI scans was moderate (r = 0.5 ± 0.1), but the correlation within fMRI scans was good (r = 0.7 ± 0.1). In the next step, the [11C]raclopride-PET/fMRI approach was used to investigate alterations in dopamine neurotransmission and its impact on functional connectivity in healthy rats and a rat model of αSYN overexpression by applying d-amphetamine as pharmacologi-cal stimulation. This study aimed to investigate the modulation of dopamine dynamics since it is involved in several vital central nervous functions such as movement, reward, sleep, attention, and learning. Furthermore, dopamine depletion in the nigrostriatal pathway is a hallmark of Parkinson’s disease, which has been reported to be involved in the impaired modulation of frontal cortical activity. Therefore, functional connectivity changes in the dopaminergic network and the default-mode network were assessed after d-amphetamine stimulation to delineate the effect of alterations of dopamine release on brain circuitry. Amphetamine-induced dopamine release decreased [11C]raclopride bind-ing in the caudate putamen, as well as functional connectivity in the dopaminergic net-work and the default-mode network affecting particular frontal cortical regions in healthy rats. Nigrostriatal dopamine depletion increased [11C]raclopride binding in the αSYN overexpressing caudate putamen. Using standard quantification methods with the cerebellum as a reference region, we did not observe any dopamine release in the αSYN overexpressing caudate putamen after d-amphetamine stimulation. Other quanti-fication approaches, independent of a reference region, are currently being tested to de-termine extrasynaptic dopamine fluctuations. D-amphetamine-induced functional con-nectivity response was decreased after the unilateral nigrostriatal dopamine depletion in both hemispheres.
In summary, the present dissertation provided preliminary evidence that MODAG-001 and its metabolite MODAG-005 are promising lead structures for the future develop-ment of a potential PET tracer targeting αSYN aggregation. However, its non-specific binding must be reduced to achieve a higher signal-to-noise ratio in human brain tissue with αSYN pathology. Since no PET tracer is still available for imaging αSYN aggrega-tion, other approaches, such as multimodal imaging to detect pre- and postsynaptic dys-functions, are very valuable linking molecular changes caused by the aggregation of αSYN to neuronal functional changes. The presented work demonstrated a relationship between dopamine depletion and a decreased frontal cortical response after ampheta-mine-induced dopamine release using a simultaneous [11C]raclopride-PET/fMRI ap-proach in a rat model of αSYN overexpression.
