Periodic Reporting for period 2 - oxDOPAMINE (Unraveling the mystery of preferential degeneration of midbrain neurons in neurodegenerative diseases)
Reporting period: 2023-01-01 to 2024-06-30
Parkinson's disease (PD) is the fastest growing neurodegenerative disease worldwide, while Neurodegeneration with Brain Iron Accumulation (NBIA) disorders are very rare. A major hallmark in both diseases is the death of dopaminergic neurons in the midbrain. There are still no causal therapies available to halt or slow down this neurodegeneration. The progressive course of this disease is difficult to understand in conventional animal or cell culture models. However, this is of crucial importance in order to develop causal therapies for early intervention in the disease processes.
In my recent work, we discovered a time-dependent pathological cascade beginning with mitochondrial oxidant stress leading to oxidation of dopamine (DA) and ultimately resulting in lysosomal dysfunction in induced pluripotent stem cell (iPSC)-derived DA neurons from PD patients (Burbulla et al., Science, 2017). This work uncovered DA oxidation (oxidation occurring when DA is improperly packaged into synaptic vesicles and stays in the cytosol) as a major mediator of dysfunction in multiple forms of PD. Importantly, this pathogenic cascade was not found in PD mouse models due to species-specific differences in DA metabolism, a fact that may - at least in part - explain relative resistance of rodent neurons to degeneration in genetic models of PD.
The mechanism of toxicity of cytosolic DA that predisposes human neurons to selective vulnerability and degeneration is unknown and studies on human neurons are needed for understanding disease progression and development of therapeutics. In fact, one of the most promising advantages of human iPSCs is their utility as a patient-specific disease model, offering the opportunity to gain deeper insights into disease mechanisms and progression.
In oxDOPAMINE we use PD and BPAN (subtype of Neurodegeneration with Brain Iron Accumulation, NBIA) patient-derived neurons to uncover the origin and nature of DA oxidation that predisposes human neurons to selective vulnerability and degeneration. We hypothesize that defective synaptic DA metabolism and iron dyshomeostasis play a critical role in the oxidation of cytosolic DA early in disease pathogenesis. A unifying feature of these pathogenic mechanisms is that impaired handling of DA contributes to toxicity of DA neurons. oxDOPAMINE will be of broad significance to a large cohort of patients with neurodegenerative diseases as it will advance our understanding of whether restoration of synaptic dysfunction and iron metabolism may prevent midbrain DA neurodegeneration, to represent a potential therapeutic target.
In my recent work, we discovered a time-dependent pathological cascade beginning with mitochondrial oxidant stress leading to oxidation of dopamine (DA) and ultimately resulting in lysosomal dysfunction in induced pluripotent stem cell (iPSC)-derived DA neurons from PD patients (Burbulla et al., Science, 2017). This work uncovered DA oxidation (oxidation occurring when DA is improperly packaged into synaptic vesicles and stays in the cytosol) as a major mediator of dysfunction in multiple forms of PD. Importantly, this pathogenic cascade was not found in PD mouse models due to species-specific differences in DA metabolism, a fact that may - at least in part - explain relative resistance of rodent neurons to degeneration in genetic models of PD.
The mechanism of toxicity of cytosolic DA that predisposes human neurons to selective vulnerability and degeneration is unknown and studies on human neurons are needed for understanding disease progression and development of therapeutics. In fact, one of the most promising advantages of human iPSCs is their utility as a patient-specific disease model, offering the opportunity to gain deeper insights into disease mechanisms and progression.
In oxDOPAMINE we use PD and BPAN (subtype of Neurodegeneration with Brain Iron Accumulation, NBIA) patient-derived neurons to uncover the origin and nature of DA oxidation that predisposes human neurons to selective vulnerability and degeneration. We hypothesize that defective synaptic DA metabolism and iron dyshomeostasis play a critical role in the oxidation of cytosolic DA early in disease pathogenesis. A unifying feature of these pathogenic mechanisms is that impaired handling of DA contributes to toxicity of DA neurons. oxDOPAMINE will be of broad significance to a large cohort of patients with neurodegenerative diseases as it will advance our understanding of whether restoration of synaptic dysfunction and iron metabolism may prevent midbrain DA neurodegeneration, to represent a potential therapeutic target.
Induced pluripotent stem cells (iPSCs) from patients with Parkinson’s disease (PD), control iPSC lines with CRISPR-edited knockout (KO) of the PD-associated gene DJ-1, PD patients with Parkin mutations, patients with BPAN (subtype NBIA) and from healthy controls were differentiated into midbrain dopaminergic neurons and mechanisms of selective vulnerability examined, in particular determinants that may affect the equilibrium between a) packaging of dopamine (DA) into synaptic vesicles, b) enzymatic degradation of cytosolic DA, and c) iron homeostasis, that may all lead to elevated levels of toxic DA oxidation.
Our preliminary results point towards alterations of the bioenergetic status, deficits in uptake of DA into synaptic vesicles as well as misregulated enzymatic degradation of DA into non-toxic DA metabolites in PD patient lines. Subsequent unbiased proteomics also uncovered key proteins/pathways involved in DA metabolism to be dysregulated in patient neurons. The affected regulation of DA metabolism may possibly explain the earlier described high amounts of oxidized DA (Burbulla et al., Science, 2017). Also, iron regulatory proteins were largely misregulated that may at least partly add to the observed phenotype of excessive DA oxidation that occurs through iron catalyzing cytosolic DA into toxic DA oxidation products.
Our preliminary results point towards alterations of the bioenergetic status, deficits in uptake of DA into synaptic vesicles as well as misregulated enzymatic degradation of DA into non-toxic DA metabolites in PD patient lines. Subsequent unbiased proteomics also uncovered key proteins/pathways involved in DA metabolism to be dysregulated in patient neurons. The affected regulation of DA metabolism may possibly explain the earlier described high amounts of oxidized DA (Burbulla et al., Science, 2017). Also, iron regulatory proteins were largely misregulated that may at least partly add to the observed phenotype of excessive DA oxidation that occurs through iron catalyzing cytosolic DA into toxic DA oxidation products.
ur observed phenotypes may, so far, at least partly explain the increased susceptibility observed in dopaminergic neurons in PD and BPAN since it can be expected that altered uptake of DA into synaptic vesicles and a dysregulation of iron metabolism add to DA neuron pathology.
To enhance our understanding of DA neuron vulnerability in PD and BPAN, we are further investigating mechanistic pathways behind the origin of DA oxidation in human nigral neurons. We will examine rescue strategies by targeting DA oxidation and improving DA packaging. Further, we will challenge the neurons by modulating enzyme activities using specific inhibitors to determine the neuron’s capacity to metabolize DA in the cytosol into the non-toxic metabolite DOPAC. We will also analyze the observed iron misregulation further, investigate upstream mechanisms driving iron accumulation and determine whether degradation of iron-rich macromolecules such as mitochondria or iron-loaded ferritin are disrupted in PD and BPAN patient neurons.
We expect that our results so far and until the end of this project will enhance the knowledge that is of significant importance for understanding selective vulnerability of midbrain dopaminergic neurons, potentially leading to the identification of potential therapeutic targets.
To enhance our understanding of DA neuron vulnerability in PD and BPAN, we are further investigating mechanistic pathways behind the origin of DA oxidation in human nigral neurons. We will examine rescue strategies by targeting DA oxidation and improving DA packaging. Further, we will challenge the neurons by modulating enzyme activities using specific inhibitors to determine the neuron’s capacity to metabolize DA in the cytosol into the non-toxic metabolite DOPAC. We will also analyze the observed iron misregulation further, investigate upstream mechanisms driving iron accumulation and determine whether degradation of iron-rich macromolecules such as mitochondria or iron-loaded ferritin are disrupted in PD and BPAN patient neurons.
We expect that our results so far and until the end of this project will enhance the knowledge that is of significant importance for understanding selective vulnerability of midbrain dopaminergic neurons, potentially leading to the identification of potential therapeutic targets.