Mechanistic disease stratification will be crucial to develop a precision medicine approach for future disease modifying therapy in sporadic Parkinson's disease (sPD). Mitochondrial and lysosomal dysfunction are key mechanisms in the pathogenesis of sPD and therefore promising targets for therapeutic intervention. We investigated mitochondrial and lysosomal function in skin fibroblasts of 100 sPD patients and 50 age-matched controls. A combination of cellular assays, RNA-seq based pathway analysis and genotyping was applied. Distinct subgroups with mitochondrial (mito-sPD) or lysosomal (lyso-sPD) dysfunction were identified. Mitochondrial dysfunction correlated with reduction in complex I and IV protein levels. RNA-seq based pathway analysis revealed marked activation of the lysosomal pathway with enrichment for lysosomal disease gene variants in lyso-sPD. Conversion of fibroblasts to induced neuronal progenitor cells and subsequent differentiation into tyrosine hydroxylase positive neurons confirmed and further enhanced both mitochondrial and lysosomal abnormalities. Treatment with ursodeoxycholic acid improved mitochondrial membrane potential and intracellular ATP levels even in sPD patient fibroblast lines with comparatively mild mitochondrial dysfunction. The results of our study suggest that in-depth phenotyping and focussed assessment of putative neuroprotective compounds in peripheral tissue are a promising approach towards disease stratification and precision medicine in sPD.

Mutations in PRKN are the most common cause of early onset Parkinson’s disease. Parkin is an E3 ubiquitin ligase, functioning in mitophagy. Mitochondrial abnormalities are present in PRKN mutant models. Patient derived neurons are a promising model in which to study pathogenic mechanisms and therapeutic targets. Here we generate induced neuronal progenitor cells from PRKN mutant patient fibroblasts with a high dopaminergic neuron yield. We reveal changing mitochondrial phenotypes as neurons undergo a metabolic switch during differentiation. Fibroblasts from 4 controls and 4 PRKN mutant patients were transformed into induced neuronal progenitor cells and subsequently differentiated into dopaminergic neurons. Mitochondrial morphology, function and mitophagy were evaluated using live cell fluorescent imaging, cellular ATP and reactive oxygen species production quantification. Direct conversion of control and PRKN mutant patient fibroblasts results in induced neuronal progenitor and their differentiation yields high percentage of dopaminergic neurons. We were able to observe changing mitochondrial phenotypes as neurons undergo a metabolic switch during differentiation. Our results show that when pre-neurons are glycolytic early in differentiation mitophagy is unimpaired by PRKN deficiency. However as neurons become oxidative phosphorylation dependent, mitophagy is severely impaired in the PRKN mutant patient neurons. These changes correlate with changes in mitochondrial function and morphology; resulting in lower neuron yield and altered neuronal morphology. Induced neuronal progenitor cell conversion can produce a high yield of dopaminergic neurons. The mitochondrial phenotype, including mitophagy status, is highly dependent on the metabolic status of the cell. Only when neurons are oxidative phosphorylation reliant the extent of mitochondrial abnormalities are identified. These data provide insight into cell specific effects of PRKN mutations, in particular in relation to mitophagy dependent disease phenotypes and provide avenues for alternative therapeutic approaches.

The insulin/insulin-like growth factor 1 (IGF1) signaling pathways are implicated in longevity and in progression of Alzheimer's disease. Previously, we showed that insulin-like growth factor 1 receptor (IGF1R) and downstream signaling transcripts are reduced in astrocytes in human brain with progression of Alzheimer's neuropathology and developed a model of IGF1 signaling impairment in human astrocytes using an IGF1R-specific monoclonal antibody, MAB391. Here, we have established a novel human astrocyte-neuron co-culture system to determine whether loss of astrocytic IGF1R affects their support for neurons. Astrocyte-neuron co-cultures were developed using human primary astrocytes and differentiated Lund Human Mesencephalic Cells (LUHMES). Neurite outgrowth assays, performed to measure astrocytic support for neurons, showed astrocytes provided contact-mediated support for neurite outgrowth. Loss of IGF1R did not affect neurite outgrowth under control conditions but when challenged with hydrogen peroxide IGF1R-impaired astrocytes were less able to protect LUHMES. To determine how loss of IGF1R affects neuronal support MAB391-treated astrocytes were FACS sorted from GFP-LUHMES and their transcriptomic profile was investigated using microarrays. Changes in transcripts involved in astrocyte energy metabolism were identified, particularly NDUFA2 and NDUFB6, which are related to complex I assembly. Loss of complex I activity in MAB391-treated astrocytes validated these findings. In conclusion, reduced IGF1 signaling in astrocytes impairs their support for neurons under conditions of stress and this is associated with defects in the mitochondrial respiratory chain in astrocytes.

Mutations in LRRK2 are the most common cause of dominantly inherited Parkinson's disease (PD). A proportion of LRRK2 PD exhibits Lewy pathology with accumulations of α-synuclein and ubiquitin in intracellular aggregates that are indistinguishable from idiopathic PD. LRRK2 is a multi-domain protein with both GTPase and kinase activities that has been shown to affect various cellular processes including protein

22Background Mutations in parkin are the most common cause of early onset Parkinson's disease. Parkin 23 is an E3 ubiquitin ligase, functioning in mitophagy. Mitochondrial abnormalities are present in parkin 24 mutant models. Patient derived neurons are a promising model in which to study pathogenic 25 mechanisms and therapeutic targets. Here we generate induced neuronal progenitor cells from parkin 26 mutant patient fibroblasts with a high dopaminergic neuron yield. We reveal changing mitochondrial 27 phenotypes as neurons undergo a metabolic switch during differentiation. Methods Fibroblasts from 28 4 controls and 4 parkin mutant patients were transformed into induced neuronal progenitor cells and 29 subsequently differentiated into dopaminergic neurons. Mitochondrial morphology, function and 30 mitophagy were evaluated using live cell fluorescent imaging, cellular ATP and reactive oxygen species 31 production quantification. Results Direct conversion of control and parkin mutant patient fibroblasts 32 results in induced neuronal progenitor and their differentiation yields high percentage of 33 dopaminergic neurons. We were able to observe changing mitochondrial phenotypes as neurons 34 undergo a metabolic switch during differentiation. Our results show that when pre-neurons are 35 glycolytic early in differentiation mitophagy is unimpaired by PRKN deficiency. However as neurons 36 become oxidative phosphorylation dependent, mitophagy is severely impaired in the PRKN mutant 37 patient neurons. These changes correlate with changes in mitochondrial function and morphology; 38 resulting in lower neuron yield and altered neuronal morphology. Conclusions Induced neuronal 39 progenitor cell conversion can produce a high yield of dopaminergic neurons. The mitochondrial 40 phenotype, including mitophagy status, is highly dependent on the metabolic status of the cell. Only 41 when neurons are oxidative phosphorylation reliant the extent of mitochondrial abnormalities are 42 identified. These data provide insight into cell specific effects of PRKN mutations, in particular in 43 relation to mitophagy dependent disease phenotypes and provide avenues for alternative therapeutic 44 approaches. 45 46 47 48 Parkinson's disease (PD) is the second most common neurodegenerative disease, with 49 approximately 10 million people affected worldwide. Only symptomatic treatment options are 50 available. Mutations in PRKN are the most common cause of early onset PD (EOPD). Parkin is an E3 51 ubiquitin ligase and functions in the mitophagy pathway 1 . Mitochondrial dysfunction is well 52 established in both familial and sporadic forms of PD (recently reviewed 2 ). Mitochondrial 53 abnormalities are present in both PRKN null Drosophila 3 and mice 4 . We and others have shown 54 mitochondrial abnormalities in peripheral cells from patients with PRKN mutations 5-8 ; these include 55 cellular ATP defects, mitochondrial membrane potential deficiencies, complex I defect and altered 56 mitochondrial morphology. Recent work suggests mitophagy is...