ABSTRACT
It has been uncertain whether specific disease-relevant biomarker phenotypes can be found using sporadic Parkinson's disease (PD) patient-derived samples, as it has been proposed that there may be a plethora of underlying causes and pathological mechanisms. Fibroblasts derived from familial PD patients harboring leucine-rich repeat kinase 2 (LRRK2), PTEN-induced putative kinase 1 (PINK1), and Parkin mutations show clear disease-relevant mitochondrial phenotypes, which are exacerbated under conditions of pharmacological stress. We utilized fibroblasts derived from non-familial sporadic PD patients (without LRRK2 mutations) or LRRK2 mutation carriers to directly compare the cellular phenotypes during and after mitochondrial stress. We then determined the effects of pharmacological LRRK2 kinase inhibition using LRRK2-in-1. We found that there were two distinct populations of sporadic PD patient-derived fibroblast lines. One group of sporadic PD lines was highly susceptible to valinomycin-induced mitochondrial depolarization, emulating the mutant LRRK2 phenotype. These lines showed elevated mitochondrial superoxide/ nitric oxide levels, displayed increased mitochondrial and lysosome co-localization, and an increased rate of mitochondrial collapse, which corresponded with changes in mitochondrial fission and fusion proteins. The application of LRRK2-in-1 reversed decreased levels of mitochondrial and lysosome co-localization and partially restored mitochondrial network associated proteins and the mitochondrial membrane potential in the fibroblasts. This study identifies novel mitochondrial biomarkers in sporadic PD patient-derived fibroblast lines, which could be used as preclinical tools in which to test novel and known neuroprotective compounds.
Subject(s)
Biomarkers/metabolism , Fibroblasts/metabolism , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/antagonists & inhibitors , Parkinson Disease/enzymology , Benzodiazepinones/pharmacology , Cell Line , Humans , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/metabolism , Lysosomes/drug effects , Lysosomes/metabolism , Membrane Potential, Mitochondrial/drug effects , Mitochondria/drug effects , Mitochondria/metabolism , Mitochondrial Dynamics/drug effects , Parkinson Disease/pathology , Protein Kinases/metabolism , Pyrimidines/pharmacology , Stress, Physiological/drug effects , Ubiquitin-Protein Ligases/metabolism , Valinomycin/pharmacologyABSTRACT
In this study, we examined protein-protein interactions between two neuronal receptors, low density lipoprotein receptor-related protein (LRP) and sorLA/LR11, and found that these receptors interact, as indicated by three independent lines of evidence: co-immunoprecipitation experiments on mouse brain extracts and mouse neuronal cells, surface plasmon resonance analysis with purified human LRP and sorLA, and fluorescence lifetime imaging microscopy (FLIM) on rat primary cortical neurons. Immunocytochemistry experiments revealed widespread co-localization of LRP and sorLA within perinuclear compartments of rat primary neurons, while FLIM analysis showed that LRP-sorLA interactions take place within a subset of these compartments.
Subject(s)
LDL-Receptor Related Proteins/metabolism , Receptors, LDL/metabolism , Animals , Binding Sites , Cells, Cultured , Embryo, Mammalian , Green Fluorescent Proteins/genetics , Humans , Immunoprecipitation/methods , LDL-Receptor Related Proteins/genetics , Mice , Microscopy, Fluorescence , Neuroblastoma , Neurons/metabolism , Protein Binding , Protein Interaction Domains and Motifs , Protein Interaction Mapping/methods , Rats , Rats, Sprague-Dawley , Receptors, LDL/genetics , Surface Plasmon Resonance/methods , Transfection/methodsABSTRACT
OFF-L-dopa dyskinesias have been a surprising side-effect of intrastriatal foetal ventral mesencephalic transplantation in patients with Parkinson's disease. It has been proposed that excessive and unregulated dopaminergic stimulation of host post-synaptic striatal neurons by the grafts could be responsible for these dyskinesias. To address this issue we transplanted foetal dopaminergic neurons from mice lacking the dopamine transporter (DATKO) or from wild-type mice, into a rat model of Parkinson's disease and L-dopa-induced dyskinesias. Both wild-type and DATKO grafts reinnervated the host striatum to a similar extent, but DATKO grafts produced a greater and more diffuse increase in extra-cellular striatal dopamine levels. Interestingly, grafts containing wild-type dopaminergic neurons improved parkinsonian signs to a similar extent as DATKO grafts, but provided a more complete reduction of L-dopa induced dyskinesias. Neither DATKO nor wild-type grafts induced OFF-L-dopa dyskinesias. Behavioural and receptor autoradiography analyses demonstrated that DATKO grafts induced a greater normalization of striatal dopaminergic receptor supersensitivity than wild-type grafts. Both graft types induced a similar downregulation and normalization of PEnk and fosb/Deltafosb in striatal neurons. In summary, DATKO grafts causing high and diffuse extra-cellular dompamine levels do not per se alter graft-induced recovery or produce OFF-L-dopa dyskinesias. Wild-type dopaminergic neurons appear to be the most effective neuronal type to restore function and reduce L-dopa-induced dyskinesias.
Subject(s)
Brain Tissue Transplantation , Dopamine Plasma Membrane Transport Proteins/deficiency , Dyskinesia, Drug-Induced/etiology , Levodopa/adverse effects , Parkinson Disease/therapy , Animals , Antiparkinson Agents/adverse effects , Antiparkinson Agents/therapeutic use , Corpus Striatum/metabolism , Corpus Striatum/pathology , Disease Models, Animal , Dopamine/metabolism , Dopamine Plasma Membrane Transport Proteins/physiology , Dyskinesia, Drug-Induced/therapy , Enkephalins/biosynthesis , Enkephalins/genetics , Female , Fetal Tissue Transplantation , Gene Expression Regulation , Graft Survival , Levodopa/therapeutic use , Mesencephalon/transplantation , Microdialysis/methods , Neurons/transplantation , Parkinson Disease/drug therapy , Parkinson Disease/metabolism , Parkinson Disease/pathology , Protein Precursors/biosynthesis , Protein Precursors/genetics , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley , Receptors, Dopamine D1/metabolism , Receptors, Dopamine D2/metabolism , Receptors, N-Methyl-D-Aspartate/metabolismABSTRACT
The development of dyskinesias and other motor complications greatly limits the use of levodopa therapy in Parkinson's disease (PD). Studies in rodent models of PD suggest that an important mechanism underlying the development of levodopa-related motor complications is alterations in striatal NMDA receptor function. We examined striatal NMDA receptors in the MPTP-lesioned primate model of PD. Quantitative immunoblotting was used to determine the subcellular abundance of NR1, NR2A and NR2B subunits in striata from unlesioned, MPTP-lesioned (parkinsonian) and MPTP-lesioned, levodopa-treated (dyskinetic) macaques. In parkinsonian macaques, NR1 and NR2B subunits in synaptosomal membranes were decreased to 66 +/- 11% and 51.2 +/- 5% of unlesioned levels respectively, while the abundance of NR2A was unaltered. Levodopa treatment eliciting dyskinesia normalized NR1 and NR2B and increased NR2A subunits to 150 +/- 12% of unlesioned levels. No alterations in receptor subunit tyrosine phosphorylation were detected. These results demonstrate that altered synaptic abundance of NMDA receptors with relative enhancement in the abundance of NR2A occurs in primate as well as rodent models of parkinsonism, and that in the macaque model, NR2A subunit abundance is further increased in dyskinesia. These data support the view that alterations in striatal NMDA receptor systems are responsible for adaptive and maladaptive responses to dopamine depletion and replacement in parkinsonism, and highlight the value of subtype selective NMDA antagonists as novel therapeutic approaches for PD.
