Limits...
Increased mitochondrial calcium sensitivity and abnormal expression of innate immunity genes precede dopaminergic defects in Pink1-deficient mice.

Akundi RS, Huang Z, Eason J, Pandya JD, Zhi L, Cass WA, Sullivan PG, Büeler H - PLoS ONE (2011)

Bottom Line: Pink1 deletion results in impaired dopamine (DA) release and decreased mitochondrial respiration in the striatum of mice.Moreover, Pink1⁻/⁻ mice had increased levels of IL-1β, IL-12 and IL-10 in the striatum after peripheral challenge with lipopolysaccharide (LPS), and Pink1⁻/⁻ embryonic fibroblasts showed decreased basal and inflammatory cytokine-induced nuclear factor kappa-β (NF-κB) activity.Differential gene expression in the nigrostriatal system of Pink1⁻/⁻ mice supports early dopaminergic dysfunction and shows that Pink1 deletion causes aberrant expression of genes that regulate innate immune responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Neurobiology, University of Kentucky College of Medicine, Lexington, Kentucky, United States of America.

ABSTRACT

Background: PTEN-induced kinase 1 (PINK1) is linked to recessive Parkinsonism (EOPD). Pink1 deletion results in impaired dopamine (DA) release and decreased mitochondrial respiration in the striatum of mice. To reveal additional mechanisms of Pink1-related dopaminergic dysfunction, we studied Ca²+ vulnerability of purified brain mitochondria, DA levels and metabolism and whether signaling pathways implicated in Parkinson's disease (PD) display altered activity in the nigrostriatal system of Pink1⁻/⁻ mice.

Methods and findings: Purified brain mitochondria of Pink1⁻/⁻ mice showed impaired Ca²+ storage capacity, resulting in increased Ca²+ induced mitochondrial permeability transition (mPT) that was rescued by cyclosporine A. A subpopulation of neurons in the substantia nigra of Pink1⁻/⁻ mice accumulated phospho-c-Jun, showing that Jun N-terminal kinase (JNK) activity is increased. Pink1⁻/⁻ mice 6 months and older displayed reduced DA levels associated with increased DA turnover. Moreover, Pink1⁻/⁻ mice had increased levels of IL-1β, IL-12 and IL-10 in the striatum after peripheral challenge with lipopolysaccharide (LPS), and Pink1⁻/⁻ embryonic fibroblasts showed decreased basal and inflammatory cytokine-induced nuclear factor kappa-β (NF-κB) activity. Quantitative transcriptional profiling in the striatum revealed that Pink1⁻/⁻ mice differentially express genes that (i) are upregulated in animals with experimentally induced dopaminergic lesions, (ii) regulate innate immune responses and/or apoptosis and (iii) promote axonal regeneration and sprouting.

Conclusions: Increased mitochondrial Ca²+ sensitivity and JNK activity are early defects in Pink1⁻/⁻ mice that precede reduced DA levels and abnormal DA homeostasis and may contribute to neuronal dysfunction in familial PD. Differential gene expression in the nigrostriatal system of Pink1⁻/⁻ mice supports early dopaminergic dysfunction and shows that Pink1 deletion causes aberrant expression of genes that regulate innate immune responses. While some differentially expressed genes may mitigate neurodegeneration, increased LPS-induced brain cytokine expression and impaired cytokine-induced NF-κB activation may predispose neurons of Pink1⁻/⁻ mice to inflammation and injury-induced cell death.

Show MeSH

Related in: MedlinePlus

Pink1−/− brain mitochondria have significantly lower Ca2+ load capacity.Brain mitochondria were isolated from 2 month-old male wildtype (WT) and Pink1−/− (PINK1-KO) mice to measure calcium load capacity. Mitochondrial protein (200 µg) from each genotype was incubated in a 2-ml reaction mixture containing 125 mM KCl buffer (37°C) along with fluorescence indicators: 100 nM Calcium Green 5-N (for measurement of extra-mitochondrial calcium concentration; excitation 506 nm, emission 532 nm) and 100 nM TMRE (for measurement of membrane potential; excitation 550 nm, emission 575 nm). Mitochondrial bioenergetic coupling was assessed by following changes in membrane potential (TMRE fluorescence, data not shown) following the addition of pyruvate + malate (PM), ADP (A) and oligomycin (O) at 1, 2 and 3 min respectively. Calcium infusion began at 5 min (rate of 160 nmol/mg protein/min) and changes in extra-mitochondrial Ca2+ were assessed (CaG5N fluorescence). In Panel A, representative traces are shown to depict the calcium uptake and storage capacity before the opening of the mitochondrial permeability transition (mPT) pore. Mitochondria from PINK1-KO mice had decreased calcium load capacity, indicated by the earlier onset of mPT (sharp rise in extra-mitochondrial Ca2+) as compared to WT mice. Calcium load capacity in PINK-KO mice was increased by incubating mitochondria with cyclosporine A (CSA, 1 µM), a specific inhibitor of the PTP, showing that the reduced calcium load capacity was due to enhanced mPT. In Panel B, quantitative measurements of maximal calcium load capacity before mPT for mitochondria isolated from WT and PINK1-KO mice are shown and expressed as nmol Ca2+ infused/mg protein. The graph in panel B illustrates a significant loss of calcium load capacity in PINK1-KO mice (*P<0.05, n = 4 mice per genotype).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3020954&req=5

pone-0016038-g004: Pink1−/− brain mitochondria have significantly lower Ca2+ load capacity.Brain mitochondria were isolated from 2 month-old male wildtype (WT) and Pink1−/− (PINK1-KO) mice to measure calcium load capacity. Mitochondrial protein (200 µg) from each genotype was incubated in a 2-ml reaction mixture containing 125 mM KCl buffer (37°C) along with fluorescence indicators: 100 nM Calcium Green 5-N (for measurement of extra-mitochondrial calcium concentration; excitation 506 nm, emission 532 nm) and 100 nM TMRE (for measurement of membrane potential; excitation 550 nm, emission 575 nm). Mitochondrial bioenergetic coupling was assessed by following changes in membrane potential (TMRE fluorescence, data not shown) following the addition of pyruvate + malate (PM), ADP (A) and oligomycin (O) at 1, 2 and 3 min respectively. Calcium infusion began at 5 min (rate of 160 nmol/mg protein/min) and changes in extra-mitochondrial Ca2+ were assessed (CaG5N fluorescence). In Panel A, representative traces are shown to depict the calcium uptake and storage capacity before the opening of the mitochondrial permeability transition (mPT) pore. Mitochondria from PINK1-KO mice had decreased calcium load capacity, indicated by the earlier onset of mPT (sharp rise in extra-mitochondrial Ca2+) as compared to WT mice. Calcium load capacity in PINK-KO mice was increased by incubating mitochondria with cyclosporine A (CSA, 1 µM), a specific inhibitor of the PTP, showing that the reduced calcium load capacity was due to enhanced mPT. In Panel B, quantitative measurements of maximal calcium load capacity before mPT for mitochondria isolated from WT and PINK1-KO mice are shown and expressed as nmol Ca2+ infused/mg protein. The graph in panel B illustrates a significant loss of calcium load capacity in PINK1-KO mice (*P<0.05, n = 4 mice per genotype).

Mentions: Recently, it has been shown that mitochondria of Pink1-deficient neurons accumulate higher basal levels of Ca2+ in the matrix (due to reduced calcium efflux capacity) and display reduced mitochondrial Ca2+ storage capacity associated with Ca2+ overload [24]. To study whether mitochondria from the brain of Pink1-deficient mice show increased sensitivity to Ca2+, we exposed preparations of Ficoll-purified whole brain mitochondria from two month-old Pink1-deficient mice and wildtype controls to increasing concentrations of Ca2+, and measured PTP opening using the Ca2+-sensitive fluorescent dye CaG5N and TMRE to monitor changes in mitochondrial membrane potential (ΔΨM) [25]. Pink1-deficient brain mitochondria displayed a significant reduction in Ca2+ buffering capacity, which could be ameliorated by the addition of the PTP inhibitor cyclosporine A (CsA) (Fig. 4A). As a consequence mitochondria from Pink1−/− mice underwent permeability transition (mPT) at significantly lower concentrations of Ca 2+ compared to those of wildtype mice (Fig. 4B). Because CaG5N is an indicator of the extra-mitochondrial calcium concentration, the results cannot be attributed to differences in the dye loading capacity between wildtype and Pink1−/− mitochondria. We also measured mitochondrial production of reactive oxygen species (ROS) [25] but found no difference in ROS production between mitochondria purified from Pink1-deficient and wildtype brain (data not shown), consistent with previous results [18].


Increased mitochondrial calcium sensitivity and abnormal expression of innate immunity genes precede dopaminergic defects in Pink1-deficient mice.

Akundi RS, Huang Z, Eason J, Pandya JD, Zhi L, Cass WA, Sullivan PG, Büeler H - PLoS ONE (2011)

Pink1−/− brain mitochondria have significantly lower Ca2+ load capacity.Brain mitochondria were isolated from 2 month-old male wildtype (WT) and Pink1−/− (PINK1-KO) mice to measure calcium load capacity. Mitochondrial protein (200 µg) from each genotype was incubated in a 2-ml reaction mixture containing 125 mM KCl buffer (37°C) along with fluorescence indicators: 100 nM Calcium Green 5-N (for measurement of extra-mitochondrial calcium concentration; excitation 506 nm, emission 532 nm) and 100 nM TMRE (for measurement of membrane potential; excitation 550 nm, emission 575 nm). Mitochondrial bioenergetic coupling was assessed by following changes in membrane potential (TMRE fluorescence, data not shown) following the addition of pyruvate + malate (PM), ADP (A) and oligomycin (O) at 1, 2 and 3 min respectively. Calcium infusion began at 5 min (rate of 160 nmol/mg protein/min) and changes in extra-mitochondrial Ca2+ were assessed (CaG5N fluorescence). In Panel A, representative traces are shown to depict the calcium uptake and storage capacity before the opening of the mitochondrial permeability transition (mPT) pore. Mitochondria from PINK1-KO mice had decreased calcium load capacity, indicated by the earlier onset of mPT (sharp rise in extra-mitochondrial Ca2+) as compared to WT mice. Calcium load capacity in PINK-KO mice was increased by incubating mitochondria with cyclosporine A (CSA, 1 µM), a specific inhibitor of the PTP, showing that the reduced calcium load capacity was due to enhanced mPT. In Panel B, quantitative measurements of maximal calcium load capacity before mPT for mitochondria isolated from WT and PINK1-KO mice are shown and expressed as nmol Ca2+ infused/mg protein. The graph in panel B illustrates a significant loss of calcium load capacity in PINK1-KO mice (*P<0.05, n = 4 mice per genotype).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3020954&req=5

pone-0016038-g004: Pink1−/− brain mitochondria have significantly lower Ca2+ load capacity.Brain mitochondria were isolated from 2 month-old male wildtype (WT) and Pink1−/− (PINK1-KO) mice to measure calcium load capacity. Mitochondrial protein (200 µg) from each genotype was incubated in a 2-ml reaction mixture containing 125 mM KCl buffer (37°C) along with fluorescence indicators: 100 nM Calcium Green 5-N (for measurement of extra-mitochondrial calcium concentration; excitation 506 nm, emission 532 nm) and 100 nM TMRE (for measurement of membrane potential; excitation 550 nm, emission 575 nm). Mitochondrial bioenergetic coupling was assessed by following changes in membrane potential (TMRE fluorescence, data not shown) following the addition of pyruvate + malate (PM), ADP (A) and oligomycin (O) at 1, 2 and 3 min respectively. Calcium infusion began at 5 min (rate of 160 nmol/mg protein/min) and changes in extra-mitochondrial Ca2+ were assessed (CaG5N fluorescence). In Panel A, representative traces are shown to depict the calcium uptake and storage capacity before the opening of the mitochondrial permeability transition (mPT) pore. Mitochondria from PINK1-KO mice had decreased calcium load capacity, indicated by the earlier onset of mPT (sharp rise in extra-mitochondrial Ca2+) as compared to WT mice. Calcium load capacity in PINK-KO mice was increased by incubating mitochondria with cyclosporine A (CSA, 1 µM), a specific inhibitor of the PTP, showing that the reduced calcium load capacity was due to enhanced mPT. In Panel B, quantitative measurements of maximal calcium load capacity before mPT for mitochondria isolated from WT and PINK1-KO mice are shown and expressed as nmol Ca2+ infused/mg protein. The graph in panel B illustrates a significant loss of calcium load capacity in PINK1-KO mice (*P<0.05, n = 4 mice per genotype).
Mentions: Recently, it has been shown that mitochondria of Pink1-deficient neurons accumulate higher basal levels of Ca2+ in the matrix (due to reduced calcium efflux capacity) and display reduced mitochondrial Ca2+ storage capacity associated with Ca2+ overload [24]. To study whether mitochondria from the brain of Pink1-deficient mice show increased sensitivity to Ca2+, we exposed preparations of Ficoll-purified whole brain mitochondria from two month-old Pink1-deficient mice and wildtype controls to increasing concentrations of Ca2+, and measured PTP opening using the Ca2+-sensitive fluorescent dye CaG5N and TMRE to monitor changes in mitochondrial membrane potential (ΔΨM) [25]. Pink1-deficient brain mitochondria displayed a significant reduction in Ca2+ buffering capacity, which could be ameliorated by the addition of the PTP inhibitor cyclosporine A (CsA) (Fig. 4A). As a consequence mitochondria from Pink1−/− mice underwent permeability transition (mPT) at significantly lower concentrations of Ca 2+ compared to those of wildtype mice (Fig. 4B). Because CaG5N is an indicator of the extra-mitochondrial calcium concentration, the results cannot be attributed to differences in the dye loading capacity between wildtype and Pink1−/− mitochondria. We also measured mitochondrial production of reactive oxygen species (ROS) [25] but found no difference in ROS production between mitochondria purified from Pink1-deficient and wildtype brain (data not shown), consistent with previous results [18].

Bottom Line: Pink1 deletion results in impaired dopamine (DA) release and decreased mitochondrial respiration in the striatum of mice.Moreover, Pink1⁻/⁻ mice had increased levels of IL-1β, IL-12 and IL-10 in the striatum after peripheral challenge with lipopolysaccharide (LPS), and Pink1⁻/⁻ embryonic fibroblasts showed decreased basal and inflammatory cytokine-induced nuclear factor kappa-β (NF-κB) activity.Differential gene expression in the nigrostriatal system of Pink1⁻/⁻ mice supports early dopaminergic dysfunction and shows that Pink1 deletion causes aberrant expression of genes that regulate innate immune responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Neurobiology, University of Kentucky College of Medicine, Lexington, Kentucky, United States of America.

ABSTRACT

Background: PTEN-induced kinase 1 (PINK1) is linked to recessive Parkinsonism (EOPD). Pink1 deletion results in impaired dopamine (DA) release and decreased mitochondrial respiration in the striatum of mice. To reveal additional mechanisms of Pink1-related dopaminergic dysfunction, we studied Ca²+ vulnerability of purified brain mitochondria, DA levels and metabolism and whether signaling pathways implicated in Parkinson's disease (PD) display altered activity in the nigrostriatal system of Pink1⁻/⁻ mice.

Methods and findings: Purified brain mitochondria of Pink1⁻/⁻ mice showed impaired Ca²+ storage capacity, resulting in increased Ca²+ induced mitochondrial permeability transition (mPT) that was rescued by cyclosporine A. A subpopulation of neurons in the substantia nigra of Pink1⁻/⁻ mice accumulated phospho-c-Jun, showing that Jun N-terminal kinase (JNK) activity is increased. Pink1⁻/⁻ mice 6 months and older displayed reduced DA levels associated with increased DA turnover. Moreover, Pink1⁻/⁻ mice had increased levels of IL-1β, IL-12 and IL-10 in the striatum after peripheral challenge with lipopolysaccharide (LPS), and Pink1⁻/⁻ embryonic fibroblasts showed decreased basal and inflammatory cytokine-induced nuclear factor kappa-β (NF-κB) activity. Quantitative transcriptional profiling in the striatum revealed that Pink1⁻/⁻ mice differentially express genes that (i) are upregulated in animals with experimentally induced dopaminergic lesions, (ii) regulate innate immune responses and/or apoptosis and (iii) promote axonal regeneration and sprouting.

Conclusions: Increased mitochondrial Ca²+ sensitivity and JNK activity are early defects in Pink1⁻/⁻ mice that precede reduced DA levels and abnormal DA homeostasis and may contribute to neuronal dysfunction in familial PD. Differential gene expression in the nigrostriatal system of Pink1⁻/⁻ mice supports early dopaminergic dysfunction and shows that Pink1 deletion causes aberrant expression of genes that regulate innate immune responses. While some differentially expressed genes may mitigate neurodegeneration, increased LPS-induced brain cytokine expression and impaired cytokine-induced NF-κB activation may predispose neurons of Pink1⁻/⁻ mice to inflammation and injury-induced cell death.

Show MeSH
Related in: MedlinePlus