Pink1/Parkin deficiency alters circulating lymphocyte populations and increases platelet-T cell aggregates in rats

Parkinson’s disease (PD) is the most common progressive neurodegenerative movement disorder and results from the selective loss of dopaminergic neurons in the substantia nigra pars compacta. Pink1 and Parkin are proteins that function together in mitochondrial quality control, and when they carry loss-of-function mutations lead to familial forms of PD. While much research has focused on central nervous system alterations in PD, peripheral contributions to PD pathogenesis are increasingly appreciated. We report Pink1/Parkin regulate glycolytic and mitochondrial oxidative metabolism in peripheral blood mononuclear cells (PBMCs) from rats. Pink1/Parkin deficiency induces changes in the circulating lymphocyte populations, namely increased CD4 + T cells and decreased CD8 + T cells and B cells. Loss of Pink1/Parkin leads to elevated platelet counts in the blood and increased platelet-T cell aggregation. Platelet-lymphocyte aggregates are associated with increased thrombosis risk, and venous thrombosis is a cause of sudden death in PD, suggesting targeting the Pink1/Parkin pathway in the periphery has therapeutic potential.


Introduction
Parkinson's disease (PD) is a prevalent, chronic and progressive neurodegenerative disorder that is clinically characterized by motor symptoms, including bradykinesia, rigidity, resting tremor, and postural instability 1 .Diagnosis is largely based on clinical symptoms, but de nitive con rmation of the disease requires pathological examination at autopsy, where progressive degeneration of nigral dopamine (DA) neurons, along with Lewy bodies in surviving neurons, is observed 2 .Currently, therapies only treat PD symptoms, mostly by enhancing DA signaling, which is required for normal movement, but do not slow PD progression or protect against neuronal cell death.Most cases of PD are idiopathic, and there is as yet no diagnostic or predictive molecular marker of disease.
Neurotoxins, such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) 3 , paraquat and rotenone 4 , which cause increased reactive oxygen species (ROS) generation and mitochondrial dysfunction, can induce parkinsonism in humans and animal models.The most widely used rodent model of PD is the 6hydroxydopamine model in which a unilateral lesion causes asymmetrical presentation of symptoms including degeneration of nigral DA neurons and gait de cits 5,6 .The identi cation of hereditary forms of PD uncovered gene mutations and variants, such as loss-of-function mutations in Pink1 and Parkin, two proteins involved in mitochondrial quality control 7 , that could be harnessed to create genetic mammalian animal models of PD 8 .Parkin is an E3 ubiquitin ligase, targeting proteins for degradation through the ubiquitin-proteasome system, as well as marking proteins on the outer mitochondrial membrane to target mitochondria for autophagic destruction (mitophagy) 9,10 .Phosphatase and tensin homolog (PTEN)-induced kinase 1 (Pink1) accumulates on the surface of mitochondria in response to stress (e.g.ROS 11 and mitochondrial depolarization 12,13 ) and recruits Parkin to promote the selective degradation of mitochondria.The Pink1/Parkin mitophagy pathway is important for mitochondrial quality control, and mitophagy interruption leads to hyperactivation of in ammatory signaling pathways and chronic systemic in ammation 14 .Increasing evidence implicates a role for immunity and in ammation in the onset and progression of PD [14][15][16] .Similar to PD in humans, the Pink1 single knockout (sKO) rat model demonstrates early motor and nonmotor de cits [17][18][19] , nigrostriatal DA loss 20 , and nigral DA neuron loss 17,21,22 .Inconsistencies have been noted in the presence of PD-relevant pathological manifestations among cohorts suggesting incomplete phenotypic penetrance 18,23 .Parkin sKO rats appear resistant to motor impairment and nigrostriatal DA neurodegeneration up to 8 months of age 17 but do show abnormalities in DA metabolites 20 .We generated a combined Pink1/Parkin double knockout rat (dKO), which display a PD-relevant phenotype, including gait abnormalities and tremor as well as α-synuclein aggregation in the striatum that coincides with loss of nigral neurons 24,25 .Indeed, similar to animal models, the phenotypes of PD are diverse in humans, and characterizing early-onset PD is challenging in humans due to differential patterns of symptom manifestation, inconsistent age of disease onset, and environmental variability.While much research has focused on central nervous system alterations in PD, peripheral contributions to PD pathogenesis are increasingly appreciated.As such, the genetic rat models enable studies into the effect of Pink1 and Parkin KO, single and combined, on peripheral pathogenic mechanisms in a mammalian model that exhibits PD-relevant pathology.
In this study, we further characterized the Pink1 sKO, Parkin sKO, and Pink1/Parkin dKO rats.Comparisons of organ weights uncovered differences between the genotypes.Our ndings suggest tissue-speci c effects of loss of Pink1 and/or Parkin.We report Pink1/Parkin regulate glycolytic and mitochondrial oxidative metabolism in peripheral blood mononuclear cells (PBMCs) from rats.Pink1/Parkin de ciency induces changes in the circulating lymphocyte populations, namely increased CD4 + T cells and decreased CD8 + T cells and B cells.Loss of Pink1/Parkin leads to elevated platelet counts in the blood and increased platelet-T cell aggregation.In PD, it is becoming clear that lymphocytes are involved in both central and peripheral in ammation 26,27 .Further, platelet-lymphocyte aggregates are associated with increased thrombosis risk 28 , and venous thrombosis is a cause of sudden death in PD 29 , suggesting targeting the Pink1/Parkin pathway in the periphery has therapeutic potential.

Results
Pink1/Parkin de ciency modulates both glycolysis and oxidative metabolism of PBMCs.
While no signi cant differences in the number of PBMCs isolated per mL of whole blood were uncovered (Supplementary Figure S1O), we investigated whether Pink1 and/or Parkin de ciency could modulate the metabolic phenotype displayed by the PBMCs.Mitochondrial respiration (OCR) and glycolysis (ECAR) were measured in PBMCs from WT, Pink1 sKO, Parkin sKO, and Pink1/Parkin dKO rats using the Seahorse Analyzer.Sequential additions of an ATP synthase inhibitor (O), ATP synthesis uncoupler (F), and mixture of complex I and III inhibitors (R/A) allowed determination of basal mitochondrial respiration, ATP production-linked rate, proton leakage, maximal mitochondrial respiration, spare respiratory capacity, and non-mitochondrial respiration.No signi cant mitochondrial respiratory alterations were uncovered in PBMCs from Parkin sKO rats (Fig. 1).In contrast, PBMCs from Pink1 sKO and Pink1/Parkin dKO rats exhibit increased respiration following uncoupling of ATP synthesis (Fig. 1A,   B) and elevated maximal mitochondrial respiration compared to those from WT rats (Fig. 1C).The glycolytic function of PBMCs from Parkin sKO rats is unaltered compared to those from WT rats (Fig. 2).
While PBMCs from Pink1/Parkin dKO rats have elevated basal and maximal glycolytic function, PBMCs from Pink1 sKO rats only show increased basal glycolytic function (Fig. 2B).The cell energy phenotype of PBMCs was determined by plotting ECAR (glycolysis) as a function of OCR (mitochondrial respiration) revealing that under basal and maximal conditions PBMCs from Pink1-and/or Parkin-de cient rats are more energetic overall (more aerobic and glycolytic) as compared to those from WT rats (Fig. 3).Thus, the Pink1/Parkin pathway is important for metabolic regulation of PBMCs in rats, and this may be driven by contributions from Pink1 > Parkin.This is signi cant since PBMC metabolism is altered in several disease conditions, including PD [30][31][32] .Pink1/Parkin de ciency alters the pro le of circulating lymphocytes in the peripheral blood.
We employed a validated ow cytometric panel 33 to characterize the major leukocyte subsets circulating in the peripheral blood using PBMCs isolated from WT, Pink1 sKO, Parkin sKO, and Pink1/Parkin dKO rats.Using antibodies targeted against a range of surface antigens our gating strategy rst removed debris and dead cells (LIVE-DEAD stain), cell clumps (FSC-H and FSC-W; exclude doublets and clustered cells), or CD45-cells (excludes erythrocytes and plasma cells).This resulted in a population of live single leukocytes which were then sequentially separated, rst for T lymphocytes by CD3 which were further delineated by CD4 and CD8, and then NK cells were identi ed by CD161 and B cells by CD45R.Changes in the circulating leukocytes, speci cally increased numbers of CD3 + T cells and decreased numbers of CD3-T cells were found due to loss of Pink1 and/or Parkin (Fig. 4).CD4 + T cells were found to be elevated in Pink1 sKO and Pink1/Parkin dKO rats compared to WT rats (Fig. 4).Pink1/Parkin dKO rats showed increased numbers of CD4 + T cells compared to Parkin sKO rats (Fig. 4).
Reduced numbers of B cells were found in Pink1/Parkin dKO rats than in WT rats (Fig. 4).Loss of the Pink1-Parkin pathway modulates the circulating lymphocyte populations in rats, important as peripheral lymphocytes exhibit substantial quantitative and qualitative changes in PD 27 .Increased platelet-T cell aggregates due to Pink1/Parkin de ciency.
Platelet-lymphocyte aggregates are associated with increased thrombosis risk 28 , and venous thrombosis is a cause of sudden death in PD 29 .We report increased platelet counts (number of CD61 + cells) in Pink1/Parkin dKO rats at 5-6 months (Fig. 5).Therefore, we hypothesized that platelet-T cell aggregates may be increased in these rats.Whole blood samples were gated based on surface receptor expression for each T cell subset and were further gated based on forward scatter vs side scatter to better de ne the population.Platelet-CD3 + T cell aggregates (CD3 + CD61 + population) were increased in Pink1/Parkin dKO rats compared to WT rats (Fig. 5).Platelet-CD4 + and platelet-CD8 + T cell aggregates (CD4 + CD61 + and CD8 + CD61 + populations, respectively) were each increased in Pink1/Parkin dKO rats compared to control WT rats (Fig. 5).Although we found increased CD3 + T cells as a percentage of total leukocytes in isolated PBMCs (Fig. 4), we found decreased CD3 + T cells as a percentage of live cells in whole blood from Pink1/Parkin dKO rats (Fig. 5).As a percentage of live CD3 + T cells in whole blood we found increased CD4 + T cells (Fig. 5), similar to our ndings of increased CD4 + T cells as a percentage of total leukocytes in isolated PBMCs from Pink1/Parkin dKO rats (Fig. 4).Further, while no signi cant change in CD8 + T cells was uncovered as a percentage of total leukocytes in isolated PBMCs (Fig. 4), we did uncover a signi cant decrease in the number of CD8 + T cells as a percentage of live CD3 + T cells in whole blood from Pink1/Parkin dKO rats (Fig. 5).

Discussion
We previously reported that maximal mitochondrial respiration is impaired in PBMCs from Pink1/Parkin dKO rats at 12 months of age 25 .Here, we compared the metabolic alterations in PBMCs derived from Pink1 sKO, Parkin sKO, and Pink1/Parkin dKO rats at 5-6 months of age to uncover the effect of age as well as role that Pink1 and Parkin play individually.We found that PBMCs from Pink1/Parkin dKO rats at 5-6 months exhibit elevated maximal mitochondrial respiration, revealing an age-dependence on mitochondrial functional alterations.Similar to Pink1/Parkin dKO rats, we found that PBMCs from Pink1 sKO rats also exhibit elevated maximal mitochondrial respiration; however, mitochondrial respiration parameters in PBMCs from Parkin sKO rats are unaltered, suggesting it is Pink1 driving this effect.
PBMCs from younger (2-2.5 months old) Pink1 sKO rats were previously shown to exhibit elevated mitochondrial respiration compared to PBMCs from WT controls 34 .We also found that maximal glycolysis is increased in PBMCs from Pink1 sKO and Pink1/Parkin dKO rats, while basal glycolysis is higher only in Pink1/Parkin dKO rats.Again, PBMCs from Parkin sKO rats show glycolytic function comparable to WT.In younger (2-2.5 months old) Pink1 sKO rats, PBMCs were shown to exhibit elevated basal glycolysis compared to PBMCs from WT controls 34 .The lack of basal glycolytic changes in Pink1 sKO rats in our study could be due to age-related effects on PBMC glycolytic function or due to sex effects since we studied only males, while the previous work was from males and females compiled together.Overall, the energetic phenotype of PBMCs from the genetic PD model rats was increased (Pink1/Parkin dKO = Pink1 sKO > Parkin sKO > WT), indicating they are more aerobic and glycolytic.Bioenergetics have been studied in PBMCs from PD patients and point to increased energetics in general.In one study no differences in mitochondrial respiration were uncovered between PBMCs from control and PD patients, a marked elevation in glycolysis was found in PD patient PBMCs 30 .However, in another study while mitochondrial basal respiration was normal, maximal respiration and spare respiratory capacity were in increased in PBMCs from PD patients, and this correlated with clinical disease measures 31 .Consistent with this, another study showed elevated mitochondrial activity in PBMCs from PD patients 32 .
Our prior study using isolated PBMCs revealed increased CD4 + T cells and decreased CD8 + T cells in Pink1/Parkin dKO rats at 12 months 25 .To assess the effects of Pink1 and Parkin alone as well as age on the circulating lymphocyte populations in rats, we isolated PBMCs from Pink1 sKO, Parkin sKO, and Pink1/Parkin dKO rats at 5-6 months of age, an age when Pink1 sKO and Pink1/Parkin dKO rats exhibit motor symptoms 17,22,24 .We found that the percentage of CD3 + T cells was higher in Pink1 sKO, Parkin sKO, and Pink1/Parkin dKO rats as compared to WT controls.In particular, the levels of CD3 + CD4 + T cells were elevated in Pink1 sKO and Pink1/Parkin dKO rats compared to WT rats (also elevated in Pink1/Parkin dKO rats compared to Parkin sKO rats).T lymphocytes are key modulators of both humoral and cellular adaptive immune responses, and their role in PD is increasingly appreciated.While some report an increased percentage of CD4 + T cells in PD patients 35,36 , others have found decreased numbers of circulating CD4 + T cells [37][38][39][40] or insigni cant changes [41][42][43] .While no signi cant changes in the circulating CD8 + T cells were uncovered in the isolated rat PBMCs, when we assess whole blood from Pink1/Parkin dKO rats we found decreased numbers of CD8 + T cells compared to WT rats.The percentage of CD8 + T cells circulating in PD patients in the reported studies is quite heterogeneous.In PD patients, the levels of circulating CD8 + T cells have been found to be decreased 39,44 , increased 39,45,46 , or not changing 40,42,43 .The variations in reported lymphocyte pathology in PD patients could be associated with various factors, including age, sex, ethnicity, disease duration, and disease severity.
Further, the in uence of medication cannot be overlooked in PD patients.Additionally, studies of T cells in genetic PD patients and strati cation of such ndings by the genetic component will aid our understanding of the role of Pink1 and Parkin in human PD.
While no changes in B cell numbers were found in Pink1/Parkin dKO rats at 12 months 25 , at 6 months, we found reduced numbers of B cells in Pink1/Parkin dKO compared to WT rats.B cells were found to be reduced in transgenic α-synuclein mouse models of PD, where it was shown regulatory B cells play a protective role, potentially attenuating in ammation and dopaminergic neuron loss 47 .It is increasingly appreciated that B cells are reduced in PD 37,[48][49][50] and a more pro-in ammatory state in the B cell compartment has been consistently described 39,47,50,51 .B lymphocytes perform a variety of roles as part of the adaptive immune system and recent evidence suggests B cells are likely to interact with the central nervous system in complex ways via the meningeal lymphatic system 52,53 and via egress through channels in the skull bone marrow 54,55 .These ndings suggest B cells may play a role in in ammation in neurodegenerative diseases, including PD, both peripherally and centrally, warranting further study.
We previously reported a signi cant elevation in the number of platelets in the blood of Pink1/Parkin dKO rats at 9 and 12 months of age 25 .Here, we found that the number of CD61 + platelets are increased in Pink1/Parkin dKO rats at 6 months of age.Platelets, small non-nucleated blood cells, are gaining recognition for novel functions beyond their traditional role in hemostasis and wound closure, revealing them to be important players during immune responses and tissue remodeling.Further, platelet dysfunction is linked to several pathologies, including neurodegeneration.Platelet structural and functional alterations are evident in PD.Platelets from PD patients show reduced mitochondrial electron transport chain (ETC) complex activities 56,57 , lower vesicular monoamine transporter 2 mRNA 58 , and decreased glutamate uptake 59 .Contradictory ndings have been reported regarding platelet counts with some reports for decreased number [60][61][62] and others of unchanged counts 63 in PD patients; however, increases in mean platelet volume are found 60 .Platelet aggregation induced by agonists (ADP and epinephrine) was signi cantly decreased in PD patients, and exogenous human α-synuclein acts as a mild platelet antiaggregant in vitro 64 .The highest concentration of α-synuclein per mg of cellular protein in the blood is found in platelets 65,66 .In addition to α-synuclein, platelets express various other PDrelevant proteins, including tyrosine hydroxylase 67 , dopamine transporter 68 , Pink1 69 , and Parkin 70 .Of note, we previously reported elevated levels of plasma α-synuclein in the Pink1/Parkin dKO rats 25 .
The role of platelets in the regulation of immune cells such as T cells is becoming increasingly appreciated.Platelets participate in in ammation by producing pro-in ammatory mediators, which are stored in their vesicles (granules) prior to release 71,72 .Platelets communicate with immune cells, including T cells, via release of such mediator as well as through release of neurotransmitters (e.g., serotonin, epinephrine, dopamine, and histamine) [73][74][75] .Additionally, platelets and T cells can form direct contacts with each other.In fact, platelet-T cell aggregates correlate with markers of platelet aggregation, immune activation, and disease progression 28,76-79 .The role of platelet-T cell aggregates during PD and in animal models of PD remain underexplored.Thus, we assessed platelet-T cell aggregation in the Pink1/Parkin dKO rat model.We found that Pink1/Parkin dKO rats have a higher percentage of platelet-CD3 + T cell aggregates, including both increased platelet-CD4 + T cell and platelet-CD8 + T cell aggregation.Platelet-lymphocyte aggregates are associated with increased thrombosis risk, and venous thrombosis is a cause of sudden death in PD 28,29 .Thus, additional studies will inform on the contribution of platelet-lymphocyte aggregation to disease progression in the Pink1/Parkin dKO rat model and we believe studies to assess platelet-lymphocyte aggregation in PD patients are warranted.
In summary, we describe changes in the circulating leukocytes, speci cally increased numbers of CD4 + T cells and decreased numbers of CD8 + T cells and B cells in Pink1/Parkin dKO rats.Increased CD4 + T cells were found in Pink1 sKO rats, suggesting Pink1 might drive this effect.In contrast to our ndings of reduced PBMC mitochondrial respiration at 12 months 25 , we found elevated mitochondrial respiration at 6 months in dKO rats, which appears to be largely driven by loss of Pink1.Further, we uncovered elevated PBMC glycolytic function, which taken together reveals an immunometabolic shift towards a more energetic phenotype is induced by loss of Pink/Parkin.Platelet numbers were elevated, and platelet-T cell aggregation was increased in Pink1/Parkin dKO rats, involving both CD4 + and CD8 + T cells.These ndings further support a role for the peripheral immune system and highlight a previously unappreciated role for platelet-T cell aggregates in Pink1/Parkin-linked PD pathogenesis.

Animal subjects
Rats with targeted disruption of the Park6 gene (Pink1 single knockout, sKO), the Park2 gene (Parkin sKO), and the background strain (Long Evans Hooded) were obtained from SAGE Labs (now Inotiv) 17 .Pink1/Parkin double KO (dKO) rats were generated in our lab from the sKO rats as described previously 24,25 .The rats were maintained on a 12-hour light/dark cycle in a temperature-controlled environment with free access to standard rat chow and water.Experimental rats were bred in-house in accordance with institutionally approved breeding protocols.All breeding and experimental procedures described herein were approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee and carried out in accordance with approved protocols and regulations.Study methods within were conducted and are reported in accordance with the ARRIVE guidelines.Rats (male, 5-6 months of age) were rendered unconscious by 5% iso urane inhalation using an anesthetic chamber, body weight was recorded, and the unconscious rats were euthanized by decapitation using a guillotine, following decapitation trunk blood was collected using vacutainer tubes containing 7.2 mg K 2 EDTA and the tissues were quickly removed and weighed.The characteristics of Pink1/Parkin de cient and control rats are presented in Supplementary Data S1 and S2.The body weights of Pink1/Parkin dKO rats were signi cantly more than Pink1 sKO and WT rats, but not than Parkin sKO rats (Figure S1A).The body weights of Pink1 sKO and Parkin sKO rats were not signi cantly different from each other or from wildtype (WT) rats (Figure S1A).Because of the signi cant difference in body weights for Pink1/Parkin dKO rats compared to Pink1 sKO and WT rats, in addition to absolute weight of tissues (Figure S2), it is important to consider tissue weights normalized to body weight (Figure S1B-N).The brain weight was signi cantly lower in Parkin sKO rats than Pink1 sKO rats (Figure S1B).While Pink1 sKO and Pink1/Parkin dKO rats were comparable, their heart weights were signi cantly higher than Parkin sKO and WT rats (Figure S1C).Pink1 sKO rats had signi cantly heavier liver (Figure S1D), spleen (Figure S1E), and testis (Figure S1F) than Parkin sKO, Pink1/Parkin dKO, and WT rats, which were all similar to each other.Parkin sKO rats had signi cantly lighter kidneys than Pink1 sKO, Pink1/Parkin dKO, and WT rats, which were comparable to each other (Figure S1G).No signi cant differences were found for lung (Figure S1H), quadricep (Figure S1I), and hamstring (Figure S1J).Pink1 sKO and Parkin sKO rats had signi cantly increased anterior tibialis mass than WT rats (Figure S1K).The extensor digitorum longus was heavier in Parkin sKO rats than in Pink1 sKO, Pink1/Parkin dKO, and WT rats (Figure S1L).The gastrocnemius weight in Pink1 sKO and Pink1/Parkin dKO rats was signi cantly higher than in Parkin sKO and WT rats (Figure S1M).WT rats have signi cantly lighter soleus than Pink1 sKO, Parkin sKO, and Pink1/Parkin dKO rats (Figure S1N).The differences that we uncovered between the genotypes reveals tissue-speci c effects of loss of Pink1 and/or Parkin.

Isolation of PBMCs
Whole blood was centrifuged at 900 x g for 20 min at RT to separate erythrocytes, plasma, and the buffy coat.The buffy coat was removed, diluted with 1x PBS, and overlayed on Ficoll-Hypaqe prior to centrifugation at 400 x g for 30 min at RT.The PBMC layer was collected, diluted with 1x PBS, and centrifuged at 500 x g for 7 min at RT.The resulting pellet was lysed with 1x RBC lysis buffer, diluted with 1x PBS, and centrifuged at 400 x g for 7 min at RT.The PBMC pellet was resuspended in 1x PBS and counted using a Beckman Coulter z1 particle counter.PBMCs were cryopreserved in Fetal Bovine Serum with 10% DMSO and stored in liquid nitrogen.Cells were thawed within 6 weeks of isolation and underwent bioenergetic and ow cytometric assessments.
Descriptive data are presented as violin plots showing the median and quartiles.Group comparisons were conducted using one-way analysis of variance (ANOVA) or two-way ANOVA with Tukey's multiple comparisons test or Sidak's multiple comparisons test, as appropriate for variance of data.Statistical analysis was performed using GraphPad Prism version 10.1.1 (GraphPad Software, San Diego, CA).

Figure 1 .
Figure 1.Elevated mitochondrial respiration in PBMCs isolated from Pink1 sKO and Pink1/Parkin dKO rats at 5-6 months.(A) Graphical representation of the OCR responses over time and (B) quanti cation of the mean OCR in A; sequential injections are indicated as O (the ATP synthase inhibitor oligomycin), F (the ATP synthesis uncoupler FCCP), R/A (a mixture of the complex I and III inhibitors rotenone and antimycin A, respectively).(C) Mitochondrial respiratory parameters calculated from the OCR shown for basal mitochondrial respiration (baseline minus R/A), ATP linked respiration (baseline minus O), proton leak (O minus R/A), maximal mitochondrial respiration (F minus R/A), and spare respiratory capacity (F minus baseline).Two-way ANOVA with Tukey's multiple comparisons test was used to determine statistical signi cance (p < 0.01**, 0.001***; n = 7 (WT) and 8 (Pink1 sKO, Parkin sKO, Pink1/Parkin dKO).

Figure 2 .
Figure 2. Increased glycolytic function in PBMCs isolated from Pink1 sKO and Pink1/Parkin dKO rats at 5-6 months.(A) Graphical representation of the ECAR responses over time and (B) quanti cation of the mean OCR in A for basal glycolysis (baseline) and maximal glycolysis (O), as well as glycolytic reserve (O minus baseline).Data after FCCP and R/A injection were not used for ECAR analysis.Twoway ANOVA with Tukey's multiple comparisons test was used to determine statistical signi cance (p < 0.05*, 0.01**; n = 7 (WT) and 8 (Pink1 sKO, Parkin sKO, Pink1/Parkin dKO).

Figure 3 .
Figure 3. Loss of Pink1/Parkin increases the metabolic phenotype of PBMCs from rats at 5-6 months.Overall energetic phenotype of the PBMCs shown as OCR as a function of ECAR for (A) basal and (B) maximal metabolism.

Figure
Figure 2

Figure 3 Loss
Figure 3