The PPAR-γ agonist pioglitazone modulates inflammation and induces neuroprotection in parkinsonian monkeys

<p>Abstract</p> <p>Background</p> <p>Activation of the peroxisome proliferator-activated receptor gamma (PPAR-γ) has been proposed as a possible neuroprotective strategy to slow down the progression of early Parkinson's disease (PD). Here we report preclinical data on the use of the PPAR-γ agonist pioglitazone (Actos^®; Takeda Pharmaceuticals Ltd.) in a paradigm resembling early PD in nonhuman primates.</p> <p>Methods</p> <p>Rhesus monkeys that were trained to perform a battery of behavioral tests received a single intracarotid arterial injection of 20 ml of saline containing 3 mg of the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Twenty-four hours later the monkeys were assessed using a clinical rating scale, matched accordingly to disability, randomly assigned to one of three groups [placebo (n = 5), 2.5 (n = 6) or 5 (n = 5) mg/kg of pioglitazone] and their treatments started. Three months after daily oral dosing, the animals were necropsied.</p> <p>Results</p> <p>We observed significant improvements in clinical rating score (<it>P </it>= 0.02) in the animals treated with 5 mg/kg compared to placebo. Behavioral recovery was associated with preservation of nigrostriatal dopaminergic markers, observed as higher tyrosine hydroxylase (TH) putaminal optical density (<it>P </it>= 0.011), higher stereological cell counts of TH-ir (<it>P </it>= 0.02) and vesicular monoamine transporter-2 (VMAT-2)-ir nigral neurons (<it>P </it>= 0.006). Stereological cell counts of Nissl stained nigral neurons confirmed neuroprotection (<it>P </it>= 0.017). Pioglitazone-treated monkeys also showed a dose-dependent modulation of CD68-ir inflammatory cells, that was significantly decreased for 5 mg/kg treated animals compared to placebo (<it>P </it>= 0.018). A separate experiment to assess CSF penetration of pioglitazone revealed that 5 mg/kg p.o. induced consistently higher levels than 2.5 mg/kg and 7.5 mg/kg. p.o.</p> <p>Conclusions</p> <p>Our results indicate that oral administration of pioglitazone is neuroprotective when administered early after inducing a parkinsonian syndrome in rhesus monkeys and supports the concept that PPAR-γ is a viable target against neurodegeneration.</p

Nonuniform Cardiac Denervation Observed by 11C-meta-Hydroxyephedrine PET in 6-OHDA-Treated Monkeys

Parkinson's disease presents nonmotor complications such as autonomic dysfunction that do not respond to traditional anti-parkinsonian therapies. The lack of established preclinical monkey models of Parkinson's disease with cardiac dysfunction hampers development and testing of new treatments to alleviate or prevent this feature. This study aimed to assess the feasibility of developing a model of cardiac dysautonomia in nonhuman primates and preclinical evaluations tools. Five rhesus monkeys received intravenous injections of 6-hydroxydopamine (total dose: 50 mg/kg). The animals were evaluated before and after with a battery of tests, including positron emission tomography with the norepinephrine analog 11C-meta-hydroxyephedrine. Imaging 1 week after neurotoxin treatment revealed nearly complete loss of specific radioligand uptake. Partial progressive recovery of cardiac uptake found between 1 and 10 weeks remained stable between 10 and 14 weeks. In all five animals, examination of the pattern of uptake (using Logan plot analysis to create distribution volume maps) revealed a persistent region-specific significant loss in the inferior wall of the left ventricle at 10 (P<0.001) and 14 weeks (P<0.01) relative to the anterior wall. Blood levels of dopamine, norepinephrine (P<0.05), epinephrine, and 3,4-dihydroxyphenylacetic acid (P<0.01) were notably decreased after 6-hydroxydopamine at all time points. These results demonstrate that systemic injection of 6-hydroxydopamine in nonhuman primates creates a nonuniform but reproducible pattern of cardiac denervation as well as a persistent loss of circulating catecholamines, supporting the use of this method to further develop a monkey model of cardiac dysautonomia

Microglial phenotypes in Parkinson's disease and animal models of the disease

Over the last decade the important concept has emerged that microglia, similar to other tissue macrophages, assume different phenotypes and serve several effector functions, generating the theory that activated microglia can be organized by their pro-inflammatory or anti-inflammatory and repairing functions. Importantly, microglia exist in a heterogenous population and their phenotypes are not permanently polarized into two categories; they exist along a continuum where they acquire different profiles based on their local environment. In Parkinson's disease (PD), neuroinflammation and microglia activation are considered neuropathological hallmarks, however their precise role in relation to disease progression is not clear, yet represent a critical challenge in the search of disease-modifying strategies. This review will critically address current knowledge on the activation states of microglia as well as microglial phenotypes found in PD and in animal models of PD, focusing on the expression of surface molecules as well as pro-inflammatory and anti-inflammatory cytokine production during the disease process. While human studies have reported an elevation of both pro- or anti-inflammatory markers in the serum and CSF of PD patients, animal models have provided insights on dynamic changes of microglia phenotypes in relation to disease progression especially prior to the development of motor deficits. We also review recent evidence of malfunction at multiple steps of NFκB signaling that may have a causal interrelationship with pathological microglia activation in animal models of PD. Finally, we discuss the immune-modifying strategies that have been explored regarding mechanisms of chronic microglial activation

Microglial Phenotypes and Their Relationship to the Cannabinoid System: Therapeutic Implications for Parkinson’s Disease

Parkinson&rsquo;s disease is a neurodegenerative disorder, the motor symptoms of which are associated classically with Lewy body formation and nigrostriatal degeneration. Neuroinflammation has been implicated in the progression of this disease, by which microglia become chronically activated in response to &alpha;-synuclein pathology and dying neurons, thereby acquiring dishomeostatic phenotypes that are cytotoxic and can cause further neuronal death. Microglia have a functional endocannabinoid signaling system, expressing the cannabinoid receptors in addition to being capable of synthesizing and degrading endocannabinoids. Alterations in the cannabinoid system&mdash;particularly an upregulation in the immunomodulatory CB2 receptor&mdash;have been demonstrated to be related to the microglial activation state and hence the microglial phenotype. This paper will review studies that examine the relationship between the cannabinoid system and microglial activation, and how this association could be manipulated for therapeutic benefit in Parkinson&rsquo;s disease

Characterization of a Cul9–Parkin double knockout mouse model for Parkinson’s disease

Acknowledgements We thank Viktoriya Nikolova at the UNC Mouse Behavioural Phenotyping Laboratory for her technical assistance. This work was supported by a Rapid Response Innovation Award (ID 9056) from the Michael J Fox Foundation for Parkinson’s Research and by the NIH Grant GM118331 to M.D. V.J. was supported by Training in translational Research in Neurology NIH Fellowship 2T32NS007480. The Tansey laboratory is supported by NIH/NIA 1R01 AG057247, NIH/NINDS 5R01 NS092122, NIH/NIA 3RF1 AG051514-01, and the Norman Fixel Institute for Neurological Diseases to M.G.T. The UNC Mouse Behavioural Phenotyping Laboratory is supported by a grant from the National Institute of Child Health and Human Development (NICHD), U54-HD079124. The Neuroscience Microscopy Core Facility is supported, in part, by funding from the NIH-NINDS Neuroscience Center Support Grant P30 NS045892 and the NIH-NICHD Intellectual and Developmental Disabilities Research Center Support Grant U54 HD079124. Contributions E.H. conducted the in vitro experiments and prepared the mice cohorts. V.S. and A.N. helped with generating the Cul9, Parkin double KO mice. V.J and M.J.T. performed the stereological analysis and quantification of dopaminergic neurons. S.M. conducted and analysed the neurobehavioral assessments. M.D. outlined and supervised the project. E.H. and M.D. produced the final version of the manuscript.Peer reviewedPublisher PD

Induced Pluripotent Stem Cell-Derived Neural Cells Survive and Mature in the Nonhuman Primate Brain

The generation of induced pluripotent stem cells (iPSCs) opens up the possibility for personalized cell therapy. Here, we show that transplanted autologous rhesus monkey iPSC-derived neural progenitors survive for up to 6 months and differentiate into neurons, astrocytes, and myelinating oligodendrocytes in the brains of MPTP-induced hemiparkinsonian rhesus monkeys with a minimal presence of inflammatory cells and reactive glia. This finding represents a significant step toward personalized regenerative therapies

6-OHDA-treated monkeys have reduced TH-ir in cardiac nerve bundles and fibers.

<p>(<b>A,B</b>) Microphotographs of H&E stained nerve bundles identified as a collection of nerve fibers (white asterisk) and intact epineurium indicated by white arrows. (<b>C</b>) Global TH-ir in cardiac nerve bundle was reduced for both AAT (t(8) = 3.508, <i>P</i> = 0.008) and OD quantification (t(8) = 3.403, <i>P</i> = 0.009) in 6-OHDA compared to control animals. (<b>D,E</b>) Microphotographs of TH-ir in matching nerve bundles. (<b>F</b>) Regional wall quantification showed reduced AAT of TH-ir in anterior (t(8) = 5.913, <i>P</i><0.0001), inferior (t(8) = 3.096, <i>P</i> = 0.015), and septal (t(8) = 3.009, <i>P</i> = 0.017) walls in 6-OHDA-treated monkeys compared to controls. In the control group there were also significant differences in OD quantification of TH (F(3,16) = 5.213, <i>P</i> = 0.011) with more TH-ir in the septal wall compared to the lateral (<i>P</i> = 0.043) and inferior walls (<i>P</i> = 0.12) (<i>post hoc</i> Bonferroni multiple comparisons analysis; not shown). (<b>G,H,J,K</b>) Microphotographs of cardiac TH positive fibers in either transverse (<b>G,H</b>) or longitudinal orientation (<b>J,K</b>) in control and 6-OHDA monkeys; black arrows indicate TH-ir fibers. (<b>I,L</b>) TH-ir fiber area density was significantly reduced in 6-OHDA compared to control animals for global (<b>I</b>) analysis (t(8) = 4.934, <i>P</i> = 0.001) and between all walls of the left ventricle (<b>L</b>) including the anterior (t(8) = 4.324, <i>P</i> = 0.003), inferior (t(8) = 5.37, <i>P</i> = 0.001), lateral (t(8) = 5.081,<i>P</i> = 0.001) and septal walls (t(8) = 3.866, <i>P</i> = 0.005). Scale bar  = 25 µm. <i>*P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001. AAT, area above threshold; OD, optical density; TH, tyrosine hydroxylase.</p

Cardiac α-synuclein was moderately affected by 6-OHDA.

<p>(<b>A–D</b>) Microphotographs of nerve bundles immunostained for soluble α-synuclein (<b>A,B</b>), phosphorylated α-synuclein (<b>C,D</b>) and proteinase K resistant α-synuclein (<b>E,F</b>) in control (<b>A,C,E</b>) and 6-OHDA-treated (<b>B,D,F</b>) monkeys. Insets (<b>a–c</b>) display immunostained nigral tissue from a Thy1-α-synuclein mouse processed in parallel for each specific antibody and used as a positive control to validate staining. (<b>G</b>) Regional reduction of soluble α-synuclein expression was found in the septal wall (t(8) = 2.787, <i>P</i> = 0.024) of 6-OHDA compared to control monkeys. Within each group, soluble α-synuclein scores did not show significant differences by wall in control animals (ANOVA; F(3,16) = 3.115, <i>P</i> = 0.056) and in 6-OHDA-treated animals (ANOVA: F(3,16) = 0.772, <i>P</i> = 0.526). (<b>H</b>) Average scores of soluble α-synuclein-ir in nerve bundles correlated with AAT TH-positive nerve bundles (r² = 0.570, <i>P</i> = 0.012). Scale bar  = 25 µm, inset scale bar  = 100 µm. <i>*P</i><0.05. AAT, area above threshold; TH, tyrosine hydroxylase.</p

Measures of cardiac TH and PGP9.5 expression correlated with <i>in vivo</i> MHED uptake.

<p>Nerve bundle AAT quantification of TH-ir correlated with TH-ir fibers (<b>A</b>) and AAT of PGP9.5-ir nerve bundles (<b>B</b>). Area density quantification of TH-ir fibers highly correlated with PGP9.5-ir fibers (<b>C</b>). <i>In vivo</i> MHED uptake correlated with TH-ir (<b>D</b>) and PGP9.5-ir fiber area density (<b>E</b>). Despite the significant interaction between MHED and TH-ir fibers, MHED uptake did not correlate with AAT quantification of TH-ir nerve bundles (r² = 0.446, <i>P</i> = 0.070) (not shown). AAT, area above threshold; MHED, meta-hydroxyephedrine; PGP9.5, protein gene product 9.5; TH, tyrosine hydroxylase.</p