Low dose (0.3mg/kg) and high dose (3mg/kg) TAK-063 reduced striatal PDE10A protein abundance.
Both low (0.3 mg/kg) and high (3 mg/kg) doses of orally administered TAK-063 reduced PDE10A protein abundance in the mouse striatum within one hour (Fig. 1A), providing evidence that PDE10A was efficiently blocked by the inhibitor. PDE10A protein level was reduced to 75 ± 13.7 and 48.8 ± 3.6 respectively, in mice receiving 0.3 mg/kg and 3 mg/kg TAK-063, as compared to vehicle treated mice.
Effects of TAK-063 on neurological deficit score, infarct size, cerebral edema and blood-brain barrier permeability.
To ensure reproducible brain injury among the experimental groups, cerebral blood flow (CBF) was monitored and analyzed in real-time via laser Doppler flow (LDF) measurement using a flexible probe attached to the animals’ skulls above the core of the MCA region (Fig. 1B, 3A). We noted that TAK-063 doses used in this study resulted in a slightly higher cerebral blood flow during reperfusion compared to the control group animals, however, this increase was not significant. Twenty-four hours after 90 min ischemia, four-point neurological scoring was performed for the evaluation of neurological deficits. Infarct volume and brain edema were evaluated by creysl violet staining. In this model of combined subcortical-cortical infarction, low and high dose TAK-063 significantly reduced stroke-related neurological deficits, infarct volume and brain edema (Fig. 1C-E). Besides, BBB permeability assessed by serum IgG extravasation was decreased by low dose, but not high dose TAK-063 (Fig. 1F).
PDE10A inhibition increases regional microcirculation in ischemic core region.
To analyze the hemodynamic effects induced by orally administered TAK-063, we further evaluated regional cerebral microcirculation in the ischemic core (Fig. 2B), ischemic periphery (Fig. 2C) and ipsilateral non-ischemic tissue (Fig. 2D) by Laser Speckle Imaging (LSI). For this aim, mice were subjected to 90 min MCAO which is followed by secondary hypoperfusion that develops within 90 min after reperfusion onset . Low dose TAK-063 which was administered at the beginning of the reperfusion significantly increased regional microcirculation in the ischemic core region starting 30 min after reperfusion onset, while high dose TAK-063 increased microcirculation in a more delayed fashion starting 100 min after reperfusion onset. Effects of TAK-063 on cerebral microcirculation in the ischemic periphery did not achieve significance. Hence, the microcirculatory effects of low dose TAK-063 were more pronounced than those of high dose TAK-063.
Effect of PDE10A inhibition on disseminate neuronal injury.
We next examined the effects of PDE10A inhibition in a model of mild focal cerebral ischemia induced by 30 min MCAO, which induces disseminate neuronal injury in the striatum developing over one to three days . In this model, low dose and high dose TAK-063 increased neuronal survival and decreased the number of DNA-fragmented (that is, irreversibly injured) cells in the ischemic striatum (Fig. 3B-C). Hence, PDE10A inhibition efficiently provided protection against a wide range of ischemic injury severities, preventing combined subcortical-cortical infarction and disseminate neuronal death at the same time.
PDE10A inhibition with TAK-063 regulates survival related proteins.
Western blot analysis was used to evaluate the effect of PDE10A inhibition with TAK-063 on cellular survival and apoptosis related protein levels in the ischemic striatum. Seventy-two hours after 30 min MCAO, PDE10A protein abundance was still reduced in the ischemic striatum (Fig. 4A). Both doses of TAK-063 increased the level of phosphorylated (that is, activated) Akt (Thr308) (Fig. 4B) and phosphorylated (that is, activated) GSK3 α/β (Fig. 4C) and decreased the level of phosphorylated (that is, inactive) mTOR (mammalian target of rapamycin) (Fig. 4D). Low dose TAK-063 also significantly increased the level of phosphorylated (that is, activated) ERK-1/2 (Fig. 4E) and decreased the level of phosphorylated (that is, inactive) PTEN (Fig. 4F). These data indicated that TAK-063 elicited a robust response of pro-survival proteins that contributed to post-ischemic neuroprotection induced by PDE10A inhibition.
In line with the stabilization of post-ischemic reperfusion and the stabilization of BBB integrity, the abundance of hypoxia inducible factor-1α (HIF-1α) (Fig. 4G) and matrix metalloproteinase-9 (MMP-9) (Fig. 4H) were significantly decreased by TAK-063. Moreover, we also evaluated the expression of pro-apoptotic Bax and anti-apoptotic Bcl-xL in the ipsilesional striatum. Both doses of TAK-063 significantly decreased Bax protein abundance and increased anti-apoptotic Bcl-xL protein abundance (Fig. 4I,J).
TAK-063 altered the cytokine/chemokine expression profile after focal cerebral ischemia.
The stabilization of BBB integrity by TAK-063 subsequently prompted us to evaluate the levels of cytokines and chemokines in the ischemic brain. The relative expression levels of 40 different cytokines and chemokines (CXCL13/BLC/BCA-1, C5a, G-CSF, GM-CSF, CCL1/I-309, CCL11/Eotaxin, ICAM-1, IFN-gamma, IL-1 alpha/IL-1F1, IL-1 beta/IL-1F2, IL-1ra/IL-1F3, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12 p70, IL-13, IL-16, IL-17, IL-23, IL-27, CXCL10/IP-10, CXCL11/I-TAC, CXCL1/KC, M-CSF, CCL2/JE/MCP-1, CCL12/MCP-5, CXCL9/MIG, CCL3/MIP-1 alpha, CCL4/MIP-1 beta, CXCL2/MIP-2, CCL5/RANTES, CXCL12/SDF-1, CCL17/TARC, TIMP-1, TNF-alpha, and TREM-1) in the ipsilesional striatum were evaluated using an antibody array (Fig. 5A). These analyses revealed a broad anti-inflammatory response. As such, the expression of G-CSF, I-309, INF-γ, IL-1α, IL-1β, IL-1ra, IL-2, IL-3, IL-7, IL-13, IL-16, IL-17, IL-23, IL-27, IP-10, I-TAC, KC, M-CSF, JE, MCP-5, MIG, MIP-1α, MIP-1β, MIP-2, RANTES, TNF-α, and TREM-1 was decreased by TAK-063 (Fig. 5B). BLC, C5/C5a, IL-4, SDF-1, TARC, and TIMP-1 were not influenced by TAK-063 (Fig. 5B).
TAK-063 mediated PDE10A inhibition altered protein profile in the ipsilesional striatum.
To understand the changes in the protein profile after TAK-063 administration, ischemic tissues were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 1721 proteins were identified in vehicle and TAK-063 treated groups. Forty different proteins were significantly altered by low dose/high dose TAK-063 treatment (Fig. 6). These proteins are: 26S proteasome non-ATPase regulatory subunit 2 (PSMD2), 40S ribosomal protein S18 (RPS18-PS5), acyl-CoA thioesterase 7 (ACOT7), ADP/ATP translocase 1 (ADT1), ankyrin repeat and sterile alpha motif domain-containing protein 1B (ANKS1B), centromere-associated protein E (CENPE), cytoplasmic FMR1-interacting protein 1 (CYFP1), dedicator of cytokinesis protein 4 (DOCK4), dihydrolipoyl dehydrogenase, mitochondrial (DLDH), DNA polymerase epsilon catalytic subunit A (DPOE1), DNA polymerase zeta catalytic subunit (REV3L), dynactin subunit 4 (DCTN4), electron transfer flavoprotein subunit beta (ETFB), extracellular matrix protein 2 (ECM2), filamin-A (FLNA), flotillin (FLOT2), hydroxyacylglutathione hydrolase, mitochondrial (HAGH), importin subunit alpha-4 (IMA4), LanC-like protein 2 (LANCL2), long-chain-fatty-acid–CoA ligase ACSBG1 (ACBG1), microtubule-associated protein RP/EB family member 2 (MAPRE2), NADH-ubiquinone oxidoreductase 18 kDa subunit (NDUFS4), ornithine aminotransferase, mitochondrial (OAT), phosphatidylinositol 4-phosphate 5-kinase type-1 gamma (PIP5K1C), phosphofurin acidic cluster sorting protein 1 (PACS1), proteasome subunit beta type-1 (PSMB1), protein S100-B (S100B), pyridoxal phosphate homeostasis protein (PLPHP), Ras-related protein Rab-8B (RAB8B), Rho GTPase-activating protein 23 (ARHGAP23), Rho-associated protein kinase 2 (ROCK2), secretory carrier-associated membrane protein (SCAMP1), serpin B6 (SERPINB6), sodium leak channel non-selective protein (NALCN), solute carrier family 25 member 11 (SLC25A11), solute carrier family 25 member 12 (SLC25A12), T-complex protein 1 subunit epsilon (TCPE), tyrosine-protein phosphatase non-receptor type substrate 1 (SHPS1), tyrosine–tRNA ligase, cytoplasmic (SYYC), voltage-dependent anion-selective channel protein 3 (VDAC3).
Identified proteins were clustered into groups based on their molecular function and biological process using the PANTHER (protein annotation through evolutionary relationship) classification system (http://www.pantherdb.org/). Molecular function classification included proteins with binding, catalytic and transporter activity. PANTHER classification based on biological process included five predominant groups; cellular process, metabolic process, localization, biological regulation, and response to stimulus. In addition to this, six signaling pathways were identified as cytoskeletal regulation by Rho GTPase, dopamine receptor mediated signaling pathway, Huntington disease, integrin signaling pathway, Parkinson’s disease, and Ubiquitin proteasome pathway.