Opposing effects of ß-2 and ß-1 adrenergic receptor signaling on neuroinflammation and dopaminergic neuron survival in α-synuclein-mediated neurotoxicity.

BACKGROUND: Noradrenergic neurons in the locus coeruleus (LC) are the primary source of norepinephrine (NE) in the brain and degeneration of these neurons is reported in the early stages of Parkinson's disease (PD), even prior to dopaminergic neuron degeneration in the substantia nigra (SN), which is a hallmark of PD pathology. NE depletion is generally associated with increased PD pathology in neurotoxin-based PD models. The effect of NE depletion in other models of PD-like α-synuclein-based models is largely unexplored. In PD models and in human patients, ß-adrenergic receptors' (AR) signaling is associated with a reduction of neuroinflammation and PD pathology. However, the effect of NE depletion in the brain and the extent of NE and ß-ARs signaling involvement in neuroinflammation, and dopaminergic neuron survival is poorly understood. METHODS: Two mouse models of PD, a 6OHDA neurotoxin-based model and a human α-synuclein (hα-SYN) virus-based model of PD, were used. DSP-4 was used to deplete NE levels in the brain and its effect was confirmed by HPLC with electrochemical detection. A pharmacological approach was used to mechanistically understand the impact of DSP-4 in the hα-SYN model of PD using a norepinephrine transporter (NET) and a ß-AR blocker. Epifluorescence and confocal imaging were used to study changes in microglia activation and T-cell infiltration after ß1-AR and ß2-AR agonist treatment in the hα-SYN virus-based model of PD. RESULTS: Consistent with previous studies, we found that DSP-4 pretreatment increased dopaminergic neuron loss after 6OHDA injection. In contrast, DSP-4 pretreatment protected dopaminergic neurons after hα-SYN overexpression. DSP-4-mediated protection of dopaminergic neurons after hα-SYN overexpression was dependent on ß-AR signaling since using a ß-AR blocker prevented DSP-4-mediated dopaminergic neuron protection in this model of PD. Finally, we found that the ß-2AR agonist, clenbuterol, reduced microglia activation, T-cell infiltration, and dopaminergic neuron degeneration, whereas xamoterol a ß-1AR agonist showed increased neuroinflammation, blood brain barrier permeability (BBB), and dopaminergic neuron degeneration in the context of hα-SYN-mediated neurotoxicity. CONCLUSIONS: Our data demonstrate that the effects of DSP-4 on dopaminergic neuron degeneration are model specific, and suggest that in the context of α-SYN-driven neuropathology, ß2-AR specific agonists may have therapeutic benefit in PD.

Compartmentalized Actions of the Plasminogen Activator Inhibitors, PAI-1 and Nsp, in Ischemic Stroke.

Tissue plasminogen activator (tPA) is a multifunctional protease. In blood tPA is best understood for its role in fibrinolysis, whereas in the brain tPA is reported to regulate blood-brain barrier (BBB) function and to promote neurodegeneration. Thrombolytic tPA is used for the treatment of ischemic stroke. However, its use is associated with an increased risk of hemorrhagic transformation. In blood the primary regulator of tPA activity is plasminogen activator inhibitor 1 (PAI-1), whereas in the brain, its primary inhibitor is thought to be neuroserpin (Nsp). In this study, we compare the effects of PAI-1 and Nsp deficiency in a mouse model of ischemic stroke and show that tPA has both beneficial and harmful effects that are differentially regulated by PAI-1 and Nsp. Following ischemic stroke Nsp deficiency in mice leads to larger strokes, increased BBB permeability, and increased spontaneous intracerebral hemorrhage. In contrast, PAI-1 deficiency results in smaller infarcts and increased cerebral blood flow recovery. Mechanistically, our data suggests that these differences are largely due to the compartmentalized action of PAI-1 and Nsp, with Nsp deficiency enhancing tPA activity in the CNS which increases BBB permeability and worsens stroke outcomes, while PAI-1 deficiency enhances fibrinolysis and improves recovery. Finally, we show that treatment with a combination therapy that enhances endogenous fibrinolysis by inhibiting PAI-1 with MDI-2268 and reduces BBB permeability by inhibiting tPA-mediated PDGFRα signaling with imatinib significantly reduces infarct size compared to vehicle-treated mice and to mice with either treatment alone.

CD73 Promotes Age-Dependent Accretion of Atherosclerosis.

OBJECTIVE: CD73 is an ectonucleotidase which catalyzes the conversion of AMP (adenosine monophosphate) to adenosine. Adenosine has been shown to be anti-inflammatory and vasorelaxant. The impact of ectonucleotidases on age-dependent atherosclerosis remains unclear. Our aim was to investigate the role of CD73 in age-dependent accumulation of atherosclerosis. Approach and results: Mice doubly deficient in CD73 and ApoE (apolipoprotein E; (cd73-/-/apoE-/-) were generated, and the extent of aortic atherosclerotic plaque was compared with apoE-/- controls at 12, 20, 32, and 52 weeks. By 12 weeks of age, cd73-/-/apoE-/- mice exhibited a significant increase in plaque (1.4±0.5% of the total vessel surface versus 0.4±0.1% in apoE-/- controls, P<0.005). By 20 weeks of age, this difference disappeared (2.9±0.4% versus 3.3±0.7%). A significant reversal in phenotype emerged at 32 weeks (9.8±1.2% versus 18.3±1.4%; P<0.0001) and persisted at the 52 week timepoint (22.4±2.1% versus 37.0±2.1%; P<0.0001). The inflammatory response to aging was found to be comparable between cd73-/-/apoE-/- mice and apoE-/- controls. A reduction in lipolysis in CD73 competent mice was observed, even with similar plasma lipid levels (cd73-/-/apoE-/- versus apoE-/- at 12 weeks [16.2±0.7 versus 9.5±1.4 nmol glycerol/well], 32 weeks [24.1±1.5 versus 7.4±0.4 nmol/well], and 52 weeks [13.8±0.62 versus 12.7±2.0 nmol/well], P<0.001). CONCLUSIONS: At early time points, CD73 exerts a subtle antiatherosclerotic influence, but with age, the pattern reverses, and the presence of CD73 promoted suppression of lipid catabolism.

Imatinib treatment reduces brain injury in a murine model of traumatic brain injury.

Current therapies for Traumatic brain injury (TBI) focus on stabilizing individuals and on preventing further damage from the secondary consequences of TBI. A major complication of TBI is cerebral edema, which can be caused by the loss of blood brain barrier (BBB) integrity. Recent studies in several CNS pathologies have shown that activation of latent platelet derived growth factor-CC (PDGF-CC) within the brain can promote BBB permeability through PDGF receptor α (PDGFRα) signaling, and that blocking this pathway improves outcomes. In this study we examine the efficacy for the treatment of TBI of an FDA approved antagonist of the PDGFRα, Imatinib. Using a murine model we show that Imatinib treatment, begun 45 min after TBI and given twice daily for 5 days, significantly reduces BBB dysfunction. This is associated with significantly reduced lesion size 24 h, 7 days, and 21 days after TBI, reduced cerebral edema, determined from apparent diffusion co-efficient (ADC) measurements, and with the preservation of cognitive function. Finally, analysis of cerebrospinal fluid (CSF) from human TBI patients suggests a possible correlation between high PDGF-CC levels and increased injury severity. Thus, our data suggests a novel strategy for the treatment of TBI with an existing FDA approved antagonist of the PDGFRα.

Activation of PDGF-CC by tissue plasminogen activator impairs blood-brain barrier integrity during ischemic stroke.

Thrombolytic treatment of ischemic stroke with tissue plasminogen activator (tPA) is markedly limited owing to concerns about hemorrhagic complications and the requirement that tPA be administered within 3 h of symptoms. Here we report that tPA activation of latent platelet-derived growth factor-CC (PDGF-CC) may explain these limitations. Intraventricular injection of tPA or active PDGF-CC, in the absence of ischemia, leads to significant increases in cerebrovascular permeability. In contrast, co-injection of neutralizing antibodies to PDGF-CC with tPA blocks this increased permeability, indicating that PDGF-CC is a downstream substrate of tPA within the neurovascular unit. These effects are mediated through activation of PDGF-alpha receptors (PDGFR-alpha) on perivascular astrocytes, and treatment of mice with the PDGFR-alpha antagonist imatinib after ischemic stroke reduces both cerebrovascular permeability and hemorrhagic complications associated with late administration of thrombolytic tPA. These data demonstrate that PDGF signaling regulates blood-brain barrier permeability and suggest potential new strategies for stroke treatment.

Calcineurin initiates smooth muscle differentiation in neural crest stem cells.

The process of vascular smooth muscle cell (vSMC) differentiation is critical to embryonic angiogenesis. However, despite its importance, the vSMC differentiation program remains largely undefined. Murine gene disruption studies have identified several gene products that are necessary for vSMC differentiation, but these methodologies cannot establish whether or not a factor is sufficient to initiate the differentiation program. A gain-of-function system consisting of normal vSMC progenitor cells would serve as a useful complement to whole animal loss-of-function studies. We use such a system here, namely freshly isolated rat neural crest stem cells (NCSCs), to show that activation of the calcineurin signaling pathway is sufficient to drive these cells toward a smooth muscle fate. In addition, we present data suggesting that transforming growth factor (TGF)-beta1, which also causes NCSCs to differentiate into smooth muscle, activates calcineurin signaling in NCSCs, leading to a model in which activation of calcineurin signaling is the mechanism by which TGF-beta1 causes SMC differentiation in these cells.