Lynda J. Peterson

Oxidative Stress and Microglial Cells in Parkinson's Disease

Significant evidence has now been accumulated that microglial cells play a central role in the degeneration of DA neurons in animal models of PD. The oxidative stress response by microglial cells, most notably the activity of the enzyme NADPH oxidase, appears to play a central role in the pathology of PD. This oxidative stress response occurs in microglia through the activation of the ERK signaling pathway by proinflammatory stimuli, leading to the phosphorylation and translocation of the p47phox and p67phox cytosolic subunits, the activation of membrane-bound PHOX, and the production of ROS. Therapeutic anti-inflammatories which prevent DA neurodegeneration in PD, including anti-inflammatory cytokines, morphinan compounds, NADPH oxidase inhibitors, NF-κB inhibitors, and β2-AR agonists, all function to inhibit the activation of the PHOX in microglial cells. These observations suggest a central role for the oxidative stress response in microglial cells as a mediator or regulator of DA neurodegeneration in PD.

β2-Adrenergic Receptor Activation Prevents Rodent Dopaminergic Neurotoxicity by Inhibiting Microglia via a Novel Signaling Pathway

The role of the β2 adrenergic receptor (β2AR) in the regulation of chronic neurodegenerative inflammation within the CNS is poorly understood. The purpose of this study was to determine neuroprotective effects of long-acting β2AR agonists such as salmeterol in rodent models of Parkinson’s disease. Results showed salmeterol exerted potent neuroprotection against both LPS and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/1-methyl-4-phenylpyridinium–induced dopaminergic neurotoxicity both in primary neuron-glia cultures (at subnanomolar concentrations) and in mice (1–10 μg/kg/day doses). Further studies demonstrated that salmeterol-mediated neuroprotection is not a direct effect on neurons; instead, it is mediated through the inhibition of LPS-induced microglial activation. Salmeterol significantly inhibited LPS-induced production of microglial proinflammatory neurotoxic mediators, such as TNF-α, superoxide, and NO, as well as the inhibition of TAK1-mediated phosphorylation of MAPK and p65 NF-κB. The anti-inflammatory effects of salmeterol required β2AR expression in microglia but were not mediated through the conventional G protein-coupled receptor/cAMP pathway. Rather, salmeterol failed to induce microglial cAMP production, could not be reversed by either protein kinase A inhibitors or an exchange protein directly activated by cAMP agonist, and was dependent on β-arrestin2 expression. Taken together, our results demonstrate that administration of extremely low doses of salmeterol exhibit potent neuroprotective effects by inhibiting microglial cell activation through a β2AR/β-arrestin2–dependent but cAMP/protein kinase A-independent pathway.

Transcriptional Factor NF-κB as a Target for Therapy in Parkinson's Disease

Parkinsons disease (PD) is a neurodegenerative condition characterized by chronic inflammation. Nuclear factor κB (NF-κB) is a family of inducible transcription factors that are expressed in a wide variety of cells and tissues, including microglia, astrocytes, and neurons, and the classical NF-κB pathway plays a key role in the activation and regulation of inflammatory mediator production during inflammation. Activation of the classical NF-κB pathway is mediated through the activity of the IKK kinase complex, which consists of a heterotrimer of IKKα, IKKβ, and IKKγ subunits. Targeting NF-κB has been proposed as an approach to the treatment of acute and chronic inflammatory conditions, and the use of inhibitors specific for either IKKβ or IKKγ has now been found to inhibit neurodegeneration of TH+ DA-producing neurons in murine and primate models of Parkinsons disease. These studies suggest that targeting the classical pathway of NF-κB through the inhibition of the IKK complex can serve as a useful therapeutic approach to the treatment of PD.

CB1 Cannabinoid Receptors Promote Maximal FAK Catalytic Activity By Stimulating Cooperative Signaling Between Receptor Tyrosine Kinases and Integrins in Neuronal Cells

Tyrosine phosphorylation (Tyr-P) of focal adhesion kinase (FAK) regulates FAK activation. Phosphorylated FAK Tyr 397 binds Src family kinases (Src), which in turn directly phosphorylate FAK Tyr 576/577 to produce maximal FAK enzymatic activity. CB₁ cannabinoid receptors (CB₁) are abundantly expressed in the nervous system and influence FAK activation by presently unknown mechanisms. The current investigation determined that CB₁-stimulated maximal FAK catalytic activity is mediated by Gi/o proteins in N18TG2 neuronal cells, and that G12/13 regulation of Rac1 and RhoA occurs concomitantly. Immunoblotting analyses using antibodies against FAK phospho-Tyr 397 and phospho-Tyr 576/577 demonstrated that the time-course of CB₁-stimulated FAK 576/577 Tyr-P occurred in three phases: Phase I (0-2 min) maximal Tyr-P, Phase II (5-20 min) rapid decline in Tyr-P, and Phase III (>20 min) plateau in Tyr-P at submaximal levels. In contrast, FAK 397 Tyr-P was monophasic and significantly lower in magnitude. FAK 397 Tyr-P and Phase I FAK 576/577 Tyr-P involved protein tyrosine phosphatase (PTP1B and Shp1/Shp2)-mediated Src activation, Protein Kinase A (PKA) inhibition, and integrin activation. Phase I maximal FAK 576/577 Tyr-P also required cooperative signaling between receptor tyrosine kinases (RTKs) and integrins. The integrin antagonist RGDS peptide, Flk-1 vascular endothelial growth factor receptor (VEGFR) antagonist SU5416, and epidermal growth factor receptor (EGFR) antagonist AG 1478 blocked Phase I FAK 576/577 Tyr-P. CB₁ agonists failed to stimulate FAK Tyr-P in the absence of integrin activation upon suspension in serum-free culture media. In contrast, cells grown on the integrin ligands fibronectin and laminin displayed increased FAK 576/577 Tyr-P that was augmented by CB₁ agonists and blocked by the Src inhibitor PP2 and Flk-1 VEGFR antagonist SU5416. Taken together, these studies have identified a complex integrative pathway utilized by CB₁ to stimulate maximal FAK 576/577 Tyr-P in neuronal cells.

Inflammatory Responses and Regulation in Parkinson’s Disease

Parkinson’s disease (PD) is a slow, progressive neurodegenerative disorder affecting an estimated 6 million people worldwide (Litteljohn, Mangano et al. 2011). The etiology of the disease is characterized by increasing loss over time of dopaminergic neurons (DA-neurons) in the substantia nigra (SN) as well as the depletion of dopamine in the striatum, which eventually leads to pathological and clinical symptoms (Jenner and Olanow 2006). PET imaging and post-mortem analyses of the brains of PD patients indicate that the appearance of symptoms, including tremor, bradykinesia, rigidity, slowness of movement, and postural instability (Jellinger 2001), generally are manifest once 60% of DA-neurons have died and a 70% threshold decrease in normal DA activity has been reached (Klockgether 2004; Litteljohn, Mangano et al. 2011). Epidemiological studies indicate that only about 10% of PD cases are early onset , i.e. prior to the age of 50 and occur mainly in familial clusters (Mizuno, Hattori et al. 2001). These cases have established genetic bases due to mutations in several recently identified genes, including parkin, leucine-rich repeat kinase 2 (LRRK2), α-synuclein, PINK-1, or DJ-1 (Polymeropoulos, Lavedan et al. 1997; Lucking, Durr et al. 2000; Abou-Sleiman, Healy et al. 2003; Farrer, Haugarvoll et al. 2006; Sun, Latourelle et al. 2006; Jiang, Wu et al. 2007; Weng, Chou et al. 2007; Bonifati, Wu-Chou et al. 2008). The majority of PD cases (approximately 90%) are late onset and idiopathic (Tanner 2003). Although the etiology of idiopathic PD is uncertain, this form of PD particularly affects the elderly, with average onset of clinical symptoms between 60 and 65 years of age (Litteljohn, Mangano et al. 2011). Idiopathic PD is thus age-associated, with approximately 1% of the population being affected by 65-70 year of age, increasing to 4-5% at 85 years (Fahn 2003; Tansey, McCoy et al. 2007). However, the causes of idiopathic non-familial PD are probably multifactorial, with some form of genetic predisposition, environmental insults and/or aging all likely to be important factors in disease initiation and progression (Nagatsu and Sawada 2006; Dickson 2007; Vilar, Coelho et al. 2007; Singh, Singh et al. 2008). While the exact cause of chronic neurodegeneration of PD is not known, increasing evidence suggests that chronic inflammation is the fundamental process mediating the progressive nature of the neurodegeneration characteristic of PD (McGeer, Yasojima et al. 2001; Hirsch and Hunot 2009). In animal PD models, long-term inflammation induces progressive loss of DA neurons within the SN brain region (Gao, Jiang et al. 2002; Dauer and Przedborski 2003; Bartels and Leenders 2007). Neuroinflammation, which is characterized by activation of