The Delta-Subunit Selective GABAA Receptor Modulator, DS2, Improves Stroke Recovery via an Anti-inflammatory Mechanism

I guess you are just going to have to wait until human followup is done. Maybe 50 years from now unless WE destroy the existing stroke associations and run them like the Michael J. Fox Foundation which is dedicated to finding a cure for Parkinson's disease. 

The Delta-Subunit Selective GABA_A Receptor Modulator, DS2, Improves Stroke Recovery via an Anti-inflammatory Mechanism

Silke Neumann^1,2†, Lily Boothman-Burrell^2†, Emma K. Gowing², Thomas A. Jacobsen³, Philip K. Ahring⁴, Sarah L. Young¹, Karin Sandager-Nielsen³ and Andrew N. Clarkson^2*

• ¹Department of Pathology, Dunedin School of Medicine, University of Otago, Dunedin, New Zealand
• ²Department of Anatomy, Brain Health Research Centre, Brain Research New Zealand, University of Otago, Dunedin, New Zealand
• ³Saniona A/S, Copenhagen, Denmark
• ⁴School of Pharmacy, University of Sydney, Sydney, NSW, Australia

Inflammatory processes are known to contribute to tissue damage in the central nervous system (CNS) across a broad range of neurological conditions, including stroke. Gamma amino butyric acid (GABA), the main inhibitory neurotransmitter in the CNS, has been implicated in modulating peripheral immune responses by acting on GABA_A receptors on antigen-presenting cells and lymphocytes. Here, we investigated the effects and mechanism of action of the delta-selective compound, DS2, to improve stroke recovery and modulate inflammation. We report a decrease in nuclear factor (NF)-κB activation in innate immune cells over a concentration range in vitro. Following a photochemically induced motor cortex stroke, treatment with DS2 at 0.1 mg/kg from 1 h post-stroke significantly decreased circulating tumor necrosis factor (TNF)-α, interleukin (IL)-17, and IL-6 levels, reduced infarct size and improved motor function in mice. Free brain concentrations of DS2 were found to be lower than needed for robust modulation of central GABA_A receptors and were not affected by the presence and absence of elacridar, an inhibitor of both P-glycoprotein and breast cancer resistance protein (BCRP). Finally, as DS2 appears to dampen peripheral immune activation and only shows limited brain exposure, we assessed the role of DS2 to promote functional recovery after stroke when administered from 3-days after the stroke. Treatment with DS2 from 3-days post-stroke improved motor function on the grid-walking, but not on the cylinder task. These data highlight the need to further develop subunit-selective compounds to better understand change in GABA receptor signaling pathways both centrally and peripherally. Importantly, we show that GABA compounds such as DS2 that only shows limited brain exposure can still afford significant protection and promote functional recovery most likely via modulation of peripheral immune cells and could be given as an adjunct treatment.

Introduction

Stroke is the leading cause of lasting disability, with patients experiencing varied levels of functional recovery, and with more than 50% of survivors being discharged into care (Dobkin, 2008; Go et al., 2014). Changes in neuronal excitability, loss of gamma amino butyric acid (GABA) inhibition, enhanced glutamatergic signaling, and changes in neuronal connections and plasticity all contribute to impairment after stroke (Wittenberg and Schaechter, 2009; Clarkson et al., 2010, 2011, 2015, 2019; Carmichael, 2012; Krakauer et al., 2012). In addition, it is well documented that the full expansion of the infarction and ongoing impairment in the weeks to months following a stroke is underpinned by inflammation and the infiltration of peripheral immune cells that cross the blood–brain-barrier (BBB) (Gelderblom et al., 2009, 2015; Doyle et al., 2015). To date, drug therapies have attempted to minimize the extent of cell death, however, all drug therapies that have been trialled have failed to translate into the clinic. Therefore, new drug therapies need to be developed in order to support the recovery of stroke patients.

Changes in inflammatory processes is a hallmark for many pathologies including obesity, diabetes and stroke. Although acute inflammation is beneficial for the repairing and healing process, chronic inflammation contributes to tissue damage. Immune cells play a critical role in contributing to brain damage initiated by ischemic stroke. As a consequence of stroke, immune cells migrate to the brain in response to danger signals (damage-associated molecular patterns, DAMPs), in an effort to repair the damage (Brait et al., 2010; Gelderblom et al., 2015). However, these cells can also promote further inflammation and damage. In addition, the injured brain has an immune-suppressive effect that promotes life-threating infections, which threaten the survival of stroke patients (Liesz et al., 2015).

Traditionally considered a disease refined to the brain, it is becoming increasingly clear that the immune system heavily impacts the pathology of stroke. Local microglia, endothelial cells, neurons, and astrocytes recognize danger signals released from dying cells, which in turn stimulate the production of pro-inflammatory cytokines that attract circulating immune cells to migrate to the central nervous system (Offner et al., 2006; Gelderblom et al., 2009). Together, local and infiltrating cells, contribute to further neural cell death by producing pro-inflammatory cytokines, reactive oxygen species and by activating matrix metalloproteinases (Amantea et al., 2015). In particular, microglia have been shown to be chronically activated even when the initial DAMPs have been cleared (Huh et al., 2003; McGeer et al., 2003). This results in prolonged neuroinflammation that is associated with delayed recovery in stroke patients (Liguz-Lecznar and Kossut, 2013), and impaired memory, sensory learning and plasticity (Greifzu et al., 2011; Doyle et al., 2015).

Gamma amino butyric acid, known for its role as inhibitory neurotransmitter in the CNS, has a similar inhibiting effect on immune cells thereby creating a link between the CNS and the peripheral inflammatory response (Reyes-Garcia et al., 2007). Both, innate and adaptive immune cells express functional GABA receptors and possess enzymes to synthesize and catabolize GABA (Wheeler et al., 2011; Fuks et al., 2012). This includes microglia, macrophages, dendritic cells and T-cells. GABA signals through GABA_A and GABA_B receptors, both of which are expressed on immune cells (Kuhn et al., 2004; Wheeler et al., 2011; Fuks et al., 2012). The composition of the five subunits that make up GABA_A receptors likely varies for the various immune cells, which in turn will account for differences in potency and efficacy of drug treatments targeting GABA receptors and GABA itself (Fuks et al., 2012). GABA is known to act on GABA_A receptors in both millimolar and nanomolar to micromolar concentrations depending on the location (synaptic versus extrasynaptic) and functional composition of the receptors (Mody, 2001; Semyanov et al., 2003; Glykys and Mody, 2007). Of importance, submicromolar GABA concentrations have not only been found around neurons in the brain, but have also been detected in blood and hormone-producing cells in the intestine (Petty et al., 1999; Braun et al., 2004; Wendt et al., 2004). In addition to being exposed to chronic low levels of GABA, these peripheral tissues and receptors are likely to also be modulated following treatment with various GABA modulators. With the development of subunit specific GABA modulators, we may be able to find and develop compounds that could selectively regulate the function of peripheral immune cells.

Extrasynaptic GABA_A receptors, which are located outside the synapse typically contain either the δ- or α5-subunit and are highly sensitive to low GABA concentrations (Mody, 2001). Recent evidence has shown that modulation of extrasynaptic GABA_A receptors plays an important role in minimizing the extent of damage when given early (within hours) to increase tonic GABA currents after a stroke. In addition, this modulation can also facilitate an improvement in motor function when treatment is initiated at a delay (days) to dampen tonic GABA currents after the initial insult (Clarkson et al., 2010, 2019). As little is known about the role of δ-containing GABA_A receptor after stroke, we were interested in testing the therapeutic effects of the δ-subunit-selective GABA_A receptor modulator DS2 (4-chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridin-3-yl]benzamide). DS2 positively modulates δ-containing GABA_A receptors (Wafford et al., 2009), however, DS2 has not been investigated in a clinical disease model. Therefore, we aimed to assess the potential of DS2 to improve stroke recovery and to modulate inflammatory responses in innate immune cells. Herein, we show that positive allosteric modulation of δ-containing GABA_A receptors with DS2 affords significant protection and improves motor function in a mouse model of stroke. Investigation into a potential mechanism of action revealed that DS2 reduces the activation of NF-κB in LPS-stimulated macrophages and reduces the expression of activation markers on bone marrow-derived dendritic cells (BMDCs). Interestingly, we show that DS2 only has limited brain exposure, indicating that DS2-mediated effects in vivo are most likely attributed to modulation of peripheral immune cells.

More at link.