No abstract

Background: Traumatic brain injury (TBI) remains one of the greatest public health concerns with increasing morbidity and mortality rates worldwide. Our group reported stimulation of astrocyte mitochondrial metabolism by P2Y1 receptor agonists significantly reduced cerebral edema and reactive gliosis in a TBI model. Subsequent data on the pharmacokinetics (PK) and rapid metabolism of these compounds suggested neuroprotection was likely mediated by a metabolite, AST-004, which binding data indicated was an adenosine A3 receptor (A3R) agonist. Methods: The neuroprotective efficacy of AST-004 was tested in a controlled closed cortical injury (CCCI) model of TBI in mice. Results: Twenty-four (24) hours post-injury, mice subjected to CCCI and treated with AST-004 (0.22mg/kg) exhibited significantly less secondary brain injury. These effects were quantified with less cell death (PSVue794 fluorescence) and loss of blood brain barrier breakdown (Evans Blue extravasation assay), compared to vehicle treated TBI mice. TBI treated mice also exhibited significantly reduced neuroinflammatory markers, glial-fibrillary acidic protein (GFAP, astrogliosis) and ionized Ca2+ binding adaptor molecule 1 (Iba1, microgliosis), both at the mRNA (gRT-PCR) and protein (western blot and immunofluorescence) levels, respectively. Four (4) weeks post-injury, AST-004 treated TBI mice presented significantly reduced impairment of long-term memory. Spatial memory was assessed with a contextual fear conditioning behavior assay (freezing behavior after shock). Finally, AST-004 treatments were found to increase in vivo ATP production in astrocytes (gfap-targeted luciferase activity), consistent with the proposed mechanism of action. Conclusions: These data reveal AST-004 as a novel A3R agonist that increases astrocyte energy production and enhances their neuroprotective efficacy after brain injury.

We investigated the contribution of excitatory transient receptor potential canonical (TRPC) cation channels to posttraumatic hyperexcitability in the brain 7 days following controlled cortical impact model of traumatic brain injury (TBI) to the parietal cortex in male adult mice. We investigated if TRPC1/TRPC4/TRPC5 channel expression is upregulated in excitatory neurons after TBI in contribution to epileptogenic hyperexcitability in key hippocampal and cortical circuits that have substantial cholinergic innervation. This was tested by measuring TRPC1/TRPC4/TRPC5 protein and messenger RNA (mRNA) expression, assays of cholinergic function, neuronal Ca2+ imaging in brain slices, and seizure susceptibility after TBI. We found region-specific increases in expression of TRPC1, TRPC4, and TRPC5 subunits in the hippocampus and cortex following TBI. The dentate gyrus, CA3 region, and cortex all exhibited robust upregulation of TRPC4 mRNA and protein. TBI increased cFos activity in dentate gyrus granule cells (DGGCs) and layer 5 pyramidal neurons both at the time of TBI and 7 days post-TBI. DGGCs displayed greater magnitude and duration of acetylcholine-induced rises in intracellular Ca2+ in brain slices from mice subjected to TBI. The TBI mice also exhibited greater seizure susceptibility in response to pentylenetetrazol-induced kindling. Blockade of TRPC4/TRPC5 channels with M084 reduced neuronal hyperexcitation and impeded epileptogenic progression of kindling. We observed that the time-dependent upregulation of TRPC4/TRPC5-containing channels alters cholinergic responses and activity of principal neurons acting to increase proexcitatory sensitivity. The underlying mechanism includes acutely decreased acetylcholinesterase function, resulting in greater Gq/11-coupled muscarinic receptor activation of TRPC channels. Overall, our evidence suggests that TBI-induced plasticity of TRPC channels strongly contributes to overt hyperexcitability and primes the hippocampus and cortex for seizures.