ABSTRACT
Stroke leads to brain damage with subsequent slow and incomplete recovery of lost brain functions. Enriched housing of stroke-injured rats provides multi-modal sensorimotor stimulation, which improves recovery, although the specific mechanisms involved have not been identified. In rats housed in an enriched environment for two weeks after permanent middle cerebral artery occlusion, we found increased sigma-1 receptor expression in peri-infarct areas. Treatment of rats subjected to permanent or transient middle cerebral artery occlusion with 1-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl)piperazine dihydrochloride, an agonist of the sigma-1 receptor, starting two days after injury, enhanced the recovery of lost sensorimotor function without decreasing infarct size. The sigma-1 receptor was found in the galactocerebroside enriched membrane microdomains of reactive astrocytes and in neurons. Sigma-1 receptor activation increased the levels of the synaptic protein neurabin and neurexin in membrane rafts in the peri-infarct area, while sigma-1 receptor silencing prevented sigma-1 receptor-mediated neurite outgrowth in primary cortical neuronal cultures. In astrocytic cultures, oxygen and glucose deprivation induced sigma-1 receptor expression and actin dependent membrane raft formation, the latter blocked by sigma-1 receptor small interfering RNA silencing and pharmacological inhibition. We conclude that sigma-1 receptor activation stimulates recovery after stroke by enhancing cellular transport of biomolecules required for brain repair, thereby stimulating brain plasticity. Pharmacological targeting of the sigma-1 receptor provides new opportunities for stroke treatment beyond the therapeutic window of neuroprotection.
Subject(s)
Brain/metabolism , Infarction, Middle Cerebral Artery/pathology , Infarction, Middle Cerebral Artery/physiopathology , Neuronal Plasticity/physiology , Receptors, sigma/metabolism , Recovery of Function/physiology , Animals , Astrocytes/drug effects , Brain/drug effects , Caveolin 1/genetics , Caveolin 1/metabolism , Cell Hypoxia/drug effects , Cells, Cultured , Disease Models, Animal , Dose-Response Relationship, Drug , Environment , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , Glucose/deficiency , Infarction, Middle Cerebral Artery/drug therapy , Infarction, Middle Cerebral Artery/metabolism , Male , Movement/drug effects , Neurites/drug effects , Neurites/physiology , Neuronal Plasticity/drug effects , Neurons/cytology , Neurons/metabolism , Nootropic Agents/pharmacology , Nootropic Agents/therapeutic use , Piperazines/pharmacology , Piperazines/therapeutic use , Protein Transport/drug effects , Psychomotor Performance/drug effects , RNA, Small Interfering/pharmacology , Rats , Rats, Inbred SHR , Receptors, sigma/genetics , Recovery of Function/drug effects , Statistics, Nonparametric , Transfection/methods , Sigma-1 ReceptorABSTRACT
Amyloid beta-derived diffusible ligands (ADDLs) comprise the neurotoxic subset of soluble Abeta(1-42) oligomers, now widely considered to be the molecular cause of memory malfunction and neurodegeneration in Alzheimer's disease (AD). We have developed a screening cascade which identifies small molecule modulators of ADDL-mediated neurotoxicity. The primary screen involves a fluorescence resonance energy transfer (FRET)-based assay which selects inhibitors of Abeta1-42 oligomer assembly. The identified hits were further characterized by assessing their ability to inhibit the assembly and binding of ADDLs to cultures of primary hippocampal neurons. This approach has led to the identification of a number of small molecules which inhibit ADDL assembly and their subsequent binding to neurons. Here we describe our small molecule discovery efforts to identify ADDL assembly blocker and ADDL binding inhibitors, and to transform validated hits into pre-clinical lead compounds.
Subject(s)
Alzheimer Disease/drug therapy , Amyloid beta-Peptides/metabolism , Antipsychotic Agents/therapeutic use , Amyloid beta-Peptides/antagonists & inhibitors , Amyloid beta-Peptides/chemistry , Animals , Antipsychotic Agents/chemistry , Drug Design , Humans , Small Molecule LibrariesABSTRACT
To facilitate drug discovery directed toward platelet-specific targets, we developed a platelet isolation and fluorophore-loading method that yields functionally responsive platelets in which we were able to detect agonist-induced calcium flux using a microfluidics-based screening platform. The platelet preparation protocol was designed to minimize preparation-induced platelet activation and to optimize signal strength. Measurement of platelet activation, as monitored by ratiometric determination of agonist-induced calcium flux in fluor-loaded human platelets, was optimized in a macrosample cuvette format in preparation for detection in a microfluidic chip-based assay. For the microfluidic device used in these studies, a cell density of 1 to 2 x 10(6) platelets per milliliter and a nominal flow rate of 5 to 10 nl per second provided optimal event resolution of 5 to 20 platelets traversing the detection volume per unit time. Platelets responded in a dose-dependent manner to adenosine diphosphate and protease-activating peptide (PAR) 1 thrombin receptor-activating peptide (TRAP). The work presented here constitutes proof-of-principle experiments demonstrating the enabling application of a microfluidic device to conduct high-throughput signaling studies and drug discovery screening against human platelet targets.
