RESUMEN
Ca2+/calmodulin-dependent protein kinase II alpha subunit (CaMKIIα) is a key neuronal signaling protein and an emerging drug target. The central hub domain regulates the activity of CaMKIIα by organizing the holoenzyme complex into functional oligomers, yet pharmacological modulation of the hub domain has never been demonstrated. Here, using a combination of photoaffinity labeling and chemical proteomics, we show that compounds related to the natural substance γ-hydroxybutyrate (GHB) bind selectively to CaMKIIα. By means of a 2.2-Å x-ray crystal structure of ligand-bound CaMKIIα hub, we reveal the molecular details of the binding site deep within the hub. Furthermore, we show that binding of GHB and related analogs to this site promotes concentration-dependent increases in hub thermal stability believed to alter holoenzyme functionality. Selectively under states of pathological CaMKIIα activation, hub ligands provide a significant and sustained neuroprotection, which is both time and dose dependent. This is demonstrated in neurons exposed to excitotoxicity and in a mouse model of cerebral ischemia with the selective GHB analog, HOCPCA (3-hydroxycyclopent-1-enecarboxylic acid). Together, our results indicate a hitherto unknown mechanism for neuroprotection by a highly specific and unforeseen interaction between the CaMKIIα hub domain and small molecule brain-penetrant GHB analogs. This establishes GHB analogs as powerful tools for investigating CaMKII neuropharmacology in general and as potential therapeutic compounds for cerebral ischemia in particular.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Oxibato de Sodio/metabolismo , Sitios de Unión , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Ácidos Carboxílicos/farmacología , Cristalografía por Rayos X , Ciclopentanos/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Neuroprotección , Unión Proteica , Dominios Proteicos , Transducción de SeñalRESUMEN
Stroke-induced cognitive impairments remain of significant concern, with very few treatment options available. The involvement of glycosaminoglycans in neuroregenerative processes is becoming better understood and recent advancements in technology have allowed for cost-effective synthesis of novel glycomimetics. The current study evaluated the therapeutic potential of two novel glycomimetics, compound A and G, when administered systemically five-days post-photothrombotic stroke to the PFC. As glycosaminoglycans are thought to facilitate growth factor function, we also investigated the combination of our glycomimetics with intracerebral, recombinant human brain-derived neurotrophic factor (rhBDNF). C56BL/6J mice received sham or stroke surgery and experimental treatment (day-5), before undergoing the object location recognition task (OLRT). Four-weeks post-surgery, animals received prelimbic injections of the retrograde tracer cholera toxin B (CTB), before tissue was collected for quantification of thalamo-PFC connectivity and reactive astrogliosis. Compound A or G treatment alone modulated a degree of reactive astrogliosis yet did not influence spatial memory performance. Contrastingly, compound G+rhBDNF treatment significantly improved spatial memory, dampened reactive astrogliosis and limited stroke-induced loss of connectivity between the PFC and midline thalamus. As rhBDNF treatment had negligible effects, these findings support compound A acted synergistically to enhance rhBDNF to restrict secondary degeneration and facilitate functional recovery after PFC stroke.
Asunto(s)
Memoria Espacial , Accidente Cerebrovascular , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Gliosis/complicaciones , Glicosaminoglicanos , Ratones , Accidente Cerebrovascular/complicacionesRESUMEN
Stroke-induced cognitive impairments are of significant concern, however mechanisms that underpin these impairments remain poorly understood and researched. To further characterise cognitive impairments in our frontal cortex stroke model, and to align our assessments with what is used clinically, we tested young C57BL/6J mice trained in operant touchscreen chambers to complete the trial-unique nonmatched-to-location (TUNL) task. Based on baseline performance, animals were given either stroke (n = 12) or sham (n = 12) surgery using a photothrombosis model, bilaterally targeting the frontal cortex. Upon recovery, post-stroke spatial working memory was assessed by varying the degree of separation and delay within TUNL trials. Seven weeks after surgery, animals received a prelimbic injection of the retrograde tracer cholera toxin B (CTB) to access thalamo-PFC connectivity. Tissue was then processed histologically and immunohistochemically to assess infarct volume, astrogliosis and thalamocortical connectivity. Assessment of TUNL probes revealed sensitivity to a frontal cortex stroke (separation: p = 0.0003, delay: p < 0.0001), with stroke animals taking significantly longer (p = 0.0170) during reacquisition of the TUNL task, relative to shams. CTB-positive cell counts revealed a stroke-induced loss of thalamo-PFC connectivity. In addition, quantification of reactive astrogliosis revealed a positive correlation between the degree of astrogliosis expanding into white matter tracts and the development of cognitive impairments. This study reveals a stroke-induced impairment in mice completing the TUNL task. Our findings also demonstrate a significant loss of thalamo-PFC connections and a correlation between white matter reactive astrogliosis and cognitive impairment. Future experiments will investigate therapeutic interventions in the hope of promoting functional improvement in cognition.
Asunto(s)
Lóbulo Frontal/patología , Trastornos de la Memoria/etiología , Memoria a Corto Plazo , Accidente Cerebrovascular/patología , Animales , Técnica del Anticuerpo Fluorescente , Masculino , Trastornos de la Memoria/patología , Ratones , Ratones Endogámicos C57BL , Memoria Espacial , Accidente Cerebrovascular/complicacionesRESUMEN
Stroke remains a leading cause of disability worldwide. Recently, we have established an animal model of stroke that results in delayed impairment in spatial memory, allowing us to better investigate cognitive deficits. Young and aged brains show different recovery profiles after stroke; therefore, we assessed aged-related differences in poststroke cognition. As neurotrophic support diminishes with age, we also investigated the involvement of brain-derived neurotrophic factor (BDNF) in these differences. Young (3-6 months old) and aged (16-21 months old) mice were trained in operant touchscreen chambers to complete a visual pairwise discrimination (VD) task. Stroke or sham surgery was induced using the photothrombotic model to induce a bilateral prefrontal cortex stroke. Five days poststroke, an additional cohort of aged stroke animals were treated with intracerebral hydrogels loaded with the BDNF decoy, TrkB-Fc. Following treatment, animals underwent the reversal and rereversal task to identify stroke-induced cognitive deficits at days 17 and 37 poststroke, respectively. Assessment of sham animals using Cox regression and log-rank analyses showed aged mice exhibit an increased impairment on VD reversal and rereversal learning compared to young controls. Stroke to young mice revealed no impairment on either task. In contrast, stroke to aged mice facilitated a significant improvement in reversal learning, which was dampened in the presence of the BDNF decoy, TrkB-Fc. In addition, aged stroke control animals required significantly less consecutive days and correction trials to master the reversal task, relative to aged shams, an effect dampened by TrkB-Fc. Our findings support age-related differences in recovery of cognitive function after stroke. Interestingly, aged stroke animals outperformed their sham counterparts, suggesting reopening of a critical window for recovery that is being mediated by BDNF.
Asunto(s)
Cognición/fisiología , Recuperación de la Función/fisiología , Aprendizaje Inverso/fisiología , Accidente Cerebrovascular/psicología , Animales , Aprendizaje Discriminativo/fisiología , Modelos Animales de Enfermedad , Masculino , Ratones , Receptor trkB/metabolismo , Accidente Cerebrovascular/metabolismoRESUMEN
Stroke remains a leading cause of adult disability with treatments limited to thrombolytic therapies that are severely limited by a narrow therapeutic window. The potential of hundreds of other therapeutic agents cannot be evaluated due to their poor ability to cross the blood-brain barrier. Recently, biopolymer hydrogels have shown promise at overcoming these obstacles via the delivering of therapeutic molecules (pharmacological, mRNA, stem cells, etc.) to injured nervous tissue to afford functional recovery in rodent models of stroke. To date, we have tested different biopolymer hydrogels in mouse models of stroke for their ability to promote post-stroke recovery and for in situ delivery of growth factors, small pharmacological compounds, siRNAs, and stem cells. Here, we describe practical instructions on how to prepare various biopolymer hydrogels in house with further guidance on how to use them for intracerebral administration of therapeutic agents in preclinical stroke models.
Asunto(s)
Hidrogeles , Accidente Cerebrovascular , Ratones , Animales , Hidrogeles/uso terapéutico , Accidente Cerebrovascular/tratamiento farmacológico , Células MadreRESUMEN
Ischemic stroke remains a leading cause of disability worldwide, with limited treatment options available. This study investigates GABAC receptors as novel pharmacological targets for stroke recovery. The expression of ρ1 and ρ2 mRNA in mice were determined in peri-infarct tissue following photothrombotic motor cortex stroke. (R)-4-amino-cyclopent-1-enyl butylphosphinic acid (R)-4-ACPBPA and (S)-4-ACPBPA were assessed using 2-elecotrode voltage electrophysiology in Xenopus laevis oocytes. Stroke mice were treated for 4 weeks with either vehicle, the α5-selective negative allosteric modulator, L655,708, or the ρ1/2 antagonists, (R)-4-ACPBPA and (S)-4-ACPBPA respectively from 3 days post-stroke. Infarct size and expression levels of GAT3 and reactive astrogliosis were determined using histochemistry and immunohistochemistry respectively, and motor function was assessed using both the grid-walking and cylinder tasks. After stroke, significant increases in ρ1 and ρ2 mRNAs were observed on day 3, with ρ2 showing a further increase on day 7. (R)- and (S)-4-ACPBPA are both potent antagonists at ρ2 and only weak inhibitors of α5ß2γ2 receptors. Treatment with either L655,708, (S)-4-ACPBPA (ρ1/2 antagonist; 5 mM only), or (R)-4-ACPBPA (ρ2 antagonist; 2.5 and 5 mM) from 3 days after stroke resulted in a significant improvement in motor recovery on the grid-walking task, with L655,708 and (R)-4-ACPBPA also showing an improvement in the cylinder task. Infarct size was unaffected, and only (R)-4-ACPBPA significantly increased peri-infarct GAT3 expression and decreased the level of reactive astrogliosis. Importantly, inhibiting GABAC receptors affords significant improvement in motor function after stroke. Targeting the ρ-subunit could provide a novel delayed treatment option for stroke recovery.
RESUMEN
Stroke remains the leading cause of long-term disability with limited options available to aid in recovery. Significant effort has been made to try and minimize neuronal damage following stroke with use of neuroprotective agents, however, these treatments have yet to show clinical efficacy. Regenerative interventions have since become of huge interest as they provide the potential to restore damaged neural tissue without being limited by a narrow therapeutic window. Neurotrophins, such as brain-derived neurotrophic factor (BDNF), and their high affinity receptors are actively produced throughout the brain and are involved in regulating neuronal activity and normal day-to-day function. Furthermore, neurotrophins are known to play a significant role in both protection and recovery of function following neurodegenerative diseases such as stroke and traumatic brain injury (TBI). Unfortunately, exogenous administration of these neurotrophins is limited by a lack of blood-brain-barrier (BBB) permeability, poor half-life, and rapid degradation. Therefore, we have focused this review on approaches that provide a direct and sustained neurotrophic support using pharmacological therapies and mimetics, physical activity, and potential drug delivery systems, including discussion around advantages and limitations for use of each of these systems. Finally, we discuss future directions of biomaterial drug-delivery systems, including the incorporation of heparan sulfate (HS) in conjunction with neurotrophin-based interventions.