GHB analogs confer neuroprotection through specific interaction with the CaMKIIα hub domain

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.

The calcium/calmodulin-dependent protein kinase II alpha subunit (CaMKIIα) is a central mediator of synaptic plasticity and responds to minute fluctuations in calcium (Ca²⁺) (1). The CaMKIIα holoenzyme is a large protein assembly of 12 to 14 subunits, each consisting of a kinase domain flexibly linked to the central hub domain. The hub domain is conserved through evolution (2). It organizes the holoenzyme into oligomeric structures (3, 4), yet displays remarkable dynamics (5). This correlates well with an emerging functional importance in activation-triggered destabilization and release of vertical dimers that may enable spreading of activity (6, 7). Furthermore, the hub domain has been reported to interact directly with the kinase domains (8–10) to confer allosteric control of kinase activity (9). The importance of preserving hub integrity is further evident from a human patient with a mutation in the hub (p.His477Tyr) causing defective oligomerization and severe neurodevelopmental defects (11). Thus far, pharmacological modulation of the hub domain has never been demonstrated but would constitute an attractive approach to regulate overall kinase function in cases of CaMKIIα aberrant activity.Functionally, CaMKIIα is activated in a highly cooperative manner, initiated by increases in intracellular Ca²⁺, Ca²⁺/CaM binding, and autophosphorylation at residue Thr286 in the regulatory segment (12). This is then accompanied by translocation of CaMKIIα to the postsynaptic density (PSD) (13). In cases of excessive stimuli, such as ischemic brain injury or glutamate-mediated excitotoxicity, Thr286 autophosphorylation permits Ca²⁺/CaM-independent autonomous activity which can persist for hours (14–16) and cause cell death (17).The natural brain substance γ-hydroxybutyrate (GHB) is a metabolite of γ-aminobutyric acid (GABA) which has been reported to be neuroprotective in mammals (18–20). GHB binds with high affinity to an until-now unknown specific binding protein highly expressed in forebrain regions (21). This site is distinct from GABA_B receptors also known to bind GHB, albeit with low affinity (22). We here reveal CaMKIIα as the long-sought-after specific GHB high-affinity binding site (SI Appendix, Fig. S1). Moreover, we show that the highly selective and brain-penetrant GHB analog 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) (23) confers significant neuroprotection in pathological states of CaMKII activation, such as after an ischemic injury. This is plausibly explained by the pronounced effect of GHB analogs on CaMKIIα hub stabilization upon binding and consequently functional regulation of the holoenzyme, although a causal link remains to be fully proven.     

Three-dimensional MRI of cerebral projections in rat brain in vivo after intracortical injection of MnCl2

In this study we investigated the potential of in vivo MRI detection of axonal Mn2+ transport for tracing neuronal projections originating in the sensorimotor cortex in healthy and lesioned rat brains. Special attention was given to the potential of visualizing neuronal sprouting of central nervous system across the midline. After injecting unchelated MnCl2 into the forelimb area of sensorimotor cortex of 18 healthy and 10 lesioned rats corticofugal projections could be traced through the internal capsule to the cerebral peduncle and the pyramidal decussation. Although the neuronal tract was visible as early as 6 h after MnCl2 injection, best contrast was achieved after 24-48 h. Beside the cortico-spinal tract, the cortico-thalamic fibres were also visualized by anterograde Mn2+ transport. Cortico-striatal fibres were partially masked by the very high signal near the MnCl2 injection site but could be discerned as well. Slight, diffuse signal enhancement of cortical tissue contralateral to the MnCl2 injection site in healthy rat brains suggests interhemispheric connections or passive diffusion of Mn2+. However, enhanced fibre tract contrast connecting both hemispheres was visible 16 weeks after onset of focal photothrombotic cortical injury. In conclusion our study has shown that we were able to visualize reproducibly the main descending corticofugal projections and interhemispheric connections by non-invasive MRI after localized injection of MnCl2. The appearance of interhemispheric Mn2+-enhanced fibres after photothrombotic focal injury indicates that the method may bear potential to follow non-invasively gross plastic changes of connectivity in the brain after injury.     

Tacrolimus (FK506) Increases Neuronal Expression of GAP-43 and Improves Functional Recovery after Spinal Cord Injury in Rats

Tacrolimus (FK506), a widely used immunosuppressant drug, has neurite-promoting activity in cultured PC12 cells and peripheral neurons. The present study investigated whether tacrolimus affects the expression of the neuronal growth-associated protein, GAP-43, as well as functional recovery after photothrombotic spinal cord injury in the rat. In injured animals receiving tacrolimus, the number of neurons expressing GAP-43 mRNA and protein approximately doubled compared to that in injured animals receiving vehicle alone. This increase in GAP-43-positive cells was paralleled by a significant improvement in neurological function evaluated by open-field and inclined plane tests. Another FKBP-12 ligand (V-10,367) had similar effects on GAP-43 expression and functional outcome, indicating that the observed effects of tacrolimus do not involve inhibition of the phosphatase calcineurin. Thus, tacrolimus, a drug which is already approved for use in humans, as well as other FKBP-12 ligands which do not inhibit calcineurin, could potentially enhance functional outcome after CNS injury in humans.     

Thinning,movement, and volume loss of residual cortical tissue occurs after stroke in the adult rat as identified by histological and magnetic resonance imaging analysis

Plasticity of residual cortical tissue has been identified as an important mediator of functional post-stroke recovery. Many studies have been directed toward describing biochemical, electrophysiological, and cytoarchitectural changes in residual cortex and correlating them with functional changes. Additionally, after neonatal stroke the thickness of residual tissue can change, the tissue can move, and tissue can fill in the stroke core. The purpose of the present study was to systematically investigate and document possible gross morphological changes in peri-infarct tissue after forelimb motor cortex stroke in the adult rat. Rats received a unilateral forelimb motor cortex stroke of equivalent size by pial strip devascularization or photothrombotic occlusion and were then examined using histology or magnetic resonance imaging (MRI) at 1 h, 1, 3, 7, 14, or 31 days post-stroke. Middle cerebral artery occlusion was used as a control stroke procedure. Decreases in cortical thickness, volume, and neural density were found to extend far beyond the stroke infarct and included most of the sensorimotor regions of the stroke and intact hemispheres. Movement of residual tissue towards the infarct was observed and confirmed using anatomical markers placed in intact cortical tissue at the time of stroke induction. The results are discussed in relation to the idea that extensive time-dependent morphological changes that occur in residual tissue must be considered when evaluating plasticity-related cortical changes associated with post-stroke recovery of function.     

Cholinergic upregulation by optogenetic stimulation of nucleus basalis after photothrombotic stroke in forelimb somatosensory cortex improves endpoint and motor but not sensory control of skilled reaching in mice

A network of cholinergic neurons in the basal forebrain innerve the forebrain and are proposed to contribute to a variety of functions including cortical plasticity, attention, and sensorimotor behavior. This study examined the contribution of the nucleus basalis cholinergic projection to the sensorimotor cortex on recovery on a skilled reach-to-eat task following photothrombotic stroke in the forelimb region of the somatosensory cortex. Mice were trained to perform a single pellet skilled reaching task and their pre and poststroke performance, from Day 4 to Day 28 poststroke, was assessed frame-by-frame by video analysis with endpoint, movement and sensorimotor integration measures. Somatosensory forelimb lesions produced impairments in endpoint and movement component measures of reaching and increased the incidence of fictive eating, a sensory impairment in mistaking a missed reach for a successful reach. Upregulated acetylcholine (ACh) release, as measured by local field potential recording, elicited via optogenetic stimulation of the nucleus basalis improved recovery of reaching and improved movement scores but did not affect sensorimotor integration impairment poststroke. The results show that the mouse cortical forelimb somatosensory region contributes to forelimb motor behavior and suggest that ACh upregulation could serve as an adjunct to behavioral therapy for acute treatment of stroke.     

Neural stem cell transplantation promotes behavioral recovery in a photothrombosis stroke model

Stem cell-based therapy provides a promising approach for treat stroke. Neural stem cells isolated from mice hippocampus possessing the capacity of differentiate into neurons and astrocytes both in vitro and vivo. Here, we investigated the capability of neural stem cell transplantation in photothrombosis stroke model. Nissl staining revealed that the cortical infarct significantly decreased by 16.32% (Vehicle: 27.93le: an mm³, n=6, NSC: 23.37le: ai mm³, n=6, P<0.05) in the NSC group compared with the vehicle. More over transplantation of neural stem cells significantly (P<0.01) improved neurological performance compared with vehicle. These results indicate that transplantation of neural stem cell is an effective therapy in ischemic stroke.