IGF-1 deficiency impairs neurovascular coupling in mice: implications for cerebromicrovascular aging

Aging is associated with marked deficiency in circulating IGF-1, which has been shown to contribute to age-related cognitive decline. Impairment of moment-to-moment adjustment of cerebral blood flow (CBF) via neurovascular coupling is thought to play a critical role in the genesis of age-related cognitive impairment. To establish the link between IGF-1 deficiency and cerebromicrovascular impairment, neurovascular coupling mechanisms were studied in a novel mouse model of IGF-1 deficiency (Igf1f/f -TBG-Cre-AAV8) and accelerated vascular aging. We found that IGF-1-deficient mice exhibit neurovascular uncoupling and show a deficit in hippocampal-dependent spatial memory test, mimicking the aging phenotype. IGF-1 deficiency significantly impaired cerebromicrovascular endothelial function decreasing NO mediation of neurovascular coupling. IGF-1 deficiency also impaired glutamate-mediated CBF responses, likely due to dysregulation of astrocytic expression of metabotropic glutamate receptors and impairing mediation of CBF responses by eicosanoid gliotransmitters. Collectively, we demonstrate that IGF-1 deficiency promotes cerebromicrovascular dysfunction and neurovascular uncoupling mimicking the aging phenotype, which are likely to contribute to cognitive impairment

Improving a Natural CaMKII Inhibitor by Random and Rational Design

CaM-KIIN has evolved to inhibit stimulated and autonomous activity of the Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) efficiently, selectively, and potently (IC50 ∼100 nM). The CN class of peptides, derived from the inhibitory region of CaM-KIIN, provides powerful new tools to study CaMKII functions. The goal of this study was to identify the residues required for CaMKII inhibition, and to assess if artificial mutations could further improve the potency achieved during evolution.First, the minimal region with full inhibitory potency was identified (CN19) by determining the effect of truncated peptides on CaMKII activity in biochemical assays. Then, individual residues of CN19 were mutated. Most individual Ala substitutions decreased potency of CaMKII inhibition, however, P3A, K13A, and R14A increased potency. Importantly, this initial Ala scan suggested a specific interaction of the region around R11 with the CaMKII substrate binding site, which was exploited for further rational mutagenesis to generate an optimized pseudo-substrate sequence. Indeed, the potency of the optimized peptide CN19o was >250fold improved (IC50 <0.4 nM), and CN19o has characteristics of a tight-binding inhibitor. The selectivity for CaMKII versus CaMKI was similarly improved (to almost 100,000fold for CN19o). A phospho-mimetic S12D mutation decreased potency, indicating potential for regulation by cellular signaling. Consistent with importance of this residue in inhibition, most other S12 mutations also significantly decreased potency, however, mutation to V or Q did not.These results provide improved research tools for studying CaMKII function, and indicate that evolution fine-tuned CaM-KIIN not for maximal potency of CaMKII inhibition, but for lower potency that may be optimal for dynamic regulation of signal transduction

Peripheral blood mononuclear cell activation sustains seizure activity

Objective: The influx of immune cells and serum proteins from the periphery into the brain due to a dysfunctional blood-brain barrier (BBB) has been proposed to contribute to the pathogenesis of seizures in various forms of epilepsy and encephalitis. We evaluated the pathophysiological impact of activated peripheral blood mononuclear cells (PBMCs) and serum albumin on neuronal excitability in an in vitro brain preparation. Methods: A condition of mild endothelial activation induced by arterial perfusion of lipopolysaccharide (LPS) was induced in the whole brain preparation of guinea pigs maintained in vitro by arterial perfusion. We analyzed the effects of co-perfusion of human recombinant serum albumin with human PBMCs activated with concanavalin A on neuronal excitability, BBB permeability (measured by FITC-albumin extravasation), and microglial activation. Results: Bioplex analysis in supernatants of concanavalin A–stimulated PBMCs revealed increased levels of several inflammatory mediators, in particular interleukin (IL)-1β, tumor necrosis factor (TNF)-α, interferon (INF)-γ, IL-6, IL-10, IL-17A, and MIP3α. LPS and human albumin arterially co-perfused with either concanavalin A–activated PBMCs or the cytokine-enriched supernatant of activated PBMCs (1) modulated calcium-calmodulin–dependent protein kinase II at excitatory synapses, (2) enhanced BBB permeability, (3) induced microglial activation, and (4) promoted seizure-like events. Separate perfusions of either nonactivated PBMCs or concanavalin A–activated PBMCs without LPS/human albumin (hALB) failed to induce inflammatory and excitability changes. Significance: Activated peripheral immune cells, such as PBMCs, and the extravasation of serum proteins in a condition of BBB impairment contribute to seizure generation

Diabetic hyperglycaemia activates CaMKII and arrhythmias by O-linked glycosylation

Ca(2+)-Calmodulin dependent protein kinase II (CaMKII) is a regulatory node in heart and brain, and its chronic activation can be pathological. CaMKII activation seen in heart failure can directly induce pathological changes in ion channels, Ca(2+) handling and gene transcription.(1) Here we discover a novel mechanism linking CaMKII and hyperglycemic signaling in diabetes mellitus, which is a key risk factor for heart(2) and neurodegenerative diseases.(3,4) Acute hyperglycemia causes covalent modification of CaMKII by O-linked N-acetylglucosamine (O-GlcNAc). O-GlcNAc modification of CaMKII at Ser-279 activates CaMKII autonomously, creating molecular memory even after [Ca(2+)] declines. O-GlcNAc modified CaMKII is increased in heart and brain from diabetic humans and rats. In cardiomyocytes, increased [glucose] significantly enhances CaMKII-dependent activation of spontaneous sarcoplasmic reticulum (SR) Ca(2+) release events that can contribute to cardiac mechanical dysfunction and arrhythmias.(1) These effects were prevented by pharmacological inhibition of O-GlcNAc signaling or genetic ablation of CaMKIIδ. In intact perfused hearts, arrhythmias were enhanced by increased [glucose] via O-GlcNAc-and CaMKII-dependent pathways. In diabetic animals, acute blockade of O-GlcNAc inhibited arrhythmogenesis. Thus, O-GlcNAc modification of CaMKII is a novel signaling event in pathways that may contribute critically to cardiac and neuronal pathophysiology in diabetes and other diseases