Selective and brain-penetrant HCN1 inhibitors reveal links between synaptic integration, cortical function, and working memory.

Hyperpolarization-activated and cyclic-nucleotide-gated 1 (HCN1) ion channels are proposed to be critical for cognitive function through regulation of synaptic integration. However, resolving the precise role of HCN1 in neurophysiology and exploiting its therapeutic potential has been hampered by minimally selective antagonists with poor potency and limited in vivo efficiency. Using automated electrophysiology in a small-molecule library screen and chemical optimization, we identified a primary carboxamide series of potent and selective HCN1 inhibitors with a distinct mode of action. In cognition-relevant brain circuits, selective inhibition of native HCN1 produced on-target effects, including enhanced excitatory postsynaptic potential summation, while administration of a selective HCN1 inhibitor to rats recovered decrement working memory. Unlike prior non-selective HCN antagonists, selective HCN1 inhibition did not alter cardiac physiology in human atrial cardiomyocytes or in rats. Collectively, selective HCN1 inhibitors described herein unmask HCN1 as a potential target for the treatment of cognitive dysfunction in brain disorders.

A versatile fluorescence-quenched substrate for quantitative measurement of glucocerebrosidase activity within live cells.

Loss of activity of the lysosomal glycosidase ß-glucocerebrosidase (GCase) causes the lysosomal storage disease Gaucher disease (GD) and has emerged as the greatest genetic risk factor for the development of both Parkinson disease (PD) and dementia with Lewy bodies. There is significant interest into how GCase dysfunction contributes to these diseases, however, progress toward a full understanding is complicated by presence of endogenous cellular factors that influence lysosomal GCase activity. Indeed, such factors are thought to contribute to the high degree of variable penetrance of GBA mutations among patients. Robust methods to quantitatively measure GCase activity within lysosomes are therefore needed to advance research in this area, as well as to develop clinical assays to monitor disease progression and assess GCase-directed therapeutics. Here, we report a selective fluorescence-quenched substrate, LysoFQ-GBA, which enables measuring endogenous levels of lysosomal GCase activity within living cells. LysoFQ-GBA is a sensitive tool for studying chemical or genetic perturbations of GCase activity using either fluorescence microscopy or flow cytometry. We validate the quantitative nature of measurements made with LysoFQ-GBA using various cell types and demonstrate that it accurately reports on both target engagement by GCase inhibitors and the GBA allele status of cells. Furthermore, through comparisons of GD, PD, and control patient-derived tissues, we show there is a close correlation in the lysosomal GCase activity within monocytes, neuronal progenitor cells, and neurons. Accordingly, analysis of clinical blood samples using LysoFQ-GBA may provide a surrogate marker of lysosomal GCase activity in neuronal tissue.

CD22 Blockage Restores Age-Related Impairments of Microglia Surveillance Capacity.

Microglia, the innate immune cells of the brain, are essential for maintaining homeostasis by their ramified, highly motile processes and for orchestrating the immune response to pathological stimuli. They are implicated in several neurodegenerative diseases like Alzheimer's and Parkinson's disease. One commonality of these diseases is their strong correlation with aging as the highest risk factor and studying age-related alterations in microglia physiology and associated signaling mechanism is indispensable for a better understanding of age-related pathomechanisms. CD22 has been identified as a modifier of microglia phagocytosis in a recent study, but not much is known about the function of CD22 in microglia. Here we show that CD22 surface levels are upregulated in aged versus adult microglia. Furthermore, in the amyloid mouse model PS2APP, Aß-containing microglia also exhibit increased CD22 signal. To assess the impact of CD22 blockage on microglia morphology and dynamics, we have established a protocol to image microglia process motility in acutely prepared brain slices from CX3CR1-GFP reporter mice. We observed a significant reduction of microglial ramification and surveillance capacity in brain slices from aged versus adult mice. The age-related decrease in surveillance can be restored by antibody-mediated CD22 blockage in aged mice, whereas surveillance in adult mice is not affected by CD22 inhibition. Moreover to complement the results obtained in mice, we show that human iPSC-derived macrophages exhibit an increased phagocytic capacity upon CD22 blockage. Downstream analysis of antibody-mediated CD22 inhibition revealed an influence on BMP and TGFß associated gene networks. Our results demonstrate CD22 as a broad age-associated modulator of microglia functionality with potential implications for neurodegenerative disorders.

VEGF/VEGFR2 signaling regulates hippocampal axon branching during development.

Axon branching is crucial for proper formation of neuronal networks. Although originally identified as an angiogenic factor, VEGF also signals directly to neurons to regulate their development and function. Here we show that VEGF and its receptor VEGFR2 (also known as KDR or FLK1) are expressed in mouse hippocampal neurons during development, with VEGFR2 locally expressed in the CA3 region. Activation of VEGF/VEGFR2 signaling in isolated hippocampal neurons results in increased axon branching. Remarkably, inactivation of VEGFR2 also results in increased axon branching in vitro and in vivo. The increased CA3 axon branching is not productive as these axons are less mature and form less functional synapses with CA1 neurons. Mechanistically, while VEGF promotes the growth of formed branches without affecting filopodia formation, loss of VEGFR2 increases the number of filopodia and enhances the growth rate of new branches. Thus, a controlled VEGF/VEGFR2 signaling is required for proper CA3 hippocampal axon branching during mouse hippocampus development.

EphrinB2 regulates VEGFR2 during dendritogenesis and hippocampal circuitry development.

Vascular endothelial growth factor (VEGF) is an angiogenic factor that play important roles in the nervous system, although it is still unclear which receptors transduce those signals in neurons. Here, we show that in the developing hippocampus VEGFR2 (also known as KDR or FLK1) is expressed specifically in the CA3 region and it is required for dendritic arborization and spine morphogenesis in hippocampal neurons. Mice lacking VEGFR2 in neurons (Nes-cre Kdrlox/-) show decreased dendritic arbors and spines as well as a reduction in long-term potentiation (LTP) at the associational-commissural - CA3 synapses. Mechanistically, VEGFR2 internalization is required for VEGF-induced spine maturation. In analogy to endothelial cells, ephrinB2 controls VEGFR2 internalization in neurons. VEGFR2-ephrinB2 compound mice (Nes-cre Kdrlox/+ Efnb2lox/+) show reduced dendritic branching, reduced spine head size and impaired LTP. Our results demonstrate the functional crosstalk of VEGFR2 and ephrinB2 in vivo to control dendritic arborization, spine morphogenesis and hippocampal circuitry development.

GRIP1 Binds to ApoER2 and EphrinB2 to Induce Activity-Dependent AMPA Receptor Insertion at the Synapse.

Regulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor trafficking in response to neuronal activity is critical for synaptic function and plasticity. Here, we show that neuronal activity induces the binding of ephrinB2 and ApoER2 receptors at the postsynapse to regulate de novo insertion of AMPA receptors. Mechanistically, the multi-PDZ adaptor glutamate-receptor-interacting protein 1 (GRIP1) binds ApoER2 and bridges a complex including ApoER2, ephrinB2, and AMPA receptors. Phosphorylation of ephrinB2 in a serine residue (Ser-9) is essential for the stability of such a complex. In vivo, a mutation on ephrinB2 Ser-9 in mice results in a complete disruption of the complex, absence of ApoER2 downstream signaling, and impaired activity-induced and ApoER2-mediated AMPA receptor insertion. Using compound genetics, we show the requirement of this complex for long-term potentiation (LTP). Together, our findings uncover a cooperative ephrinB2 and ApoER2 signaling at the synapse, which serves to modulate activity-dependent AMPA receptor dynamic changes during synaptic plasticity.

APP Deletion Accounts for Age-Dependent Changes in the Bioenergetic Metabolism and in Hyperphosphorylated CaMKII at Stimulated Hippocampal Presynaptic Active Zones.

Synaptic release sites are characterized by exocytosis-competent synaptic vesicles tightly anchored to the presynaptic active zone (PAZ) whose proteome orchestrates the fast signaling events involved in synaptic vesicle cycle and plasticity. Allocation of the amyloid precursor protein (APP) to the PAZ proteome implicated a functional impact of APP in neuronal communication. In this study, we combined state-of-the-art proteomics, electrophysiology and bioinformatics to address protein abundance and functional changes at the native hippocampal PAZ in young and old APP-KO mice. We evaluated if APP deletion has an impact on the metabolic activity of presynaptic mitochondria. Furthermore, we quantified differences in the phosphorylation status after long-term-potentiation (LTP) induction at the purified native PAZ. We observed an increase in the phosphorylation of the signaling enzyme calmodulin-dependent kinase II (CaMKII) only in old APP-KO mice. During aging APP deletion is accompanied by a severe decrease in metabolic activity and hyperphosphorylation of CaMKII. This attributes an essential functional role to APP at hippocampal PAZ and putative molecular mechanisms underlying the age-dependent impairments in learning and memory in APP-KO mice.

Measurement of ROS Levels and Membrane Potential Dynamics in the Intact Carotid Body Ex Vivo.

Reactive oxygen species (ROS) generated by the NADPH oxidase have been proposed to play an important role in the carotid body (CB) oxygen sensing process (Cross et al. 1990). Up to now it remains unclear whether hypoxia causes an increase or decrease of CB ROS levels. We transfected CBs with the ROS sensitive HSP-FRET construct and subsequently measured the intracellular redox state by means of Förster resonance energy transfer (FRET) microscopy. In a previous study we found both increasing and decreasing ROS levels under hypoxic conditions. The transition from decreasing to increasing ROS levels coincided with the change of the caging system from ambient environment caging (AEC) to individually ventilated caging (IVC) (Bernardini A, Brockmeier U, Metzen E, Berchner-Pfannschmidt U, Harde E, Acker-Palmer A, Papkovsky D, Acker H, Fandrey J, Type I cell ROS kinetics under hypoxia in the intact mouse carotid body ex vivo: a FRET based study. Am J Physiol Cell Physiol. doi: 10.1152/ajpcell.00370.2013 , 2014). In this work we analyze hypoxia induced ROS reaction of animals from an IVC system that had been exposed to AEC conditions for 5 days. The results further support the hypothesis of an important impact of the caging system on CB ROS reaction.