Functional Organization of Glycolytic Metabolon on Mitochondria.

Glucose, the primary cellular energy source, is metabolized through glycolysis initiated by the rate-limiting enzyme Hexokinase (HK). In energy-demanding tissues like the brain, HK1 is the dominant isoform, primarily localized on mitochondria, crucial for efficient glycolysis-oxidative phosphorylation coupling and optimal energy generation. This study unveils a unique mechanism regulating HK1 activity, glycolysis, and the dynamics of mitochondrial coupling, mediated by the metabolic sensor enzyme O-GlcNAc transferase (OGT). OGT catalyzes reversible O-GlcNAcylation, a post-translational modification, influenced by glucose flux. Elevated OGT activity induces dynamic O-GlcNAcylation of HK1's regulatory domain, subsequently promoting the assembly of the glycolytic metabolon on the outer mitochondrial membrane. This modification enhances HK1's mitochondrial association, orchestrating glycolytic and mitochondrial ATP production. Mutations in HK1's O-GlcNAcylation site reduce ATP generation, affecting synaptic functions in neurons. The study uncovers a novel pathway that bridges neuronal metabolism and mitochondrial function via OGT and the formation of the glycolytic metabolon, offering new prospects for tackling metabolic and neurological disorders.

HCN4 knockdown in dorsal hippocampus promotes anxiety-like behavior in mice.

Hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels mediate the Ih current in the murine hippocampus. Disruption of the Ih current by knockout of HCN1, HCN2 or tetratricopeptide repeat-containing Rab8b-interacting protein has been shown to affect physiological processes such as synaptic integration and maintenance of resting membrane potentials as well as several behaviors in mice, including depressive-like and anxiety-like behaviors. However, the potential involvement of the HCN4 isoform in these processes is unknown. Here, we assessed the contribution of the HCN4 isoform to neuronal processing and hippocampus-based behaviors in mice. We show that HCN4 is expressed in various regions of the hippocampus, with distinct expression patterns that partially overlapped with other HCN isoforms. For behavioral analysis, we specifically modulated HCN4 expression by injecting recombinant adeno-associated viral (rAAV) vectors mediating expression of short hairpin RNA against hcn4 (shHcn4) into the dorsal hippocampus of mice. HCN4 knockdown produced no effect on contextual fear conditioning or spatial memory. However, a pronounced anxiogenic effect was evident in mice treated with shHcn4 compared to control littermates. Our findings suggest that HCN4 specifically contributes to anxiety-like behaviors in mice.

ß-adrenergic signaling broadly contributes to LTP induction.

Long-lasting forms of long-term potentiation (LTP) represent one of the major cellular mechanisms underlying learning and memory. One of the fundamental questions in the field of LTP is why different molecules are critical for long-lasting forms of LTP induced by diverse experimental protocols. Further complexity stems from spatial aspects of signaling networks, such that some molecules function in the dendrite and some are critical in the spine. We investigated whether the diverse experimental evidence can be unified by creating a spatial, mechanistic model of multiple signaling pathways in hippocampal CA1 neurons. Our results show that the combination of activity of several key kinases can predict the occurrence of long-lasting forms of LTP for multiple experimental protocols. Specifically Ca2+/calmodulin activated kinase II, protein kinase A and exchange protein activated by cAMP (Epac) together predict the occurrence of LTP in response to strong stimulation (multiple trains of 100 Hz) or weak stimulation augmented by isoproterenol. Furthermore, our analysis suggests that activation of the ß-adrenergic receptor either via canonical (Gs-coupled) or non-canonical (Gi-coupled) pathways underpins most forms of long-lasting LTP. Simulations make the experimentally testable prediction that a complete antagonist of the ß-adrenergic receptor will likely block long-lasting LTP in response to strong stimulation. Collectively these results suggest that converging molecular mechanisms allow CA1 neurons to flexibly utilize signaling mechanisms best tuned to temporal pattern of synaptic input to achieve long-lasting LTP and memory storage.

Acetyl-CoA synthetase regulates histone acetylation and hippocampal memory.

Metabolic production of acetyl coenzyme A (acetyl-CoA) is linked to histone acetylation and gene regulation, but the precise mechanisms of this process are largely unknown. Here we show that the metabolic enzyme acetyl-CoA synthetase 2 (ACSS2) directly regulates histone acetylation in neurons and spatial memory in mammals. In a neuronal cell culture model, ACSS2 increases in the nuclei of differentiating neurons and localizes to upregulated neuronal genes near sites of elevated histone acetylation. A decrease in ACSS2 lowers nuclear acetyl-CoA levels, histone acetylation, and responsive expression of the cohort of neuronal genes. In adult mice, attenuation of hippocampal ACSS2 expression impairs long-term spatial memory, a cognitive process that relies on histone acetylation. A decrease in ACSS2 in the hippocampus also leads to defective upregulation of memory-related neuronal genes that are pre-bound by ACSS2. These results reveal a connection between cellular metabolism, gene regulation, and neural plasticity and establish a link between acetyl-CoA generation 'on-site' at chromatin for histone acetylation and the transcription of key neuronal genes.

Dendritic diameter influences the rate and magnitude of hippocampal cAMP and PKA transients during ß-adrenergic receptor activation.

In the hippocampus, cyclic-adenosine monophosphate (cAMP) and cAMP-dependent protein kinase (PKA) form a critical signaling cascade required for long-lasting synaptic plasticity, learning and memory. Plasticity and memory are known to occur following pathway-specific changes in synaptic strength that are thought to result from spatially and temporally coordinated intracellular signaling events. To better understand how cAMP and PKA dynamically operate within the structural complexity of hippocampal neurons, we used live two-photon imaging and genetically-encoded fluorescent biosensors to monitor cAMP levels or PKA activity in CA1 neurons of acute hippocampal slices. Stimulation of ß-adrenergic receptors (isoproterenol) or combined activation of adenylyl cyclase (forskolin) and inhibition of phosphodiesterase (IBMX) produced cAMP transients with greater amplitude and rapid on-rates in intermediate and distal dendrites compared to somata and proximal dendrites. In contrast, isoproterenol produced greater PKA activity in somata and proximal dendrites compared to intermediate and distal dendrites, and the on-rate of PKA activity did not differ between compartments. Computational models show that our observed compartmental difference in cAMP can be reproduced by a uniform distribution of PDE4 and a variable density of adenylyl cyclase that scales with compartment size to compensate for changes in surface to volume ratios. However, reproducing our observed compartmental difference in PKA activity required enrichment of protein phosphatase in small compartments; neither reduced PKA subunits nor increased PKA substrates were sufficient. Together, our imaging and computational results show that compartment diameter interacts with rate-limiting components like adenylyl cyclase, phosphodiesterase and protein phosphatase to shape the spatial and temporal components of cAMP and PKA signaling in CA1 neurons and suggests that small neuronal compartments are most sensitive to cAMP signals whereas large neuronal compartments accommodate a greater dynamic range in PKA activity.

Sleep deprivation causes memory deficits by negatively impacting neuronal connectivity in hippocampal area CA1.

Brief periods of sleep loss have long-lasting consequences such as impaired memory consolidation. Structural changes in synaptic connectivity have been proposed as a substrate of memory storage. Here, we examine the impact of brief periods of sleep deprivation on dendritic structure. In mice, we find that five hours of sleep deprivation decreases dendritic spine numbers selectively in hippocampal area CA1 and increased activity of the filamentous actin severing protein cofilin. Recovery sleep normalizes these structural alterations. Suppression of cofilin function prevents spine loss, deficits in hippocampal synaptic plasticity, and impairments in long-term memory caused by sleep deprivation. The elevated cofilin activity is caused by cAMP-degrading phosphodiesterase-4A5 (PDE4A5), which hampers cAMP-PKA-LIMK signaling. Attenuating PDE4A5 function prevents changes in cAMP-PKA-LIMK-cofilin signaling and cognitive deficits associated with sleep deprivation. Our work demonstrates the necessity of an intact cAMP-PDE4-PKA-LIMK-cofilin activation-signaling pathway for sleep deprivation-induced memory disruption and reduction in hippocampal spine density.

Gravin orchestrates protein kinase A and ß2-adrenergic receptor signaling critical for synaptic plasticity and memory.

A kinase-anchoring proteins (AKAPs) organize compartmentalized pools of protein kinase A (PKA) to enable localized signaling events within neurons. However, it is unclear which of the many expressed AKAPs in neurons target PKA to signaling complexes important for long-lasting forms of synaptic plasticity and memory storage. In the forebrain, the anchoring protein gravin recruits a signaling complex containing PKA, PKC, calmodulin, and PDE4D (phosphodiesterase 4D) to the ß2-adrenergic receptor. Here, we show that mice lacking the α-isoform of gravin have deficits in PKA-dependent long-lasting forms of hippocampal synaptic plasticity including ß2-adrenergic receptor-mediated plasticity, and selective impairments of long-term memory storage. Furthermore, both hippocampal ß2-adrenergic receptor phosphorylation by PKA, and learning-induced activation of ERK in the CA1 region of the hippocampus are attenuated in mice lacking gravin-α. We conclude that gravin compartmentalizes a significant pool of PKA that regulates learning-induced ß2-adrenergic receptor signaling and ERK activation in the hippocampus in vivo, thereby organizing molecular interactions between glutamatergic and noradrenergic signaling pathways for long-lasting synaptic plasticity, and memory storage.

Mitotic kinesin CENP-E promotes microtubule plus-end elongation.

Centromere protein CENP-E is a dimeric kinesin (Kinesin-7 family) with critical roles in mitosis, including establishment of microtubule (MT)-chromosome linkage and movement of mono-oriented chromosomes on kinetochore microtubules for proper alignment at metaphase [1-9]. We performed studies to test the hypothesis that CENP-E promotes MT elongation at the MT plus ends. A human CENP-E construct was engineered, expressed, and purified, and it yielded the CENP-E-6His dimeric motor protein. The results show that CENP-E promotes MT plus-end-directed MT gliding at 11 nm/s. The results from real-time microscopy assays indicate that 60.3% of polarity-marked MTs exhibited CENP-E-promoted MT plus-end elongation. The MT extension required ATP turnover, and MT plus-end elongation occurred at 1.48 µm/30 min. Immunolocalization studies revealed that 80.8% of plus-end-elongated MTs showed CENP-E at the MT plus end. The time dependence of CENP-E-promoted MT elongation in solution best fit a single exponential function (k(obs) = 5.1 s(-1)), which is indicative of a mechanism in which α,ß-tubulin subunit addition is tightly coupled to ATP turnover. Based on these results, we propose that CENP-E, as part of its function in chromosome kinetochore-MT linkage, plays a direct role in MT elongation.