Motor Domain Phosphorylation Modulates Kinesin-1 Transport

Disruptions in microtubule motor transport are associated with a variety of neurodegenerative diseases. Post-translational modification of the cargo-binding domain of the light and heavy chains of kinesin has been shown to regulate transport, but less is known about how modifications of the motor domain affect transport. Here we report on the effects of phosphorylation of a mammalian kinesin motor domain by the kinase JNK3 at a conserved serine residue (Ser-175 in the B isoform and Ser-176 in the A and C isoforms). Phosphorylation of this residue has been implicated in Huntington disease, but the mechanism by which Ser-175 phosphorylation affects transport is unclear. The ATPase, microtubule-binding affinity, and processivity are unchanged between a phosphomimetic S175D and a nonphosphorylatable S175A construct. However, we find that application of force differentiates between the two. Placement of negative charge at Ser-175, through phosphorylation or mutation, leads to a lower stall force and decreased velocity under a load of 1 piconewton or greater. Sedimentation velocity experiments also show that addition of a negative charge at Ser-175 favors the autoinhibited conformation of kinesin. These observations imply that when cargo is transported by both dynein and phosphorylated kinesin, a common occurrence in the cell, there may be a bias that favors motion toward the minus-end of microtubules. Such bias could be used to tune transport in healthy cells when properly regulated but contribute to a disease state when misregulated.     

Effects of age and size on critical swimming speed of juvenile Chinese sturgeon Acipenser sinensis at seasonal temperatures

Changes in the critical swimming speed (U_crit, cm s^?1) with ontogeny of 2·5–12·5 month‐old juvenile anadromous Chinese sturgeon Acipenser sinesis were measured in a modified Blazka‐type swimming tunnel. The absolute U_crit increased with length, mass and age; the relative U^′_crit (body lengths, s^?1), however, decreased. Juvenile A. sinesis did not display a parr–smolt transformation at the length or age threshold to tolerate full‐strength seawater.     

Nicotinic acid and DP1 blockade: studies in mouse models of atherosclerosis

The use of nicotinic acid to treat dyslipidemia is limited by induction of a “flushing” response, mediated in part by the interaction of prostaglandin D₂ (PGD₂) with its G-protein coupled receptor, DP1 (Ptgdr). The impact of DP1 blockade (genetic or pharmacologic) was assessed in experimental murine models of atherosclerosis. In Ptgdr^−/−ApoE^−/− mice versus ApoE^−/− mice, both fed a high-fat diet, aortic cholesterol content was modestly higher (1.3- to 1.5-fold, P < 0.05) in Ptgdr^−/−ApoE^−/− mice at 16 and 24 weeks of age, but not at 32 weeks. In multiple ApoE^−/− mouse studies, a DP1-specific antagonist, L-655, generally had a neutral to beneficial effect on aortic lipids in the presence or absence of nicotinic acid treatment. In a separate study, a modest increase in some atherosclerotic measures was observed with L-655 treatment in Ldlr^−/− mice fed a high-fat diet for 8 weeks; however, this effect was not sustained for 16 or 24 weeks. In the same study, treatment with nicotinic acid alone generally decreased plasma and/or aortic lipids, and addition of L-655 did not negate those beneficial effects. These studies demonstrate that inhibition of DP1, with or without nicotinic acid treatment, does not lead to consistent or sustained effects on plaque burden in mouse atherosclerotic models.     

Lactate administration reproduces specific brain and liver exercise‐related changes

The effects of exercise are not limited to muscle, and its ability to mitigate some chronic diseases is under study. A more complete understanding of how exercise impacts non‐muscle tissues might facilitate design of clinical trials and exercise mimetics. Here, we focused on lactate's ability to mediate changes in liver and brain bioenergetic‐associated parameters. In one group of experiments, C57BL/6 mice underwent 7 weeks of treadmill exercise sessions at intensities intended to exceed the lactate threshold. Over time, the mice dramatically increased their lactate threshold. To ensure that plasma lactate accumulated during the final week, the mice were run to exhaustion. In the liver, mRNA levels of gluconeogenesis‐promoting genes increased. While peroxisome proliferator‐activated receptor‐gamma co‐activator 1 alpha (PGC‐1α) expression increased, there was a decrease in PGC‐1β expression, and overall gene expression changes favored respiratory chain down‐regulation. In the brain, PGC‐1α and PGC‐1β were unchanged, but PGC‐1‐related co‐activator expression and mitochondrial DNA copy number increased. Brain tumor necrosis factor alpha expression fell, whereas vascular endothelial growth factor A expression rose. In another group of experiments, exogenously administered lactate was found to reproduce some but not all of these observed liver and brain changes. Our data suggest that lactate, an exercise byproduct, could mediate some of the effects exercise has on the liver and the brain, and that lactate itself can act as a partial exercise mimetic.

     

Betaine attenuates Alzheimer‐like pathological changes and memory deficits induced by homocysteine

Hyperhomocysteinemia (Hhcy) may induce memory deficits with β‐amyloid (Aβ) accumulation and tau hyperphosphorylation. Simultaneous supplement of folate and vitamin B12 partially restored the plasma homocysteine level and attenuated tau hyperphosphorylation, Aβ accumulation and memory impairments induced by Hhcy. However, folate and vitamin B12 treatment have no effects on Hhcy which has the methylenetetrahydrofolate reductase genotype mutation. In this study, we investigated the effects of simultaneous supplement of betaine on Alzheimer‐like pathological changes and memory deficits in hyperhomocysteinemic rats after a 2‐week induction by vena caudalis injection of homocysteine (Hcy). We found that supplementation of betaine could ameliorate the Hcy‐induced memory deficits, enhance long‐term potentiation (LTP) and increase dendritic branches numbers and the density of the dendritic spines, with up‐regulation of NR1, NR2A, synaptotagmin, synaptophysin, and phosphorylated synapsin I protein levels. Supplementation of betaine also attenuated the Hcy‐induced tau hyperphosphorylation at multiple AD‐related sites through activation protein phosphatase‐2A (PP2A) with decreased inhibitory demethylated PP2A_C at Leu309 and phosphorylated PP2A_C at Tyr307. In addition, supplementation of betaine also decreased Aβ production with decreased presenilin‐1 protein levels. Our data suggest that betaine could be a promising candidate for arresting Hcy‐induced AD‐like pathological changes and memory deficits.     

Mass spectrometry images acylcarnitines,phosphatidylcholines, and sphingomyelin in MDA-MB-231 breast tumor models

The lipid compositions of different breast tumor microenvironments are largely unknown due to limitations in lipid imaging techniques. Imaging lipid distributions would enhance our understanding of processes occurring inside growing tumors, such as cancer cell proliferation, invasion, and metastasis. Recent developments in MALDI mass spectrometry imaging (MSI) enable rapid and specific detection of lipids directly from thin tissue sections. In this study, we performed multimodal imaging of acylcarnitines, phosphatidylcholines (PC), a lysophosphatidylcholine (LPC), and a sphingomyelin (SM) from different microenvironments of breast tumor xenograft models, which carried tdTomato red fluorescent protein as a hypoxia-response element-driven reporter gene. The MSI molecular lipid images revealed spatially heterogeneous lipid distributions within tumor tissue. Four of the most-abundant lipid species, namely PC(16:0/16:0), PC(16:0/18:1), PC(18:1/18:1), and PC(18:0/18:1), were localized in viable tumor regions, whereas LPC(16:0/0:0) was detected in necrotic tumor regions. We identified a heterogeneous distribution of palmitoylcarnitine, stearoylcarnitine, PC(16:0/22:1), and SM(d18:1/16:0) sodium adduct, which colocalized primarily with hypoxic tumor regions. For the first time, we have applied a multimodal imaging approach that has combined optical imaging and MALDI-MSI with ion mobility separation to spatially localize and structurally identify acylcarnitines and a variety of lipid species present in breast tumor xenograft models.     

Auxiliary KChIP4a Suppresses A-type K+ Current through Endoplasmic Reticulum (ER) Retention and Promoting Closed-state Inactivation of Kv4 Channels

In the brain and heart, auxiliary Kv channel-interacting proteins (KChIPs) co-assemble with pore-forming Kv4 α-subunits to form a native K⁺ channel complex and regulate the expression and gating properties of Kv4 currents. Among the KChIP1–4 members, KChIP4a exhibits a unique N terminus that is known to suppress Kv4 function, but the underlying mechanism of Kv4 inhibition remains unknown. Using a combination of confocal imaging, surface biotinylation, and electrophysiological recordings, we identified a novel endoplasmic reticulum (ER) retention motif, consisting of six hydrophobic and aliphatic residues, 12–17 (LIVIVL), within the KChIP4a N-terminal KID, that functions to reduce surface expression of Kv4-KChIP complexes. This ER retention capacity is transferable and depends on its flanking location. In addition, adjacent to the ER retention motif, the residues 19–21 (VKL motif) directly promote closed-state inactivation of Kv4.3, thus leading to an inhibition of channel current. Taken together, our findings demonstrate that KChIP4a suppresses A-type Kv4 current via ER retention and enhancement of Kv4 closed-state inactivation.     

Exosomes from Retinal Astrocytes Contain Antiangiogenic Components That Inhibit Laser-induced Choroidal Neovascularization

Exosomes released from different types of host cells have different biological effects. We report that exosomes released from retinal astroglial cells (RACs) suppress retinal vessel leakage and inhibit choroidal neovascularization (CNV) in a laser-induced CNV model, whereas exosomes released from retinal pigmental epithelium do not. RAC exosomes inhibit the migration of macrophages and the tubule forming of mouse retinal microvascular endothelial cells. Further, we analyzed antiangiogenic components in RAC exosomes using an angiogenesis array kit and detected several endogenous inhibitors of angiogenesis exclusively present in RAC exosomes, such as endostatin. Moreover, blockade of matrix metalloproteinases in the cleavage of collagen XVIII to form endostatin using FN-439 reverses RAC exosome-mediated retinal vessel leakage. This study demonstrates that exosomes released from retinal tissue cells have different angiogenic effects, with exosomes from RACs containing antiangiogenic components that might protect the eye from angiogenesis and maintain its functional integrity. In addition, by identifying additional components and their functions of RAC exosomes, we might improve the antiangiogenic therapy for CNV in age-related macular degeneration and diabetic retinopathy.     

Understanding How Noncatalytic Carbohydrate Binding Modules Can Display Specificity for Xyloglucan

Plant biomass is central to the carbon cycle and to environmentally sustainable industries exemplified by the biofuel sector. Plant cell wall degrading enzymes generally contain noncatalytic carbohydrate binding modules (CBMs) that fulfil a targeting function, which enhances catalysis. CBMs that bind β-glucan chains often display broad specificity recognizing β1,4-glucans (cellulose), β1,3-β1,4-mixed linked glucans and xyloglucan, a β1,4-glucan decorated with α1,6-xylose residues, by targeting structures common to the three polysaccharides. Thus, CBMs that recognize xyloglucan target the β1,4-glucan backbone and only accommodate the xylose decorations. Here we show that two closely related CBMs, CBM65A and CBM65B, derived from EcCel5A, a Eubacterium cellulosolvens endoglucanase, bind to a range of β-glucans but, uniquely, display significant preference for xyloglucan. The structures of the two CBMs reveal a β-sandwich fold. The ligand binding site comprises the β-sheet that forms the concave surface of the proteins. Binding to the backbone chains of β-glucans is mediated primarily by five aromatic residues that also make hydrophobic interactions with the xylose side chains of xyloglucan, conferring the distinctive specificity of the CBMs for the decorated polysaccharide. Significantly, and in contrast to other CBMs that recognize β-glucans, CBM65A utilizes different polar residues to bind cellulose and mixed linked glucans. Thus, Gln¹⁰⁶ is central to cellulose recognition, but is not required for binding to mixed linked glucans. This report reveals the mechanism by which β-glucan-specific CBMs can distinguish between linear and mixed linked glucans, and show how these CBMs can exploit an extensive hydrophobic platform to target the side chains of decorated β-glucans.     

Cross-talk from β-Adrenergic Receptors Modulates α2A-Adrenergic Receptor Endocytosis in Sympathetic Neurons via Protein Kinase A and Spinophilin

Inter-regulation of adrenergic receptors (ARs) via cross-talk is a long appreciated but mechanistically unclear physiological phenomenon. Evidence from the AR literature and our own extensive studies on regulation of α_2AARs by the scaffolding protein spinophilin have illuminated a potential novel mechanism for cross-talk from β to α₂ARs. In the present study, we have characterized a mode of endogenous AR cross-talk in native adrenergic neurons whereby canonical βAR-mediated signaling modulates spinophilin-regulated α_2AAR endocytosis through PKA. Our findings demonstrate that co-activation of β and α_2AARs, either by application of endogenous agonist or by simultaneous stimulation with distinct selective agonists, results in acceleration of endogenous α_2AAR endocytosis in native neurons. We show that receptor-independent PKA activation by forskolin is sufficient to accelerate α_2AAR endocytosis and that α_2AAR stimulation alone drives accelerated endocytosis in spinophilin-null neurons. Endocytic response acceleration by β/α_2AAR co-activation is blocked by PKA inhibition and lost in spinophilin-null neurons, consistent with our previous finding that spinophilin is a substrate for phosphorylation by PKA that disrupts its interaction with α_2AARs. Importantly, we show that α₂AR agonist-mediated α_2AAR/spinophilin interaction is blocked by βAR co-activation in a PKA-dependent fashion. We therefore propose a novel mechanism for cross-talk from β to α₂ARs, whereby canonical βAR-mediated signaling coupled to PKA activation results in phosphorylation of spinophilin, disrupting its interaction with α_2AARs and accelerating α_2AAR endocytic responses. This mechanism of cross-talk has significant implications for endogenous adrenergic physiology and for therapeutic targeting of β and α_2AARs.