Ischemic axonal injury up-regulates MARK4 in cortical neurons and primes tau phosphorylation and aggregation
Eric Y. HaydenJennifer PutmanStefanie NuñezWoo Shik ShinMandavi R. OberoiMalena CharretonSuman DuttaZizheng LiYutaro KomuroMary T. JoyGal BitanAllan MacKenzie‐GrahamLin JiangJason D. Hinman
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Ischemic injury to white matter tracts is increasingly recognized to play a key role in age-related cognitive decline, vascular dementia, and Alzheimer's disease. Knowledge of the effects of ischemic axonal injury on cortical neurons is limited yet critical to identifying molecular pathways that link neurodegeneration and ischemia. Using a mouse model of subcortical white matter ischemic injury coupled with retrograde neuronal tracing, we employed magnetic affinity cell sorting with fluorescence-activated cell sorting to capture layer-specific cortical neurons and performed RNA-sequencing. With this approach, we identified a role for microtubule reorganization within stroke-injured neurons acting through the regulation of tau. We find that subcortical stroke-injured Layer 5 cortical neurons up-regulate the microtubule affinity-regulating kinase, Mark4, in response to axonal injury. Stroke-induced up-regulation of Mark4 is associated with selective remodeling of the apical dendrite after stroke and the phosphorylation of tau in vivo. In a cell-based tau biosensor assay, Mark4 promotes the aggregation of human tau in vitro. Increased expression of Mark4 after ischemic axonal injury in deep layer cortical neurons provides new evidence for synergism between axonal and neurodegenerative pathologies by priming of tau phosphorylation and aggregation.Keywords:
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Repetitive stimulation of the dorsal thalamus at 7-14 Hz produces an increasing number of spikes at an increasing frequency in neocortical neurons during the first few stimuli. Possible mechanisms underlying these cortical augmenting responses were analyzed with a computer model that included populations of thalamocortical cells, thalamic reticular neurons, up to two layers of cortical pyramidal cells, and cortical inhibitory interneurons. Repetitive thalamic stimulation produced a low-threshold intrathalamic augmentation in the model based on the deinactivation of the low-threshold Ca2+ current in thalamocortical cells, which in turn induced cortical augmenting responses. In the cortical model, augmenting responses were more powerful in the "input" layer compared with those in the "output" layer. Cortical stimulation of the network model produced augmenting responses in cortical neurons in distant cortical areas through corticothalamocortical loops and low-threshold intrathalamic augmentation. Thalamic stimulation was more effective in eliciting augmenting responses than cortical stimulation. Intracortical inhibition had an important influence on the genesis of augmenting responses in cortical neurons: A shift in the balance between intracortical excitation and inhibition toward excitation transformed an augmenting responses to long-lasting paroxysmal discharge. The predictions of the model were compared with in vivo recordings from neurons in cortical area 4 and thalamic ventrolateral nucleus of anesthetized cats. The known intrinsic properties of thalamic cells and thalamocortical interconnections can account for the basic properties of cortical augmenting responses.
Thalamic reticular nucleus
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Reticular connective tissue
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Some new data on neuronal and synaptic organization of sensorimotor cortical area in cat are obtained by a complex of morphological and electrophysiological methods. These data permit considering that direct afferent inhibition is ensured by thalamo-cortical neurons and neurons forming the callosal and association links. The recurrent and lateral inhibition are structurally realized through the ascending recurrent axon collaterals of pyramidal neurons forming links either with short-axon or with long-axon interneurons. Cortico-thalamic (cortico-fugal) inhibition may be performed either via descending cortico-thalamic neurons or via cortico-cortical ipsi- and contralateral neurons. The above mentioned neuronal chains may be considered as structural elements of more complex neuronal sets which ensure the inhibition at the cortical inputs, outputs and intracortically.
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Tyrosine hydroxylase catalyzes the initial and rate-limiting step in the biosynthesis of the neurotransmitter dopamine. The phosphorylation state of Ser40 and Ser31 is believed to exert a direct effect on the enzymatic activity of tyrosine hydroxylase. Interestingly, some studies report that Ser31 phosphorylation affects Ser40 phosphorylation, while Ser40 phosphorylation has no effect on Ser31 phosphorylation, a process named hierarchical phosphorylation. Here, we provide a detailed investigation into the signal transduction mechanisms regulating Ser40 and Ser31 phosphorylation in dopaminergic mouse MN9D and Neuro2A cells. We find that cyclic nucleotide signaling drives Ser40 phosphorylation, and that Ser31 phosphorylation is strongly regulated by ERK signaling. Inhibition of ERK1/2 with UO126 or PD98059 reduced Ser31 phosphorylation, but surprisingly had no effect on Ser40 phosphorylation, contradicting a role for Ser31 in the regulation of Ser40. Moreover, to elucidate a possible hierarchical mechanism controlling tyrosine hydroxylase phosphorylation, we introduced tyrosine hydroxylase variants in Neuro2A mouse neuroblastoma cells that mimic either phosphorylated or unphosphorylated serine residues. When we introduced a Ser40Ala tyrosine hydroxylase variant, Ser31 phosphorylation was completely absent. Additionally, neither the tyrosine hydroxylase variant Ser31Asp, nor the variant Ser31Ala had any significant effect on basal Ser40 phosphorylation levels. These results suggest that tyrosine hydroxylase is not controlled by hierarchical phosphorylation in the sense that first Ser31 has to be phosphorylated and subsequently Ser40, but, conversely, that Ser40 phosphorylation is essential for Ser31 phosphorylation. Overall our study suggests that Ser40 is the crucial residue to target so as to modulate tyrosine hydroxylase activity.
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O-GlcNAcylation of proteins is a recently discovered post-translational modification of nuclear and cytoplasmic proteins. This modification is similar to protein phosphorylation rather than to classical protein glycosylation of membrane and secreted proteins. Both O-GlcNAcylation and phosphorylation modify the hydroxyl group of serine or threonine residues of tau, the effect of O-GlcNAcylation on phosphorylation of tau was studied. The level of O-GlcNAcylation in differenciated PC12 cells was modulated by changing the concentration of the donor of O-GlcNAcylation and activities of the key enzymes, then, the consequent changes of tau phosphorylation at various phosphorylation sites were examined by using Western blot developed with phosphorylation-dependent and site-specific tau antibodies. It was found that O-GlcNAcylation modulated phosphorylation of tau at many phosphorylation sites and in a site-specific manner. Increased protein O-GlcNAcylation induced a decrease in tau phosphorylation at most of phosphorylation sites, and vise versa. These results suggest that O-GlcNAcylation negatively modulates tau phosphorylation at most phosphorylation sites. Therefore, these studies provide novel insight into the regulation of tau phosphorylation and the molecular mechenism of abnormal hyperphosphorylation of tau in Alzheimer disease brain.
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Protein synthesis in mammalian cells can be regulated through phosphorylation/dephosphorylation of the α subunit of initiation factor 2, eIF‐2. Two specific kinases have been identified that apparently phosphorylate the same site(s). Controversy exists as to whether serine‐48 is a phosphorylation site in addition to serine‐51. A recent publication is discussed that, in this author's view, answers the question of the phosphorylation sites. It is suggested that phosphorylation procedes sequentially with serine‐51 being the first and serine‐48 the second phosphorylation site. Phosphorylation of both sites is required for inhibition of protein synthesis.
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Tropomyosin (Tm) is an alpha‐helical coiled coil dimer involved in regulating striated muscle contraction. Although sarcomeric Tm is known to be phosphorylated at the penultimate amino acid Ser‐283, little is known regarding its phosphorylation levels in healthy and cardiac diseased states. The objective of this study was to study Tm phosphorylation levels with respect to age in wild type mice and in mice with familial hypertrophic cardiomyopathy (FHC). Phosphorylation levels were determined by western blotting of 2‐D gel electrophoresis of myofibrillar protein preparations. Results show that cardiac Tm phosphorylation is high during the perinatal period and decreases throughout adulthood in wild type mice. We also found that walls of the different cardiac chambers exhibit differential Tm phosphorylation. Two transgenic mouse models harboring FHC mutations in alpha‐Tm (Glu180Gly, and Asp175Asn) were studied to determine the effect of this disease on cardiac Tm phosphorylation. Results show that Tm phosphorylation is altered in both FHC models. Unlike in wild type mice, Tm phosphorylation levels do not decrease after birth, but rather remain constant. Depending on the role of Tm phosphorylation, modulation of phosphorylation levels in FHC patients may be potentially of therapeutic value. This project was funded by NHLBI HL81680 grant to DFW. HNS was supported by a fellowship from Dubai Medical College.
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Thalamocortical relay neurons whose axons project into a penicillininduced cortical epileptogenic focus generate bursts of action potentials during spontaneous interictal epileptiform discharges. These bursts originate in intracortical axons and propagate antidromically into thalamic neurons. Repetitive spike generation in cortical axons and presynaptic terminals could produce a potent excitatory drive and contribute to the generation of the large depolarization shifts which are seen in cortical elements during focal epileptogenesis.
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Pandemic
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2019-20 coronavirus outbreak
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Background and Purpose The biological significance of the multi‐site phosphorylation of Bcl‐2 at its loop region (T69, S70 and S87) has remained controversial for decades. This is a major obstacle for understanding apoptosis and anti‐tumour drug development. Experimental Approach We established a mathematical model into which a phosphorylation and de‐phosphorylation process of Bcl‐2 was integrated. Paclitaxel‐treated breast cancer cells were used as experimental models. Changes in the kinetics of binding with its critical partners, induced by phosphorylation of Bcl‐2 were experimentally obtained by surface plasmon resonance, using a phosphorylation‐mimicking mutant EEE‐Bcl‐2 (T69E, S70E and S87E). Key Results Mathematical simulations combined with experimental validation showed that phosphorylation regulates Bcl‐2 with different dynamics depending on the extent of Bcl‐2 phosphorylation and the phosphorylated Bcl‐2‐induced changes in binding kinetics. In response to Bcl‐2 homology 3 (BH3)‐only protein Bmf stress, Bcl‐2 phosphorylation switched from diminishing to enhancing the Bcl‐2 anti‐apoptotic ability with increased phosphorylation of Bcl‐2, and the turning point was 50% Bcl‐2 phosphorylation induced by 0.2 μM paclitaxel treatment. In contrast, Bcl‐2 phosphorylation enhanced the anti‐apoptotic ability of Bcl‐2 towards other BH3‐only proteins Bim, Bad and Puma, throughout the entire phosphorylation procedure. Conclusions and Implications The model could accurately predict the effects of anti‐tumour drugs that involve the Bcl‐2 family pathway, as shown with ABT‐199 or etoposide.
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