Tau phosphorylation suppresses oxidative stress-induced mitophagy via FKBP8 receptor modulation.

Neurodegenerative diseases are often characterized by mitochondrial dysfunction. In Alzheimer's disease, abnormal tau phosphorylation disrupts mitophagy, a quality control process through which damaged organelles are selectively removed from the mitochondrial network. The precise mechanism through which this occurs remains unclear. Previously, we showed that tau which has been mutated at Thr-231 to glutamic acid to mimic an Alzheimer's-relevant phospho-epitope expressed early in disease selectively inhibits oxidative stress-induced mitophagy in C. elegans. Here, we use immortalized mouse hippocampal neuronal cell lines to extend that result into mammalian cells. Specifically, we show that phosphomimetic tau at Ser-396/404 (EC) or Thr-231/Ser-235 (EM) partly inhibits mitophagy induction by paraquat, a potent inducer of mitochondrial oxidative stress. Moreover, a combination of immunologic and biochemical approaches demonstrates that the levels of the mitophagy receptor FKBP8, significantly decrease in response to paraquat in cells expressing EC or EM tau mutants, but not in cells expressing wildtype tau. In contrast, paraquat treatment results in a decrease in the levels of the mitophagy receptors FUNDC1 and BNIP3 in the presence of both wildtype tau and the tau mutants. Interestingly, FKBP8 is normally trafficked to the endoplasmic reticulum during oxidative stress induced mitophagy, and our results support a model where this trafficking is impacted by disease-relevant tau, perhaps through a direct interaction. We provide new insights into the molecular mechanisms underlying tau pathology in Alzheimer's disease and highlight FKBP8 receptor as a potential target for mitigating mitochondrial dysfunction in neurodegenerative diseases.

Site-specific phosphorylation of tau impacts mitochondrial function and response to stressors.

Phosphorylation of tau at sites associated with Alzheimer's disease (AD) likely plays a role in the disease progression. Mitochondrial impairment, correlating with increased presence of phosphorylated tau, has been identified as a contributing factor to neurodegenerative processes in AD. However, how tau phosphorylated at specific sites impacts mitochondrial function has not been fully defined. We examined how AD-relevant phosphomimetics of tau impact selected aspects of mitochondrial biology. To mimic phosphorylation at AD-associated sites, the serine/threonine (Ser/Thr) sites in wild-type green fluorescent protein (GFP)-tagged tau (T4) were converted to glutamic acid (E) to make pseudo-phosphorylated GFP-tagged Ser-396/404 (2EC) and GFP-tagged Thr-231/Ser-235 (2EM) constructs. These constructs were expressed in immortalized mouse hippocampal neuronal cell lines, and their impact on specific mitochondrial functions and responses to stressors were measured. Phosphomimetic tau altered mitochondrial distribution. Specifically, mitochondria accumulated in the soma of cells expressing either 2EC or 2EM and neurite-like extensions in 2EC cells were shorter. Additionally, adenosine triphosphate levels were reduced in both 2EC- and 2EM-expressing cells, and reactive oxygen species (ROS) production increased in 2EC cells during oxidation of succinate when compared to T4-expressing cells. Thapsigargin reduced mitochondrial membrane potential and increased ROS production in both 2EC and 2EM cells relative to T4 cells, with no significant difference in the effects of rotenone. These results show that tau phosphorylation at specific AD-relevant epitopes negatively affects mitochondria, with the extent of dysfunction and stress response varying according to the sites of phosphorylation. Altogether, these findings show that phosphorylated tau increases mitochondrial susceptibility to stressors and extend our understanding of potential mechanisms whereby phosphorylated tau promotes mitochondria dysfunction in tauopathies, including AD.

Deletion of Transglutaminase 2 from Mouse Astrocytes Significantly Improves Their Ability to Promote Neurite Outgrowth on an Inhibitory Matrix.

Astrocytes are the primary support cells of the central nervous system (CNS) that help maintain the energetic requirements and homeostatic environment of neurons. CNS injury causes astrocytes to take on reactive phenotypes with an altered overall function that can range from supportive to harmful for recovering neurons. The characterization of reactive astrocyte populations is a rapidly developing field, and the underlying factors and signaling pathways governing which type of reactive phenotype that astrocytes take on are poorly understood. Our previous studies suggest that transglutaminase 2 (TG2) has an important role in determining the astrocytic response to injury. Selectively deleting TG2 from astrocytes improves functional outcomes after CNS injury and causes widespread changes in gene regulation, which is associated with its nuclear localization. To begin to understand how TG2 impacts astrocytic function, we used a neuron-astrocyte co-culture paradigm to compare the effects of TG2-/- and wild-type (WT) mouse astrocytes on neurite outgrowth and synapse formation. Neurons were grown on a control substrate or an injury-simulating matrix comprised of inhibitory chondroitin sulfate proteoglycans (CSPGs). Compared to WT astrocytes, TG2-/- astrocytes supported neurite outgrowth to a significantly greater extent only on the CSPG matrix, while synapse formation assays showed mixed results depending on the pre- and post-synaptic markers analyzed. We hypothesize that TG2 regulates the supportive functions of astrocytes in injury conditions by modulating gene expression through interactions with transcription factors and transcription complexes. Based on the results of a previous yeast two-hybrid screen for TG2 interactors, we further investigated the interaction of TG2 with Zbtb7a, a ubiquitously expressed transcription factor. Co-immunoprecipitation and colocalization analyses confirmed the interaction of TG2 and Zbtb7a in the nucleus of astrocytes. Overexpression or knockdown of Zbtb7a levels in WT and TG2-/- astrocytes revealed that Zbtb7a robustly influenced astrocytic morphology and the ability of astrocytes to support neuronal outgrowth, which was significantly modulated by the presence of TG2. These findings support our hypothesis that astrocytic TG2 acts as a transcriptional regulator to influence astrocytic function, with greater influence under injury conditions that increase its expression, and Zbtb7a likely contributes to the overall effects observed with astrocytic TG2 deletion.

Site-specific phosphorylation of tau impacts mitochondrial biology and response to stressors.

Phosphorylation of tau at sites associated with Alzheimer's disease (AD) likely plays a role in the disease progression. Mitochondrial impairment, correlating with increased presence of phosphorylated tau, has been identified as a contributing factor to neurodegenerative processes in AD. However, how tau phosphorylated at specific sites impacts mitochondrial function has not been fully defined. We examined how AD-relevant phosphomimetics of tau impact selected aspects of mitochondrial biology. To mimic phosphorylation at AD-associated sites, the Ser/Thr sites in wild-type GFP tagged-tau (T4) were converted to glutamic acid (E) to make pseudophosphorylated GFP tagged-Ser-396/404 (2EC) and GFP tagged-Thr-231/Ser-235 (2EM) constructs. These constructs were expressed in neuronal HT22 cells and their impact on specific mitochondrial functions and responses to stressors were measured. Phosphomimetic tau altered mitochondrial distribution. Specifically, mitochondria accumulated in the soma of cells expressing either 2EC or 2EM, and neurite-like extensions in 2EC cells were shorter. Additionally, ATP levels were reduced in both 2EC and 2EM expressing cells, and ROS production increased in 2EC cells during oxidation of succinate when compared to T4 expressing cells. Thapsigargin reduced mitochondrial membrane potential (Ψ m ) and increased ROS production in both 2EC and 2EM cells relative to T4 cells, with no significant difference in the effects of rotenone. These results show that tau phosphorylation at specific AD-relevant epitopes negatively affects mitochondria, with the extent of dysfunction and stress response varying according to the sites of phosphorylation. Altogether, these findings extend our understanding of potential mechanisms whereby phosphorylated tau promotes mitochondria dysfunction in tauopathies, including AD. Funding information: R01 AG067617.

Deletion of transglutaminase 2 from astrocytes significantly improves their ability to promote neurite outgrowth on an inhibitory matrix.

Astrocytes are the primary support cells of the central nervous system (CNS) that help maintain the energetic requirements and homeostatic environment of neurons. CNS injury causes astrocytes to take on reactive phenotypes with altered overall function that can range from supportive to harmful for recovering neurons. The characterization of reactive astrocyte populations is a rapidly developing field, and the underlying factors and signaling pathways governing which type of reactive phenotype that astrocytes take on is poorly understood. Our previous studies suggest that transglutaminase 2 (TG2) has an important role in determining the astrocytic response to injury. TG2 is upregulated in astrocytes across multiple injury models, and selectively deleting TG2 from astrocytes improves functional outcomes after CNS injury and causes widespread changes in gene regulation, which is associated with its nuclear localization. The underlying molecular mechanisms by which TG2 causes these functional changes are unknown, and its interactions in the nucleus of astrocytes has not yet been described. To begin to understand how TG2 impacts astrocytic function, we used a neuron-astrocyte co-culture paradigm to compare the effects of TG2-/- and wild type (WT) astrocytes on neurite outgrowth and synapse formation. We assayed neurons on both a growth-supportive substrate and an injury-simulating matrix comprised of inhibitory chondroitin sulfate proteoglycans (CSPGs). Compared to WT astrocytes, TG2-/- astrocytes supported neurite outgrowth to a significantly greater extent only on the CSPG matrix, while synapse formation assays showed mixed results depending on the pre- and post-synaptic markers analyzed. We hypothesize that TG2 regulates the supportive functions of astrocytes in injury conditions by modulating the expression of a wide range of genes through interactions with transcription factors and transcription complexes. Based on results of a previous yeast two-hybrid screen for TG2 interactors, we further investigated the interaction of TG2 with Zbtb7a, a ubiquitously expressed transcription factor. Coimmunoprecipitation and colocalization analyses confirmed the interaction of TG2 and Zbtb7a in the nucleus of astrocytes. Genetic overexpression or knockdown of Zbtb7a levels in TG2-/- and WT astrocytes revealed that Zbtb7a robustly influenced astrocytic morphology and the ability of astrocytes to support neuronal outgrowth, which was significantly modulated by the presence of TG2. These findings support our hypothesis that astrocytic TG2 acts as a transcriptional regulator to influence astrocytic function, with greater influence under injury conditions that increase its expression, and Zbtb7a likely contributes to the overall effects observed with astrocytic TG2 deletion.

BAG3 Regulation of RAB35 Mediates the Endosomal Sorting Complexes Required for Transport/Endolysosome Pathway and Tau Clearance.

BACKGROUND: Declining proteostasis with aging contributes to increased susceptibility to neurodegenerative diseases, including Alzheimer's disease (AD). Emerging studies implicate impairment of the endosome-lysosome pathway as a significant factor in the pathogenesis of these diseases. Previously, we demonstrated that BAG3 regulates phosphorylated tau clearance. However, we did not fully define how BAG3 regulates endogenous tau proteostasis, especially in the early stages of disease progression. METHODS: Mass spectrometric analyses were performed to identify neuronal BAG3 interactors. Multiple biochemical assays were used to investigate the BAG3-HSP70-TBC1D10B (EPI64B)-RAB35-HRS regulatory networks. Live-cell imaging was used to study the dynamics of the endosomal pathway. Immunohistochemistry and immunoblotting were performed in human AD brains and in P301S tau transgenic mice with BAG3 overexpressed. RESULTS: The primary group of neuronal BAG3 interactors identified are involved in the endocytic pathway. Among them were key regulators of small GTPases, such as the RAB35 GTPase-activating protein TBC1D10B. We demonstrated that a BAG3-HSP70-TBC1D10B complex attenuates the ability of TBC1D10B to inactivate RAB35. Thus, BAG3 interacts with TBC1D10B to support the activation of RAB35 and recruitment of HRS, initiating endosomal sorting complex required for transport-mediated endosomal tau clearance. Furthermore, TBC1D10B shows significantly less colocalization with BAG3 in AD brains than in age-matched controls. Overexpression of BAG3 in P301S tau transgenic mice increased the colocalization of phosphorylated tau with the endosomal sorting complex required for transport III protein CHMP2B and reduced the levels of the mutant human tau. CONCLUSIONS: We identified a novel BAG3-TBC1D10B-RAB35 regulatory axis that modulates endosomal sorting complex required for transport-dependent protein degradation machinery and tau clearance. Dysregulation of BAG3 could contribute to the pathogenesis of AD.

The role of BAG3 in health and disease: A "Magic BAG of Tricks".

The multi-domain structure of Bcl-2-associated athanogene 3 (BAG3) facilitates its interaction with many different proteins that participate in regulating a variety of biological pathways. After revisiting the BAG3 literature published over the past ten years with Citespace software, we classified the BAG3 research into several clusters, including cancer, cardiomyopathy, neurodegeneration, and viral propagation. We then highlighted recent key findings in each cluster. To gain greater insight into the roles of BAG3, we analyzed five different published mass spectrometry data sets of proteins that co-immunoprecipitate with BAG3. These data gave us insight into universal, as well as cell-type-specific BAG3 interactors in cancer cells, cardiomyocytes, and neurons. Finally, we mapped variable BAG3 SNPs and also mutation data from previous publications to further explore the link between the domains and function of BAG3. We believe this review will provide a better understanding of BAG3 and direct future studies towards understanding BAG3 function in physiological and pathological conditions.

Deletion or Inhibition of Astrocytic Transglutaminase 2 Promotes Functional Recovery after Spinal Cord Injury.

Following CNS injury, astrocytes become "reactive" and exhibit pro-regenerative or harmful properties. However, the molecular mechanisms that cause astrocytes to adopt either phenotype are not well understood. Transglutaminase 2 (TG2) plays a key role in regulating the response of astrocytes to insults. Here, we used mice in which TG2 was specifically deleted in astrocytes (Gfap-Cre+/- TG2fl/fl, referred to here as TG2-A-cKO) in a spinal cord contusion injury (SCI) model. Deletion of TG2 from astrocytes resulted in a significant improvement in motor function following SCI. GFAP and NG2 immunoreactivity, as well as number of SOX9 positive cells, were significantly reduced in TG2-A-cKO mice. RNA-seq analysis of spinal cords from TG2-A-cKO and control mice 3 days post-injury identified thirty-seven differentially expressed genes, all of which were increased in TG2-A-cKO mice. Pathway analysis revealed a prevalence for fatty acid metabolism, lipid storage and energy pathways, which play essential roles in neuron-astrocyte metabolic coupling. Excitingly, treatment of wild type mice with the selective TG2 inhibitor VA4 significantly improved functional recovery after SCI, similar to what was observed using the genetic model. These findings indicate the use of TG2 inhibitors as a novel strategy for the treatment of SCI and other CNS injuries.

Angioplasty facilitated permanent pacing in de novo superior vena cava obstruction.

The finding of complete occlusion of the superior venal cava during the performance of cardiac pacing is rare. We report a case where performance of angioplasty aided the successful completion of permanent pacemaker implantation.