Mitochondrial energy state controls AMPK-mediated foraging behavior in C. elegans.

Organisms surveil and respond to their environment using behaviors entrained by metabolic cues that reflect food availability. Mitochondria act as metabolic hubs and at the center of mitochondrial energy production is the protonmotive force (PMF), an electrochemical gradient generated by metabolite consumption. The PMF serves as a central integrator of mitochondrial status, but its role in governing metabolic signaling is poorly understood. We used optogenetics to dissipate the PMF in Caenorhabditis elegans tissues to test its role in food-related behaviors. Our data demonstrate that PMF reduction in the intestine is sufficient to initiate locomotor responses to acute food deprivation. This behavioral adaptation requires the cellular energy regulator AMP-activated protein kinase (AMPK) in neurons, not in the intestine, and relies on mitochondrial dynamics and axonal trafficking. Our results highlight a role for intestinal PMF as an internal metabolic cue, and we identify a bottom-up signaling axis through which changes in the PMF trigger AMPK activity in neurons to promote foraging behavior.

Mitochondrial complex I ROS production and redox signaling in hypoxia.

Mitochondria are a main source of cellular energy. Oxidative phosphorylation (OXPHOS) is the major process of aerobic respiration. Enzyme complexes of the electron transport chain (ETC) pump protons to generate a protonmotive force (Δp) that drives OXPHOS. Complex I is an electron entry point into the ETC. Complex I oxidizes nicotinamide adenine dinucleotide (NADH) and transfers electrons to ubiquinone in a reaction coupled with proton pumping. Complex I also produces reactive oxygen species (ROS) under various conditions. The enzymatic activities of complex I can be regulated by metabolic conditions and serves as a regulatory node of the ETC. Complex I ROS plays diverse roles in cell metabolism ranging from physiologic to pathologic conditions. Progress in our understanding indicates that ROS release from complex I serves important signaling functions. Increasing evidence suggests that complex I ROS is important in signaling a mismatch in energy production and demand. In this article, we review the role of ROS from complex I in sensing acute hypoxia.

All-optical spatiotemporal mapping of ROS dynamics across mitochondrial microdomains in situ.

Hydrogen peroxide (H2O2) functions as a second messenger to signal metabolic distress through highly compartmentalized production in mitochondria. The dynamics of reactive oxygen species (ROS) generation and diffusion between mitochondrial compartments and into the cytosol govern oxidative stress responses and pathology, though these processes remain poorly understood. Here, we couple the H2O2 biosensor, HyPer7, with optogenetic stimulation of the ROS-generating protein KillerRed targeted into multiple mitochondrial microdomains. Single mitochondrial photogeneration of H2O2 demonstrates the spatiotemporal dynamics of ROS diffusion and transient hyperfusion of mitochondria due to ROS. This transient hyperfusion phenotype required mitochondrial fusion but not fission machinery. Measurement of microdomain-specific H2O2 diffusion kinetics reveals directionally selective diffusion through mitochondrial microdomains. All-optical generation and detection of physiologically-relevant concentrations of H2O2 between mitochondrial compartments provide a map of mitochondrial H2O2 diffusion dynamics in situ as a framework to understand the role of ROS in health and disease.

Selective disruption of Drp1-independent mitophagy and mitolysosome trafficking by an Alzheimer's disease relevant tau modification in a novel Caenorhabditis elegans model.

Accumulation of inappropriately phosphorylated tau into neurofibrillary tangles is a defining feature of Alzheimer's disease, with Tau pT231 being an early harbinger of tau pathology. Previously, we demonstrated that expressing a single genomic copy of human phosphomimetic mutant tau (T231E) in Caenorhabditis elegans drove age-dependent neurodegeneration. A critical finding was that T231E, unlike wild-type tau, completely and selectively suppressed oxidative stress-induced mitophagy. Here, we used dynamic imaging approaches to analyze T231E-associated changes in mitochondria and mitolysosome morphology, abundance, trafficking, and stress-induced mitophagy as a function of mitochondrial fission mediator dynamin-related protein 1, which has been demonstrated to interact with hyper phosphorylated tau and contribute to Alzheimer's disease pathogenesis, as well as Pink1, a well-recognized mediator of mitochondrial quality control that works together with Parkin to support stress-induced mitophagy. T231E impacted both mitophagy and mitolysosome neurite trafficking with exquisite selectivity, sparing macroautophagy as well as lysosome and autolysosome trafficking. Both oxidative-stress-induced mitophagy and the ability of T231E to suppress it were independent of drp-1, but at least partially dependent on pink-1. Organelle trafficking was more complicated, with drp-1 and pink-1 mutants exerting independent effects, but generally supported the idea that the mitophagy phenotype is of greater physiologic impact in T231E. Collectively, our results refine the mechanistic pathway through which T231E causes neurodegeneration, demonstrating pathologic selectivity for mutations that mimic tauopathy-associated post-translational modifications, physiologic selectivity for organelles that contain damaged mitochondria, and molecular selectivity for dynamin-related protein 1-independent, Pink1-dependent, perhaps adaptive, and mitophagy.

A reversible mitochondrial complex I thiol switch mediates hypoxic avoidance behavior in C. elegans.

C. elegans react to metabolic distress caused by mismatches in oxygen and energy status via distinct behavioral responses. At the molecular level, these responses are coordinated by under-characterized, redox-sensitive processes, thought to initiate in mitochondria. Complex I of the electron transport chain is a major site of reactive oxygen species (ROS) production and is canonically associated with oxidative damage following hypoxic exposure. Here, we use a combination of optogenetics and CRISPR/Cas9-mediated genome editing to exert spatiotemporal control over ROS production. We demonstrate a photo-locomotory remodeling of avoidance behavior by local ROS production due to the reversible oxidation of a single thiol on the complex I subunit NDUF-2.1. Reversible thiol oxidation at this site is necessary and sufficient for the behavioral response to hypoxia, does not respond to ROS produced at more distal sites, and protects against lethal hypoxic exposure. Molecular modeling suggests that oxidation at this thiol residue alters the ability for NDUF-2.1 to coordinate electron transfer to coenzyme Q by destabilizing the Q-binding pocket, causing decreased complex I activity. Overall, site-specific ROS production regulates behavioral responses and these findings provide a mechanistic target to suppress the detrimental effects of hypoxia.

Site-specific mitochondrial dysfunction in neurodegeneration.

Mitochondria are essential for neuronal survival and mitochondrial dysfunction is a hallmark of neurodegeneration. The loss in mitochondrial energy production, oxidative stress, and changes in calcium handling are associated with neurodegenerative diseases; however, different sites and types of mitochondrial dysfunction are linked to distinct neuropathologies. Understanding the causal or correlative relationship between changes in mitochondria and neuropathology will lead to new therapeutic strategies. Here, we summarize the evidence of site-specific mitochondrial dysfunction and mitochondrial-related clinical trials for neurodegenerative diseases. We further discuss potential therapeutic approaches, such as mitochondrial transplantation, restoration of mitochondrial function, and pharmacological alleviation of mitochondrial dysfunction.

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.

Q134R: Small chemical compound with NFAT inhibitory properties improves behavioral performance and synapse function in mouse models of amyloid pathology.

Inhibition of the protein phosphatase calcineurin (CN) ameliorates pathophysiologic and cognitive changes in aging rodents and mice with aging-related Alzheimer's disease (AD)-like pathology. However, concerns over adverse effects have slowed the transition of common CN-inhibiting drugs to the clinic for the treatment of AD and AD-related disorders. Targeting substrates of CN, like the nuclear factor of activated T cells (NFATs), has been suggested as an alternative, safer approach to CN inhibitors. However, small chemical inhibitors of NFATs have only rarely been described. Here, we investigate a newly developed neuroprotective hydroxyquinoline derivative (Q134R) that suppresses NFAT signaling, without inhibiting CN activity. Q134R partially inhibited NFAT activity in primary rat astrocytes, but did not prevent CN-mediated dephosphorylation of a non-NFAT target, either in vivo, or in vitro. Acute (≤1 week) oral delivery of Q134R to APP/PS1 (12 months old) or wild-type mice (3-4 months old) infused with oligomeric Aß peptides led to improved Y maze performance. Chronic (≥3 months) oral delivery of Q134R appeared to be safe, and, in fact, promoted survival in wild-type (WT) mice when given for many months beyond middle age. Finally, chronic delivery of Q134R to APP/PS1 mice during the early stages of amyloid pathology (i.e., between 6 and 9 months) tended to reduce signs of glial reactivity, prevented the upregulation of astrocytic NFAT4, and ameliorated deficits in synaptic strength and plasticity, without noticeably altering parenchymal Aß plaque pathology. The results suggest that Q134R is a promising drug for treating AD and aging-related disorders.

Broad Kinase Inhibition Mitigates Early Neuronal Dysfunction in Tauopathy.

Tauopathies are a group of more than twenty known disorders that involve progressive neurodegeneration, cognitive decline and pathological tau accumulation. Current therapeutic strategies provide only limited, late-stage symptomatic treatment. This is partly due to lack of understanding of the molecular mechanisms linking tau and cellular dysfunction, especially during the early stages of disease progression. In this study, we treated early stage tau transgenic mice with a multi-target kinase inhibitor to identify novel substrates that contribute to cognitive impairment and exhibit therapeutic potential. Drug treatment significantly ameliorated brain atrophy and cognitive function as determined by behavioral testing and a sensitive imaging technique called manganese-enhanced magnetic resonance imaging (MEMRI) with quantitative R1 mapping. Surprisingly, these benefits occurred despite unchanged hyperphosphorylated tau levels. To elucidate the mechanism behind these improved cognitive outcomes, we performed quantitative proteomics to determine the altered protein network during this early stage in tauopathy and compare this model with the human Alzheimer's disease (AD) proteome. We identified a cluster of preserved pathways shared with human tauopathy with striking potential for broad multi-target kinase intervention. We further report high confidence candidate proteins as novel therapeutically relevant targets for the treatment of tauopathy. Proteomics data are available via ProteomeXchange with identifier PXD023562.

Tau-mediated dysregulation of RNA: Evidence for a common molecular mechanism of toxicity in frontotemporal dementia and other tauopathies.

Frontotemporal dementias (FTDs) encompass several disorders commonly characterized by progressive frontotemporal lobar degeneration and dementia. Pathologically, TDP-43, FUS, dipeptide repeats, and tau constitute the protein aggregates in FTD, which in turn coincide with heterogeneity in clinical variants. The underlying molecular etiology explaining the formation of each type of protein aggregate remains unclear; however, dysregulated RNA metabolism rises as a common pathogenic factor. Alongside with TDP-43 and FUS, which bind to and regulate RNA dynamics, emerging data suggest that tau may also regulate RNA metabolism and translation. The complex mechanisms that drive translational selectivity in turn regulate the broad clinical presentation of FTDs. Here, we focus on the enigmatic relationship between tau and RNA and review the mechanisms of tau-mediated dysregulation of RNA in tauopathies such as FTD.

Proteomic Techniques to Examine Neuronal Translational Dynamics.

Impairments in translation have been increasingly implicated in the pathogenesis and progression of multiple neurodegenerative diseases. Assessing the spatiotemporal dynamics of translation in the context of disease is a major challenge. Recent developments in proteomic analyses have enabled the resolution of nascent peptides in a short timescale on the order of minutes. In addition, a quantitative analysis of translation has progressed in vivo, showing remarkable potential for coupling these techniques with cognitive and behavioral outcomes. Here, we review these modern approaches to measure changes in translation and ribosomal function with a specific focus on current applications in the mammalian brain and in the study of neurodegenerative diseases.

Tau drives translational selectivity by interacting with ribosomal proteins.

There is a fundamental gap in understanding the consequences of tau-ribosome interactions. Tau oligomers and filaments hinder protein synthesis in vitro, and they associate strongly with ribosomes in vivo. Here, we investigated the consequences of tau interactions with ribosomes in transgenic mice, in cells, and in human brain tissues to identify tau as a direct modulator of ribosomal selectivity. First, we performed microarrays and nascent proteomics to measure changes in protein synthesis. Using regulatable rTg4510 tau transgenic mice, we determined that tau expression differentially shifts both the transcriptome and the nascent proteome, and that the synthesis of ribosomal proteins is reversibly dependent on tau levels. We further extended these results to human brains and found that tau pathologically interacts with ribosomal protein S6 (rpS6 or S6), a crucial regulator of translation. Consequently, protein synthesis under translational control of rpS6 was reduced under tauopathic conditions in Alzheimer's disease brains. Our data establish tau as a driver of RNA translation selectivity. Moreover, since regulation of protein synthesis is critical for learning and memory, aberrant tau-ribosome interactions in disease could explain the linkage between tauopathies and cognitive impairment.

Manganese-Enhanced Magnetic Resonance Imaging: Overview and Central Nervous System Applications With a Focus on Neurodegeneration.

Manganese-enhanced magnetic resonance imaging (MEMRI) rose to prominence in the 1990s as a sensitive approach to high contrast imaging. Following the discovery of manganese conductance through calcium-permeable channels, MEMRI applications expanded to include functional imaging in the central nervous system (CNS) and other body systems. MEMRI has since been employed in the investigation of physiology in many animal models and in humans. Here, we review historical perspectives that follow the evolution of applied MRI research into MEMRI with particular focus on its potential toxicity. Furthermore, we discuss the more current in vivo investigative uses of MEMRI in CNS investigations and the brief but decorated clinical usage of chelated manganese compound mangafodipir in humans.