Mitophagy: Molecular Mechanisms, New Concepts on Parkin Activation and the Emerging Role of AMPK/ULK1 Axis

Mitochondria are multifunctional subcellular organelles essential for cellular energy homeostasis and apoptotic cell death. It is, therefore, crucial to maintain mitochondrial fitness. Mitophagy, the selective removal of dysfunctional mitochondria by autophagy, is critical for regulating mitochondrial quality control in many physiological processes, including cell development and differentiation. On the other hand, both impaired and excessive mitophagy are involved in the pathogenesis of different ageing-associated diseases such as neurodegeneration, cancer, myocardial injury, liver disease, sarcopenia and diabetes. The best-characterized mitophagy pathway is the PTEN-induced putative kinase 1 (PINK1)/Parkin-dependent pathway. However, other Parkin-independent pathways are also reported to mediate the tethering of mitochondria to the autophagy apparatuses, directly activating mitophagy (mitophagy receptors and other E3 ligases). In addition, the existence of molecular mechanisms other than PINK1-mediated phosphorylation for Parkin activation was proposed. The adenosine5′-monophosphate (AMP)-activated protein kinase (AMPK) is emerging as a key player in mitochondrial metabolism and mitophagy. Beyond its involvement in mitochondrial fission and autophagosomal engulfment, its interplay with the PINK1–Parkin pathway is also reported. Here, we review the recent advances in elucidating the canonical molecular mechanisms and signaling pathways that regulate mitophagy, focusing on the early role and spatial specificity of the AMPK/ULK1 axis.

Old Plays, New Narratives

When a theatrical performance is digitally broadcast live to cinemas, the limitations of temporal and spatial specificity are removed and the theatrical experience is simultaneously opened up to a wider audience and inherently altered. One such production, Coriolanus (Donmar Warehouse, 2013-14), starring an actor with a particularly enthusiastic online fan community, was broadcast to cinemas by National Theatre Live, where fans recorded it on digital devices, extracted clips and produced animated gifs, which they captioned to reinterpret the play, sharing them online, removed from their original context. The transformation of theatre texts to cinemas to social media platforms raises exciting questions related to how fans interact with culture both as consumers and as producers of new media texts. How do the different transformations (technical and actively fan-produced) affect both the narrative and the cultural experience? How do new texts function as surrogates for, and extensions of, the ‘official' narrative, as well as new interactive narratives in their own right? This paper addresses these questions in the context of a specific theatrical event as it crossed the boundary from a live, co-located experience first into cinema, and then into interactive hypertexts and memes. Drawing on theories of fandom and participatory culture, as well as post-Web 2.0 analyses of Internet behaviours, the paper examines fan production of new media texts and how they both transmit and transform the source narrative via interpretation, re-interpretation, and misinterpretation. Image Credit: Still of fromhiddleswithlove (2014)

Cerebral blood flow is associated with matrix metalloproteinase levels during the early symptomatic phase of concussion

Concussion is associated with disrupted cerebral blood flow (CBF), although there appears to be substantial inter-individual variability in CBF response. At present, the mechanisms of variable CBF response remain incompletely understood, but one potential contributor is matrix metalloproteinase (MMP) expression. In more severe forms of acquired brain injury, MMP up-regulation contributes to CBF impairments via increased blood-brain barrier permeability. A similar relationship is hypothesized for concussion, where recently concussed individuals with higher MMP levels have lower CBF. To test this hypothesis, 35 concussed athletes were assessed longitudinally at early symptomatic injury (median: 5 days post-injury) and at medical clearance (median: 24 days post-injury), along with 71 athletic controls. For all athletes, plasma MMPs were measured and arterial spin labelling was used to measure CBF. Consistent with our hypothesis, higher concentrations of MMP-2 and MMP-3 were correlated with lower global CBF. The correlations between MMPs and global CBF were also significantly diminished for concussed athletes at medical clearance and for athletic controls. These results indicate an inverse relationship between plasma MMP levels and CBF that is specific to the symptomatic phase of concussion. Analyses of regional CBF further showed that correlations with MMP levels exhibited some spatial specificity, with greatest effects in occipital, parietal and temporal lobes. These findings provide new insights into the mechanisms of post-concussion cerebrovascular dysfunction.

Mitochondria-localized AMPK responds to local energetics and contributes to exercise and energetic stress-induced mitophagy

Mitochondria form a complex, interconnected reticulum that is maintained through coordination among biogenesis, dynamic fission, and fusion and mitophagy, which are initiated in response to various cues to maintain energetic homeostasis. These cellular events, which make up mitochondrial quality control, act with remarkable spatial precision, but what governs such spatial specificity is poorly understood. Herein, we demonstrate that specific isoforms of the cellular bioenergetic sensor, 5′ AMP-activated protein kinase (AMPKα1/α2/β2/γ1), are localized on the outer mitochondrial membrane, referred to as mitoAMPK, in various tissues in mice and humans. Activation of mitoAMPK varies across the reticulum in response to energetic stress, and inhibition of mitoAMPK activity attenuates exercise-induced mitophagy in skeletal muscle in vivo. Discovery of a mitochondrial pool of AMPK and its local importance for mitochondrial quality control underscores the complexity of sensing cellular energetics in vivo that has implications for targeting mitochondrial energetics for disease treatment.

Human Appropriation of Net Primary Production: From a Planet to a Pixel

Human appropriation of net primary production (HANPP) is a substantial improvement upon 20th century attempts at developing an ecological footprint indicator because of its measurability in relation to net primary production, its close relationship to other key footprint measures, such as carbon and water, and its spatial specificity. This paper explores HANPP across four geographical scales: through literature review, the planet; through reanalysis of existing data, variations among the world’s countries; and through novel analyses, U.S. counties and the 30 m pixel scale for one U.S. county. Results show that HANPP informs different sustainability narratives at different scales. At the planetary scale, HANPP is a critical planetary limit that improves upon areal land use indicators. At the country macroscale, HANPP indicates the degree to which meeting the needs of the domestic population for provisioning ecosystem services (food, feed, biofiber, biofuel) presses against the domestic ecological endowment of net primary production. At the county mesoscale, HANPP reveals the dependency of metropolitan areas upon regional specialized rural forestry and agroecosystems to which they are teleconnected through trade and transport infrastructures. At the pixel microscale, HANPP provides the basis for deriving spatial patterns of remaining net primary production upon which biodiversity and regulatory and cultural ecosystem services are dependent. HANPP is thus a sustainability indicator that can fulfill similar needs as carbon, water and other footprints.

Analysis of subcellular transcriptomes by RNA proximity labeling with Halo-seq

Thousands of RNA species display nonuniform distribution within cells. However, quantification of the spatial patterns adopted by individual RNAs remains difficult, in part by a lack of quantitative tools for subcellular transcriptome analysis. In this study, we describe an RNA proximity labeling method that facilitates the quantification of subcellular RNA populations with high spatial specificity. This method, termed Halo-seq, pairs a light-activatable, radical generating small molecule with highly efficient Click chemistry to efficiently label and purify spatially defined RNA samples. We compared Halo-seq with previously reported similar methods and found that Halo-seq displayed a higher efficiency of RNA labeling, indicating that it is well suited to the investigation of small, precisely localized RNA populations. We then used Halo-seq to quantify nuclear, nucleolar, and cytoplasmic transcriptomes, characterize their dynamic nature following perturbation, and identify RNA sequence features associated with their composition. Specifically, we found that RNAs containing AU-rich elements are relatively enriched in the nucleus. This enrichment becomes stronger upon treatment with the nuclear export inhibitor leptomycin B, both expanding the role of HuR in RNA export and generating a comprehensive set of transcripts whose export from the nucleus depends on HuR.

Outmigration Survival of a Threatened Steelhead Population through a Tidal Estuary

Juvenile steelhead are exposed to numerous threats in heterogeneous, estuarine environments, yet understanding of survival patterns and processes during this migratory stage is often limited by studies that use surrogate species or are restricted in duration and spatial specificity. Lack of detailed survival information in this critical migratory stage limits the effectiveness of management to maintain juvenile life history diversity in threatened populations. We used acoustic telemetry with multistate release-recapture models to investigate survival patterns during a key stage of the juvenile emigration of anadromous steelhead through the Sacramento-San Joaquin River Delta of California, United States, over multiple years, including three drought years. Survival was highly variable both within and among the six years of the study; estimated total survival through the Delta ranged from 0.06 (May 2014) to 0.69 (March 2011). Survival in the upstream reaches was associated with river discharge into the Delta, while survival through the lower reaches was associated with migration route. The lack of a single factor associated with survival in all reaches counteracts preconceived ideas of survival processes. Hydrodynamic manipulation and habitat improvements are recommended to support this anadromous population in a changing climate.

Optical Imaging-Based Guidance of Viral Microinjections and Insertion of a Laminar Electrophysiology Probe Into a Predetermined Barrel in Mouse Area S1BF

Wide-field Optical Imaging of Intrinsic Signals (OI-IS; Grinvald et al., 1986) is a method for imaging functional brain hemodynamic responses, mainly used to image activity from the surface of the cerebral cortex. It localizes small functional modules – such as cortical columns – with great spatial resolution and spatial specificity relative to the site of increases in neuronal activity. OI-IS is capable of imaging responses either through an intact or thinned skull or following a craniotomy. Therefore, it is minimally invasive, which makes it ideal for survival experiments. Here we describe OI-IS-based methods for guiding microinjections of optogenetics viral vectors in proximity to small functional modules (S1 barrels) of the cerebral cortex and for guiding the insertion of electrodes for electrophysiological recording into such modules. We validate our proposed methods by tissue processing of the cerebral barrel field area, revealing the track of the electrode in a predetermined barrel. In addition, we demonstrate the use of optical imaging to visualize the spatial extent of the optogenetics photostimulation, making it possible to estimate one of the two variables that conjointly determine which region of the brain is stimulated. Lastly, we demonstrate the use of OI-IS at high-magnification for imaging the upper recording contacts of a laminar probe, making it possible to estimate the insertion depth of all contacts relative to the surface of the cortex. These methods support the precise positioning of microinjections and recording electrodes, thus overcoming the variability in the spatial position of fine-scale functional modules.

Estimating the glutamate transporter surface density in mouse hippocampal astrocytes

Glutamate transporters preserve the spatial specificity of synaptic transmission by limiting glutamate diffusion away from the synaptic cleft, and prevent excitotoxicity by keeping the extracellular concentration of glutamate at low nanomolar levels. Glutamate transporters are abundantly expressed in astrocytes. Previous estimates in the rat hippocampus suggest that the surface density of glutamate transporters in astrocytic membranes is ~10,800 μm−2. Here, we estimate their surface density in astrocytic membranes of the mouse hippocampus, at different ages. By using realistic 3D Monte Carlo reaction-diffusion models, we show that varying the local glutamate transporter expression in astrocytes can alter profoundly the activation of extrasynaptic AMPA and NMDA receptors. Our findings show that the average density of astrocyte membranes and their surface density of glutamate transporters is higher in mice compared to rats, and increases with mouse age. There are stark differences in the density of expression of these molecules in different subcellular compartments, indicating that the extent to which astrocytes limit extrasynaptic glutamate diffusion depends not only on the level of astrocytic coverage, but also on the identity of the astrocyte compartment in contact with the synapse. Together, these findings provide information on the spatial distribution of glutamate transporters in the mouse hippocampus, which can be used in mathematical models of the spatiotemporal profile of extracellular glutamate after synaptic release.