Miro1-dependent mitochondrial positioning drives the rescaling of presynaptic Ca2+ signals during homeostatic plasticity

Mitochondrial trafficking is influenced by neuronal activity, but it remains unclear how mitochondrial positioning influences neuronal transmission and plasticity. Here, we use live cell imaging with the genetically encoded presynaptically targeted Ca2+ indicator, SyGCaMP5, to address whether presynaptic Ca2+ responses are altered by mitochondria in synaptic terminals. We find that presynaptic Ca2+ signals, as well as neurotransmitter release, are significantly decreased in terminals containing mitochondria. Moreover, the localisation of mitochondria at presynaptic sites can be altered during long‐term activity changes, dependent on the Ca2+‐sensing function of the mitochondrial trafficking protein, Miro1. In addition, we find that Miro1‐mediated activity‐dependent synaptic repositioning of mitochondria allows neurons to homeostatically alter the strength of presynaptic Ca2+ signals in response to prolonged changes in neuronal activity. Our results support a model in which mitochondria are recruited to presynaptic terminals during periods of raised neuronal activity and are involved in rescaling synaptic signals during homeostatic plasticity

Quantum dot conjugated nanobodies for multiplex imaging of protein dynamics at synapses

Neurons communicate with each other through synapses, which show enrichment for specialized receptors. Although many studies have explored spatial enrichment and diffusion of these receptors in dissociated neurons using single particle tracking, much less is known about their dynamic properties at synapses in complex tissue like brain slices. Here we report the use of smaller and highly specific quantum dots conjugated with a recombinant single domain antibody fragment (VHH fragment) against green fluorescent protein to provide information on diffusion of adhesion molecules at the growth cone and neurotransmitter receptors at synapses. Our data reveals that QD-nanobodies can measure neurotransmitter receptor dynamics at both excitatory and inhibitory synapses in primary neuronal cultures as well as in ex vivo rat brain slices. We also demonstrate that this approach can be applied to tagging multiple proteins to simultaneously monitor their behavior. Thus, we provide a strategy for multiplex imaging of tagged membrane proteins to study their clustering, diffusion and transport both in vitro as well as in native tissue environments such as brain slices

Miro1 Regulates Activity-Driven Positioning of Mitochondria within Astrocytic Processes Apposed to Synapses to Regulate Intracellular Calcium Signaling

It is fast emerging that maintaining mitochondrial function is important for regulating astrocyte function, although the specific mechanisms that govern astrocyte mitochondrial trafficking and positioning remain poorly understood. The mitochondrial Rho-GTPase 1 protein (Miro1) regulates mitochondrial trafficking and detachment from the microtubule transport network to control activity-dependent mitochondrial positioning in neurons. However, whether Miro proteins are important for regulating signaling-dependent mitochondrial dynamics in astrocytic processes remains unclear. Using live-cell confocal microscopy of rat organotypic hippocampal slices, we find that enhancing neuronal activity induces transient mitochondrial remodeling in astrocytes, with a concomitant, transient reduction in mitochondrial trafficking, mediated by elevations in intracellular Ca(2+). Stimulating neuronal activity also induced mitochondrial confinement within astrocytic processes in close proximity to synapses. Furthermore, we show that the Ca(2+)-sensing EF-hand domains of Miro1 are important for regulating mitochondrial trafficking in astrocytes and required for activity-driven mitochondrial confinement near synapses. Additionally, activity-dependent mitochondrial positioning by Miro1 reciprocally regulates the levels of intracellular Ca(2+) in astrocytic processes. Thus, the regulation of intracellular Ca(2+) signaling, dependent on Miro1-mediated mitochondrial positioning, could have important consequences for astrocyte Ca(2+) wave propagation, gliotransmission, and ultimately neuronal function

α-thalassaemia

Alpha-thalassaemia is inherited as an autosomal recessive disorder characterised by a microcytic hypochromic anaemia, and a clinical phenotype varying from almost asymptomatic to a lethal haemolytic anaemia

Utilisation of an operative difficulty grading scale for laparoscopic cholecystectomy

Background A reliable system for grading operative difficulty of laparoscopic cholecystectomy would standardise description of findings and reporting of outcomes. The aim of this study was to validate a difficulty grading system (Nassar scale), testing its applicability and consistency in two large prospective datasets. Methods Patient and disease-related variables and 30-day outcomes were identified in two prospective cholecystectomy databases: the multi-centre prospective cohort of 8820 patients from the recent CholeS Study and the single-surgeon series containing 4089 patients. Operative data and patient outcomes were correlated with Nassar operative difficultly scale, using Kendall’s tau for dichotomous variables, or Jonckheere–Terpstra tests for continuous variables. A ROC curve analysis was performed, to quantify the predictive accuracy of the scale for each outcome, with continuous outcomes dichotomised, prior to analysis. Results A higher operative difficulty grade was consistently associated with worse outcomes for the patients in both the reference and CholeS cohorts. The median length of stay increased from 0 to 4 days, and the 30-day complication rate from 7.6 to 24.4% as the difficulty grade increased from 1 to 4/5 (both p < 0.001). In the CholeS cohort, a higher difficulty grade was found to be most strongly associated with conversion to open and 30-day mortality (AUROC = 0.903, 0.822, respectively). On multivariable analysis, the Nassar operative difficultly scale was found to be a significant independent predictor of operative duration, conversion to open surgery, 30-day complications and 30-day reintervention (all p < 0.001). Conclusion We have shown that an operative difficulty scale can standardise the description of operative findings by multiple grades of surgeons to facilitate audit, training assessment and research. It provides a tool for reporting operative findings, disease severity and technical difficulty and can be utilised in future research to reliably compare outcomes according to case mix and intra-operative difficulty

Population‐based cohort study of outcomes following cholecystectomy for benign gallbladder diseases

Background The aim was to describe the management of benign gallbladder disease and identify characteristics associated with all‐cause 30‐day readmissions and complications in a prospective population‐based cohort. Methods Data were collected on consecutive patients undergoing cholecystectomy in acute UK and Irish hospitals between 1 March and 1 May 2014. Potential explanatory variables influencing all‐cause 30‐day readmissions and complications were analysed by means of multilevel, multivariable logistic regression modelling using a two‐level hierarchical structure with patients (level 1) nested within hospitals (level 2). Results Data were collected on 8909 patients undergoing cholecystectomy from 167 hospitals. Some 1451 cholecystectomies (16·3 per cent) were performed as an emergency, 4165 (46·8 per cent) as elective operations, and 3293 patients (37·0 per cent) had had at least one previous emergency admission, but had surgery on a delayed basis. The readmission and complication rates at 30 days were 7·1 per cent (633 of 8909) and 10·8 per cent (962 of 8909) respectively. Both readmissions and complications were independently associated with increasing ASA fitness grade, duration of surgery, and increasing numbers of emergency admissions with gallbladder disease before cholecystectomy. No identifiable hospital characteristics were linked to readmissions and complications. Conclusion Readmissions and complications following cholecystectomy are common and associated with patient and disease characteristics

Miro1-dependent mitochondrial dynamics in parvalbumin interneurons

Parvalbumin (PV+) interneurons constitute a small proportion of the total neuronal population (less than 2% in the hippocampus), yet they possess crucial roles in shaping neuronal network activity (Freund and Buzsáki, 1996; Jonas et al., 2004; Pelkey et al., 2017). PV+ interneurons inhibit their postsynaptic targets efficiently by applying fast perisomatic inhibition and have been directly implicated in the generation of network activity at the gamma (γ) band frequency (30–80 Hz) (Antonoudiou et al., 2020; Cardin et al., 2009; Hájos et al., 2004; Mann et al., 2005; Sohal et al., 2009). Network oscillations at γ-band frequency are believed to facilitate information transmission through circuit synchronization and local gain control that may be instrumental in multiple cognitive processes such as attention, learning, and memory (Akam and Kullmann, 2010; Fries, 2015; Howard et al., 2003; Montgomery and Buzsaki, 2007; Sohal, 2016). Importantly, these oscillations are thought to be metabolically very costly, and it has therefore been postulated that PV+ interneurons require substantial amounts of energy via ATP hydrolysis to sustain the high firing rate and dissipate ion gradients during neuronal transmission (Attwell and Laughlin, 2001; Kann, 2011; Kann, 2016; Kann and Kovács, 2007; Kann et al., 2014). Thus, it is crucial to understand the metabolic expenditure and the involvement of mitochondria in PV+ interneurons. Indeed, electron microscopy, histochemical, and transcriptomic approaches have revealed that PV+ interneurons have a higher density of energy-producing mitochondria and elevated expression levels of electron transport chain components (Adams et al., 2015; Gulyás et al., 2006; Nie and Wong-Riley, 1995; Paul et al., 2017). The spatiotemporal organization of mitochondria is essential for the precise provision of ATP and Ca2+-buffering for neuronal transmission and communication (Devine and Kittler, 2018; MacAskill and Kittler, 2010). Miro1 is a mitochondrial adaptor protein, responsible for coupling mitochondria to the cytoskeleton and for their bidirectional trafficking in axons and dendrites (Birsa et al., 2013; Guo et al., 2005; López-Doménech et al., 2016; López-Doménech et al., 2018; Macaskill et al., 2009; Nguyen et al., 2014; Saotome et al., 2008; Wang and Schwarz, 2009). Global deletion of Miro1 (encoded by the Rhot1 gene) is perinatal lethal, while the conditional removal of Miro1 from cortical and hippocampal pyramidal cells alters the occupancy of dendritic mitochondria due to impairment in trafficking, resulting in dendritic degeneration and cell death (López-Doménech et al., 2016). In contrast, the significance of mitochondrial trafficking and distribution in PV+ interneurons, and the role of Miro1, is completely unexplored and especially interesting as their axon is highly branched with a cumulative length reaching up to 50 mm in the hippocampus (Hu et al., 2014). In this study, we generated a transgenic mouse line where mitochondria are fluorescently labelled in PV+ interneurons. We crossed this line with the Miro1 floxed mouse (Rhot1flox/flox), to generate a model where Miro1 was conditionally knocked-out exclusively in PV+ interneurons. Using two-photon live-imaging of ex vivo organotypic brain slices, we demonstrated a reduction in mitochondrial trafficking in the absence of Miro1 in PV+ interneurons in the hippocampus. The impairment in Miro1-directed mitochondrial transport led to an accumulation of mitochondria in the soma and their depletion from axonal presynaptic terminals in acute hippocampal brain slices. Loss of Miro1 resulted in alterations in axonal but not dendritic branching in PV+ interneurons. While the ability of PV+ interneurons to apply long-lasting inhibition to post-synaptic targets remained intact, the changes in Miro1-dependent mitochondrial dynamics were accompanied by an increased frequency of γ-oscillations in hippocampal brain slices and a reduction in anxiety-related emotional behavior. Thus, we show that Miro1-dependent mitochondrial positioning is essential for correct PV+ interneuron function, network activity, and anxiolytic animal behavior