Role of the cytoskeleton in steroidogenesis

: Steroidogenesis in the adrenal cortex or gonads is a complicated process, modulated by various elements either at the tissue or molecular level. The substrate—cholesterol is first delivered to the outer membrane of mitochondria, undergoing a series of enzymatic reactions along with the material exchange between the mitochondria and the ER (endoplasmic reticulum) and ultimately yield various steroids: aldosterone, cortisol, testosterone and estrone. Several valves are set to adjust the amount of production to the needs. e.g. StAR(steroidogenic acute regulator) is in charge of the rate-limiting step—traffic of cholesterol from outer membrane to inner membrane of mitochondria. And the “needs” is partly reflected by trophic signals like ACTH、LH and downstream pathways-- intracellular cAMP pathway, which represents the endocrinal regulation of steroid synthesis, too. The coordinated activities of these related factors are all associated with another crucial cellular constituent—the cytoskeleton, which plays a crucial role in the cellular architecture and substrate trafficking. Though considerable studies have been performed regarding steroid synthesis, details about the upstream signaling pathways and mechanisms of the regulation by cytoskeleton network still remain unclear. The metabolism and interplays of the pivotal cellular organelles with cytoskeleton are worth exploring as well. In this review, we summarize research of different time span, describing the roles of specific cytoskeleton elements in steroidogenesis and related signaling pathways involved in the steroid synthesis. In addition, we discussed the inner cytoskeletal network involved in steroidogenic processes such as mitochondrial movement, organelle interactions and cholesterol trafficking.

Live imaging reveals the cellular events downstream of SARM1 activation

SARM1 is an inducible NAD+ hydrolase that triggers axon loss and neuronal cell death in the injured and diseased nervous system. While SARM1 activation and enzyme function are well defined, the cellular events downstream of SARM1 activity but prior to axonal demise are much less well understood. Defects in calcium, mitochondria, ATP, and membrane homeostasis occur in injured axons, but the relationships among these events have been difficult to disentangle because prior studies analyzed large collections of axons in which cellular events occur asynchronously. Here we used live imaging of mouse sensory neurons with single axon resolution to investigate the cellular events downstream of SARM1 activity. Our studies support a model in which SARM1 NADase activity leads to an ordered sequence of events from loss of cellular ATP, to defects in mitochondrial movement and depolarization, followed by calcium influx, externalization of phosphatidylserine, and loss of membrane permeability prior to catastrophic axonal self-destruction.

Precise and Long-Term Tracking of Mitochondria in Neurons using a Bioconjugatable and Photostable AIE Luminogen

Tracking mitochondrial movement in neurons is an attractive research field as dysregulation of mitochondrial motion is associated with multiple neurological diseases. To attain the precise trajectory of a single mitochondrion and achieve long-term imaging of mitochondria in neurons, specific and photostable fluorescent probes with a long emission lifetime are required. Existing mitochondrial targeting fluorescent dyes suffer from poor photostability, high toxicity, “always-on” behavior, and aggregation-caused quenching effect, which limit their use in studying mitochondria in neurons. To overcome these challenges, we designed and synthesized an aggregation-induced emission (AIE)-active luminogen, TPAP-C5-yne, which consists of an activated alkyne terminus for bioconjugation with amines, and a cationic pyridinium moiety to selectively target mitochondria. For the first time using TPAP-C5-yne, we successfully tracked and analyzed the motion of a single mitochondrion in live primary hippocampal neurons accurately using real-time fluorescence images acquired by a sensitive EMCCD camera. In addition, long-term imaging of mitochondria in live neurons for a week is achieved by TPAP-C5-yne, which was not feasible with a commercially available mitochondrial targeting probe before.

FHL2 anchors mitochondria to actin and adapts mitochondrial dynamics to glucose supply

Mitochondrial movement and distribution are fundamental to their function. Here we report a mechanism that regulates mitochondrial movement by anchoring mitochondria to the F-actin cytoskeleton. This mechanism is activated by an increase in glucose influx and the consequent O-GlcNAcylation of TRAK (Milton), a component of the mitochondrial motor-adaptor complex. The protein four and a half LIM domains protein 2 (FHL2) serves as the anchor. FHL2 associates with O-GlcNAcylated TRAK and is both necessary and sufficient to drive the accumulation of F-actin around mitochondria and to arrest mitochondrial movement by anchoring to F-actin. Disruption of F-actin restores mitochondrial movement that had been arrested by either TRAK O-GlcNAcylation or forced direction of FHL2 to mitochondria. This pathway for mitochondrial immobilization is present in both neurons and non-neuronal cells and can thereby adapt mitochondrial dynamics to changes in glucose availability.

Mitochondria dysfunction in Charcot Marie Tooth 2B Peripheral Sensory Neuropathy

Recent evidence has uncovered an important role of Rab7 in regulating mitochondrial morphology and function. Missense mutation(s) of Rab7 underlies the pathogenesis of Charcot Marie Tooth 2B (CMT2B) peripheral neuropathy. Herein, we investigated how mitochondrial morphology and function were impacted by the CMT2B associated Rab7V162M mutation in fibroblasts from human CMT2B patients as well as in a knockin mouse model. In contrast to recently published results from studies of using heterologous overexpression systems, our results have demonstrated significant mitochondrial fragmentation in fibroblasts of both human CMT2B patients and CMT2B mouse embryonic fibroblasts (MEFs). Furthermore, we have shown that mitochondria were fragmented and axonal mitochondrial movement was dysregulated in primary cultured E18 dorsal root ganglion (DRG) sensory neurons, but not in E18 hippocampal and cortical primary neurons. We also show that inhibitors to either the mitochondrial fission protein Drp1 or to the nucleotide binding to Rab7 normalized the mitochondrial deficits in both MEFs and E18 cultured DRG neurons. Our study has revealed, for the first time, that expression of CMT2B Rab7 mutation at physiological level enhances Drp1 activity to promote mitochondrial fission, that may potentially underlie selective vulnerability of peripheral sensory neurons in CMT2B pathogenesis.

Adult-onset mitochondrial movement disorders: a national picture from the Italian Network

Introduction Both prevalence and clinical features of the various movement disorders in adults with primary mitochondrial diseases are unknown. Methods Based on the database of the “Nation-wide Italian Collaborative Network of Mitochondrial Diseases”, we reviewed the clinical, genetic, neuroimaging and neurophysiological data of adult patients with primary mitochondrial diseases (n = 764) where ataxia, myoclonus or other movement disorders were part of the clinical phenotype. Results Ataxia, myoclonus and movement disorders were present in 105/764 adults (13.7%), with the onset coinciding or preceding the diagnosis of the mitochondrial disease in 49/105 (46.7%). Ataxia and parkinsonism were the most represented, with an overall prevalence at last follow-up of 59.1% and 30.5%, respectively. Hyperkinetic movement disorders were reported in 15.3% at last follow-up, being the less common reported movement disorders. The pathogenic m.8344A > G and POLG variants were always associated with a movement disorder, while LHON variants and mtDNA single deletions were more commonly found in the subjects who did not present a movement disorder. The most common neuroimaging features were cortical and/or cerebellar atrophy, white matter hyperintensities, basal ganglia abnormalities and nigro-striatal degeneration. Almost 70% of patients with parkinsonism responded to dopaminergic therapy, mainly levodopa, and 50% with myoclonus were successfully treated with levetiracetam. Conclusion Movement disorders, mainly ataxia and parkinsonism, are important findings in adult primary mitochondrial diseases. This study underlies the importance of looking for a mitochondrial etiology in the diagnostic flowchart of a movement disorder and may help direct genetic screening in daily practice.

Mitochondrial Arrest on the Microtubule Highway—A Feature of Heart Failure and Diabetic Cardiomyopathy?

A pathophysiological consequence of both type 1 and 2 diabetes is remodelling of the myocardium leading to the loss of left ventricular pump function and ultimately heart failure (HF). Abnormal cardiac bioenergetics associated with mitochondrial dysfunction occurs in the early stages of HF. Key factors influencing mitochondrial function are the shape, size and organisation of mitochondria within cardiomyocytes, with reports identifying small, fragmented mitochondria in the myocardium of diabetic patients. Cardiac mitochondria are now known to be dynamic organelles (with various functions beyond energy production); however, the mechanisms that underpin their dynamism are complex and links to motility are yet to be fully understood, particularly within the context of HF. This review will consider how the outer mitochondrial membrane protein Miro1 (Rhot1) mediates mitochondrial movement along microtubules via crosstalk with kinesin motors and explore the evidence for molecular level changes in the setting of diabetic cardiomyopathy. As HF and diabetes are recognised inflammatory conditions, with reports of enhanced activation of the NLRP3 inflammasome, we will also consider evidence linking microtubule organisation, inflammation and the association to mitochondrial motility. Diabetes is a global pandemic but with limited treatment options for diabetic cardiomyopathy, therefore we also discuss potential therapeutic approaches to target the mitochondrial-microtubule-inflammatory axis.

Live Imaging Reveals the Cellular Events Downstream of SARM1 Activation

SARM1 is an inducible NAD+ hydrolase that triggers axon loss and neuronal cell death in the injured and diseased nervous system. While SARM1 activation and enzyme function are well defined, the cellular events downstream of SARM1 activity but prior to axonal demise are much less well understood. Defects in calcium, mitochondria, ATP, and membrane homeostasis occur in injured axons, but the relationships among these events have been difficult to disentangle because prior studies analyzed large collections of axons in which cellular events occur asynchronously. Here we used live imaging with single axon resolution to investigate the cellular events downstream of SARM1 activity. Our studies support a model in which SARM1 NADase activity leads to an ordered sequence of events from loss of cellular ATP, to defects in mitochondrial movement and depolarization, followed by calcium influx, externalization of phosphatidylserine, and loss of membrane permeability prior to catastrophic axonal self destruction.

TRAK proteins encode distinct MIRO-dependent and MIRO-independent mechanisms for associating with the mitochondrial outer membrane

Mitochondria are essential to proper cell function, mislocalization of mitochondria leads to disease. Previous research has indicated that MYO19, an unconventional myosin, localizes to the mitochondria outer membrane (MOM) and enables actin-based mitochondrial movement along the cytoskeleton. MOM insertion of MYO19 is assisted by the small GTPase MIRO, a "molecular switch" that facilitates MYO19/mitochondria interactions. MIRO serves as a recruiter of MYO19 to the MOM, rather than the tether/receptor that mediates attachment, as MYO19 contains a second, MIRO-independent mitochondrial association domain. MIRO proteins have previously been reported to serve as attachment points for the microtubule-based motors through interactions with the adaptor protein, TRAK. Past research has identified a MIRO-binding domain of TRAK that directly participates in the interaction with MIRO. We chose to investigate whether the interactions between TRAK and MOM paralleled our hypothesized mechanism for MYO19/MOM interactions by examining the MIRO-mediated enhancement of TRAK protein localization to mitochondria, by identifying the location of a MIRO-independent mitochondrial association domain in the c-terminus of TRAK proteins, and by examining the steady-state binding kinetics of various TRAK constructs to mitochondria. We interpret these data to indicate that MIRO proteins serve in the initial recruitment of TRAK proteins to mitochondria, but that the MIRO-independent domain plays a significant role in long-term association between TRAK and MOM.