Neutrophil extracellular trap formation and gene programs distinguish TST/IGRA sensitization outcomes among Mycobacterium tuberculosis exposed persons living with HIV.

Persons living with HIV (PLWH) have an increased risk for tuberculosis (TB). After prolonged and repeated exposure, some PLWH never develop TB and show no evidence of immune sensitization to Mycobacterium tuberculosis (Mtb) as defined by persistently negative tuberculin skin tests (TST) and interferon gamma release assays (IGRA). This group has been identified and defined as HIV+ persistently TB, tuberculin and IGRA negative (HITTIN). To investigate potential innate mechanisms unique to individuals with the HITTIN phenotype we compared their neutrophil Mtb infection response to that of PLWH, with no TB history, but who test persistently IGRA positive, and tuberculin positive (HIT). Neutrophil samples from 17 HITTIN (PMNHITTIN) and 11 HIT (PMNHIT) were isolated and infected with Mtb H37Rv for 1h and 6h. RNA was extracted and used for RNAseq analysis. Since there was no significant differential transcriptional response at 1h between infected PMNHITTIN and PMNHIT, we focused on the 6h timepoint. When compared to uninfected PMN, PMNHITTIN displayed 3106 significantly upregulated and 3548 significantly downregulated differentially expressed genes (DEGs) (absolute cutoff of a log2FC of 0.2, FDR < 0.05) whereas PMNHIT demonstrated 3816 significantly upregulated and 3794 significantly downregulated DEGs following 6h Mtb infection. Contrasting the log2FC 6h infection response to Mtb from PMNHITTIN against PMNHIT, 2285 genes showed significant differential response between the two groups. Overall PMNHITTIN had a lower fold change response to Mtb infection compared to PMNHIT. According to pathway enrichment, Apoptosis and NETosis were differentially regulated between HITTIN and HIT PMN responses after 6h Mtb infection. To corroborate the blunted NETosis transcriptional response measured among HITTIN, fluorescence microscopy revealed relatively lower neutrophil extracellular trap formation and cell loss in PMNHITTIN compared to PMNHIT, showing that PMNHITTIN have a distinct response to Mtb.

DRP1 mutations associated with EMPF1 encephalopathy alter mitochondrial membrane potential and metabolic programs.

Mitochondria and peroxisomes are dynamic signaling organelles that constantly undergo fission, driven by the large GTPase dynamin-related protein 1 (DRP1; encoded by DNM1L). Patients with de novo heterozygous missense mutations in DNM1L present with encephalopathy due to defective mitochondrial and peroxisomal fission (EMPF1) - a devastating neurodevelopmental disease with no effective treatment. To interrogate the mechanisms by which DRP1 mutations cause cellular dysfunction, we used human-derived fibroblasts from patients who present with EMPF1. In addition to elongated mitochondrial morphology and lack of fission, patient cells display lower coupling efficiency, increased proton leak and upregulation of glycolysis. Mitochondrial hyperfusion also results in aberrant cristae structure and hyperpolarized mitochondrial membrane potential. Peroxisomes show a severely elongated morphology in patient cells, which is associated with reduced respiration when cells are reliant on fatty acid oxidation. Metabolomic analyses revealed impaired methionine cycle and synthesis of pyrimidine nucleotides. Our study provides insight into the role of mitochondrial dynamics in cristae maintenance and the metabolic capacity of the cell, as well as the disease mechanism underlying EMPF1.

Exploring the connection between autophagy and heat-stress tolerance in Drosophila melanogaster.

Mechanisms aimed at recovering from heat-induced damages are closely associated with the ability of ectotherms to survive exposure to stressful temperatures. Autophagy, a ubiquitous stress-responsive catabolic process, has recently gained renewed attention as one of these mechanisms. By increasing the turnover of cellular structures as well as the clearance of long-lived protein and protein aggregates, the induction of autophagy has been linked to increased tolerance to a range of abiotic stressors in diverse ectothermic organisms. However, whether a link between autophagy and heat-tolerance exists in insect models remains unclear despite broad ecophysiological implications thereof. Here, we explored the putative association between autophagy and heat-tolerance using Drosophila melanogaster as a model. We hypothesized that (i) heat-stress would cause an increase of autophagy in flies' tissues, and (ii) rapamycin exposure would trigger a detectable autophagic response in adults and increase their heat-tolerance. In line with our hypothesis, we report that flies exposed to heat-stress present signs of protein aggregation and appear to trigger an autophagy-related homoeostatic response as a result. We further show that rapamycin feeding causes the systemic effect associated with target of rapamycin (TOR) inhibition, induces autophagy locally in the fly gut, and increases the heat-stress tolerance of individuals. These results argue in favour of a substantial contribution of autophagy to the heat-stress tolerance mechanisms of insects.

Rooibos tea-in the cross fire of ROS, mitochondrial dysfunction and loss of proteostasis-positioned for healthy aging.

Impaired mitochondrial function and loss of cellular proteostasis control are key hallmarks of aging and are implicated in the development of neurodegenerative diseases. A common denominator is the cell's inability to handle reactive oxygen species (ROS), leading to major downstream oxidative damage that exacerbates neuronal dysfunction. Although we have progressed in understanding the molecular defects associated with neuronal aging, many unanswered questions remain. How much ROS is required to serve cellular function before it becomes detrimental and how does the cell's oxidative status impact mitochondrial function and protein degradation through autophagy? How does ROS regulate autophagy? Aspalathus linearis, also commonly known as rooibos, is an endemic South African plant that is gaining globally acclaim for its antioxidant properties and its role as functional medicinal beverage. In this article we dissect the role of rooibos in the context of the cell's ROS handling capacity, mitochondrial function and autophagy activity. By addressing the dynamic relationship between these critical interconnected systems, and by evaluating the functional properties of rooibos, we unravel its position for preserving cell viability and promoting healthy aging.

Neurons die with heightened but functional macro- and chaperone mediated autophagy upon increased amyloid-ß induced toxicity with region-specific protection in prolonged intermittent fasting.

Alzheimer's disease (AD) is a devastating neurodegenerative condition with significant socio-economic impact that is exacerbated by the rapid increase in population aging, particularly impacting already burdened health care systems of poorly resourced countries. Accumulation of the amyloid-ß (Aß) peptide, generated through amyloid precursor protein (APP) processing, manifesting in senile plaques, is a well-established neuropathological feature. Aß plays a key role in driving synaptic dysfunction, neuronal cell loss, glial cell activation and oxidative stress associated with the pathogenesis of AD. Thus, the enhanced clearance of Aß peptide though modulation of the mechanisms that regulate intracellular Aß metabolism and clearance during AD progression have received major attention. Autophagy, a lysosome-based major proteolytic pathway, plays a crucial role in intracellular protein quality control and has been shown to contribute to the clearance of Aß peptide. However, to what extent autophagy activity remains upregulated and functional in the process of increasing Aß neurotoxicity is largely unclear. Here, we investigated the extent of neuronal toxicity in vitro by characterising autophagic flux, the expression profile of key amyloidogenic proteins, and proteins associated with prominent subtypes of the autophagy pathway to dissect the interplay between the engagement of proteolytic pathways and cell death onset in the context of APP overexpression. Moreover, we assessed the neuroprotective effects of a caloric restriction regime in vivo on the modulation of autophagy in specific brain regions. Our results reveal that autophagy is upregulated in the presence of high levels of APP and Aß and remains heightened and functional despite concomitant apoptosis induction, suggestive of a mismatch between autophagy cargo generation and clearance capacity. These findings were confirmed when implementing a prolonged intermittent fasting (IF) intervention in a model of paraquat-induced neuronal toxicity, where markers of autophagic activity were increased, while apoptosis onset and lipid peroxidation were robustly decreased in brain regions associated with neurodegeneration. This work highlights that especially caloric restriction mimetics and controlled prolonged IF may indeed be a highly promising therapeutic strategy at all stages of AD-associated pathology progression, for a cell-inherent and cell specific augmentation of Aß clearance through the powerful engagement of autophagy and thereby robustly contributing to neuronal protection.

Interactions between developmental and adult acclimation have distinct consequences for heat tolerance and heat stress recovery.

Developmental and adult thermal acclimation can have distinct, even opposite, effects on adult heat resistance in ectotherms. Yet, their relative contribution to heat-hardiness of ectotherms remains unclear despite the broad ecological implications thereof. Furthermore, the deterministic relationship between heat knockdown and recovery from heat stress is poorly understood but significant for establishing causal links between climate variability and population dynamics. Here, using Drosophila melanogaster in a full-factorial experimental design, we assessed the heat tolerance of flies in static stress assays, and document how developmental and adult acclimation interact with a distinct pattern to promote survival to heat stress in adults. We show that warmer adult acclimation is the initial factor enhancing survival to constant stressful high temperatures in flies, but also that the interaction between adult and developmental acclimation becomes gradually more important to ensure survival as the stress persists. This provides an important framework revealing the dynamic interplay between these two forms of acclimation that ultimately enhance thermal tolerance as a function of stress duration. Furthermore, by investigating recovery rates post-stress, we also show that the process of heat-hardening and recovery post-heat knockdown are likely to be based on set of (at least partially) divergent mechanisms. This could bear ecological significance as a trade-off may exist between increasing thermal tolerance and maximizing recovery rates post-stress, constraining population responses when exposed to variable and stressful climatic conditions.

Identification of a novel WAS mutation in a South African patient presenting with atypical Wiskott-Aldrich syndrome: a case report.

BACKGROUND: The X-linked recessive primary immunodeficiency disease (PIDD) Wiskott-Aldrich syndrome (WAS) is identified by an extreme susceptibility to infections, eczema and thrombocytopenia with microplatelets. The syndrome, the result of mutations in the WAS gene which encodes the Wiskott-Aldrich protein (WASp), has wide clinical phenotype variation, ranging from classical WAS to X-linked thrombocytopaenia and X-linked neutropaenia. In many cases, the diagnosis of WAS in first affected males is delayed, because patients may not present with the classic signs and symptoms, which may intersect with other thrombocytopenia causes. CASE PRESENTATION: Here, we describe a three-year-old HIV negative boy presenting with recurrent infections, skin rashes, features of autoimmunity and atopy. However, platelets were initially reported as normal in numbers and morphology as were baseline immune investigations. An older male sibling had died in infancy from suspected immunodeficiency. Uncertainty of diagnosis and suspected severe PIDD prompted urgent further molecular investigation. Whole exome sequencing identified c. 397 G > A as a novel hemizygous missense mutation located in exon 4 of WAS. CONCLUSION: With definitive molecular diagnosis, we could target treatment and offer genetic counselling and prenatal diagnostic testing to the family. The identification of novel variants is important to confirm phenotype variations of a syndrome.

Wild-type and mutant (G2019S) leucine-rich repeat kinase 2 (LRRK2) associate with subunits of the translocase of outer mitochondrial membrane (TOM) complex.

Leucine-rich repeat kinase 2 (LRRK2) is important in various cellular processes including mitochondrial homeostasis and mutations in this gene lead to Parkinson's disease (PD). However, the full spectrum of LRRK2's functions remain to be elucidated. The translocase of outer mitochondrial membrane (TOM) complex is essential for the import of almost all nuclear-encoded mitochondrial proteins and is fundamental for cellular survival. Using co-immunoprecipitation, super-resolution structured illumination microscopy (SR-SIM), and 3D virtual reality (VR) assisted co-localization analysis techniques we show that wild-type and mutant (G2019S) LRRK2 associate and co-localize with subunits of the TOM complex, either under basal (dimethyl sulfoxide, DMSO) or stress-induced (carbonyl cyanide m-chlorophenyl hydrazine, CCCP) conditions. Interestingly, LRRK2 interacted with TOM40 under both DMSO and CCCP conditions, and when the PD causing mutation, G2019S was introduced, the association was not altered. Moreover, overexpression of G2019S LRRK2 resulted in the formation of large, perinuclear aggregates that co-localized with the TOM complex. Taken together, this is the first study to show that both WT and mutant LRRK2 associate with the TOM complex subunits. These findings provide additional evidence for LRRK2's role in mitochondrial function which has important implications for its role in PD pathogenesis.

WD40-repeat 47, a microtubule-associated protein, is essential for brain development and autophagy.

The family of WD40-repeat (WDR) proteins is one of the largest in eukaryotes, but little is known about their function in brain development. Among 26 WDR genes assessed, we found 7 displaying a major impact in neuronal morphology when inactivated in mice. Remarkably, all seven genes showed corpus callosum defects, including thicker (Atg16l1, Coro1c, Dmxl2, and Herc1), thinner (Kif21b and Wdr89), or absent corpus callosum (Wdr47), revealing a common role for WDR genes in brain connectivity. We focused on the poorly studied WDR47 protein sharing structural homology with LIS1, which causes lissencephaly. In a dosage-dependent manner, mice lacking Wdr47 showed lethality, extensive fiber defects, microcephaly, thinner cortices, and sensory motor gating abnormalities. We showed that WDR47 shares functional characteristics with LIS1 and participates in key microtubule-mediated processes, including neural stem cell proliferation, radial migration, and growth cone dynamics. In absence of WDR47, the exhaustion of late cortical progenitors and the consequent decrease of neurogenesis together with the impaired survival of late-born neurons are likely yielding to the worsening of the microcephaly phenotype postnatally. Interestingly, the WDR47-specific C-terminal to LisH (CTLH) domain was associated with functions in autophagy described in mammals. Silencing WDR47 in hypothalamic GT1-7 neuronal cells and yeast models independently recapitulated these findings, showing conserved mechanisms. Finally, our data identified superior cervical ganglion-10 (SCG10) as an interacting partner of WDR47. Taken together, these results provide a starting point for studying the implications of WDR proteins in neuronal regulation of microtubules and autophagy.

Melatonin improves cardiac and mitochondrial function during doxorubicin-induced cardiotoxicity: A possible role for peroxisome proliferator-activated receptor gamma coactivator 1-alpha and sirtuin activity?

Mitochondrial dysfunction is a central element in the development of doxorubicin (DXR)-induced cardiotoxicity. In this context, melatonin is known to influence mitochondrial homeostasis and function. This study aimed to investigate the effects of melatonin on cardiac function, tumor growth, mitochondrial fission and fusion, PGC1-α and sirtuin activity in an acute model of DXR-induced cardiotoxicity. During the in vitro study, H9c2 rat cardiomyoblasts were pre-treated with melatonin (10 µM, 24 h) followed by DXR exposure (3 µM, 24 h). Following treatment, cellular ATP levels and mitochondrial morphology were assessed. In the in vivo study, female Sprague Dawley rats (16 weeks old), were inoculated with a LA7 rat mammary tumor cell line and tumors were measure daily. Animals were injected with DXR (3 × 4 mg/kg) and/or received melatonin (6 mg/kg) for 14 days in their drinking water. Rat hearts were used to conduct isolated heart perfusions to assess cardiac function and thereafter, heart tissue was used for immunoblot analysis. DXR treatment increased cell death and mitochondrial fission which were reduced with melatonin treatment. Cardiac output increased in rats treated with DXR + melatonin compared to DXR-treated rats. Tumor volumes was significantly reduced in DXR + melatonin-treated rats on Day 8 in comparison to DXR-treated rats. Furthermore, DXR + melatonin treatment increased cellular ATP levels, PGC1-α and SIRT1 expression which was attenuated by DXR treatment. These results indicate that melatonin treatment confers a dual cardio-protective and oncostatic effect by improving mitochondrial function and cardiac function whilst simultaneously retarding tumor growth during DXR-induced cardiotoxicity.

Autophagy is essential for the maintenance of amino acids and ATP levels during acute amino acid starvation in MDAMB231 cells.

Autophagy plays a major role in the adaptive metabolic response of cancer cells during adverse conditions such as nutrient deprivation. However, specific data that assess metabolite profiles in context with adenosine triphosphate (ATP) availability and cell death susceptibility remain limited. Human breast cancer cells, MDAMB231, and normal breast epithelial cells, MCF12A, were subjected to short-term amino acid starvation and the cellular apoptotic and autophagic responses assessed. The role of autophagy in the control of cellular amino acid, ATP, free fatty acid, and glucose levels during amino acid starvation were compared. We demonstrate that breast cancer cells have an increased metabolic demand contributing to significant amino acid and ATP depletion in a nutrient-poor environment. Upregulation of autophagy was important for the generation of amino acids and free fatty acids and maintenance of cellular ATP levels. In contrast to normal cells, breast cancer cells were unable to maintain the response after 12 hours of amino acid starvation. Regulation of autophagic activity in these environments had indirect consequences on cell death susceptibility. Overall, our data provide support for autophagy as an important survival mechanism capable of providing metabolic substrates when cancer cells are faced with nutrient-deprived environments. SIGNIFICANCE OF STUDY: The results obtained in this study helps to expand our current knowledge on how cells respond to environmental changes; the biochemical and metabolic consequences and the physiological processes activated in response. The environmental stress applied in this study is relevant to tumour physiology, and results can be translated to cancer therapeutic and clinical research areas, ultimately assisting in the specific targeting of cancer cells while avoiding harm to normal cells.

Virtual reality assisted microscopy data visualization and colocalization analysis.

BACKGROUND: Confocal microscopes deliver detailed three-dimensional data and are instrumental in biological analysis and research. Usually, this three-dimensional data is rendered as a projection onto a two-dimensional display. We describe a system for rendering such data using a modern virtual reality (VR) headset. Sample manipulation is possible by fully-immersive hand-tracking and also by means of a conventional gamepad. We apply this system to the specific task of colocalization analysis, an important analysis tool in biological microscopy. We evaluate our system by means of a set of user trials. RESULTS: The user trials show that, despite inaccuracies which still plague the hand tracking, this is the most productive and intuitive interface. The inaccuracies nevertheless lead to a perception among users that productivity is low, resulting in a subjective preference for the gamepad. Fully-immersive manipulation was shown to be particularly effective when defining a region of interest (ROI) for colocalization analysis. CONCLUSIONS: Virtual reality offers an attractive and powerful means of visualization for microscopy data. Fully immersive interfaces using hand tracking show the highest levels of intuitiveness and consequent productivity. However, current inaccuracies in hand tracking performance still lead to a disproportionately critical user perception.

Ascribing novel functions to the sarcomeric protein, myosin binding protein H (MyBPH) in cardiac sarcomere contraction.

Myosin binding protein H (MyBPH) is a protein of unknown function, which shares sequence and structural similarities with myosin binding protein C (cMyBPC), a protein frequently implicated in hypertrophic cardiomyopathy (HCM). Given the similarity between cMyBPC and MyBPH, we proposed that MyBPH, like cMyBPC, could be involved in HCM pathogenesis and we therefore sought to determine its function. We identified MyBPH-interacting proteins by using yeast two-hybrid (Y2H) analysis. The role of MyBPH and cMyBPC in cardiac cell contractility was analysed by measuring the planar cell surface area of differentiated H9c2 rat cardiomyocytes in response to ß-adrenergic stress after siRNA knockdown of MyBPH and cMyBPC. Individual knockdown of either protein had no effect on cardiac contractility, while concurrent knockdowns reduced cardiac contractility. These proteins therefore functionally compensate for one another and are critical for cardiac contractility. We further show that both proteins co-localise with the autophagosomal membrane protein LC3, suggesting that both proteins are involved in autophagosomal membrane maturation processes. The results of this study ascribe novel functions to MyBPH, which may contribute to our understanding of its role in the sarcomere. This study provides evidence for a potential role of MyBPH in HCM, which warrants further investigation.

Evidence for a common biological pathway linking three Parkinson's disease-causing genes: parkin, PINK1 and DJ-1.

Parkinson's disease (PD) is characterised by the loss of dopaminergic neurons in the midbrain. Autosomal recessive, early-onset cases of PD are predominantly caused by mutations in the parkin, PINK1 and DJ-1 genes. Animal and cellular models have verified a direct link between parkin and PINK1, whereby PINK1 phosphorylates and activates parkin at the outer mitochondrial membrane, resulting in removal of dysfunctional mitochondria via mitophagy. Despite the overwhelming evidence for this interaction, few studies have been able to identify a link for DJ-1 with parkin or PINK1. The aim of this review is to summarise the functions of these three proteins, and to analyse the existing evidence for direct and indirect interactions between them. DJ-1 is able to rescue the phenotype of PINK1-knockout Drosophila models, but not of parkin-knockouts, suggesting that DJ-1 may act in a parallel pathway to that of the PINK1/parkin pathway. To further elucidate a commonality between these three proteins, bioinformatics analysis established that Miro (RHOT1) interacts with parkin and PINK1, and HSPA4 interacts with all three proteins. Furthermore, 30 transcription factors were found to be common amongst all three proteins, with many of them being involved in transcriptional regulation. Interestingly, expression of these proteins and their associated transcription factors are found to be significantly down-regulated in PD patients compared to healthy controls. In summary, this review provides insight into common pathways linking three PD-causing genes and highlights some key questions, the answers to which may provide critical insight into the disease process.

Mitochondrial impairment observed in fibroblasts from South African Parkinson's disease patients with parkin mutations.

Parkinson's disease (PD), defined as a neurodegenerative disorder, is characterized by the loss of dopaminergic neurons in the substantia nigra in the midbrain. Loss-of-function mutations in the parkin gene are a major cause of autosomal recessive, early-onset PD. Parkin has been implicated in the maintenance of healthy mitochondria, although previous studies show conflicting findings regarding mitochondrial abnormalities in fibroblasts from patients harboring parkin-null mutations. The aim of the present study was to determine whether South African PD patients with parkin mutations exhibit evidence for mitochondrial dysfunction. Fibroblasts were cultured from skin biopsies obtained from three patients with homozygous parkin-null mutations, two heterozygous mutation carriers and two wild-type controls. Muscle biopsies were obtained from two of the patients. The muscle fibers showed subtle abnormalities such as slightly swollen mitochondria in focal areas of the fibers and some folding of the sarcolemma. Although no differences in the degree of mitochondrial network branching were found in the fibroblasts, ultrastructural abnormalities were observed including the presence of electron-dense vacuoles. Moreover, decreased ATP levels which are consistent with mitochondrial dysfunction were observed in the patients' fibroblasts compared to controls. Remarkably, these defects did not manifest in one patient, which may be due to possible compensatory mechanisms. These results suggest that parkin-null patients exhibit features of mitochondrial dysfunction. Involvement of mitochondria as a key role player in PD pathogenesis will have important implications for the design of new and more effective therapies.

Mitochondrial catastrophe during doxorubicin-induced cardiotoxicity: a review of the protective role of melatonin.

Anthracyclines, such as doxorubicin, are among the most valuable treatments for various cancers, but their clinical use is limited due to detrimental side effects such as cardiotoxicity. Doxorubicin-induced cardiotoxicity is emerging as a critical issue among cancer survivors and is an area of much significance to the field of cardio-oncology. Abnormalities in mitochondrial functions such as defects in the respiratory chain, decreased adenosine triphosphate production, mitochondrial DNA damage, modulation of mitochondrial sirtuin activity and free radical formation have all been suggested as the primary causative factors in the pathogenesis of doxorubicin-induced cardiotoxicity. Melatonin is a potent antioxidant, is nontoxic, and has been shown to influence mitochondrial homeostasis and function. Although a number of studies support the mitochondrial protective role of melatonin, the exact mechanisms by which melatonin confers mitochondrial protection in the context of doxorubicin-induced cardiotoxicity remain to be elucidated. This review focuses on the role of melatonin on doxorubicin-induced bioenergetic failure, free radical generation, and cell death. A further aim is to highlight other mitochondrial parameters such as mitophagy, autophagy, mitochondrial fission and fusion, and mitochondrial sirtuin activity, which lack evidence to support the role of melatonin in the context of cardiotoxicity.

5-ALA localises to the autophagy compartment and increases its fluorescence upon autophagy enhancement through caloric restriction and spermidine treatment in human glioblastoma.

Glioblastoma Multiforme (GBM) is the most invasive and prevalent Central Nervous System (CNS) malignancy. It is characterised by diffuse infiltrative growth and metabolic dysregulation that impairs the extent of surgical resection (EoR), contributing to its poor prognosis. 5-Aminolevulinic acid (5-ALA) fluorescence-guided surgical resection (FGR) takes advantage of the preferential generation of 5-ALA-derived fluorescence signal in glioma cells, thereby improving visualisation and enhancing the EoR. However, despite 5-ALA FGR is a widely used technique in the surgical management of malignant gliomas, the infiltrative tumour margins usually show only vague or no visible fluorescence and thus a significant amount of residual tumour tissue may hence remain in the resection cavity, subsequently driving tumour recurrence. To investigate the molecular mechanisms that govern the preferential accumulation of 5-ALA in glioma cells, we investigated the precise subcellular localisation of 5-ALA signal using Correlative Light and Electron Microscopy (CLEM) and colocalisation analyses in U118MG glioma cells. Our results revealed strong 5-ALA signal localisation in the autophagy compartment - specifically autolysosomes and lysosomes. Flow cytometry was employed to investigate whether autophagy enhancement through spermidine treatment (SPD) or nutrient deprivation/caloric restriction (CR) would enhance 5-ALA fluorescence signal generation. Indeed, SPD, CR and a combination of SPD/CR treatment significantly increased 5-ALA signal intensity, with a most robust increase in signal intensity observed in the combination treatment of SPD/CR. When using 3-D glioma spheroids to assess the effect of 5-ALA on cellular ultrastructure, we demonstrate that 5-ALA exposure leads to cytoplasmic disruption, vacuolarisation and large-scale mitophagy induction. These findings not only suggest a critical role for the autophagy compartment in 5-ALA engagement and signal generation but also point towards a novel and practically feasible approach to enhance 5-ALA fluorescence signal intensity. The findings may highlight that indeed autophagy control may serve as a promising avenue to promote an improved resection and GBM prognosis.

Cogs in the autophagic machine-equipped to combat dementia-prone neurodegenerative diseases.

Neurodegenerative diseases are often characterized by hydrophobic inclusion bodies, and it may be the case that the aggregate-prone proteins that comprise these inclusion bodies are in fact the cause of neurotoxicity. Indeed, the appearance of protein aggregates leads to a proteostatic imbalance that causes various interruptions in physiological cellular processes, including lysosomal and mitochondrial dysfunction, as well as break down in calcium homeostasis. Oftentimes the approach to counteract proteotoxicity is taken to merely upregulate autophagy, measured by an increase in autophagosomes, without a deeper assessment of contributors toward effective turnover through autophagy. There are various ways in which autophagy is regulated ranging from the mammalian target of rapamycin (mTOR) to acetylation status of proteins. Healthy mitochondria and the intracellular energetic charge they preserve are key for the acidification status of lysosomes and thus ensuring effective clearance of components through the autophagy pathway. Both mitochondria and lysosomes have been shown to bear functional protein complexes that aid in the regulation of autophagy. Indeed, it may be the case that minimizing the proteins associated with the respective neurodegenerative pathology may be of greater importance than addressing molecularly their resulting inclusion bodies. It is in this context that this review will dissect the autophagy signaling pathway, its control and the manner in which it is molecularly and functionally connected with the mitochondrial and lysosomal system, as well as provide a summary of the role of autophagy dysfunction in driving neurodegenerative disease as a means to better position the potential of rapamycin-mediated bioactivities to control autophagy favorably.

The Highs and Lows of Memantine-An Autophagy and Mitophagy Inducing Agent That Protects Mitochondria.

Memantine is an FDA-approved, non-competitive NMDA-receptor antagonist that has been shown to have mitochondrial protective effects, improve cell viability and enhance clearance of Aß42 peptide. Currently, there are uncertainties regarding the precise molecular targets as well as the most favourable treatment concentrations of memantine. Here, we made use of an imaging-based approach to investigate the concentration-dependent effects of memantine on mitochondrial fission and fusion dynamics, autophagy and mitochondrial quality control using a neuronal model of CCCP-induced mitochondrial injury so as to better unpack how memantine aids in promoting neuronal health. GT1-7 murine hypothalamic cells were cultured under standard conditions, treated with a relatively high and low concentration (100 µM and 50 µM) of memantine for 48 h. Images were acquired using a Zeiss 780 PS1 platform. Utilising the mitochondrial event localiser (MEL), we demonstrated clear concentration-dependent effects of memantine causing a protective response to mitochondrial injury. Both concentrations maintained the mitochondrial network volume whilst the low concentration caused an increase in mitochondrial number as well as increased fission and fusion events following CCCP-induced injury. Additionally, we made use of a customised Python-based image processing and analysis pipeline to quantitatively assess memantine-dependent changes in the autophagosomal and lysosomal compartments. Our results revealed that memantine elicits a differential, concentration-dependent effect on autophagy pathway intermediates. Intriguingly, low but not high concentrations of memantine lead to the induction of mitophagy. Taken together, our findings have shown that memantine is able to protect the mitochondrial network by preserving its volume upon mitochondrial injury with high concentrations of memantine inducing macroautophagy, whereas low concentrations lead to the induction of mitophagy.