The Multifaceted Interactions of Dictyostelium Atg1 with Mitochondrial Function, Endocytosis, Growth, and Development.

Autophagy is a degradative recycling process central to the maintenance of homeostasis in all eukaryotes. By ensuring the degradation of damaged mitochondria, it plays a key role in maintaining mitochondrial health and function. Of the highly conserved autophagy proteins, autophagy-related protein 1 (Atg1) is essential to the process. The involvement of these proteins in intracellular signalling pathways, including those involving mitochondrial function, are still being elucidated. Here the role of Atg1 was investigated in the simple model organism Dictyostelium discoideum using an atg1 null mutant and mutants overexpressing or antisense-inhibiting atg1. When evaluated against the well-characterised outcomes of mitochondrial dysfunction in this model, altered atg1 expression resulted in an unconventional set of phenotypic outcomes in growth, endocytosis, multicellular development, and mitochondrial homeostasis. The findings here show that Atg1 is involved in a tightly regulated signal transduction pathway coordinating energy-consuming processes such as cell growth and multicellular development, along with nutrient status and energy production. Furthermore, Atg1's effects on energy homeostasis indicate a peripheral ancillary role in the mitochondrial signalling network, with effects on energy balance rather than direct effects on electron transport chain function. Further research is required to tease out these complex networks. Nevertheless, this study adds further evidence to the theory that autophagy and mitochondrial signalling are not opposing but rather linked, yet strictly controlled, homeostatic mechanisms.

Plate-Based Assays for the Characterization of Mitochondrial and Cellular Phenotypes.

The mitochondria are essential to eukaryotic life, acting as key drivers of energy generation while also being involved in the regulation of many cellular processes including apoptosis, cell proliferation, calcium homeostasis, and metabolism. Mitochondrial diseases which disrupt these processes lead to a diverse range of pathologies and lack consistency in symptom presentation. In disease, mitochondrial activity and energy homeostasis can be adapted to cellular requirements, and studies using Dictyostelium and human lymphoblastoid cell lines have shown that such changes can be facilitated by the key cellular and energy regulators, TORC1 and AMPK. Fluorescence-based assays are increasingly utilized to measure mitochondrial and cell signalling function in mitochondrial disease research. Here, we describe a streamlined method for the simultaneous measurement of mitochondrial mass, membrane potential, and reactive oxygen species production using MitoTracker Green™ FM, MitoTracker Red™ CMXRos, and DCFH-DA probes. This protocol has been adapted for both Dictyostelium and human lymphoblastoid cell lines. We also describe a method for assessing TORC1 and AMPK activity simultaneously in lymphoblastoid cells. These techniques allow for the characterization of mitochondrial defects in a rapid and easy to implement manner.

Dysregulated Gene Expression in Lymphoblasts from Parkinson's Disease.

Parkinson's disease is the second largest neurodegenerative disease worldwide and is caused by a combination of genetics and environment. It is characterized by the death of neurons in the substantia nigra of the brain but is not solely a disease of the brain, as it affects multiple tissues and organs. Studying Parkinson's disease in accessible tissues such as skin and blood has increased our understanding of the disease's pathogenesis. Here, we used lymphoblast cell lines generated from Parkinson's disease patient and healthy age- and sex-matched control groups and obtained their whole-cell transcriptomes and proteomes. Our analysis revealed, in both the transcriptomes and the proteomes of PD cells, a global downregulation of genes involved in protein synthesis, as well as the upregulation of immune processes and sphingolipid metabolism. In contrast, we discovered an uncoupling of mRNA and protein expression in processes associated with mitochondrial respiration in the form of a general downregulation in associated transcripts and an upregulation in proteins. Complex V was different to the other oxidative phosphorylation complexes in that the levels of its associated transcripts were also lower, but the levels of their encoded polypeptides were not elevated. This may suggest that further layers of regulation specific to Complex V are in play.

Cytopathological Outcomes of Knocking down Expression of Mitochondrial Complex II Subunits in Dictyostelium discoideum.

Mitochondrial Complex II is composed of four core subunits and mutations to any of the subunits result in lowered Complex II activity. Surprisingly, although mutations in any of the subunits can yield similar clinical outcomes, there are distinct differences in the patterns of clinical disease most commonly associated with mutations in different subunits. Thus, mutations to the SdhA subunit most often result in mitochondrial disease phenotypes, whilst mutations to the other subunits SdhB-D more commonly result in tumour formation. The reason the clinical outcomes are so different is unknown. Here, we individually antisense-inhibited three of the Complex II subunits, SdhA, SdhB or SdhC, in the simple model organism Dictyostelium discoideum. Whilst SdhB and SdhC knockdown resulted in growth defects on bacterial lawns, antisense inhibition of SdhA expression resulted in a different pattern of phenotypic defects, including impairments of growth in liquid medium, enhanced intracellular proliferation of the bacterial pathogen Legionella pneumophila and phagocytosis. Knockdown of the individual subunits also produced different abnormalities in mitochondrial function with only SdhA knockdown resulting in broad mitochondrial dysfunction. Furthermore, these defects were shown to be mediated by the chronic activation of the cellular energy sensor AMP-activated protein kinase. Our results are in agreement with a role for loss of function of SdhA but not the other Complex II subunits in impairing mitochondrial oxidative phosphorylation and they suggest a role for AMP-activated protein kinase in mediating the cytopathological outcomes.

Dictyostelium discoideum: A Model System for Neurological Disorders.

BACKGROUND: The incidence of neurological disorders is increasing due to population growth and extended life expectancy. Despite advances in the understanding of these disorders, curative strategies for treatment have not yet eventuated. In part, this is due to the complexities of the disorders and a lack of identification of their specific underlying pathologies. Dictyostelium discoideum has provided a useful, simple model to aid in unraveling the complex pathological characteristics of neurological disorders including Alzheimer's disease, Parkinson's disease, Huntington's disease, neuronal ceroid lipofuscinoses and lissencephaly. In addition, D. discoideum has proven to be an innovative model for pharmaceutical research in the neurological field. SCOPE OF REVIEW: This review describes the contributions of D. discoideum in the field of neurological research. The continued exploration of proteins implicated in neurological disorders in D. discoideum may elucidate their pathological roles and fast-track curative therapeutics.

Lymphoblastoid Cell Lines as Models to Study Mitochondrial Function in Neurological Disorders.

Neurological disorders, including neurodegenerative diseases, are collectively a major cause of death and disability worldwide. Whilst the underlying disease mechanisms remain elusive, altered mitochondrial function has been clearly implicated and is a key area of study in these disorders. Studying mitochondrial function in these disorders is difficult due to the inaccessibility of brain tissue, which is the key tissue affected in these diseases. To overcome this issue, numerous cell models have been used, each providing unique benefits and limitations. Here, we focussed on the use of lymphoblastoid cell lines (LCLs) to study mitochondrial function in neurological disorders. LCLs have long been used as tools for genomic analyses, but here we described their use in functional studies specifically in regard to mitochondrial function. These models have enabled characterisation of the underlying mitochondrial defect, identification of altered signalling pathways and proteins, differences in mitochondrial function between subsets of particular disorders and identification of biomarkers of the disease. The examples provided here suggest that these cells will be useful for development of diagnostic tests (which in most cases do not exist), identification of drug targets and testing of pharmacological agents, and are a worthwhile model for studying mitochondrial function in neurological disorders.

Transcription of the Dictyostelium discoideum mitochondrial genome occurs from a single initiation site.

Transcription of the mitochondrial genome in Dictyostelium discoideum gives rise to eight major polycistronic RNA species that can be detected by Northern hybridization. In order to determine whether these transcripts could possibly derive from processing of even larger transcripts, reverse transcriptase polymerase chain reactions (RT-PCRs) were performed in an attempt to amplify the intervening regions between the eight major transcripts. All but one intervening region were successfully reverse transcribed and amplified, indicating that even larger transcripts existed and that the eight major transcripts detected previously may be the products of transcript processing. Southern hybridization analyses of DNA fragments representing the sequences between the eight major transcripts with in vitro capped mitochondrial RNA identified the 5' end of only one of the eight major transcripts as a genuine transcription start site. The ability to initiate transcription from DNA sequences upstream of the identified transcription initiation site was demonstrated in bacterial cells expressing the Dictyostelium mitochondrial RNA polymerase. We conclude that transcription of the Dictyostelium mitochondrial genome is initiated at a single site, generating a large polycistronic transcript that is very efficiently, probably cotranscriptionally processed into mature RNA species. This is the first report on a protist mitochondrial DNA that is, although much larger in size than its metazoan counterparts, transcribed from a single transcription initiation site.