Exploiting Drosophila as a model system for studying REEP1-linked HSP in vivo

Hereditary Spastic Paraplegia (HSP) is a genetic group of neurodegenerative disorders characterized by progressive degeneration of corticospinal tracts. Mutations in the SPG31 gene, encoding REEP1, are the third most common cause of autosomal dominant form of HSP. Recent studies have reported that REEP1, an integral ER membrane protein, interacts with the microtubule cytoskeleton to coordinate ER shaping. However it precise molecular function is still unknown. To better understand the function of REEP1, we generated a model (Drosophila melanogaster) for the in vivo analysis of the fly REEP1 homolog (D-REEP1). Drosophila and human REEP1 proteins display remarkable homology and conservation of domain organization. We analyzed D-REEP1 loss of function and gain of function transgenic lines as well as animals expressing pathological forms of the protein. Our in vivo data in Drosophila have shown a strong involvement of D-REEP1 in the regulation of lipid droplets (LDs) number and size in neuronal and non neuronal tissues. Loss of D-REEP1 results in larvae leaner and smaller than their wild type counterparts while endoplasmic reticulum membranes are elongated when compared to controls. These ER defects are associated with a decrease in lipid droplets number and low triglycerides content. On the contrary over expression of wild type D-REEP1 produces a reduction in the size of lipid droplets. The lack of animal models available for REEP1 studies and experimental data concerning the functional alteration caused by pathological mutations of REEP1 prompted to generate transgenic lines carrying D-REEP1 pathological mutations and to analyse the consequence of their expression in vivo. Two missense mutations (P19R, D56N) affecting the trans-membrane domains of REEP1 and a novel mutation (A132V) located in the C-terminal part of the protein have been assessed.The mutations in the trans membranes domains relocate REEP1 from the ER to the membrane of lipid droplets when expressed in mammalian cells. In vivo expression of Drosophila P19R caused oversized LDs in the brain and axons and increased levels of triacylgycerides. LDs are believed to originate from the endoplasmic reticulum, although the exact molecular mechanisms of their biogenesis is still not known. Based on the findings described above and the knowledge about REEP family, we hypothesize that REEP1 probably play an important role in membrane remodelling and possibly affects the lipid droplets metabolism. While, pathological forms of REEP1 could perturb the biogenesis and/or turnover of lipid droplets and eventually produce an imbalance in neuronal lipid metabolis

Naringenin Ameliorates Drosophila ReepA Hereditary Spastic Paraplegia-Linked Phenotypes

Defects in the endoplasmic reticulum (ER) membrane shaping and interaction with other organelles seem to be a crucial mechanism underlying Hereditary Spastic Paraplegia (HSP) neurodegeneration. REEP1, a transmembrane protein belonging to TB2/HVA22 family, is implicated in SPG31, an autosomal dominant form of HSP, and its interaction with Atlastin/SPG3A and Spastin/SPG4, the other two major HSP linked proteins, has been demonstrated to play a crucial role in modifying ER architecture. In addition, the Drosophila ortholog of REEP1, named ReepA, has been found to regulate the response to ER neuronal stress. Herein we investigated the role of ReepA in ER morphology and stress response. ReepA is upregulated under stress conditions and aging. Our data show that ReepA triggers a selective activation of Ire1 and Atf6 branches of Unfolded Protein Response (UPR) and modifies ER morphology. Drosophila lacking ReepA showed Atf6 and Ire1 activation, expansion of ER sheet-like structures, locomotor dysfunction and shortened lifespan. Furthermore, we found that naringenin, a flavonoid that possesses strong antioxidant and neuroprotective activity, can rescue the cellular phenotypes, the lifespan and locomotor disability associated with ReepA loss of function. Our data highlight the importance of ER homeostasis in nervous system functionality and HSP neurodegenerative mechanisms, opening new opportunities for HSP treatment

Proteasome dysfunction induces excessive proteome instability and loss of mitostasis that can be mitigated by enhancing mitochondrial fusion or autophagy

The ubiquitin-proteasome pathway (UPP) is central to proteostasis network (PN) functionality and proteome quality control. Yet, the functional implication of the UPP in tissue homeodynamics at the whole organism level and its potential cross-talk with other proteostatic or mitostatic modules are not well understood. We show here that knock down (KD) of proteasome subunits in Drosophila flies, induced, for most subunits, developmental lethality. Ubiquitous or tissue specific proteasome dysfunction triggered systemic proteome instability and activation of PN modules, including macroautophagy/autophagy, molecular chaperones and the antioxidant cncC (the fly ortholog of NFE2L2/Nrf2) pathway. Also, proteasome KD increased genomic instability, altered metabolic pathways and severely disrupted mitochondrial functionality, triggering a cncC-dependent upregulation of mitostatic genes and enhanced rates of mitophagy. Whereas, overexpression of key regulators of antioxidant responses (e.g., cncC or foxo) could not suppress the deleterious effects of proteasome dysfunction; these were alleviated in both larvae and adult flies by modulating mitochondrial dynamics towards increased fusion or by enhancing autophagy. Our findings reveal the extensive functional wiring of genomic, proteostatic and mitostatic modules in higher metazoans. Also, they support the notion that age-related increase of proteotoxic stress due to decreased UPP activity deregulates all aspects of cellular functionality being thus a driving force for most age-related diseases. Abbreviations: ALP: autophagy-lysosome pathway; ARE: antioxidant response element; Atg8a: autophagy-related 8a; ATPsynβ: ATP synthase, β subunit; C-L: caspase-like proteasomal activity; cncC: cap-n-collar isoform-C; CT-L: chymotrypsin-like proteasomal activity; Drp1: dynamin related protein 1; ER: endoplasmic reticulum; foxo: forkhead box, sub-group O; GLU: glucose; GFP: green fluorescent protein; GLY: glycogen; Hsf: heat shock factor; Hsp: Heat shock protein; Keap1: kelch-like ECH-associated protein 1; Marf: mitochondrial assembly regulatory factor; NFE2L2/Nrf2: nuclear factor, erythroid 2 like 2; Opa1: optic atrophy 1; PN: proteostasis network; RNAi: RNA interference; ROS: reactive oxygen species; ref(2)P: refractory to sigma P; SQSTM1: sequestosome 1; SdhA: succinate dehydrogenase, subunit A; T-L: trypsin-like proteasomal activity; TREH: trehalose; UAS: upstream activation sequence; Ub: ubiquitin; UPR: unfolded protein response; UPP: ubiquitin-proteasome pathway.</p

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Exploiting Drosophila as a model system for studying REEP1-linked HSP in vivo

Hereditary Spastic Paraplegia (HSP) is a genetic group of neurodegenerative disorders characterized by progressive degeneration of corticospinal tracts. Mutations in the SPG31 gene, encoding REEP1, are the third most common cause of autosomal dominant form of HSP. Recent studies have reported that REEP1, an integral ER membrane protein, interacts with the microtubule cytoskeleton to coordinate ER shaping. However it precise molecular function is still unknown. To better understand the function of REEP1, we generated a model (Drosophila melanogaster) for the in vivo analysis of the fly REEP1 homolog (D-REEP1). Drosophila and human REEP1 proteins display remarkable homology and conservation of domain organization. We analyzed D-REEP1 loss of function and gain of function transgenic lines as well as animals expressing pathological forms of the protein. Our in vivo data in Drosophila have shown a strong involvement of D-REEP1 in the regulation of lipid droplets (LDs) number and size in neuronal and non neuronal tissues. Loss of D-REEP1 results in larvae leaner and smaller than their wild type counterparts while endoplasmic reticulum membranes are elongated when compared to controls. These ER defects are associated with a decrease in lipid droplets number and low triglycerides content. On the contrary over expression of wild type D-REEP1 produces a reduction in the size of lipid droplets. The lack of animal models available for REEP1 studies and experimental data concerning the functional alteration caused by pathological mutations of REEP1 prompted to generate transgenic lines carrying D-REEP1 pathological mutations and to analyse the consequence of their expression in vivo. Two missense mutations (P19R, D56N) affecting the trans-membrane domains of REEP1 and a novel mutation (A132V) located in the C-terminal part of the protein have been assessed.The mutations in the trans membranes domains relocate REEP1 from the ER to the membrane of lipid droplets when expressed in mammalian cells. In vivo expression of Drosophila P19R caused oversized LDs in the brain and axons and increased levels of triacylgycerides. LDs are believed to originate from the endoplasmic reticulum, although the exact molecular mechanisms of their biogenesis is still not known. Based on the findings described above and the knowledge about REEP family, we hypothesize that REEP1 probably play an important role in membrane remodelling and possibly affects the lipid droplets metabolism. While, pathological forms of REEP1 could perturb the biogenesis and/or turnover of lipid droplets and eventually produce an imbalance in neuronal lipid metabolismLe Paraplegie Spastiche Ereditarie (HSP) sono un gruppo eterogeneo di malattie neurodegenerative, caratterizzate da progressiva spasticità degli arti inferiori, e degenerazione del tratto corticospinale. Mutazioni a carico del gene SPG31, codificante per la proteina REEP1, sono la terza causa più comune di forme dominanti di HSP. Studi recenti suggeriscono che REEP1, una proteina integrale della membrana del reticolo endoplasmatico (ER), sia coinvolto nel rimodellamento delle membrane del ER attraverso l’interazione con i microtubuli del citoscheletro. Tuttavia la precisa funzione biologica e il meccanismo patologica di questa proteina sono ancora sconosciuti. Questa tesi ha come oggetto lo studio in vivo della funzione di REEP1 utilizzando come organismo modello Drosophila melanogaster. A tale scopo abbiamo identificato l’omologo in Drosophila di REEP1 (D-REEP1) e generato delle linee transgeniche per la modulazione dell’espressione genica in vivo sia della proteina wild type sia di alcune sue varianti patologiche. Analisi in vivo suggeriscono che D-REEP1 sia coinvolto nella regolazione del numero e della dimensione dei lipid droplets (LDs) in tessuti neuronali e non neuronali. L’assenza di D-REEP1 causa una riduzione delle dimensioni larvali e ad un allungamento delle membrane del reticolo endoplasmatico. Le alterazioni morfologiche del reticolo endoplasmatico sono associate ad una diminuzione del numero totale dei LDs e alla riduzione del contenuto dei trigliceridi. Al contrariola sovra-espressione di D-REEP1 in vivo induce una riduzione delle dimensioni dei LDs La mancanza di studi su organismi modelli e dati sperimentali per valutare le possibili alterazioni funzionali causate delle mutazioni patologiche di D-REEP1, ha portato a creare delle linee transgeniche di Drosophila per forme mutate di D-REEP1. In tal modo si è voluto valutare gli effetti, sia in vivo, che in vitro, di due mutazioni missenso (P19R, D56N) localizzate nei domini transmembrana ed una mutazione nuova (A132V), non ancora pubblicata, localizzata nella parte C-terminale di D-REEP1. Le analisi in vitro hanno dimostrato che le mutazioni situate nei domini transmembrana determinano una alterata localizzazione subcellulare di REEP1. Inoltre, la sovrespressione in vivo di D-REEP1-P19R determina un aumento delle dimensioni dei LDs nel sistema nervoso di Drosophila. Seppure si ritiene che la biogenesi dei lipidi avviene a livello del reticolo endoplasmatico, appare tuttora sconosciuto l’esatto meccanismo molecolare coinvolto. I dati da noi ottenuti e le conoscenze attuali riguardo la famiglia delle proteine REEP suggeriscono che, agendo sulla curvatura delle membrane del ER o reclutando particolari proteine dei LDs, REEP1 sia probabilmente importante nella generazione dei lipid droplets con possibili effetti sul metabolismo lipidic

Hereditary Spastic Paraplegia and Future Therapeutic Directions: Beneficial Effects of Small Compounds Acting on Cellular Stress

Hereditary spastic paraplegia (HSP) is a group of inherited neurodegenerative conditions that share a characteristic feature of degeneration of the longest axons within the corticospinal tract, which leads to progressive spasticity and weakness of the lower limbs. Mutations of over 70 genes produce defects in various biological pathways: axonal transport, lipid metabolism, endoplasmic reticulum (ER) shaping, mitochondrial function, and endosomal trafficking. HSPs suffer from an adequate therapeutic plan. Currently the treatments foreseen for patients affected by this pathology are physiotherapy, to maintain the outgoing tone, and muscle relaxant therapies for spasticity. Very few clinical studies have been conducted, and it's urgent to implement preclinical animal studies devoted to pharmacological test and screening, to expand the rose of compounds potentially attractive for clinical trials. Small animal models, such as Drosophila melanogaster and zebrafish, have been generated, analyzed, and used as preclinical model for screening of compounds and their effects. In this work, we briefly described the role of HSP-linked proteins in the organization of ER endomembrane system and in the regulation of ER homeostasis and stress as a common pathological mechanism for these HSP forms. We then focused our attention on the pharmacodynamic and pharmacokinetic features of some recently identified molecules with antioxidant property, such as salubrinal, guanabenz, N-acetyl cysteine, methylene blue, rapamycin, and naringenin, and on their potential use in future clinical studies. Expanding the models and the pharmacological screening for HSP disease is necessary to give an opportunity to patients and clinicians to test new molecules