Carmen Aguado

Laforin, the most common protein mutated in Lafora disease, regulates autophagy

Lafora disease (LD) is an autosomal recessive, progressive myoclonus epilepsy, which is characterized by the accumulation of polyglucosan inclusion bodies, called Lafora bodies, in the cytoplasm of cells in the central nervous system and in many other organs. However, it is unclear at the moment whether Lafora bodies are the cause of the disease, or whether they are secondary consequences of a primary metabolic alteration. Here we describe that the major genetic lesion that causes LD, loss-of-function of the protein laforin, impairs autophagy. This phenomenon is confirmed in cell lines from human patients, mouse embryonic fibroblasts from laforin knockout mice and in tissues from such mice. Conversely, laforin expression stimulates autophagy. Laforin regulates autophagy via the mammalian target of rapamycin kinase-dependent pathway. The changes in autophagy mediated by laforin regulate the accumulation of diverse autophagy substrates and would be predicted to impact on the Lafora body accumulation and the cell stress seen in this disease that may eventually contribute to cell death.

Lafora bodies and neurological defects in malin-deficient mice correlate with impaired autophagy

Lafora disease (LD), a fatal neurodegenerative disorder characterized by the presence of intracellular inclusions called Lafora bodies (LBs), is caused by loss-of-function mutations in laforin or malin. Previous studies suggested a role of these proteins in the regulation of glycogen biosynthesis, in glycogen dephosphorylation and in the modulation of the intracellular proteolytic systems. However, the contribution of each of these processes to LD pathogenesis is unclear. We have generated a malin-deficient (Epm2b-/-) mouse with a phenotype similar to that of LD patients. By 3-6 months of age, Epm2b-/- mice present neurological and behavioral abnormalities that correlate with a massive presence of LBs in the cortex, hippocampus and cerebellum. Sixteen-day-old Epm2b-/- mice, without detectable LBs, show an impairment of macroautophagy (hereafter called autophagy), which remains compromised in adult animals. These data demonstrate similarities between the Epm2a-/- and Epm2b-/- mice that provide further insights into LD pathogenesis. They illustrate that the dysfunction of autophagy is a consequence of the lack of laforin-malin complexes and a common feature of both mouse models of LD. Because this dysfunction precedes other pathological manifestations, we propose that decreased autophagy plays a primary role in the formation of LBs and it is critical in LD pathogenesis.

Intracellular protein degradation in mammalian cells: recent developments.

In higher organisms, dietary proteins are broken down into amino acids within the digestive tract but outside the cells, which incorporate the resulting amino acids into their metabolism. However, under certain conditions, an organism loses more nitrogen than is assimilated in the diet. This additional loss was found in the past century to come from intracellular proteins and started an intensive research that produced an enormous expansion of the field and a dispersed literature. Therefore, our purpose is to provide an updated summary of the current knowledge on the proteolytic machinery involved in intracellular protein degradation and its physiological and pathological relevance, especially addressed to newcomers in the field who may find further details in more specialized reviews. However, even providing a general overview, this is an extremely wide field and, therefore, we mainly focus on mammalian cells, while other cells will be mentioned only for comparison purposes.

Role of AMP-activated protein kinase in autophagy and proteasome function

In this work, we have examined the possible role of AMP-activated protein kinase (a key energy sensor) in regulating intracellular protein degradation. We have found that AICAR, a known activator of AMPK, has a dual effect. On one hand, it inhibits autophagy by a mechanism independent of AMPK activity; AICAR decreases class III PI3-kinase binding to beclin-1 and this effect counteracts and reverses the known positive effect of AMPK activity on autophagy. On the other hand, AICAR inhibits the proteasomal degradation of proteins by an AMPK-dependent mechanism. This is a novel function of AMPK that allows the regulation of proteasomal activity under conditions of energy demand.

Dynamics of an F-actin aggresome generated by the actin-stabilizing toxin jasplakinolide

In this study, we report the formation of several cytoplasmic inclusion bodies composed of filamentous actin (F-actin) and generated by experimental treatments using depolymerizing or stabilizing actin toxins in neuronal and non-neuronal mammalian cell lines. The actin-stabilizing toxin jasplakinolide (Jpk) induced, in a microtubule-dependent manner, a single, large F-actin aggregate, which contained β- and γ-actin, ADF/cofilin, cortactin, and the actin nucleator Arp2/3. This aggregate was tightly associated with the Golgi complex and mitochondria, and was surrounded by vimentin intermediate filaments, microtubules and MAP4. Therefore, the Jpk-induced single, large F-actin aggregate fits the established criteria for being considered an aggresome. Lysosomes and/or autophagic vacuoles, proteasomes and microtubules were found to directly participate in the dissolution of this F-actin aggresome. Finally, the model reported here is simple, highly reproducible and reversible, and it provides an opportunity to test pharmacological agents that interfere with the formation, maintenance and/or disappearance of F-actin-enriched pathological inclusion bodies.

Alterations in ROS Activity and Lysosomal pH Account for Distinct Patterns of Macroautophagy in LINCL and JNCL Fibroblasts

Neuronal Ceroid Lipofuscinoses (NCL) are lysosomal storage disorders characterized by the accumulation of lipofuscin within lysosomes. Late infantile (LINCL) and juvenile (JNCL) are their most common forms and are caused by loss-of-function mutations in tripeptidyl peptidase 1 (TPP1), a lysosomal endopeptidase, and CLN3 protein (CLN3p), whose location and function is still controversial. LINCL patients suffer more severely from NCL consequences than JNCL patients, in spite of having in common an abnormal accumulation of material with a similar composition in the lysosomes. To identify distinctive characteristics that could explain the differences in the severity of LINCL and JNCL pathologies, we compared the protein degradation mechanisms in patientś fibroblasts. Pulse-chase experiments show a significant decrease in protein degradation by macroautophagy in fibroblasts bearing TPP1 (CLN2) and CLN3p (CLN3) mutations. In CLN2 fibroblasts, LC3-II levels and other procedures indicate an impaired formation of autophagosomes, which confirms the pulse-chase experiments. This defect is linked to an accumulation of Reactive Oxygen Species (ROS), an upregulation of the Akt-mTOR signalling pathway and increased activities of the p38α and ERK1/2 MAPKs. In CLN3 fibroblasts, LC3-II analysis indicates impairment in autophagosome maturation and there is also a defect in fluid phase endocytosis, two alterations that can be related to an observed increase of 0.5 units in lysosomal pH. CLN3 fibroblasts also accumulate ROS but to a lower extent than CLN2. TPP1 activity is completely abrogated in CLN2 and partially diminished in CLN3 fibroblasts. TPP1 cleaves small hydrophobic proteins like subunit c of mitochondrial ATP synthase and the lack or a lower activity of this enzyme can contribute to lipofuscin accumulation. These alterations in TPP1 activity lead to an increased ROS production, especially in CLN2 in which it is aggravated by a decrease in catalase activity. This could explain the earlier appearance of the symptoms in the LINCL form.

Annexin A5 stimulates autophagy and inhibits endocytosis

Macroautophagy is a major lysosomal catabolic process activated particularly under starvation in eukaryotic cells. A new organelle, the autophagosome, engulfs cytoplasmic substrates, which are degraded after fusion with endosomes and/or lysosomes. During a shotgun proteome analysis of purified lysosomal membranes from mouse fibroblasts, a Ca2+-dependent phospholipid-binding protein, annexin A5, was found to increase on lysosomal membranes under starvation. This suggests a role for this protein, an abundant annexin with a still unknown intracellular function, in starvation-induced lysosomal degradation. Transient overexpression and silencing experiments showed that annexin A5 increased lysosomal protein degradation, and colocalisation experiments, based on GFP sensitivity to lysosomal acidic pH, indicated that this was mainly the result of inducing autophagosome–lysosome fusion. Annexin A5 also inhibited the endocytosis of a fluid-phase marker and cholera toxin, but not receptor-mediated endocytosis. Therefore, we propose a double and opposite role of annexin A5 in regulating the endocytic and autophagic pathways and the fusion of autophagosomes with lysosomes and endosomes.

Characterization of Human GTPBP3, a GTP-Binding Protein Involved in Mitochondrial tRNA Modification

ABSTRACT Human GTPBP3 is an evolutionarily conserved, multidomain protein involved in mitochondrial tRNA modification. Characterization of its biochemical properties and the phenotype conferred by GTPBP3 inactivation is crucial to understanding the role of this protein in tRNA maturation and its effects on mitochondrial respiration. We show that the two most abundant GTPBP3 isoforms exhibit moderate affinity for guanine nucleotides like their bacterial homologue, MnmE, although they hydrolyze GTP at a 100-fold lower rate. This suggests that regulation of the GTPase activity, essential for the tRNA modification function of MnmE, is different in GTPBP3. In fact, potassium-induced dimerization of the G domain leads to stimulation of the GTPase activity in MnmE but not in GTPBP3. The GTPBP3 N-terminal domain mediates a potassium-independent dimerization, which appears as an evolutionarily conserved property of the protein family, probably related to the construction of the binding site for the one-carbon-unit donor in the modification reaction. Partial inactivation of GTPBP3 by small interfering RNA reduces oxygen consumption, ATP production, and mitochondrial protein synthesis, while the degradation of these proteins slightly increases. It also results in mitochondria with defective membrane potential and increased superoxide levels. These phenotypic traits suggest that GTPBP3 defects contribute to the pathogenesis of some oxidative phosphorylation diseases.

PTEN Increases Autophagy and Inhibits the Ubiquitin-Proteasome Pathway in Glioma Cells Independently of its Lipid Phosphatase Activity

Two major mechanisms of intracellular protein degradation, autophagy and the ubiquitin-proteasome pathway, operate in mammalian cells. PTEN, which is frequently mutated in glioblastomas, is a tumor suppressor gene that encodes a dual specificity phosphatase that antagonizes the phosphatidylinositol 3-kinase class I/AKT/mTOR pathway, which is a key regulator of autophagy. Here, we investigated in U87MG human glioma cells the role of PTEN in the regulation of autophagy and the ubiquitin-proteasome pathway, because both are functionally linked and are relevant in cancer progression. Since U87MG glioma cells lack a functional PTEN, we used stable clones that express, under the control of a tetracycline-inducible system (Tet-on), wild-type PTEN and two of its mutants, G129E-PTEN and C124S-PTEN, which, respectively, lack the lipid phosphatase activity only and both the lipid and the protein phosphatase activities of this protein. Expression of PTEN in U87MG glioma cells decreased proteasome activity and also reduced protein ubiquitination. On the contrary, expression of PTEN increased the autophagic flux and the lysosomal mass. Interestingly, and although PTEN negatively regulates the phosphatidylinositol 3-kinase class I/AKT/mTOR signaling pathway by its lipid phosphatase activity, both effects in U87MG cells were independent of this activity. These results suggest a new mTOR-independent signaling pathway by which PTEN can regulate in opposite directions the main mechanisms of intracellular protein degradation.

Impaired autophagy in Lafora disease

Lafora disease (LD) is a progressive, lethal, autosomal recessive, neurodegenerative disorder that manifests with myoclonus epilepsy. LD is characterized by the presence of intracellular inclusion bodies called Lafora bodies (LB), in brain, spinal cord and other tissues. More than 50% of LD is caused by mutations in EPM2A that encodes laforin. Here we review our recent findings that revealed that laforin regulates autophagy. We consider how autophagy compromise may predispose to LB formation and neurodegeneration in LD, and discuss future investigations suggested by our data.