Defending stressed mitochondria: uncovering the role of MUL1 in suppressing neuronal mitophagy.

Chronic mitochondrial stress is associated with major neurodegenerative diseases; and thus, the recovery of those mitochondria constitutes a critical step of energy maintenance in early stages of neurodegeneration. Our recent study provides the first lines of evidence showing that the MUL1-MFN2 pathway acts as an early checkpoint to maintain mitochondrial integrity by regulating mitochondrial morphology and interplay with the endoplasmic reticulum (ER). This mechanism ensures that degradation through mitophagy is restrained in neurons under early stress conditions. MUL1 deficiency increases MFN2 activity, triggering the first phase of mitochondrial hyperfusion and acting as an antagonist of ER-mitochondria (ER-Mito) tethering. Reduced ER-Mito interplay enhances the cytoplasmic Ca2+ load that induces the DNM1L/Drp1-dependent second phase of mitochondrial fragmentation and mitophagy. Our study provides new mechanistic insights into neuronal mitochondrial maintenance under stress conditions. Identifying this pathway is particularly relevant because chronic mitochondrial dysfunction and altered ER-Mito contacts have been reported in major neurodegenerative diseases.

Mul1 restrains Parkin-mediated mitophagy in mature neurons by maintaining ER-mitochondrial contacts.

Chronic mitochondrial stress associates with major neurodegenerative diseases. Recovering stressed mitochondria constitutes a critical step of mitochondrial quality control and thus energy maintenance in early stages of neurodegeneration. Here, we reveal Mul1-Mfn2 pathway that maintains neuronal mitochondrial integrity under stress conditions. Mul1 deficiency increases Mfn2 activity that triggers the first phasic mitochondrial hyperfusion and also acts as an ER-Mito tethering antagonist. Reduced ER-Mito coupling leads to increased cytoplasmic Ca2+ load that activates calcineurin and induces the second phasic Drp1-dependent mitochondrial fragmentation and mitophagy. Overexpressing Mfn2, but not Mfn1, mimics Mul1-deficient phenotypes, while expressing PTPIP51, an ER-Mito anchoring protein, suppresses Parkin-mediated mitophagy. Thus, by regulating mitochondrial morphology and ER-Mito contacts, Mul1-Mfn2 pathway plays an early checkpoint role in maintaining mitochondrial integrity. Our study provides new mechanistic insights into neuronal mitochondrial maintenance under stress conditions, which is relevant to several major neurodegenerative diseases associated with mitochondrial dysfunction and altered ER-Mito interplay.

MUL1 acts in parallel to the PINK1/parkin pathway in regulating mitofusin and compensates for loss of PINK1/parkin.

Parkinson's disease (PD) genes PINK1 and parkin act in a common pathway that regulates mitochondrial integrity and quality. Identifying new suppressors of the pathway is important for finding new therapeutic strategies. In this study, we show that MUL1 suppresses PINK1 or parkin mutant phenotypes in Drosophila. The suppression is achieved through the ubiquitin-dependent degradation of Mitofusin, which itself causes PINK1/parkin mutant-like toxicity when overexpressed. We further show that removing MUL1 in PINK1 or parkin loss-of-function mutant aggravates phenotypes caused by loss of either gene alone, leading to lethality in flies and degeneration in mouse cortical neurons. Together, these observations show that MUL1 acts in parallel to the PINK1/parkin pathway on a shared target mitofusin to maintain mitochondrial integrity. The MUL1 pathway compensates for loss of PINK1/parkin in both Drosophila and mammals and is a promising therapeutic target for PD.DOI: http://dx.doi.org/10.7554/eLife.01958.001.

Autophagy defects in Lafora disease: cause or consequence?

Lafora disease (LD) is an inherited and fatal form of neurodegenerative disorder characterized by the presence of an abnormal form of glycogen inclusions, called Lafora bodies, in neurons and other tissues. While Lafora bodies have been thought to underlie the neuropathology in LD, the specific process by which these inclusions might affect the neuronal functions was not very well understood. Here we review one of our recent studies on the LD animal model, wherein we have shown that the Lafora bodies might contribute to the impairment in the endosomal-lysosomal and autophagy pathways.

Dysfunctions in endosomal-lysosomal and autophagy pathways underlie neuropathology in a mouse model for Lafora disease.

Lafora progressive myoclonus epilepsy (also known as Lafora disease, LD) is an inherited and fatal form of a neurodegenerative disorder characterized by the presence of carbohydrate-rich inclusions called Lafora bodies. LD can be caused by defects in the laforin phosphatase or the malin ubiquitin ligase and the clinical symptoms resulting from these two defects are almost similar. In order to understand the molecular basis of LD pathogenesis and the role of Lafora bodies in neuropathology, we have studied the laforin-deficient mice as a model and show here that Lafora bodies recruit proteasomal subunit, endoplasmic reticulum chaperone GRP78/Bip, autophagic protein p62 and endosomal regulators Rab5 and Rab7. The laforin-deficient brain also reveals the proliferation of enlarged lysosomes, lipofuscin granules, amyloid-ß peptides and increased levels of insoluble form of ubiquitinated protein, indicating a significant impairment in the cellular degradative pathway. Further, abnormal dendrites and increased gliosis, especially at the vicinity of Lafora bodies, were noted in the LD brain. Taken together, our study suggests that the neuropathology in LD is not limited to Lafora bodies, that some of the neuropathological changes in LD are likely to be secondary effects caused by Lafora bodies, and that impairment in the autophagy-endosomal-lysosomal pathways might underlie some of the symptoms in LD.

Increased glucose concentration results in reduced proteasomal activity and the formation of glycogen positive aggresomal structures.

Recent studies indicate that glycogen, besides being a principal storage product, confers protection against cellular stress through an unknown physiological pathway. Abnormal glycogen inclusions have also been considered to underlie pathology in a few neurodegenerative disorders that are caused by proteolytic dysfunctions, although a link between proteolytic pathways and glycogen accumulation is yet to be established. In the present study, we investigated the subcellular localization of glycogen particles and report that their distribution is altered under physiological stress. Using a cellular model, we show that glycogen particles are recruited to the centrosomal aggresomal structures upon proteasomal or lysosomal blockade, and that this recruitment is dependent on the microtubule function. We also show that an increase in the glucose concentration leads to decreased cellular proteasomal activity and the formation of glycogen positive aggresomal structures. Proteasomal blockade also leads to the formation of diastase-resistant polyglucosan bodies. The glycogen particles in aggresomes might provide energy to the proteolytic process and/or function as a scaffold. Taken together, the findings of the present study suggest a functional link between proteasomal function and polyglucosan bodies, and also suggest that these two physiological processes could be linked in neurodegenerative disorders.

Laforin in autophagy: a possible link between carbohydrate and protein in Lafora disease?

The progressive myoclonus epilepsy of Lafora disease (LD) is a fatal form of neurodegenerative disorder associated with progressive intellectual decline and ataxia in addition to epilepsy. The disease can be caused by defects in the EPM2A gene encoding laforin phosphatase or the NHLRC1 gene encoding malin ubiquitin ligase. Laforin and malin function together as a complex in the ubiquitin-proteasome system, and hence defects in proteolytic processes are thought to underlie some of the symptoms in LD. One of the pathological hallmarks of LD is the presence of cytoplasmic polyglucosan inclusions, the Lafora bodies. While Lafora bodies are known as a lesser branched form of glycogen with high phosphate content, a physiological basis for their genesis in the cytoplasm was not well understood. Recently it was shown in a mouse model for LD that loss of laforin inhibits autophagosome formation, suggesting that laforin plays a critical role in autophagosome biogenesis. The polyglucosan inclusions could be one of the substrates of autophagy, and loss of laforin might affect their sequestration into autophagosomes leading to their aggregation as Lafora bodies. Thus, laforin's proposed role in autophagy suggests a possible link between the proteolytic system and the polyglucosan inclusions in LD.

Hyperphosphorylation and aggregation of Tau in laforin-deficient mice, an animal model for Lafora disease.

Lafora progressive myoclonous epilepsy (Lafora disease; LD) is caused by mutations in the EPM2A gene encoding a dual specificity protein phosphatase named laforin. Our analyses on the Epm2a gene knock-out mice, which developed most of the symptoms of LD, reveal the presence of hyperphosphorylated Tau protein (Ser(396) and Ser(202)) as neurofibrillary tangles (NFTs) in the brain. Intriguingly, NFTs were also observed in the skeletal muscle tissues of the knock-out mice. The hyperphosphorylation of Tau was associated with increased levels of the active form of GSK3 beta. The observations on Tau protein were replicated in cell lines using laforin overexpression and knockdown approaches. We also show here that laforin and Tau proteins physically interact and that the interaction was limited to the phosphatase domain of laforin. Finally, our in vitro and in vivo assays demonstrate that laforin dephosphorylates Tau, and therefore laforin is a novel Tau phosphatase. Taken together, our study suggests that laforin is one of the critical regulators of Tau protein, that the NFTs could underlie some of the symptoms seen in LD, and that laforin can contribute to the NFT formation in Alzheimer disease and other tauopathies.

The malin-laforin complex suppresses the cellular toxicity of misfolded proteins by promoting their degradation through the ubiquitin-proteasome system.

Lafora disease (LD), a progressive form of inherited epilepsy, is associated with widespread neurodegeneration and the formation of polyglucosan bodies in the neurons. Laforin, a protein phosphatase, and malin, an E3 ubiquitin ligase, are two of the proteins that are defective in LD. We have shown recently that laforin and malin (referred together as LD proteins) are recruited to aggresome upon proteasomal blockade, possibly to clear misfolded proteins through the ubiquitin-proteasome system (UPS). Here we test this possibility using a variety of cytotoxic misfolded proteins, including the expanded polyglutamine protein, as potential substrates. Laforin and malin, together with Hsp70 as a functional complex, suppress the cellular toxicity of misfolded proteins, and all the three members of this complex are required for this function. Laforin and malin interact with misfolded proteins and promote their degradation through the UPS. LD proteins are recruited to the polyglutamine aggregates and reduce the frequency of aggregate-positive cells. Taken together, our results suggest that the malin-laforin complex is a novel player in the neuronal response to misfolded proteins and could be potential therapeutic targets for neurodegenerative disorders associated with cytotoxic proteins.

Mannosylated self-assembled structures for molecular confinement and gene delivery applications.

This paper reports self-assembly of a lysine conjugated with a biantennary mannose to form spherical structures. These supramolecular structures are found to be hollow in nature and they afford effective encapsulation of alkaline phosphatase enzyme, plasmid DNA and a GFP reporter gene, which was transfected in COS-7 cells. Loaded hollow structures also get disrupted upon mild sonication, releasing encapsulated molecules thereby illustrating their potential for confinement and delivery applications.

Recent advances in the molecular basis of Lafora's progressive myoclonus epilepsy.

Lafora's disease (LD) is an autosomal recessive and fatal form of progressive myoclonus epilepsy with onset in late childhood or adolescence. LD is characterised by the presence of intracellular polyglucosan inclusions, called Lafora bodies, in tissues including the brain, liver and skin. Patients have progressive neurologic deterioration, leading to death within 10 years of onset. No preventive or curative treatment is available for LD. At least three genes underlie LD, of which two have been isolated and mutations characterised: EPM2A and NHLRC1. The EPM2A gene product laforin is a protein phosphatase while the NHLRC1 gene product malin is an E3 ubiquitin ligase that ubiquitinates and promotes the degradation of laforin. Analyses of the structure and function of these gene products suggest defects in post-translational modification of proteins as the common mechanism that leads to the formation of Lafora inclusion bodies, neurodegeneration and the epileptic phenotype of LD. In this review, we summarise the available information on the genetic basis of LD, and correlate these advances with the rapidly expanding information about the mechanisms of LD gained from studies on both cell biological and animal models. Finally, we also discuss a possible mechanism to explain the locus heterogeneity observed in LD.