Elsevier

Neurobiology of Aging

Volume 54, June 2017, Pages 71-83
Neurobiology of Aging

Regular article
Enhancing Mitofusin/Marf ameliorates neuromuscular dysfunction in Drosophila models of TDP-43 proteinopathies

https://doi.org/10.1016/j.neurobiolaging.2017.02.016Get rights and content

Abstract

Transactive response DNA-binding protein 43 kDa (TDP-43) is considered a major pathological protein in amyotrophic lateral sclerosis and frontotemporal lobar degeneration. The precise mechanisms by which TDP-43 dysregulation leads to toxicity in neurons are not fully understood. Using TDP-43-expressing Drosophila, we examined whether mitochondrial dysfunction is a central determinant in TDP-43 pathogenesis. Expression of human wild-type TDP-43 in Drosophila neurons results in abnormally small mitochondria. The mitochondrial fragmentation is correlated with a specific decrease in the mRNA and protein levels of the Drosophila profusion gene mitofusin/marf. Importantly, overexpression of Marf ameliorates defects in spontaneous walking activity and startle-induced climbing response of TDP-43-expressing flies. Partial inactivation of the mitochondrial profission factor, dynamin-related protein 1, also mitigates TDP-43–induced locomotor deficits. Expression of TDP-43 impairs neuromuscular junction transmission upon repetitive stimulation of the giant fiber circuit that controls flight muscles, which is also ameliorated by Marf overexpression. We show here for the first time that enhancing the profusion gene mitofusin/marf is beneficial in an in vivo model of TDP-43 proteinopathies, serving as a potential therapeutic target.

Introduction

Amyotrophic lateral sclerosis (ALS) is characterized by a progressive degeneration of upper and lower motor neurons, leading to muscle weakness. Patients present paralysis, dysphagia, dysarthria, and spasticity. Cognitive defects are quite rare whereas 20% of ALS cases also show frontotemporal dementia. Over time, complications appear with feeding difficulties and respiratory failure. No cure has yet been found and ALS is generally lethal 2–5 years after the diagnosis. About 90% of ALS patients are sporadic cases and the remaining 10% are inherited forms. Because mutations in the copper/zinc superoxide dismutase 1 (SOD1) gene were discovered first, most of our knowledge on ALS pathogenesis derives from animal models overexpressing human SOD1 mutants (Rosen, 1993).

More recently, the transactive response DNA-binding protein of 43 kDa (TDP-43), an evolutionarily conserved heterogeneous nuclear ribonucleoprotein, was recognized to be a major constituent of ubiquitinated and phosphorylated inclusions found in the nervous system of patients with ALS or with frontotemporal lobar degeneration (FTLD) (Arai et al., 2006, Neumann et al., 2006). TDP-43 might play a primary role in those neurodegenerative diseases as mutations in TDP-43 were directly linked to familial cases of ALS and FTLD (Pesiridis et al., 2009). Moreover, TDP-43 is reported to accumulate secondary to pathological proteins in a large spectrum of other neuropathologies including Alzheimer's disease, Lewy body disease, Huntington's disease and spinocerebellar ataxia (Amador-Ortiz et al., 2007, Arai et al., 2009, Freeman et al., 2008, Higashi et al., 2007, Schwab et al., 2008, Toyoshima and Takahashi, 2014). Thus, TDP-43 is a common determinant in distinct neurodegenerative disorders collectively known as TDP-43 proteinopathies.

Normally, TDP-43 primarily localizes in the nucleus, whereas cytoplasmic aggregation is observed in ALS patients. Structural analysis reveals that TDP-43 consists of a central region with 2 RNA recognition motifs followed by a C-terminus region containing a prion-like, glycine-rich domain that accounts for the propensity of TDP-43 to interact with heterogeneous nuclear ribonucleoproteins including itself. The precise mechanisms by which TDP-43 leads to toxicity in neurons are not fully elucidated, even if our understanding of the physiological role of TDP-43 has increased in the last few years. TDP-43 regulates mRNA metabolism from gene transcription to protein translation including intron splicing, mRNA transport and stability (Buratti and Baralle, 2012). Accumulating evidence also support that TDP-43 is a stress-responsive RNA-associated factor (Aulas and Vande Velde, 2015). Exposure to a stress induces relocalization of TDP-43 into stress granules that represent cytoplasmic foci where translationally stalled transcripts are stored before triage for degradation or reinitiation. RNA immunoprecipitation methods followed by deep sequencing have revealed that TDP-43 binds predominantly to mRNA in UG-rich motifs (Sephton et al., 2011). As a consequence, a prolonged misexpression or mislocalization of TDP-43 might affect many neuronal mRNA targets that control RNA metabolism, cell signaling, axonogenesis, synaptic development and transmission, and energy metabolism (Sephton et al., 2011). One important challenge would be to determine which among these processes have a direct role in ALS pathogenesis.

Within the nervous system, the main site of ATP synthesis and expenditure is the neuron, mostly to support synaptic activity. As a consequence, neurons are also the most vulnerable to mitochondrial dysfunction. Early studies at the ultrastructural level on biopsies from ALS patients have revealed morphological abnormalities in mitochondria of skeletal muscles, spinal cord, and motor cortex (Afifi et al., 1966, Atsumi, 1981, Hirano et al., 1984, Okamoto et al., 1990, Siklos et al., 1996). Major insights have arisen with studies on mutant SOD1. It was proposed that mislocalization of mutant SOD1 with mitochondria may enhance oxidative damage (Panov et al., 2011). Recently much attention was given to mitochondrial shape, size, and transport. Indeed, smaller mitochondria were observed in cellular and mouse models expressing mutant SOD1 (Cozzolino et al., 2009, Magrane et al., 2014). Altered mitochondrial transport along axons was also described in SOD1 models (Magrane et al., 2014, Marinkovic et al., 2012, Williamson and Cleveland, 1999). Wild-type or mutant TDP-43 was found to induce mitochondrial fragmentation and to impair mitochondrial transport in cultured motor neurons as well as in the intact sciatic nerve of living mice (Magrane et al., 2014, Wang et al., 2013). Fibroblasts from patients harboring the A382T mutation in TDP-43 show a fragmented mitochondrial network (Onesto et al., 2016). Several groups have reported the presence of abnormal mitochondrial clusters in transgenic TDP-43 mice, which is reminiscent of an accumulation of defective mitochondria (Magrane et al., 2014, Shan et al., 2010, Xu et al., 2010). However it remains to demonstrate that dysregulation of mitochondrial dynamics is a significant contributor to ALS.

In recent years, Drosophila has been used to identify pathways involved in ALS pathogenesis. Silencing and overexpressing TDP-43 result in similar phenotype in flies, suggesting that both loss and gain-of-function contribute to the disease. Regulated expression of TDP-43 is required for neuronal development and activity in Drosophila, as misexpression impairs neuromuscular junction (NMJ) morphology as well as locomotor performances (Diaper et al., 2013, Estes et al., 2011, Li et al., 2010). In the present study, we took advantage of genetic tools in Drosophila to study the role of mitochondrial dynamics in ALS pathogenesis. Expression of human wild-type TDP-43 (TDP-43WT) in Drosophila neurons induced abnormal mitochondrial fragmentation as previously described in vitro (Wang et al., 2013). The molecular mechanisms underlying mitochondrial fusion and fission events are relatively well-known. Mitochondrial dynamics is controlled by profusion genes, optic atrophy 1 (OPA1) and mitofusins (Mfn) for the inner and outer mitochondrial membranes, respectively, whereas fission is mediated by dynamin-related protein 1 (DRP1). We found that transcript and protein levels of Drosophila Mfn/mitochondrial assembly regulatory factor (Marf) were both reduced in TDP-43WT-expressing flies. Next, we examined whether or not manipulation of genes regulating mitochondrial dynamics may impact the phenotype. Overexpressing Marf or reducing DRP1 activity both ameliorate locomotor defects of flies expressing TDP-43WT. Repetitive activation of the giant fibers that control flight muscles revealed that TDP-43WT progressively impairs NMJ efficacy, while Marf overexpression alleviates this increased fatigability. A similar neuroprotection was observed with flies expressing the G298S mutant ALS variant of human TDP-43 (TDP-43G298S). Thus, we provide compelling evidence that imbalance between mitochondrial fusion/fission plays a significant role in TDP-43–induced pathogenesis in Drosophila.

Section snippets

Drosophila strains

All flies were raised on a standard agar/cornmeal/yeast diet at 25 °C unless mentioned in the text. Fly stocks carrying UAS-marfOE and the mutation DRP11 were a gift from Pr. H. Bellen, flies carrying UAS-marfIR and UAS-DRP1OE from Dr. M. Guo. The UAS-TDP-43, UAS-TDP-43mutNLS, and UAS-TDP-43mutNES lines were generated as previously described (Miguel et al., 2011). Flies expressing UAS-TDP-43RRM1 and the respective UAS-TDP-43WT were from Dr. J. B. Schulz (Voigt et al., 2010). Elav-GAL4 driver

TDP-43 results in mitochondrial fragmentation in Drosophila neurons

Previous works have reported that TDP-43 misexpression leads to increased number of small mitochondria in ALS models and patients (Magrane et al., 2014, Onesto et al., 2016, Wang et al., 2013). We therefore investigated whether overexpression of TDP-43 can also impact mitochondrial morphology in Drosophila. Mitochondrial size, shape, and density were analyzed by using transmission electron microscopy in brains from adult flies expressing human wild-type TDP-43 under the regulation of the

Discussion

The purpose of the study was to combine genetic and molecular approaches in Drosophila to further understand the role and importance of mitochondria in TDP-43 proteinopathies. As mitochondrial functioning strongly depends on mitochondrial dynamics, we investigated the impact of TDP-43 on mitochondrial morphology. We first demonstrated that TDP-43 reduces mitochondrial size and increases their number in Drosophila brain. This is correlated with a specific downregulation of the mitochondrial

Disclosure statement

The authors have no actual or potential conflicting or financial interest to disclose.

Acknowledgements

B. K. had a PhD fellowship from the Ministère de la Recherche. J. C. L. and M. L. are supported by a grant from the Association pour la recherche sur la Sclérose Latérale Amyotrophique et autres Maladies du Motoneurone (ARSLA). M. L. is also supported by a grant from the Région Haute-Normandie.

The authors thank Pr. H. Bellen, Dr. M. Guo, Dr. J. B. Schulz, and the Bloomington Drosophila Stock Center for mutant and transgenic flies. We greatly acknowledge Dr. J. Devaux for his expertise and

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