Investigating the spread and toxicity of glycine-alanine dipeptides in C9orf72 ALS/FTD using Drosophila melanogaster

Hexanucleotide repeat expansions of variable size in C9orf72 are the most prevalent genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The role of repeat size in disease onset and severity in humans remains controversial. Transcripts of the expansions are translated into five dipeptide repeat (DPR) proteins. Most preclinical studies have used relatively short and tagged poly-DPR constructs to investigate DPR-mediated toxicity, and shown that poly-GR, poly-PR and, to a lesser extent, poly-GA DPRs are neurotoxic. Consequently, a major emphasis has been placed on understanding poly-GR- and poly-PR-mediated toxicity. However, poly-GA is the most abundant DPR in patient tissue. Transmission of protein aggregates may be a major driver of toxicity in neurodegeneration. In this study, I show for the first time that only poly-GA DPRs can spread trans-neuronally in vivo using the adult fly brain. Repeat length and tissue age modulate this phenomenon, and exosomes and synaptic vesicles are relevant in the extracellular release of GA DPRs. I also compared the toxicity, aggregation and cellular responses of GA100 DPRs carrying or not commonly used tags. Expression of tagged GA100 was markedly less toxic. GA100 tagged with GFP and mCherry exhibited aggregation differences and failed to cause DNA damage or proteostasis stress compared to untagged GA100 and GA100FLAG. These findings highlight the need to use untagged DPRs as controls when investigating their pathobiology. Finally, I tested the role of repeat size in modulating GA toxicity, subcellular localization, aggregation and cellular responses by comparing these in flies expressing untagged GA100, GA200 and GA400 DPRs. While aggregation propensity and proteostasis stress hold a positive correlation with repeat length, and GA400 was markedly more toxic than GA100, the latter was in turn more toxic than GA200. This highlights a non-linear correlation between repeat length and toxicity. GA100 and GA200 formed numerous puncta-like aggregates both in the soma and axons of neurons and, especially GA200, exhibited spreading, whereas GA400 resided only in somata and did not spread. Surprisingly, GA200 caused more DNA damage than GA100, but this effect was not observed upon GA400 expression. Collectively, I show that GA DPRs have a unique ability to spread in vivo, and their toxicity may have been previously underestimated by the use of short and tagged constructs. Therefore, my data support the further characterization of GA DPRs of a clinically relevant composition to develop strategies with therapeutic potential for C9orf72 mutation carriers

Toxicity of C9orf72-associated dipeptide repeat peptides is modified by commonly used protein tags

Hexanucleotide repeat expansions in the C9orf72 gene are the most prevalent genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Transcripts of the expansions are translated into toxic dipeptide repeat (DPR) proteins. Most preclinical studies in cell and animal models have used protein-tagged polyDPR constructs to investigate DPR toxicity but the effects of tags on DPR toxicity have not been systematically explored. Here, we used Drosophila to assess the influence of protein tags on DPR toxicity. Tagging of 36 but not 100 arginine-rich DPRs with mCherry increased toxicity, whereas adding mCherry or GFP to GA100 completely abolished toxicity. FLAG tagging also reduced GA100 toxicity but less than the longer fluorescent tags. Expression of untagged but not GFP- or mCherry-tagged GA100 caused DNA damage and increased p62 levels. Fluorescent tags also affected GA100 stability and degradation. In summary, protein tags affect DPR toxicity in a tag- and DPR-dependent manner, and GA toxicity might be underestimated in studies using tagged GA proteins. Thus, including untagged DPRs as controls is important when assessing DPR toxicity in preclinical models

Activating transcription factor 6 derepression mediates neuroprotection in Huntington disease

Deregulated protein and Ca2+ homeostasis underlie synaptic dysfunction and neurodegeneration in Huntington disease (HD); however, the factors that disrupt homeostasis are not fully understood. Here, we determined that expression of downstream regulatory element antagonist modulator (DREAM), a multifunctional Ca2+-binding protein, is reduced in murine in vivo and in vitro HD models and in HD patients. DREAM downregulation was observed early after birth and was associated with endogenous neuroprotection. In the R6/2 mouse HD model, induced DREAM haplodeficiency or blockade of DREAM activity by chronic administration of the drug repaglinide delayed onset of motor dysfunction, reduced striatal atrophy, and prolonged life span. DREAM-related neuroprotection was linked to an interaction between DREAM and the unfolded protein response (UPR) sensor activating transcription factor 6 (ATF6). Repaglinide blocked this interaction and enhanced ATF6 processing and nuclear accumulation of transcriptionally active ATF6, improving prosurvival UPR function in striatal neurons. Together, our results identify a role for DREAM silencing in the activation of ATF6 signaling, which promotes early neuroprotection in HDThis work was funded by the Instituto de Salud Carlos III/CIBERNED (to J.R. Naranjo, B. Mellström, and A. Rábano), FISS-RIC RD12/0042/0019 (to C. Valenzuela), Madrid regional government/Neurodegmodels (to J.R. Naranjo), MINECO grants SAF2010-21784 and SAF2014-53412-R (to J.R. Naranjo), SAF2012-32209 (to M. Gutierrez-Rodriguez), SAF2010-14916 and SAF2013-45800-R (to C. Valenzuela), and a grant from the Swedish Research Council (J.Y. Li

Neuroprotection by Phytoestrogens in the Model of Deprivation and Resupply of Oxygen and Glucose In Vitro: The Contribution of Autophagy and Related Signaling Mechanisms

Phytoestrogens can have a neuroprotective effect towards ischemia-reperfusion-induced neuronal damage. However, their mechanism of action has not been well described. In this work, we investigate the type of neuronal cell death induced by oxygen and glucose deprivation (OGD) and resupply (OGDR) and pinpoint some of the signaling mechanisms whereby the neuroprotective effects of phytoestrogens occur in these conditions. First, we found that autophagy initiation affords neuronal protection upon neuronal damage induced by OGD and OGDR. The mammalian target of rapamycin/ribosomal S6 kinase (mTOR/S6K) pathway is blocked in these conditions, and we provide evidence that this is mediated by modulation of both the 50 AMP-activated protein kinase (AMPK) and phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) pathways. These are dampened up or down, respectively, under OGDR-induced neuronal damage. In contrast, the MAPK-Erk kinase/extracellular signal-regulated kinase (MEK/ERK) pathway is increased under these conditions. Regarding the pathways affected by phytoestrogens, we show that their protective properties require autophagy initiation, but at later stages, they decrease mitogen-activated protein kinase (MAPK) and AMPK activation and increase mTOR/S6K activation. Collectively, our results put forward a novel mode of action where phytoestrogens play a dual role in the regulation of autophagy by acting as autophagy initiation enhancers when autophagy is a neuroprotective and pro-survival mechanism, and as autophagy initiation inhibitors when autophagy is a pro-death mechanism. Finally, our results support the therapeutic potential of phytoestrogens in brain ischemia by modulating autophagy

Activating transcription factor 6 derepression mediates neuroprotection in Huntington disease

Deregulated protein and Ca2+ homeostasis underlie synaptic dysfunction and neurodegeneration in Huntington disease (HD); however, the factors that disrupt homeostasis are not fully understood. Here, we determined that expression of downstream regulatory element antagonist modulator (DREAM), a multifunctional Ca2+-binding protein, is reduced in murine in vivo and in vitro HD models and in HD patients. DREAM downregulation was observed early after birth and was associated with endogenous neuroprotection. In the R6/2 mouse HD model, induced DREAM haplodeficiency or blockade of DREAM activity by chronic administration of the drug repaglinide delayed onset of motor dysfunction, reduced striatal atrophy, and prolonged life span. DREAM-related neuroprotection was linked to an interaction between DREAM and the unfolded protein response (UPR) sensor activating transcription factor 6 (ATF6). Repaglinide blocked this interaction and enhanced ATF6 processing and nuclear accumulation of transcriptionally active ATF6, improving prosurvival UPR function in striatal neurons. Together, our results identify a role for DREAM silencing in the activation of ATF6 signaling, which promotes early neuroprotection in HD.A. De la Cruz holds a RECAVA contract, A. Prieto and P. Cercós hold FPI fellowships, and T. González holds a Ramón y Cajal contract. J. Casado-Vela holds a JAE-DOC (CSIC) from the Spanish Ministerio de Economía y Competitividad (MINECO), cofunded by the European Social Fund. This work was funded by the Instituto de Salud Carlos III/CIBERNED (to J.R. Naranjo, B. Mellström, and A. Rábano), FISS-RIC RD12/0042/0019 (to C. Valenzuela), Madrid regional government/Neurodegmodels (to J.R. Naranjo), MINECO grants SAF2010-21784 and SAF2014-53412-R (to J.R. Naranjo), SAF2012-32209 (to M. Gutierrez-Rodriguez), SAF2010-14916 and SAF2013-45800-R (to C. Valenzuela), and a grant from the Swedish Research Council (J.Y. Li).Peer Reviewe