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

PolyGR and polyPR knock-in mice reveal a conserved neuroprotective extracellular matrix signature in C9orf72 ALS/FTD neurons

Dipeptide repeat proteins are a major pathogenic feature of C9orf72 amyotrophic lateral sclerosis (C9ALS)/frontotemporal dementia (FTD) pathology, but their physiological impact has yet to be fully determined. Here we generated C9orf72 dipeptide repeat knock-in mouse models characterized by expression of 400 codon-optimized polyGR or polyPR repeats, and heterozygous C9orf72 reduction. (GR)400 and (PR)400 knock-in mice recapitulate key features of C9ALS/FTD, including cortical neuronal hyperexcitability, age-dependent spinal motor neuron loss and progressive motor dysfunction. Quantitative proteomics revealed an increase in extracellular matrix (ECM) proteins in (GR)400 and (PR)400 spinal cord, with the collagen COL6A1 the most increased protein. TGF-β1 was one of the top predicted regulators of this ECM signature and polyGR expression in human induced pluripotent stem cell neurons was sufficient to induce TGF-β1 followed by COL6A1. Knockdown of TGF-β1 or COL6A1 orthologues in polyGR model Drosophila exacerbated neurodegeneration, while expression of TGF-β1 or COL6A1 in induced pluripotent stem cell-derived motor neurons of patients with C9ALS/FTD protected against glutamate-induced cell death. Altogether, our findings reveal a neuroprotective and conserved ECM signature in C9ALS/FTD.</p

A monocarboxylate transporter rescues frontotemporal dementia and Alzheimer's disease models.

Brains are highly metabolically active organs, consuming 20% of a person's energy at resting state. A decline in glucose metabolism is a common feature across a number of neurodegenerative diseases. Another common feature is the progressive accumulation of insoluble protein deposits, it's unclear if the two are linked. Glucose metabolism in the brain is highly coupled between neurons and glia, with glucose taken up by glia and metabolised to lactate, which is then shuttled via transporters to neurons, where it is converted back to pyruvate and fed into the TCA cycle for ATP production. Monocarboxylates are also involved in signalling, and play broad ranging roles in brain homeostasis and metabolic reprogramming. However, the role of monocarboxylates in dementia has not been tested. Here, we find that increasing pyruvate import in Drosophila neurons by over-expression of the transporter bumpel, leads to a rescue of lifespan and behavioural phenotypes in fly models of both frontotemporal dementia and Alzheimer's disease. The rescue is linked to a clearance of late stage autolysosomes, leading to degradation of toxic peptides associated with disease. We propose upregulation of pyruvate import into neurons as potentially a broad-scope therapeutic approach to increase neuronal autophagy, which could be beneficial for multiple dementias

Bumpel leads to pyruvate import.

(A) and (B) Lactate and pyruvate transport were measured using the lactate sensor Laconic (A) or the pyruvate sensor Pyronic (B). (A) Salivary glands from third instar larvae expressing Laconic and bumpel or its control were isolated and exposed to 1 mM lactate. Traces show the mean fluorescence ratio of mTFP/Venus obtained from 6 independent experiments (6 animals, total of 71 cells in control animals and 77 cells in animals over-expressing bumpel). Slope was calculated from the first 2 min of recording in the presence of lactate. p = 0.026 by Mann Whitney test. Area under the curve was calculated from the fluorescence obtained during the 5 min of lactate exposure. p = 0.004 by unpaired t-test. (B) Salivary glands expressing Pyronic and bumpel or its control were isolated and exposed to 1 mM pyruvate. Traces show the mean fluorescence ratio of mTFP/Venus obtained from 8 independent experiments (69 cells in control animals and 68 cells overexpressing bumpel). Slope were calculated from the first 2 min of recordings in the presence of pyruvate. p = 0.026 by Mann Whitney test. Area under the curve was calculated from the fluorescence obtained in 5 min of pyruvate exposure. p = 0.0346 by unpaired t-test. (C) Western blot of GP levels, relative to actin, in flies injected with 300 mM ethyl pyruvate and 2 M sodium pyruvate, or PBS as a control, and allowed to recover for 24 hours. Two different experimental replicates were combined in the statistical analysis (marked by different symbols), and then compared by t-test, *p = 0.0360 (D) GP levels in C9 and C9 bumpel fly heads with or without downregulation of Ldh by RNAi (E) GP levels in C9 and C9 bumpel fly heads with or without downregulation of Pdha by RNAi. GP levels in both D and E were compared by one way ANOVA followed by Šídák’s multiple comparisons test. All westerns samples shown for each panel were run on the same gel. Gentoypes: (A) UAS-36 (G4C2), actinGal4/UAS-Laconic (B) UAS-36 (G4C2), actinGal4/UAS-Pyronic (C) UAS-36 (G4C2), elavGS, (D) UAS-36 (G4C2), elavGS, UAS-36 (G4C2)/LdhRNAi, elavGS, UAS-36 (G4C2), elavGS/UAS-bumpel, UAS-36 (G4C2)/LdhRNAi, elavGS/UAS-bumpel, (E) UAS-36 (G4C2), elavGS, UAS-36 (G4C2)/Pdha RNAi, elavGS, UAS-36 (G4C2), elavGS/UAS-bumpel, UAS-36 (G4C2)/PdhaRNAi, elavGS/UAS-bumpel.</p

Over-expression of bumpel reduces DPR levels.

(A) Survival curves of flies over-expressing bumpel (+RU) and their controls (-RU) during normal ageing. p = 0.06 by log rank (B) GR levels in fly heads over-expressing C9 alone or with bumpel after 7 days of induction, measured by ELISA. p = 0.0005 by unpaired t-test (C) GP levels in fly heads over-expressing C9 alone or with bumpel after 7 days of induction, measured by western blot. p = 0.0005 by unpaired t-test (D) mCD8GFP levels in fly heads over-expressing C9+mCD8GFP or with bumpel after 7 days of induction, measured by western blot. p = 0.1723 by unpaired t-test. (E) Survival curves of flies expressing 36(GR) repeats with (pink) and without (black) bumpel. p = 1.5E-44 by log rank test (F) Survival curves of flies expressing 36(PR) repeats with (pink) and without (black) bumpel. p = 2.1E-74 by log rank test (G) Survival curves of flies expressing 36(GA) repeats with (green) and without (black) bumpel. p = 3.2E-9 by log rank test. Genotypes: (A) elavGS/UAS-bumpel (B, C) UAS-36 (G4C2), elavGS, UAS-36 (G4C2), elavGS/UAS-bumpel (D) UAS-36 (G4C2)/UAS-mCD8GFP, elavGS, UAS-36 (G4C2)/UAS-mCD8GFP, elavGS/UAS-bumpel (E) UAS-36GR, elavGS, UAS-36 GR, elavGS/UAS-bumpel (F) UAS-36PR, elavGS, UAS-36PR, elavGS/UAS-bumpel. (G) UAS36GA, elavGS, UAS36GA, elavGS/UAS-bumpel.</p

Log fold change of TFEB targets genes in RNA seq of GR100 expressing flies vs control.

Log fold change of TFEB targets genes in RNA seq of GR100 expressing flies vs control.</p

S4 Fig -

Full western blots for Fig 3D (A) and 3E (B), with samples labelled. (PDF)</p

Data for Fig 2.

Brains are highly metabolically active organs, consuming 20% of a person’s energy at resting state. A decline in glucose metabolism is a common feature across a number of neurodegenerative diseases. Another common feature is the progressive accumulation of insoluble protein deposits, it’s unclear if the two are linked. Glucose metabolism in the brain is highly coupled between neurons and glia, with glucose taken up by glia and metabolised to lactate, which is then shuttled via transporters to neurons, where it is converted back to pyruvate and fed into the TCA cycle for ATP production. Monocarboxylates are also involved in signalling, and play broad ranging roles in brain homeostasis and metabolic reprogramming. However, the role of monocarboxylates in dementia has not been tested. Here, we find that increasing pyruvate import in Drosophila neurons by over-expression of the transporter bumpel, leads to a rescue of lifespan and behavioural phenotypes in fly models of both frontotemporal dementia and Alzheimer’s disease. The rescue is linked to a clearance of late stage autolysosomes, leading to degradation of toxic peptides associated with disease. We propose upregulation of pyruvate import into neurons as potentially a broad-scope therapeutic approach to increase neuronal autophagy, which could be beneficial for multiple dementias.</div