Fuchs

Pathophysiology of transposable elements in the brain

Principal investigator: Julia Fuchs, CR1 Inserm

Less than 2% of mammalian genomes are protein-coding, meaning that genetic information is transferred from DNA to RNA to protein. Until recently, the majority of the non-coding genome was considered “junk” DNA. However, it is becoming clear that non-coding DNA is crucial for cellular function and plays an essential role in gene regulation. The major component of non-coding DNA, comprising almost 50% of mammalian genomes, are transposable elements (TEs, Fig1), long considered as genomic parasites. TEs are intrinsic components of the genomes of virtually all living organisms, including humans. TEs had, and a minority still has, the ability to move and to modulate genetic information and organization and for some, to expand overall genome size.

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Fig 1: Transposable elements (TEs) are abundant in mammalian genomes
Less than 2% of the human genome is protein coding (red). Almost 50% of the human genome consist of remnants of TEs. 21% of those belong to the LINE-1 (L1) family, most of which are fossilized. An active L1 contains a ≈ 6-7kB full-length (fl) sequence with a promoter in the 5’UTR and two open reading frames (ORF1p: RNA binding protein; ORF2p: endonuclease and reverse transcriptase). A flL1 element is autonomous for mobilization as it contains all the necessary machinery for expression and insertion into a new genomic location.

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In our team, we investigate how TEs influence neuronal function and dysfunction in mice and humans. In particular, we seek to understand how the presence and expression of one class of TEs, Line-1 (L1) retrotransposons, influences on the epigenome, on gene expression and on the function of neurons. We also investigate the link between L1 retrotransposons, environmental factors and the aging process. Recent data, stemming from different laboratories including our group, suggests a novel pathogenic axis in neurodegenerative diseases involving epigenetic remodeling linked to the aging process and resulting in the dysinhibition of TEs (Fig2). We have recently shown that, in adult dopaminergic neurons, which project to the striatum and are vulnerable to neurodegeneration in Parkinson’s disease (PD), oxidative stress-induced overexpression of L1 (Fig3) causes DNA strand breaks (Fig4) leading to the degeneration of this neuronal population. Mice heterozygous for En1 (En1-het), a model for PD, show a progressive, adult-onset degeneration of midbrain dopaminergic neurons and L1 inhibition partially rescues neurodegeneration in these mice. L1 retrotransposons become derepressed in post-mitotic neurons under oxidative stress conditions, which we have shown earlier, can induce heterochromatin relaxation (Fig5). The aging process is known to be linked to oxidative stress, an increase in DNA damage and a decrease in the DNA repair capacity. However, whether neurodegeneration or accelerated aging are causally related to an increase in L1 activity is yet unknown. We are currently investigating whether heterochromatin relaxation and L1 activation are direct drivers of neurodegeneration and whether L1 activation is associated with PD and other neurodegenerative disorders. Having successfully used an FDA-approved drug (the non-nucleoside reverse transcriptase inhibitor stavudine) to prevent neurodegeneration in an acute oxidative-stress model, we propose that “anti-L1” strategies might be novel therapeutic targets in the treatment of neurodegenerative disorders.

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Fig2: An emerging pathogenic axis linking aging, epigenetics, TEs and neurodegeneration
Aging, oxidative stress and DNA damage, all intimately linked, lead to heterochromatin relaxation and derepression of TE, including LINE-1. The endonuclease of LINE-1 (ORF2p) induces DNA double strand breaks, further decompacting chromatin and, upon a certain threshold, finally leading to cell death once the DNA repair machinery is overwhelmed.

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Fig 3: Activation of L1-RNA and the encoded Orf1 protein uponacute oxidative stress
ORF1p staining (red) is increased upon oxidative stress in dopaminergic neurons (TH-positive, green) and L1-RNA (LINE-1 Tf/Gf, right lower panel, green line) preceeds the appearance of DNA damage (left lower panel, red line). 8-oxoguanine quantifies the oxidative-stress induced base changes in DNA (blue line, left lower panel).

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Fig4: DNA damage in dopaminergic neurons in the midbrain upon acute oxidative stress
The drug 6-OHDA induces acute oxidative stress selectively in dopaminergic neurons (TH-positive, green) leading to DNA damage in the form of DNA strand breaks illustrated by the increase in yH2aX-positive foci (red).

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Fig 5: Heterochromatin relaxation upon oxidative stress
Heterochromatin marker H3K9me3, among other markers not shown here, is less intense and less organized in foci after exposure to an acute oxidative stress in dopaminergic neurons (TH-positive, green).

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Another goal of our team is to adress the question whether L1 and their proteins have acquired a physiological role in adult neurons. L1 are active in post-mitotic neurons and we have observed that L1 RNA and the encoded Orf1p protein are expressed in adult neurons in the mouse brain (Fig6). We use stereotaxic injections of viral constructs, antisense oligonucleotides and Penetratin-coupled siRNA in vivo and pharmacological interventions, viral infections, plasmid and siRNA transfections in neuronal cell culture in vitro to manipulate L1 levels. Following gain- and loss-of-function of L1, we make use of the tremendous technical advances in sequencing technologies, in the adaptation of techniques like chomatin immunoprecipitation (ChIP) to small sample sizes, in methods allowing the isolation of specific neuronal populations up to the single cell level such as fluorescence-activated cell sorting (FACS) and laser microdissection, and bioinformatic approaches helping to approximate the correct mapping of sequencing reads to transposable elements. Using these techniques, we characterize the expression dynamics of specific L1 genomic loci, their chromatin environment and the impact of these dynamics on gene expression regulation in individual neuronal populations.
In summary, our scientific objective is to increase our knowledge about the role of transposable elements in brain physiology and pathology.

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Fig6: Full-length L1 elements are expressed in the adult mouse ventral midbrain and in TH+ neurons of the SNpc
A: RNA from neuronal and non-neuronal tissues was analyzed for L1 expression by RT–qPCR with primers located in the 50UTR for subfamily detection (L1 Tf/Gf, L1 A) and in Orf2. Cycle thresholds from tissues obtained from three mice were normalized to values obtained from kidney tissues using the ddCt method relative to the expression of Gapdh; error bars represent SEM.
B: Midbrain slices were analyzed by immunofluorescence against Orf1p in TH+, NeuN+, or TH NeuN+ neurons, and Orf1p fluorescence intensity distribution was measured (right). Scale bar represents 30 µm.

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Selected publications 2011-2019

- Di Nardo, A. A., Fuchs, J., Joshi, R. L., Moya, K. L., and Prochiantz, A. (2018). The Physiology of Homeoprotein Transduction. Physiol. Rev. 98, 1943–1982.

- Blaudin de Thé, F.-X., Rekaik, H., Peze-Heidsieck, E., Massiani-Beaudoin, O., Joshi, R. L., Fuchs, J., and Prochiantz, A. (2018). Engrailed homeoprotein blocks degeneration in adult dopaminergic neurons through LINE-1 repression. EMBO J. 37.

- Blaudin de Thé, F.-X., Rekaik, H., Prochiantz, A., Fuchs, J., and Joshi, R. L. (2016). Neuroprotective Transcription Factors in Animal Models of Parkinson Disease. Neural Plast. 2016, 6097107.

- Rekaik, H., Blaudin de Thé, F.-X., Fuchs, J., Massiani-Beaudoin, O., Prochiantz, A., and Joshi, R. L. (2015b). Engrailed Homeoprotein Protects Mesencephalic Dopaminergic Neurons from Oxidative Stress. Cell Rep 13, 242–250.

- Rekaik, H., Blaudin de Thé, F.-X., Prochiantz, A., Fuchs, J., and Joshi, R. L. (2015a). Dissecting the role of Engrailed in adult dopaminergic neurons--Insights into Parkinson disease pathogenesis. FEBS Lett. 589, 3786–3794.

- Nordström, U., Beauvais, G., Ghosh, A., Pulikkaparambil Sasidharan, B. C., Lundblad, M., Fuchs, J., Joshi, R. L., Lipton, J. W., Roholt, A., Medicetty, S., et al. (2015). Progressive nigrostriatal terminal dysfunction and degeneration in the engrailed1 heterozygous mouse model of Parkinson’s disease. Neurobiol. Dis. 73, 70–82.

- Prochiantz, A., Fuchs, J., and Di Nardo, A. A. (2014). Postnatal signalling with homeoprotein transcription factors. Philos. Trans. R. Soc. Lond., B, Biol. Sci. 369.

- Fuchs, J., Stettler, O., Alvarez-Fischer, D., Prochiantz, A., Moya, K. L., and Joshi, R. L. (2012). Engrailed signaling in axon guidance and neuron survival. Eur. J. Neurosci. 35, 1837–1845.

- Alvarez-Fischer, D., Fuchs, J., Castagner, F., Stettler, O., Massiani-Beaudoin, O., Moya, K. L., Bouillot, C., Oertel, W. H., Lombès, A., Faigle, W., et al. (2011). Engrailed protects mouse midbrain dopaminergic neurons against mitochondrial complex I insults. Nat. Neurosci. 14, 1260–1266.

People:

Group leader:
Julia Fuchs, CR1 INSERM

Senior researcher:
Rajiv L. Joshi, DR2 CNRS

Technical staff:
Olivia Beaudoin, IR2 CNRS

PhD students and Master students:
Eugénie Peze-Heidsieck, PhD student
Camille Ravel Godreuil, PhD student
Marguerite Jamet, CDD AI