Nuclear translocation of LINE-1 encoded ORF1p alters nuclear envelope integrity and disrupts nucleocytoplasmic transport in human neurons

Abstract

LINE-1 retrotransposons are emerging as possible culprits in neurodegenerative diseases. However, the molecular mechanisms underlying the pathogenic role of LINE-1 and their encoded proteins ORF1p and ORF2p are still not completely understood. While the endonuclease and reverse transcriptase activity of ORF2p has been associated with DNA damage and inflammation, no pathogenic role has yet been assigned to ORF1p. Using a neuronal model of oxidative stress displaying increased LINE-1 expression, we report here that ORF1p stress-dependently translocates into the nucleus, localizes to the nuclear envelope and directly binds to nuclear lamina (Lamin B1), nuclear pore complex (NUP153) components and nuclear import (KPNB1) proteins. Stress-dependent targeting of nuclear envelope components by ORF1p alters nuclear envelope integrity, disrupts nucleo-cytoplasmic transport and induces heterochromatin destructuration, which are key features associated with neurodegenerative diseases and aging. Interestingly, nuclear deformations and heterochromatin destructuration were restored by the small molecule Remodelin which also normalized nuclear ORF1p levels. This study thus reveals a retrotransposition-independent pathogenic action of ORF1p perturbing nuclear envelope barrier function.

Steady-state neuron-predominant LINE-1 encoded ORF1p protein and LINE-1 RNA increase with aging in the mouse and human brain

“…Finally, we found components of RNA polymerase II and the SWI/SNF complex as partners of ORF1p. This further indicates that ORF1p has access to the nucleus in mouse brain neurons as described for other cells 86,87 and recently in human dopaminergic neurons in vitro 28 , implying that ORF1p potentially has access to chromatin.…”

“…Finally, we found components of RNA polymerase II and the SWI/SNF complex as partners of ORF1p. This further indicates that ORF1p has access to the nucleus in mouse brain neurons as described for other cells 86,87 and recently in human dopaminergic neurons in vitro 28 , implying that ORF1p potentially has access to chromatin. These findings give rise to intriguing questions concerning the potential function of ORF1p as ORF1p is widely distributed throughout the brain under normal physiological conditions and interacts with a variety of proteins which might not directly relate to the LINE-1 life cycle.…”

“…Notably, a multitude of novel interactors belonged to the “neuronal cell body” and “neuron projections” clusters, proposing potential neuron-specific partners of ORF1p such as Grm2/5, Bai1, Epha4, Kcnn2, Grik2 and Dmd among others. A last cluster, formed by Ncoa5 (Nuclear Receptor Coactivator 5), Nxf1 (Nuclear RNA Export Factor 1), Ranbp2 and Nup133 (both nucleoporins), might imply a role for these interactions in L1-RNA nuclear export and/or a mechanism for the LINE-1 RNP to gain access to the nucleus in post- mitotic neurons, a process we described recently in human neurons 28 . Altogether, the identification of known and novel interactors of ORF1p in the mouse brain suggests roles of ORF1p in the LINE-1 life cycle (RNA binding and metabolism, RNP formation, nuclear access) but also suggests potential novel physiological roles of ORF1p in the brain related to cytoskeleton organization, cGMP signaling, neuron-specific functions (i.e.…”

“…Neurodegeneration was partially prevented by anti-LINE-1 strategies among which NRTIs 16 and similar LINE-1 protein- dependent neuronal toxicity has been shown in drosophila 30,31 and the mouse cerebellum 32 . Recently, ORF1p has been evoked as a destabilizer of the nuclear envelope with negative consequences on nucleocytoplasmic transport and chromatin organization, independently on the known endonuclease or reverse transcriptase-dependent toxic properties of ORF2p 28 .…”

“…The copyright holder for this this version posted December 13, 2023. ; https://doi.org/10.1101/2023.12.12.571308 doi: bioRxiv preprint thus be incomplete and if so, it remains unclear how cells then prevent cell toxicity associated with LINE-1 encoded protein activity. Indeed, LINE-1 encoded proteins have been demonstrated to induce genomic instability (ORF2p endonuclease-mediated 16, [19][20][21][22][23][24] ; inflammation (ORF2p reverse transcriptase-mediated [25][26][27] and/or nuclear envelope fragility (ORF1p-mediated 28 ) and these cellular activities might be causally related to organismal aging and/or cancer , autoimmune and neurological diseases 29 . For instance, LINE-1 activation can drive neurodegeneration of mouse dopaminergic neurons 16 , of drosophila neurons 30,31 and of mouse Purkinje neurons 32 which can be at least partially rescued with nucleoside analogue reverse transcriptase inhibitors (NRTIs) or other anti-LINE-1 strategies.…”