Studying the pathophysiology of REEP1 and REEP2-associated axonal disorders

• Applicant: Professor Dr. Christian Andreas Hübner
• Subject Area: Molecular and Cellular Neurology and Neuropathology ; Human Genetics
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 431954759

Many genes associated with neurodegenerative disorders manifesting at axons encode membrane shaping proteins of the endoplasmic reticulum (ER). In this project we focus on REEP1 and REEP2, closely related ER shaping proteins characterized by two reticulon homology domains (RHD) followed by an amphipathic helix. While REEP2 variants have only been associated with autosomal dominant hereditary spastic paraplegia (HSP), some REEP1 variants cause HSP while others result in hereditary motor neuropathy (HMN). To resolve why some REEP1 mutations cause HSP and others HMN, we generated mice with an in-frame-deletion of exon 5 thus modeling the HMN associated REEP1 variant c.304-2A>G, which leads to skipping of exon 5. In the first funding period we analyzed the resulting knock-in mice in comparison with Reep1 knockout mice. We can show that mice homozygous for the exon 5 deletion indeed develop HMN with degeneration of peripheral motor fibers. Because the abundance of the Reep1 variant is strongly increased in sciatic nerve lysates of knock-in mice compared to WT protein in control mice, we hypothesize that the turnover of the variant protein is delayed. Indeed, we can show that REEP1 is degraded via the proteasomal pathway and that HMN associated variants are not or less ubiquitinated compared to the WT protein. Liposome shaping assays further suggest that exon 5 deletion increases the shaping properties of REEP1. The accumulation of the exon 5 deletion variant together with its increased shaping properties may thus result in a toxic gain-of-function. In agreement, we observe a fragmented ER in spinal cord motoneurons our HMN mouse model. During the requested second funding period, we aim to finalize these experiments to further resolve the role of the ubiquitination of Reep1. We identified the E3 Ubiquitin ligase HUWE1 in the interactome of Reep1 and will now address whether HUWE1 may serve as its E3 ligase. We will perform in vitro ubiquitination assays with HUWE1 and study whether ubiquitination affects the shaping properties or REEP1 as recently shown by us for other related proteins. Moreover, we will analyze how ER functions such as the unfolded protein response, the secretory pathway, glycosylation and Calcium homeostasis are affected by Reep1 loss- or gain-of-function. Because of two very recent reports that the fission yeast orthologue Rop1 for Reep1 and Reep2 is required for the formation of the phagophore, we will study whether autophagy is affected by Reep1 or Reep2 loss-of-function. To directly visualize the consequences of Reep1 loss- or gain-of function for the ER in motoneurons and their axons, we will generate mice with robust expression of the HaloTag within the lumen of the ER. After mating to Reep1 KO and KI mouse models, this will enable us to reconstruct the ER in tissues of Reep1 KO and KI mice by dSTORM. Finally, we will continue with the analysis of Reep2 and Reep1/Reep2 double knockout mice.

• DFG Programme: Research Grants