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

Hereditary Spastic Paraplegias (HSPs) comprise a growing and heterogeneous group of diseases primarily affecting cortical motoneurons which thus lead to a progressive spastic gait disorder. Pathologically, HSPs can be classified as axonopathies and are characterized by a dying-back degeneration of the long axons of the central nervous system while the integrity of the cell body is often preserved long into the disease course. The gene SPG31 is associated with autosomal-dominantly inherited HSP and encodes the ER-resident protein REEP1. In previous works we could show that REEP1 induces membrane curvature via its Reticulon domain and that the ER in cell bodies of cortical motoneurons of REEP1-knockout mice is structurally altered. REEP2 is another member of the REEP family of proteins mutated in HSP (SPG72). Like REEP1 it is characterized by an N-terminal Reticulon domain and is predominantly expressed in neurons. Here, we propose to compare and study the roles of REEP1 and REEP2 for membrane shaping, ER structure and the consequences for the functionality of the ER. For this purpose we also generated REEP2 knockout mice. To be able to analyze the ER structure in detail we further established a reporter strain, which expresses the tdTomato protein within the lumen of the ER, which will be mated with REEP1- and REEP2-knockout mice. With the help of advanced light microscopy we will thus be able to study the ER in axons of live cultured primary neurons or tissue samples obtained from REEP1 or REEP2 knockout mice. Apart from the analysis of the ER structure we will also address, whether ER functions such as the unfolded protein response, the secretory pathway or intracellular calcium homeostasis are compromised upon disruption of REEP1 or REEP2 or both. While HSP associated SPG31 variants are predicted to either result in the absence of the variant protein or to destroy the Reticulon domain, REEP1 variants, which leave the Reticulon domain intact, manifest as a dominant hereditary motoneuropathy (dHMN5). Therefore we also established a REEP1 knockin mouse model for a HMN associated SPG31mutation. Comparison with our REEP1 knockout mouse will hopefully allow us to address whether mutations observed in HMN act via a direct toxic effect.

• DFG Programme: Research Grants