A spastic paraplegia mouse model reveals REEP1-dependent ER shaping
Christian Beetz, … , Britta Qualmann, Christian A. Hübner
Christian Beetz, … , Britta Qualmann, Christian A. Hübner
Published September 24, 2013
Citation Information: J Clin Invest. 2013;123(10):4273-4282. https://doi.org/10.1172/JCI65665.
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Research Article

A spastic paraplegia mouse model reveals REEP1-dependent ER shaping

Abstract

Axonopathies are a group of clinically diverse disorders characterized by the progressive degeneration of the axons of specific neurons. In hereditary spastic paraplegia (HSP), the axons of cortical motor neurons degenerate and cause a spastic movement disorder. HSP is linked to mutations in several loci known collectively as the spastic paraplegia genes (SPGs). We identified a heterozygous receptor accessory protein 1 (REEP1) exon 2 deletion in a patient suffering from the autosomal dominantly inherited HSP variant SPG31. We generated the corresponding mouse model to study the underlying cellular pathology. Mice with heterozygous deletion of exon 2 in Reep1 displayed a gait disorder closely resembling SPG31 in humans. Homozygous exon 2 deletion resulted in the complete loss of REEP1 and a more severe phenotype with earlier onset. At the molecular level, we demonstrated that REEP1 is a neuron-specific, membrane-binding, and membrane curvature–inducing protein that resides in the ER. We further show that Reep1 expression was prominent in cortical motor neurons. In REEP1-deficient mice, these neurons showed reduced complexity of the peripheral ER upon ultrastructural analysis. Our study connects proper neuronal ER architecture to long-term axon survival.

Authors

Christian Beetz, Nicole Koch, Mukhran Khundadze, Geraldine Zimmer, Sandor Nietzsche, Nicole Hertel, Antje-Kathrin Huebner, Rizwan Mumtaz, Michaela Schweizer, Elisabeth Dirren, Kathrin N. Karle, Andrey Irintchev, Victoria Alvarez, Christoph Redies, Martin Westermann, Ingo Kurth, Thomas Deufel, Michael M. Kessels, Britta Qualmann, Christian A. Hübner

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Figure 5

REEP1 binds and curves membranes in vitro.

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REEP1 binds and curves membranes in vitro. (A) Constructs used for lipos...
(A) Constructs used for liposome-binding assays. The hydrophobic region encompassing residues 39–78 of REEP1 is marked (gray box). The positions of hydrophobic residues mutated to serines in HisTrx-REEP1-mut are indicated. (B) Liposome cofloatation assays. Proteins were detected using anti-HisTrx antibodies in immunoblots of sucrose gradient fractions 1 (top) to 6 (bottom). Tagged REEP1 and REEP1-mut, but not the tag alone, floated with liposomes in fraction 2. (C) TEM images of freeze-fractured incubations of liposomes with the indicated recombinant proteins. Scale bars: 200 nm. (D) Distribution of liposome diameters observed by TEM of freeze-fractured liposome incubations. Note that incubation with REEP1 leads to a pronounced increase in the relative numbers of 20- to 40-nm structures, the frequencies of which are strongly diminished upon incubation with HisTrx-REEP1-mut and are largely absent in the control incubations. (E) Box plots of the full set of data partially presented in C. Note that the y axis is logarithmic. ***P < 0.001 (one-way ANOVA). Boxes contain 50% of the values; minimal, maximal, and median values are marked by vertical lines. (F) High-resolution TEM analysis of freeze-fractured liposomes incubated with REEP1 and immunogold labeled for REEP1 shows REEP1 at positively curved membranes of predominantly small liposomes (arrows). Scale bar: 80 nm. (G) Sequential video frames of liposomes incubated with HisTrx control protein and HisTrx-REEP1 (taken from Supplemental Videos 3 and 4). Arrows indicate the constriction of a large liposome into two smaller, stronger curved membrane structures upon addition of HisTrx-REEP1. Scale bars: 5 μm.