The aim to better characterize the factors that are important for axonal maintenance prompted us to model SPG31 in mice. Based on our identification of a deletion of exon 2 in REEP1 in a patient suffering from HSP, we removed the corresponding exon 2 in mice by a Cre-lox strategy. The deletion causes a frame shift resulting in a Reep1 knockout allele, which was confirmed by the absence of REEP1 protein in brain lysates of homozygously targeted mice. During early adulthood, Reep1+/– mice developed a progressive movement disorder of the hind limbs with weakness, spasticity, and degeneration of axons of the corticospinal tract mirroring the human SPG31 phenotype. Homozygous Reep1–/– mice became symptomatic 4 weeks earlier and were more severely affected, but the restriction of the pathology to upper motor neurons was preserved. The correlation between the phenotype and the number of knockout alleles establishes loss of function as the leading pathomechanism in REEP1-related HSP and suggests that HSP in patients with other REEP1 mutations also reflect loss of function and not dominant-negative effects of truncated or mutated REEP1 protein variants.
There have been conflicting data regarding the sites of Reep1
expression. While some have reported ubiquitous expression (8
), others exclusively detected Reep1
in the brain (23
). Here, we demonstrate that Reep1
, though expressed throughout the nervous system, shows particularly strong expression in cell bodies of upper motor neurons in cortical layer V. This is consistent with the main pathology seen in pure HSP. From our Western blot analysis of cultured neurons and glial cells, we further conclude that the REEP1 protein is neuron specific. In contrast, SPAST
, which is mutated in SPG4 and is associated with a similar phenotype and pathology to that of REEP1
, has a much broader expression (25
). Regarding the developmental pattern of Reep1
expression, its transcripts were already abundant in the nervous system at embryonic stages, however, Reep1+/–
mice developed normally and had neither motor deficits nor gross alterations of the brain or spinal cord axons in their first months of life. Moreover, axonal outgrowth in neurons cultured from REEP1-deficient mice was unaffected. Thus, REEP1 is most likely not crucial for brain and spinal cord development, which fits well with the lack of phenotypes of REEP1
mutations in humans during early childhood and is consistent with the view that HSP generally manifests as a neurodegenerative rather than a neurodevelopmental disease (26
Regarding the subcellular distribution of REEP1, conflicting reports have been published, which can be attributed to difficulties in the detection of endogenous REEP1. In agreement with overexpression studies in HEK293 and HeLa cells (11
), our Western blot analysis of subcellular fractions of brain lysates clearly shows that endogenous REEP1 is associated with ER membranes, whereas the mitochondria, where REEP1 was initially reported to localize (8
), were devoid of REEP1.
Our in vitro reconstitution assays show that REEP1 directly associates with lipids. Since we used liposomes, i.e., reconstituted membranes, it can further be concluded that REEP1 is not only able to bind to lipid molecules, but can also very effectively associate with lipid bilayers. Since our assay was devoid of any other proteins, it can furthermore be concluded that this high affinity for lipid bilayers does not reflect a signal-dependent transmembrane integration, but rather is inherent to REEP1.
The live microscopic analyses and quantitative examinations of in vitro reconstitutions further show that association of REEP1 with membranes promotes positive membrane curvature, causing the constriction of liposomes and a corresponding substantial increase in the abundance of small-diameter liposome structures. In line with these data, REEP1 immunogold labeling was restricted to the surface of smaller liposomes. So far, curvature-promoting properties of ER proteins have been experimentally shown in reticulons and REEP5/Yop1p (14
), which are characterized by the presence of paired hydrophobic domains with unusual lengths thought to both insert into highly curved membranes as wedges. REEP1 is set apart from the reticulons and REEP5/Yop1p because of its shorter N-terminal hydrophobic domain (11
). Our data show that the second, longer hydrophobic domain is sufficient and its integrity critical for the shaping activity of REEP1.
In order to gain insights for the first time into the pathophysiology of REEP1 deficiency at the subcellular level in vivo, we conducted a detailed morphometric analysis of the ER in layer V neurons of the motor cortex, i.e., a region in which wild-type mice show particularly strong Reep1
expression. Quantitative evaluations showed that the number of ER structures was decreased in both Reep1+/–
mice, whereas the length of individual ER compartments was increased. Reduced ER complexity corresponds to a reduced abundance of sharply curved ER membrane surfaces such as those found at the rims of ER sheets, at fenestrae, and at tubules. These dynamic ER substructures have a positive curvature with a relatively uniform diameter of about 35 to 40 nm (28
). This value strikingly mirrors our in vitro finding that liposomes with diameters of about 40 nm are the most abundant in the presence of REEP1. Supporting this, RNAi-based knockdown of the Drosophila reticulon Rtnl1
in epidermal cell cultures was recently reported to increase the size of ER structures (29
). As reticulons have also been suggested to bend membranes, these observations may correspond to reduced ER membrane curvature similar to the effects we observed upon REEP1 knockout.
Layer V motor neurons project over an extremely long distance onto spinal cord motor neurons, thus it is conceivable that these neurons depend on a particularly elaborate ER to maintain their long axonal extensions. This is supported by evidence that proteins implicated in three other major types of dominant HSPs are also linked to the ER: ATL1
, mutated in SPG3A, encodes ATLASTIN1, which was shown to directly interact with tubule-shaping proteins to promote homotypic fusion of ER membranes and maintain proper ER network formation (30
, mutated in SPG4, encodes SPASTIN, a protein that was shown to interact with ATLASTIN1 and REEP1 and organizes the structure of the ER by connecting it to microtubules (11
). Finally, RTN2
, which is mutated in SPG12 (32
), encodes a protein of the reticulon family that has been shown to reside in and shape the ER (29
Thus, REEP1 knockout in mice and comparison with the clinical, neurodegenerative phenotype of the autosomal dominantly inherited HSP variant SPG31 revealed that loss of REEP1 leads to defects in ER organization. Whether an altered ER complexity may also interfere with ER functions like the unfolded protein response and/or specific ER export functions are important questions to address in the future.