# ER Morphology in the Pathogenesis of Hereditary
## 3 ER Shaping Proteins
### Reticulon-2 (SPG12)  
Reticulons (RTNs) comprise a large family of conserved membrane-spanning proteins residing in the ER. RTNs apparently arose early in the evolution of eukaryotes, where they are ubiquitous, although the number of genes encoding these proteins varies widely among species (4 RTN genes in vertebrates (RTN1-4), 2 in yeast (RTN1 and RTN2), 1 in _Drosophila melanogaster_, 1 in _Caenorhabditis elegans_, 21 and 17 in _Arabidopsis thaliana_ and _Oryza sativa_ plants, respectively [66; 67; 68]). Mutations in RTN2 have been linked to human disease and shown to be the cause of SPG12, an autosomal dominant form of pure HSP [10]. RTNs contain a so-called'reticulon homology domain' (RHD), a sequence of 150-200 amino acid residues located at the _C_-terminus characterized by two unusually long hydrophobic regions interrupted by a hydrophilic loop, which is of great importance for the localization and function of the protein [66]. The RHD is the only structural feature common to all RTNs, whose _N_-terminal part, instead, is extremely variable both in length, which ranges from a few to over one thousand amino acids, and in sequence. The hydrophobic stretches of the RHD have been demonstrated to insert into lipid bilayers as a hairpin [69] that, working like a wedge, occupies more space on the outer leaflet of the lipid bilayer than on the inner leaflet, thereby producing positive membrane curvature. Through this mechanism, RTNs have been shown to stabilize curved membrane tubules and sheet edges contributing to shape the complex architecture of this organelle [70]. Moreover, in vivo and in vitro analyses using the _Drosophila_ RTN homolog (Reticulon-like-1, Rtnl1), have shown that this protein, in addition to generating the static curvature of the moderately curved membranes of ER tubules and sheet edges, produces also extreme curvature and constriction of dynamic tubules leading to their fission, thus acting as a counterbalance to atlastin-mediated fusion in the regulation of ER morphology [71]. Since atlastins and RTNs are involved in the maintenance of ER shape, it is reasonable to speculate that disruption of this process through pathological mutation would ultimately underpin their role in the pathogenesis of HSP. While for ATL1 there is some evidence converging on this interpretation, support for a similar conclusion about RTNs is still scarce. The main obstacles to understanding how RTNs might cause HSP disease are, first, the difficulty in sorting out the phenotypes associated with loss of RTNs function and, second, their putative general involvement in neurodegeneration. RTNs have indeed been implicated in several neurodegenerative diseases such as Alzheimer's, Parkinson's, amyotrophic lateral sclerosis, and multiple sclerosis [72; 73], although a mechanistic understanding of a possible causal link between the neurodegenerative process and RTNs function is still lacking. The four mammalian RTNs exist in a number of differentially spliced isoforms that share varying degrees of homology and have potentially redundant roles that are presently not fully understood. Indeed, knock-out mice for RTN1, RTN3, and RTN4 have been generated but all homozygous mutant individuals are viable and seemingly normal, potentially suggesting a compensatory mechanism among the RTNs. In addition, examination of the ER by electron microscopy shows that loss of the RTNs in different types of cells of these mice had no effect on its tubular structure [74; 75; 76; 77; 78]. This is consistent with a report showing that even the simultaneous loss of RTN1, RTN3, and RTN4 in U2O2 cells only partially converts tubules to sheets [79] as well as with another work where in a double-deletion mutant of the two RTN genes in _S. cerevisiae_ the morphology of the peripheral ER network was normal [69]. In these models, the contradictory effects of the loss of RTNs on ER morphology seem to indicate that their individual role in shaping this organelle is dispensable, suggesting instead that a concerted action may be required.