A cryptic promoter in the first exon of the SPG4 gene directs the synthesis of the 60-kDa spastin isoform

Background: Mutations in SPG4 cause the most common form of autosomal dominant hereditary spastic paraplegia, a neurodegenerative disease characterized by weakness and spasticity of the lower limbs due to degeneration of the corticospinal tract. SPG4 encodes spastin, a microtubule-severing ATPase belonging to the AAA family. Two isoforms of spastin, 68 and 60 kDa, respectively, are variably abundant in tissues, show different subcellular localizations and interact with distinct molecules. The isoforms arise through alternative initiation of translation from two AUG codons in exon I; however, it is unclear how regulation of their expression may be achieved. Results: We present data that rule out the hypothesis that a cap-independent mechanism may be involved in the translation of the 60-kDa spastin isoform. Instead, we provide evidence for a complex transcriptional regulation of SPG4 that involves both a TATA-less ubiquitous promoter and a cryptic promoter in exon I. The cryptic promoter covers the 5'-UTR and overlaps with the coding region of the gene. By using promoter-less constructs in various experimental settings, we found that the cryptic promoter is active in HeLa, HEK293 and motoneuronal NSC34 cells but not in SH-SY-5Y neuroblastoma cells. We showed that the cryptic promoter directs the synthesis of a SPG4 transcript that contains a shorter 5'-UTR and translates the 60-kDa spastin isoform selectively. Two polymorphisms (S44L and P45Q), leading to an early onset severe form of hereditary spastic paraplegia when present in heterozygosity with a mutant allele, fall a few nucleotides downstream of the novel transcriptional start site, opening up the possibility that they may exert their modifier effect at the transcriptional level. We provide evidence that at least one of them decreases the activity of the cryptic promoter in luciferase assays. Conclusion: We identified a cryptic promoter in exon I of the SPG4 gene that selectively drives the expression of the 60-kDa spastin isoform in a tissue-regulated manner. These data may have implications for the understanding of the biology of spastin and the pathogenic basis of hereditary spastic paraplegia.