Isoform‐specific increase of spastin stability by N‐terminal missense variants including intragenic modifiers of SPG4 hereditary spastic paraplegia

Abstract

Hereditary spastic paraplegia (HSP) is a neurodegenerative disorder selectively affecting axons of spinal cord motoneurons. Classical mutations in the most frequent HSP gene SPAST (spastin protein) act through haploinsufficiency by abolishing the activity of a C-terminal ATPase domain or by interfering with expression from the affected allele. N-terminal missense variants have been suggested to represent rare polymorphisms, to cause unusually mild phenotypes, and to aggravate the effect of a classical mutation. We confirm these associations for p.S44L but do not detect two other variants (p.E43Q; p.P45Q) in HSP patients and controls. We show that neither of several disease mechanisms associated with classical SPAST mutations applies to the N-terminal variants. Instead, all three alterations enhance the stability of one of two alternative spastin isoforms. Their phenotypic effect may thus not be mediated by haploinsufficiency but by increasing isoform competition for interacting proteins, substrates or oligomerization partners.

Sequence variants in SPAST, SPG3A and HSPD1 in hereditary spastic paraplegia

“…1). The pathogenicity of the ten sequence variants previously reported [4,7,8,14,15,18,[25][26][27] were confirmed by segregation with HSP in the families described here and by confirming the occurrence of alternative splicing for the splice site variants.…”

“…The c.122C N T (p.Pro41Leu) variant found in this study is the third, rare, N-terminal, non-synonymous sequence variant described in close proximity to the well known p.Ser44Leu variant [18]. The p.Ser44Leu variant is found in approximately 1% of controls, while the p.Glu43Gln and the p.Pro45Gln [7,10], like the p.Pro41Leu, are absent in controls [18].…”

Sequence variants in SPAST, SPG3A and HSPD1 in hereditary spastic paraplegia

“…1). The pathogenicity of the ten sequence variants previously reported [4,7,8,14,15,18,[25][26][27] were confirmed by segregation with HSP in the families described here and by confirming the occurrence of alternative splicing for the splice site variants.…”

“…The c.122C N T (p.Pro41Leu) variant found in this study is the third, rare, N-terminal, non-synonymous sequence variant described in close proximity to the well known p.Ser44Leu variant [18]. The p.Ser44Leu variant is found in approximately 1% of controls, while the p.Glu43Gln and the p.Pro45Gln [7,10], like the p.Pro41Leu, are absent in controls [18].…”

Distinct intracellular vesicle transport mechanisms are selectively modified by spastin and spastin mutations

“…Like the ΔN mutation, S44L showed no robust effects on vesicle velocity, independent of the velocity classes, when it was compared to respective controls. Previous reports of overexpression experiments indicated that S44L did not cause abnormal microtubule bundling and its localization was similar to wild‐type spastin (Errico et al, 2002; Schickel et al, 2007). Its reduced function my be explained by the amino acid exchange in its “PEST‐Sequence” between amino acids 23–45, which serves as a signal for accelerated protein degradation (Rogers et al, 1986).…”

Computational analysis of missense mutations causing Snyder-Robinson syndrome

“…Here we use the word “perturbation” to refer to any change of the WT characteristics of SMS, because the disease could be caused not only by destabilization of the 3D structure. There are many examples of a single nucleotide polymorphism that were shown to increase the stability of either RNA [Capon et al, 2004] or the corresponding protein [Allali‐Hassani et al, 2009; Schickel et al, 2007] and to enhance protein–protein interactions [Teng et al, 2009a]. The same argument can be made for any other SMS property, which if altered from its WT characteristics, could result in a pathogenic effect [Lupo et al, 2009; Najat et al, 2009] or natural difference [Merino et al, 2009].…”

Sequence variants in SPAST, SPG3A and HSPD1 in hereditary spastic paraplegia

“…1). The pathogenicity of the ten sequence variants previously reported [4,7,8,14,15,18,[25][26][27] were confirmed by segregation with HSP in the families described here and by confirming the occurrence of alternative splicing for the splice site variants.…”

“…The c.122C N T (p.Pro41Leu) variant found in this study is the third, rare, N-terminal, non-synonymous sequence variant described in close proximity to the well known p.Ser44Leu variant [18]. The p.Ser44Leu variant is found in approximately 1% of controls, while the p.Glu43Gln and the p.Pro45Gln [7,10], like the p.Pro41Leu, are absent in controls [18].…”

Distinct intracellular vesicle transport mechanisms are selectively modified by spastin and spastin mutations

“…Like the ΔN mutation, S44L showed no robust effects on vesicle velocity, independent of the velocity classes, when it was compared to respective controls. Previous reports of overexpression experiments indicated that S44L did not cause abnormal microtubule bundling and its localization was similar to wild‐type spastin (Errico et al, 2002; Schickel et al, 2007). Its reduced function my be explained by the amino acid exchange in its “PEST‐Sequence” between amino acids 23–45, which serves as a signal for accelerated protein degradation (Rogers et al, 1986).…”

Computational analysis of missense mutations causing Snyder-Robinson syndrome

“…Here we use the word “perturbation” to refer to any change of the WT characteristics of SMS, because the disease could be caused not only by destabilization of the 3D structure. There are many examples of a single nucleotide polymorphism that were shown to increase the stability of either RNA [Capon et al, 2004] or the corresponding protein [Allali‐Hassani et al, 2009; Schickel et al, 2007] and to enhance protein–protein interactions [Teng et al, 2009a]. The same argument can be made for any other SMS property, which if altered from its WT characteristics, could result in a pathogenic effect [Lupo et al, 2009; Najat et al, 2009] or natural difference [Merino et al, 2009].…”

Sequence variants in SPAST, SPG3A and HSPD1 in hereditary spastic paraplegia

“…1). The pathogenicity of the ten sequence variants previously reported [4,7,8,14,15,18,[25][26][27] were confirmed by segregation with HSP in the families described here and by confirming the occurrence of alternative splicing for the splice site variants.…”

“…The c.122C N T (p.Pro41Leu) variant found in this study is the third, rare, N-terminal, non-synonymous sequence variant described in close proximity to the well known p.Ser44Leu variant [18]. The p.Ser44Leu variant is found in approximately 1% of controls, while the p.Glu43Gln and the p.Pro45Gln [7,10], like the p.Pro41Leu, are absent in controls [18].…”

Distinct intracellular vesicle transport mechanisms are selectively modified by spastin and spastin mutations

“…Like the ΔN mutation, S44L showed no robust effects on vesicle velocity, independent of the velocity classes, when it was compared to respective controls. Previous reports of overexpression experiments indicated that S44L did not cause abnormal microtubule bundling and its localization was similar to wild‐type spastin (Errico et al, 2002; Schickel et al, 2007). Its reduced function my be explained by the amino acid exchange in its “PEST‐Sequence” between amino acids 23–45, which serves as a signal for accelerated protein degradation (Rogers et al, 1986).…”

Computational analysis of missense mutations causing Snyder-Robinson syndrome

“…Here we use the word “perturbation” to refer to any change of the WT characteristics of SMS, because the disease could be caused not only by destabilization of the 3D structure. There are many examples of a single nucleotide polymorphism that were shown to increase the stability of either RNA [Capon et al, 2004] or the corresponding protein [Allali‐Hassani et al, 2009; Schickel et al, 2007] and to enhance protein–protein interactions [Teng et al, 2009a]. The same argument can be made for any other SMS property, which if altered from its WT characteristics, could result in a pathogenic effect [Lupo et al, 2009; Najat et al, 2009] or natural difference [Merino et al, 2009].…”