Cellular distribution and subcellular localization of spatacsin and spastizin, two proteins involved in hereditary spastic paraplegia

https://doi.org/10.1016/j.mcn.2011.04.004Get rights and content

Abstract

Truncating mutations in the SPG11 and SPG15 genes cause complicated spastic paraplegia, severe neurological conditions due to loss of the functions of spatacsin and spastizin, respectively. We developed specific polyclonal anti-spatacsin (SPG11) and anti-spastizin (SPG15) antisera, which we then used to explore the intracellular and tissue localizations of these proteins. We observed expression of both proteins in human and rat central nervous system, which was particularly strong in cortical and spinal motor neurons as well as in retina. Both proteins were also expressed ubiquitously and strongly in embryos. In cultured cells, these two proteins had similar diffuse punctate, cytoplasmic and sometimes nuclear (spastizin) distributions. They partially co-localized with multiple organelles, particularly with protein-trafficking vesicles, endoplasmic reticulum and microtubules. Spastizin was also found at the mitochondria surface. This first study of the endogenous expression of spatacsin and spastizin shows similarities in their expression patterns that could account for their overlapping clinical phenotypes and involvement in a common protein complex.

Introduction

Hereditary spastic paraplegias (HSP) are clinically and genetically heterogeneous inherited neurodegenerative disorders characterized by progressive weakness and spasticity in the lower limbs (Harding, 1983, Boukhris et al., 2009, Stevanin et al., 2008a). Neuropathologically, the disease is characterized by degeneration of the terminal portion of long descending (corticospinal tracts) and ascending (dorsal columns) pathways in the spinal cord, although some studies have shown degeneration of syndrome-specific structures and loss of Betz cells in the motor cortex (Behan and Maia, 1974, Bruyn, 1992, White et al., 2000, Seidel et al., 2009, Deluca et al., 2004).

Clinically, HSP are classified as (1) “uncomplicated or pure forms” which classically involve lower limb spasticity only and (2) “complicated or complex HSP” where additional neurological symptoms, such as mental retardation, dementia, cerebellar ataxia, epilepsy, peripheral neuropathy and retinitis pigmentosa, are variably present (Harding, 1983, Harding, 1993, Fink, 2003). Mendelian modes of inheritance (autosomal dominant, autosomal recessive and X-linked recessive) are observed in HSP (Harding, 1983, Fink, 2003). So far, more than 43 distinct HSP loci (SPG) and 21 disease-associated genes have been identified (Stevanin, 2010).

Mutations in the SPG11 and SPG15 genes are responsible for the majority of autosomal recessive complicated HSP with thin corpus callosum and mental impairment (ARHSP-TCC), accounting for ~ 70% of such cases and approximately 25% of all ARHSP (Hehr et al., 2007, Boukhris et al., 2008, Stevanin et al., 2008b, Denora et al., 2009a, Denora et al., 2009b, Goizet et al., 2009). The spectrum of mutations in SPG11 and SPG15 is broad, leading to the loss of function of the corresponding proteins (Stevanin et al., 2007, Erichsen et al., 2008, Hanein et al., 2008, Paisan-Ruiz et al., 2008, Bauer et al., 2009, Denora et al., 2009a, Denora et al., 2009b). Interestingly, patients with mutations in SPG11 and SPG15 genes have very similar symptoms (Boukhris et al., 2009, Denora et al., 2009a, Denora et al., 2009b, Orlen et al., 2009).

Although many genes responsible for HSP have been identified, the mechanisms underlying the degeneration of the corticospinal tracts and syndrome-specific structures remain unknown. Abnormalities in mitochondrial functions, lipid metabolism, intracellular trafficking, axonal transport and myelination are implicated in several forms of HSP (Soderblom and Blackstone, 2006, Stevanin et al., 2008a). Very little is known about the pathogenesis of SPG11- and SPG15-associated HSP.

In this study, we investigated the endogenous expression of spatacsin (SPG11) and spastizin (SPG15) in human and rodent tissues and in cells in culture, using newly generated anti-spatacsin and anti-spastizin antisera. We found that the two proteins had similar tissue distributions in the central nervous system (CNS), including in the retina. Furthermore, we found that both proteins had a neuronal pattern of expression in the brain. Widespread and strong expression of both proteins was also detected in rat embryonic tissues inside and outside the nervous system. In cell cultures, both proteins had diffuse, cytoplasmic and punctate distributions. Partial co-localization observed with various cellular markers and electron microscopy (EM) suggests a role for these proteins in protein/vesicle trafficking and/or mitochondrial functions.

Section snippets

Validation of anti-spatacsin and anti-spastizin antibodies

Affinity-purified rabbit polyclonal anti-spatacsin (HAL: residues 531–546) and anti-spastizin (PER: residues 1207–1224) antibodies were tested on Western blots on non-human primate, murine and human cell lines (COS-7, NSC34, PC12, HEK-293 and SH-SY5Y). Both full-length proteins were detected in all cell lines at their predicted size, ~ 270 kDa and ~ 280 kDa for spatacsin and spastizin, respectively (Fig. 1A and C). Specificity was validated by peptide blocking and RNA interference. Incubation of

Discussion

The SPG11 and SPG15 genes, when mutated, give rise to similar clinical presentation in patients, i.e. early onset spastic paraplegia associated with additional neurological symptoms (Hehr et al., 2007, Stevanin et al., 2008b, Paisan-Ruiz et al., 2008, Boukhris et al., 2009, Denora et al., 2009a, Goizet et al., 2009). This observation prompted us to study these two genes, the functions of which are largely unknown, in parallel. To date, no other studies on the tissue and sub-cellular

Production and verification of anti-spatacsin and anti-spastizin antisera

Rabbit polyclonal antibodies were generated at Proteogenix (France) against peptides corresponding to residues 531–546, HALEAGIENRQLDTVN (HAL) in spatacsin and residues 1207–1224, PERLAALLAQENLSLSVP (PER) in spastizin. These peptides were positioned outside known domains and in regions conserved between both human and murine spatacsin and spastizin, in order to detect both orthologues. The antibodies were affinity purified.

Constructs for over-expression studies

We amplified the KIAA1840 cDNA by PCR using primers

Acknowledgments

We would like to thank Dr. Dominique Langui for providing assistance with electron microscopy. We also thank Dr. Serge Picaud for providing us with the anti-rhodopsin antibody. This work was supported by grants from the Verum foundation (to AB) and the French National Agency for Research (to GS and to the EUROSPA E-rare consortium). EM received a fellowship from the Fondation pour la Recherche Médicale (France, Grant Line Pomaret-Delalande) and PSD was financially supported by IRCCS Bambino

References (41)

  • M. Anheim et al.

    SPG11 spastic paraplegia. A new cause of juvenile parkinsonism

    J. Neurol.

    (2009)
  • P. Bauer et al.

    Identification of a heterozygous genomic deletion in the spatacsin gene in SPG11 patients using high-resolution comparative genomic hybridization

    Neurogenetics

    (2009)
  • W.M. Behan et al.

    Strumpell's familial spastic paraplegia: genetics and neuropathology

    J. Neurol. Neurosurg. Psychiatry

    (1974)
  • A. Boukhris et al.

    Hereditary spastic paraplegia with mental impairment and thin corpus callosum in Tunisia: SPG11, SPG15 and further genetic heterogeneity

    Arch. Neurol.

    (2008)
  • A. Boukhris et al.

    Tunisian hereditary spastic paraplegias: clinical variability supported by genetic heterogeneity

    Clin. Genet.

    (2009)
  • K. Brockmann et al.

    Complicated hereditary spastic paraplegia with thin corpus callosum (HSP-TCC) and childhood onset

    Neuropediatrics

    (2005)
  • R.P. Bruyn

    The neuropathology of hereditary spastic paraparesis

    Clin. Neurol. Neurosurg.

    (1992)
  • N.R. Cashman et al.

    Neuroblastoma × spinal cord (NSC) hybrid cell lines resemble developing motor neurons

    Dev. Dyn.

    (1992)
  • G.C. Deluca et al.

    The extent of axonal loss in the long tracts in hereditary spastic paraplegia

    Neuropathol. Appl. Neurobiol.

    (2004)
  • P.S. Denora et al.

    Screening of ARHSP-TCC patients expands the spectrum of SPG11 mutations and includes a large scale gene deletion

    Hum. Mutat.

    (2009)
  • Cited by (66)

    • Cytosolic sequestration of spatacsin by Protein Kinase A and 14-3-3 proteins

      2022, Neurobiology of Disease
      Citation Excerpt :

      Despite the large amount of genetic contributors, HSP genes have been associated with a relatively small number of cellular functions related to organelle biogenesis and membrane trafficking, lipid and mitochondria metabolism, development and myelination, as well as axonal transport and autophagy (Darios et al., 2020; Blackstone, 2018; Lo Giudice et al., 2014). Spatacsin is a 270 kDa protein, highly expressed throughout the central nervous system, particularly in the motor cortex, spinal cord, hippocampus, cerebellum, dentate nucleus and pons (Murmu et al., 2011; Sharma et al., 2015). Despite its association with multiple neurodegenerative disorders, including HSP, juvenile Parkinson's disease, Charcot-Marie-Tooth disease, amyotrophic lateral sclerosis and Kjellin's syndrome (Patto and O'kane, 2020), very little is yet known about the physiological role(s) of spatacsin and the consequences of its loss of function.

    • Ophthalmological changes in hereditary spastic paraplegia and other genetic diseases with spastic paraplegia

      2020, Journal of the Neurological Sciences
      Citation Excerpt :

      Despite the advances in the understanding of the motor phenotype of SPG11, the pathogenesis of retinal degeneration in Kjellin syndrome remains unclear. However, strong expression of spatacsin and spastizin has been reported in the retina and photoreceptor cells of an animal model [20]. Thus, it may be speculated that abnormal autophagy affects retinal pigment epithelium function, leading defective degradation of external segments of photoreceptors and accumulation of lipofuscin and related molecules in the retina of individuals with Kjellin syndrome.

    View all citing articles on Scopus
    View full text