Brain Advance Access originally published online on May 2, 2006
Brain 2006 129(7):1710-1719; doi:10.1093/brain/awl104
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The first ALS2 missense mutation associated with JPLS reveals new aspects of alsin biological function
1 IRCCS E. Medea, Laboratory of Molecular Biology Bosisio Parini Lecco 2 Stem Cell Research Institute, DIBIT-H San Raffaele Scientific Institute Milan 3 Centro Dino Ferrari Fondazione IRCCS Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Department of Neurological Sciences Milan 4 Department of Preclinical Sciences, University of Milan Milan 5 Department of Pharmaco-Biology, University of Calabria Rende 6 IRCCS E. Medea, Conegliano Research Center Conegliano 7 Dulbecco Telethon Institute at the Department of Pharmacology, University of Padova Padova, Italy 8 MRC Centre for Neurodegeneration Research, Department of Neuroscience, Institute of Psychiatry, Kings College London, UK
Correspondence to: M. T. Bassi, PhD, Laboratory of Molecular Biology, IRCCS E. Medea, Via D. L. Monza 20, 23842 Bosisio Parini Lecco, Italy E-mail: mariateresa.bassi{at}bp.lnf.it
Primary lateral sclerosis (PLS) is a rare progressive paralytic disorder that results from dysfunction of the upper motoneurons. Although PLS is a sporadic disorder of adult middle age, it has also been described in children as juvenile PLS or JPLS. The causative gene for JPLS was found to be ALS2, which is also responsible for a recessive form of amyotrophic lateral sclerosis, for infantile onset ascending hereditary spastic paralysis (IAHSP) and for a form of complicated hereditary spastic paraplegia (cHSP). ALS2 gene encodes a protein termed alsin, containing multiple guanine nucleotide exchange factor domains, specifically binding to small GTPase Rab5 and acting as a GEF for Rab5. In vitrostudies performed with full-length and truncating forms of alsin protein support its role in endosomal dynamics and trafficking of mitochondria. All ALS2 mutations so far reported generate alsin protein truncation. Here, we describe the first homozygous missense mutation in ALS2, p.G540E. The mutation, which falls within the RCC1 domain, was identified in a 34-year-old patient with typical signs of JPLS such as ascending generalized and severe spasticity involving the limbs and the bulbar region, dysphagia, limb atrophy, preserved cognition and sensation. The father and two proband's sisters were found to be heterozygous carriers of the mutation with no signs of the disease. Studies in the neuronal cell line SK-N-BE indicated that the known subcellular localization of wild-type alsin with the early endosome antigen 1, in enlarged endosomal structures, and transferrin receptor is completely lost by the mutant protein, thus indicating that this mutation leads to protein delocalization. Mutant alsin induced neuronal death itself and also significantly enhanced the apoptogenic effect of NMDA and staurosporine. This effect was associated with decreased Bcl-xL : Bax ratio. In contrast, wild-type alsin was neuroprotective and increased Bcl-xL : Bax ratio. Our results provide the first demonstration that a missense mutation in alsin is cytotoxic. In addition, the identification of Bcl-xL/Bax as target of protection by alsin and of cytotoxicity by the mutant form provides a new signalling event regulated by alsin protein that may be important to define its role in neuronal physiology and neurodegeneration. Finally, the phenotype–genotype correlation in our patient, in view of all other ALS2 mutant cases reported previously, suggests a functional interplay of long and short forms of alsin in relation to disease onset and progression.
Key Words: ALS2; alsin; missense mutation; cell death; apoptosis
Abbreviations: ALS, amyotrophic lateral sclerosis; EEA1, early endosome antigen 1; EGFP, enhanced green fluorescent protein; GEF, guanine nucleotide exchange factor; GFP, green fluorescent protein; IAHSP, infantile onset ascending hereditary spastic paralysis; JPLS, juvenile primary lateral sclerosis; LMN, lower motoneuron; NMDA, N-methyl-D-aspartic acid; TFR, transferrin receptor; UMN, upper motoneuron
Received October 2, 2005. Revised February 23, 2006. Accepted March 27, 2006.
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