NIPA1(SPG6), the Basis for Autosomal Dominant Form of Hereditary Spastic Paraplegia, Encodes a Functional Mg2+ Transporter

Mutations in the NIPA1(SPG6) gene, named for “nonimprinted in Prader-Willi/Angelman” has been implicated in one form of autosomal dominant hereditary spastic paraplegia (HSP), a neurodegenerative disorder characterized by progressive lower limb spasticity and weakness. However, the function of NIPA1 is unknown. Here, we show that reduced magnesium concentration enhances expression of NIPA1 suggesting a role in cellular magnesium metabolism. Indeed NIPA1 mediates Mg2+ uptake that is electrogenic, voltage-dependent, and saturable with a Michaelis constant of 0.69 ± 0.21 mm when expressed in Xenopus oocytes. Subcellular localization with immunofluorescence showed that endogenous NIPA1 protein associates with early endosomes and the cell surface in a variety of neuronal and epithelial cells. As expected of a magnesium-responsive gene, we find that altered magnesium concentration leads to a redistribution between the endosomal compartment and the plasma membrane; high magnesium results in diminished cell surface NIPA1 whereas low magnesium leads to accumulation in early endosomes and recruitment to the plasma membrane. The mouse NIPA1 mutants, T39R and G100R, corresponding to the respective human mutants showed a loss-of-function when expressed in oocytes and altered trafficking in transfected COS7 cells. We conclude that NIPA1 normally encodes a Mg2+ transporter and the loss-of function of NIPA1(SPG6) due to abnormal trafficking of the mutated protein provides the basis of the HSP phenotype. Mutations in the NIPA1(SPG6) gene, named for “nonimprinted in Prader-Willi/Angelman” has been implicated in one form of autosomal dominant hereditary spastic paraplegia (HSP), a neurodegenerative disorder characterized by progressive lower limb spasticity and weakness. However, the function of NIPA1 is unknown. Here, we show that reduced magnesium concentration enhances expression of NIPA1 suggesting a role in cellular magnesium metabolism. Indeed NIPA1 mediates Mg2+ uptake that is electrogenic, voltage-dependent, and saturable with a Michaelis constant of 0.69 ± 0.21 mm when expressed in Xenopus oocytes. Subcellular localization with immunofluorescence showed that endogenous NIPA1 protein associates with early endosomes and the cell surface in a variety of neuronal and epithelial cells. As expected of a magnesium-responsive gene, we find that altered magnesium concentration leads to a redistribution between the endosomal compartment and the plasma membrane; high magnesium results in diminished cell surface NIPA1 whereas low magnesium leads to accumulation in early endosomes and recruitment to the plasma membrane. The mouse NIPA1 mutants, T39R and G100R, corresponding to the respective human mutants showed a loss-of-function when expressed in oocytes and altered trafficking in transfected COS7 cells. We conclude that NIPA1 normally encodes a Mg2+ transporter and the loss-of function of NIPA1(SPG6) due to abnormal trafficking of the mutated protein provides the basis of the HSP phenotype. The NIPA1 [NT_078094] gene is named for “nonimprinted in Prader-Willi/Angelman” because it was thought to be located among about 30 imprinted genes linked to chromosome 15q11-q13 (SPG6 locus) involved in the Prader-Willi syndrome (1Butler M.G. Am. J. Med. Genet. 1990; 35: 319-332Crossref PubMed Scopus (433) Google Scholar, 2Nicholls R.D. Gottlieb W. Russell L.B. Davda M. Horsthemke B. Rinchik E.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2050-2054Crossref PubMed Scopus (69) Google Scholar, 3Nicholls R.D. Knepper J.L. Annu. Rev. Genomics Hum. Genet. 2001; 2: 153-175Crossref PubMed Scopus (501) Google Scholar, 4Butler M.G. Bittel D.C. Kibiryeva N. Talebizadeh Z. Thompson T. Pediatrics. 2004; 113: 565-573Crossref PubMed Scopus (213) Google Scholar, 5Chai J-H. Locke D.P. Greally J.M. Knoll J.H.M. Ohta T. Dunai J. Yavor A. Eichler E.E. Nicholls R.D. Am. J. Hum. Genet. 2003; 73: 898-925Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). However, NIPA1 has also been implicated in another distinct disorder termed autosomal dominant hereditary spastic paraplegia (HSP) 4The abbreviations used are: HSP, hereditary spastic paraplegia; HA, hemagglutinin; MDCT, mouse distal convoluted tubule; PBS, phosphate-buffered saline; TMD, transmembrane domain. 4The abbreviations used are: HSP, hereditary spastic paraplegia; HA, hemagglutinin; MDCT, mouse distal convoluted tubule; PBS, phosphate-buffered saline; TMD, transmembrane domain. (OMIM 608145 and 600363). HSP comprises more than 30 genetic disorders whose predominant feature is a spastic gait (6Fink J.K. Arch. Neurol. 2003; 60: 1045-1049Crossref PubMed Scopus (118) Google Scholar). Mutations in at least six genes have been associated with autosomal dominant HSP including NIPA1(SPG6). This heterogenous group presents with progressive lower limb spasticity and weakness. In the absence of other clinical features these disorders are referred to as pure or uncomplicated HSP (6Fink J.K. Arch. Neurol. 2003; 60: 1045-1049Crossref PubMed Scopus (118) Google Scholar). Fink et al. (7Fink J.K. Sharp G.B. Lange B.M. Wu C.B. Haley T. Otterud B. Peacock M. Leppert M. Neurology. 1995; 45: 325-331Crossref PubMed Scopus (30) Google Scholar, 8Fink J.K. Wu C.T. Jones S.M. Sharp G.B. Lange B.M. Lesicki A. Reinglass T. Varvil T. Otterud B. Leppert M. Am. J. Hum. Genet. 1995; 56: 188-192Crossref PubMed Scopus (36) Google Scholar) reported that uncomplicated HSP was linked to chromosome 15q, the region of NIPA1. Additional studies by this group identified a nucleotide substitution at position 134 of the NIPA1 cDNA that resulted in an amino acid substitution at position 45 of the NIPA1 protein (T45R) in SPG6-linked HSP kindred and in an unrelated kindred that was too small for linkage analysis (9Rainer S. Chai J-H. Tokarz D. Nicholls R.D. Fink J.K. Am. J. Hum. Genet. 2003; 3: 967-971Abstract Full Text Full Text PDF Scopus (165) Google Scholar). More recently, three different research groups have identified a missense substitution in NIPA1, G106R, in a number of large unrelated families (10Reed J.A. Wilkinson P.A. Patel H. Simpson M.A. Chatonnet A. Robay D. Patton M.A. Crosby A.H. Warner T.T. Neurogenetics. 2005; 6: 79-84Crossref PubMed Scopus (47) Google Scholar, 11Chen S. Song C. Guo H. Xu P. Huang W. Zhou Y. Sun J. Li C.X. Du Y. Li X. Liu Z. Geng D. Maxwell P.H. Zhang C. Wang Y. Hum. Mutat. 2005; 25: 135-141Crossref PubMed Scopus (49) Google Scholar, 12Munhoz R.P. Kawarai T. Teive H.A. Raskin S. Sato C. Liang Y. St George-Hyslop P.H. Rogaeva E. Mov. Disord. 2006; 21: 279-281Crossref PubMed Scopus (19) Google Scholar). The functional role of NIPA1 in Prader-Willi or HSP syndromes has not been determined. Magnesium is the second most abundant cation within the cell and plays an important role in many intracellular biochemical functions (13Quamme G.A. Kidney Int. 1997; 52: 1180-1195Abstract Full Text PDF PubMed Scopus (282) Google Scholar). Despite the abundance and importance of magnesium, little is known about how eukaryotic cells regulate their magnesium content. Intracellular free Mg2+ concentration is in the order of 0.5 mm, which is 1–2% of the total cellular magnesium (13Quamme G.A. Kidney Int. 1997; 52: 1180-1195Abstract Full Text PDF PubMed Scopus (282) Google Scholar). Accordingly, intracellular Mg2+ is maintained below the concentration predicted from the transmembrane electrochemical potential. Intracellular Mg2+ concentration is finely regulated likely by precise controls of Mg2+ entry, Mg2+ efflux, and intracellular storage compartments (14Dai L-J. Ritchie G. Kerstan D. Kang H.S. Cole D.E.C. Quamme G.A. Physiol. Rev. 2001; 81: 51-84Crossref PubMed Scopus (258) Google Scholar). We have shown that Mg2+ entry is through specific and regulated magnesium pathways that are regulated by intrinsic mechanisms so that the culture of cells in media containing low magnesium results in up-regulation of Mg2+ uptake into the cells. This adaptive increase in Mg2+ entry was shown to be dependent on de novo transcription since prior treatment of the epithelial cells with actinomycin D prevented the adaptation to low extracellular magnesium (15Dai L-J. Quamme G.A. J. Clin. Invest. 1991; 88: 1255-1264Crossref PubMed Scopus (60) Google Scholar). The data suggest that epithelial cells can sense the environmental magnesium and through transcription- and translation-dependent processes alter Mg2+ transport and maintain magnesium balance. In an attempt to identify genes underlying cellular changes resulting from adaptation to low extracellular magnesium, we used oligonucleotide microarray analysis to screen for magnesium-regulated transcripts in epithelial cells (16Goytain A. Quamme G.A. BMC Genomics. 2005; 6: 48Crossref PubMed Scopus (135) Google Scholar). One transcript, NIPA2, was significantly up-regulated by extracellular magnesium suggesting that the synthesis was regulated by changes in cell magnesium. 5A. Goytain, R. M. Hines, A. El-Husseini, and G. A. Quamme, unpublished observations. 5A. Goytain, R. M. Hines, A. El-Husseini, and G. A. Quamme, unpublished observations. We showed that NIPA2 mediated Mg2+ transport when expressed in Xenopus laevis oocytes. An in silico search demonstrated a second member of this family of proteins, NIPA1. The goal of the present study was to see if NIPA1 mediates Mg2+ transport using electrophysiological and fluorescence studies. Furthermore, cellular distribution and subcellular localization was determined by Western blot analysis and immunofluorescence microscopy. Redistribution of NIPA1 protein was evaluated in response to changes in cellular magnesium. Finally, the NIPA1-T39R and G100R mutants, associated with HSP, were created and tested. Our data indicate that NIPA1 protein mediates Mg2+ transport and is regulated by magnesium indicating that it may play a role in control of cellular magnesium homeostasis. The T39R and G100R mutations resulted in altered intracellular trafficking of NIPA1 protein and diminished Mg2+ transport suggesting a role in the HSP phenotype. Construction of Expression Vectors Encoding NIPA1—A mouse NIPA1 cDNA clone was purchased from RIKEN, catalog number B430207K20. The human NIPA1 cDNA is longer than the mouse due an were to The the region of the cDNA by of and of mouse and in which was with and was with were from using the was to NIPA1 cDNA by with an oligonucleotide containing the was into the the with resulting in the COS7 cells were as below and transfected with using Expression of NIPA1 in Xenopus and of Mg2+ Xenopus was from and cDNA and with in the of using the transcription of with and were as and at (16Goytain A. Quamme G.A. BMC Genomics. 2005; 6: 48Crossref PubMed Scopus (135) Google Scholar). were were using the and and with analysis (16Goytain A. Quamme G.A. BMC Genomics. 2005; 6: 48Crossref PubMed Scopus (135) Google Scholar). was used to Mg2+ into oocytes using the fluorescence (16Goytain A. Quamme G.A. BMC Genomics. 2005; 6: 48Crossref PubMed Scopus (135) Google Scholar). were with acid prior to The was on an with a and an determined. were at for fluorescence for the was using a with for at and at and at are as the which the intracellular Mg2+ and were maintained for on a low magnesium number with that is with mouse Quamme G.A. Am. J. Physiol. Google Scholar). mouse distal convoluted and neuronal cells were in with mm and in a of at were from and using to that N. Huang Kang R. Wang A. J. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). cells were in mm, or high mm, for or prior to or for as reported (16Goytain A. Quamme G.A. BMC Genomics. 2005; 6: 48Crossref PubMed Scopus (135) Google Scholar). of the culture were to the of NIPA1 by were with using fluorescence to the The of mouse NIPA1 and The of NIPA1 were to the mouse NIPA1 cDNA was determined by The NIPA1 amino acid is in the data and were identified using and the data was predicted by the on analysis J. J. PubMed Scopus Google Scholar). Western was the extracellular of the human NIPA1 protein using a acid NIPA1 was in mm mm containing at a concentration about Western analysis was by the with at by three with PBS, The were with for three with the were with to the of cells were at for in were three with phosphate-buffered containing The were NIPA1, and that were in mouse and were from were in of in for and were used to for in the to in were on with were in and in in were through oocytes and were in and for using the NIPA1 and were using a to a and or were from of and used for of of NIPA1 cDNA a the that gene expression is involved with control of epithelial cell magnesium was to microarray analysis to identify that were up-regulated with low magnesium (15Dai L-J. Quamme G.A. J. Clin. Invest. 1991; 88: 1255-1264Crossref PubMed Scopus (60) Google Scholar). As was to identify transport proteins, we these to reported for One of the cDNA identified by an increase in was NIPA2 protein search of the data was and another member of this NIPA1, was As NIPA1 was not on the mouse and used at the of microarray we showed that the NIPA1 transcript, NIPA2 is regulated by magnesium using NIPA1 in distal convoluted MDCT, epithelial in low magnesium with cells that NIPA1 is regulated by magnesium. of human and mouse of the NIPA1 is the least member of the family it is located in with NIPA2 on chromosome The human and mouse NIPA1 were to be in amino acid by a of on the The human NIPA1 and mouse NIPA1 between and to the other three respective human and mouse amino acid are present in the and indicating that of the NIPA1 from NIPA2, and was an early in using the predicted a with predicted transmembrane are the mouse T39R and G100R that to the respective human and located in the predicted and NIPA1 Mg2+ in Xenopus NIPA1 encodes a functional Mg2+ transporter as we mouse NIPA1 it into Xenopus oocytes and using analysis and Mg2+ using fluorescence The electrophysiological data for a with in NIPA1 whereas were in control or total cells from the of oocytes. was a in with a increase in magnesium which the predicted by the the of is the of The at a were saturable a Michaelis constant of 0.69 ± 0.21 mm that was of the not fluorescence that the were due to Mg2+ magnesium the of at was not at to the fluorescence of the and not at to for the using a specific surface localization of NIPA1 protein in whereas was in oocytes The second of most is Accordingly, a variety of extracellular were used to the of the expressed NIPA1 NIPA1 was for Mg2+ The of was than Mg2+ and the of other and were small with by were for changes in by the respective cation using from oocytes Accordingly, cation transport was for Finally, a of is the to be by not We if the that were not by NIPA1 large of mm and mm were in the of mm Mg2+ whereas not transport as by the in for these data indicate that transport and the to be by other of NIPA1 we show that NIPA1 transcripts are expressed among the mouse tested. Western blot analysis was to the endogenous NIPA1 protein expression in the mouse of NIPA1 a specific was by of was an at the expected of in and a of in the and in the small was in the and that may suggest in these cells. were in the and at about and a in that may the of the The was for three As the NIPA1 is to magnesium, we determined NIPA1 protein expression in cells in low magnesium media to magnesium. the mouse cell MDCT, as a epithelial Western blot analysis about a increase in protein in cells in low magnesium the predominant increase was in the with in the The a of the NIPA1 may be the Subcellular of NIPA1 the subcellular localization of NIPA1 we immunofluorescence using the specific in COS7 cells. NIPA1 with and that are early and Furthermore, the of NIPA1 is with the of NIPA1 in early the localization of NIPA1 to early a form of was NIPA1 with the expressed in endosomes In to the endosomal NIPA1 was the cell surface at the as by of of the resulted in the absence of not As endogenous NIPA1 was with the this of distribution was not the of protein resulting from This of distribution is with the that NIPA1 through the early likely also endosomes and the plasma membrane. from the COS7 cells are because the intracellular was to with of distribution were with and cells not and neuronal cells NIPA1 was in a in the and of the and was from The that endogenous NIPA1 is present at the surface is in with the functional studies with oocytes. in Magnesium to Subcellular Redistribution of NIPA1 NIPA1 is involved with cellular magnesium we that may be subcellular redistribution of protein in response to changes in magnesium COS7 cells were in high magnesium, mm for or or in for or and subcellular localization was determined with immunofluorescence as The most changes in NIPA1 subcellular distribution within the surface and the early endosomes reduced in the culture in high to cells in culture media containing 0.5 mm magnesium, NIPA1 protein was reduced at the surface and in the early endosomes This trafficking from the surface to the early compartment was at and at the to high magnesium The accumulation of NIPA1 protein was large to as of protein in the early endosomes the cells in low magnesium for and to an increase in NIPA1 protein in the early not it was more within the endosomes with that with magnesium was a recruitment of NIPA1 protein to the surface that was at and at of magnesium. the surface with NIPA1 was so that the cell was suggesting that it was in the plasma in Mutations of NIPA1 et al. (9Rainer S. Chai J-H. Tokarz D. Nicholls R.D. Fink J.K. Am. J. Hum. Genet. 2003; 3: 967-971Abstract Full Text Full Text PDF Scopus (165) Google Scholar) identified a nucleotide substitution at position 134 of the NIPA1 cDNA that resulted in an amino acid substitution at position 45 of the NIPA1 protein (T45R) in a HSP This to position of the mouse NIPA1. The T39R at a at the of the transmembrane and the This be expected to the by three amino that may alter protein trafficking or transport we with and the associated into oocytes and and fluorescence The T39R to diminished Mg2+ transport by about with the NIPA1 as determined by and fluorescence This be by a of to to the plasma or by that normally are between these the NIPA1 and the were with so that cell surface expression be evaluated in transfected COS7 cells by immunofluorescence The was expressed in the early endosomes as as the cell surface in a as was with immunofluorescence with the NIPA1 The plasma was than and was of the in the the and are identified by was also an increase in endosomal that suggest abnormal protein within the endosomes (10Reed J.A. Wilkinson P.A. Patel H. Simpson M.A. Chatonnet A. Robay D. Patton M.A. Crosby A.H. Warner T.T. Neurogenetics. 2005; 6: 79-84Crossref PubMed Scopus (47) Google S. Song C. Guo H. Xu P. Huang W. Zhou Y. Sun J. Li C.X. Du Y. Li X. Liu Z. Geng D. Maxwell P.H. Zhang C. Wang Y. Hum. Mutat. 2005; 25: 135-141Crossref PubMed Scopus (49) Google R.P. Kawarai T. Teive H.A. Raskin S. Sato C. Liang Y. St George-Hyslop P.H. Rogaeva E. Mov. Disord. 2006; 21: 279-281Crossref PubMed Scopus (19) Google and their respective have identified a missense G106R, in large unrelated families with The human to mouse G100R This is located in a amino acid region within the of the so that it be expected to significantly alter the of the protein (10Reed J.A. Wilkinson P.A. Patel H. Simpson M.A. Chatonnet A. Robay D. Patton M.A. Crosby A.H. Warner T.T. Neurogenetics. 2005; 6: 79-84Crossref PubMed Scopus (47) Google Scholar). We with and the associated was into oocytes. The G100R Mg2+ transport as determined by or fluorescence and In with these the was in the and was little trafficking of the transport protein to the cell surface and As with the T39R was an increase in endosomal protein We conclude that diminished surface expression of and the absence of protein results in the and of Mg2+ of these We show that NIPA1, the basis of of HSP, mediates Mg2+ The that NIPA1 is a magnesium transporter is expression of NIPA1 in Xenopus oocytes with with The to the with a of as predicted by the with in magnesium Mg2+ are and as be expected of a NIPA1 mediates Mg2+ as determined by fluorescence with the NIPA1 and protein are altered with changes in Mg2+ with (16Goytain A. Quamme G.A. BMC Genomics. 2005; 6: 48Crossref PubMed Scopus (135) Google Scholar). we that NIPA1 trafficking to the early endosomes and plasma is with diminished extracellular as be expected of a magnesium-regulated Finally, we show that the and mutations to a of Mg2+ transport in oocytes the altered NIPA1. We conclude that NIPA1 encodes a Mg2+ is not is how a NIPA1 loss-of-function can to a that is to spastic et al. (9Rainer S. Chai J-H. Tokarz D. Nicholls R.D. Fink J.K. Am. J. Hum. Genet. 2003; 3: 967-971Abstract Full Text Full Text PDF Scopus (165) Google Scholar) and Chai et al. J-H. Locke D.P. Greally J.M. Knoll J.H.M. Ohta T. Dunai J. Yavor A. Eichler E.E. Nicholls R.D. Am. J. Hum. Genet. 2003; 73: 898-925Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar) have reported the of NIPA1 and in human and mouse J-H. Locke D.P. Greally J.M. Knoll J.H.M. Ohta T. Dunai J. Yavor A. Eichler E.E. Nicholls R.D. Am. J. Hum. Genet. 2003; 73: 898-925Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, S. Chai J-H. Tokarz D. Nicholls R.D. Fink J.K. Am. J. Hum. Genet. 2003; 3: 967-971Abstract Full Text Full Text PDF Scopus (165) Google Scholar). The from within J-H. Locke D.P. Greally J.M. Knoll J.H.M. Ohta T. Dunai J. Yavor A. Eichler E.E. Nicholls R.D. Am. J. Hum. Genet. 2003; 73: 898-925Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). The are expressed in are in J-H. Locke D.P. Greally J.M. Knoll J.H.M. Ohta T. Dunai J. Yavor A. Eichler E.E. Nicholls R.D. Am. J. Hum. Genet. 2003; 73: 898-925Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, S. Chai J-H. Tokarz D. Nicholls R.D. Fink J.K. Am. J. Hum. Genet. 2003; 3: 967-971Abstract Full Text Full Text PDF Scopus (165) Google Scholar). Our results using Western analysis are with these observations. a specific that we in we show that the endogenous NIPA1 protein is present in many abundant in the form in cells to be about the predicted from the amino acid that that it may be a and or for their In response to low magnesium, was a increase in NIPA1 protein in mouse cells. the increase was in the form with in the We that the form of the NIPA1 transporter is the protein is expressed in epithelial cells in low magnesium that is associated with Mg2+ on the that the form in the cell is the we that the NIPA1 transporter is in the the NIPA1 protein was in the early endosomal compartment and the was little protein in the or the endosomes and This localization that the NIPA1 protein plays a role at the surface membrane. In of this is the redistribution of NIPA1 in response to magnesium. was an trafficking from the surface and in the endosomes in cells in high magnesium, whereas was a of NIPA1 to the with low magnesium. with the Western was a increase in total protein in epithelial cells in low magnesium and an of NIPA1 protein in early endosomes a We conclude that high magnesium leads to diminished surface expression of NIPA1 protein and in endosomes whereas low magnesium results in an increase in early endosomal NIPA1 protein and to the surface membrane. changes in protein expression likely in for the associated changes in Mg2+ Finally, we show that the and mutations to a of Mg2+ transport in oocytes the altered NIPA1. The missense mutations G100R and to a loss-of-function suggesting that and are if not in of the NIPA1 Mg2+ transport and are among NIPA1 the that the is More recently, has been another reported in a family with the HSP S. Kawarai T. E. S. Sato C. A. G. Liang Y. H. Rogaeva E. St George-Hyslop P. Mov. Disord. 2006; 21: PubMed Scopus (19) Google Scholar). The changes in transport to be determined. are in the other of the the NIPA2 or the and so that the protein is likely different between these The of these in the of the to be is also that leads to loss-of-function whereas mediates Mg2+ be to see if are in clinical between these groups of HSP The present show that NIPA1 a Mg2+ a Mg2+ transporter to the of HSP is In to the progressive lower limb with uncomplicated HSP have and in the (9Rainer S. Chai J-H. Tokarz D. Nicholls R.D. Fink J.K. Am. J. Hum. Genet. 2003; 3: 967-971Abstract Full Text Full Text PDF Scopus (165) Google Scholar). in magnesium in these have not been reported has the of magnesium been of is HSP present with in their or than or (6Fink J.K. Arch. Neurol. 2003; 60: 1045-1049Crossref PubMed Scopus (118) Google Scholar). may suggest an or control of NIPA1 gene because HSP is a and heterogenous group of neurodegenerative it is likely that other and genes can to the of the in the HSP P. P. S. Fink J.K. 2005; PubMed Scopus Google Scholar). et al. P. P. S. Fink J.K. 2005; PubMed Scopus Google Scholar) have shown that early and in due to suggesting of the the lower analysis of the not constant that the NIPA1 HSP from the control Accordingly, it is to on the function of NIPA1 in the and or other cells it is We conclude from the present studies that NIPA1 normally encodes a Mg2+ transporter and the loss-of function of NIPA1(SPG6) due to of the mutated protein provides the basis for the HSP phenotype. with

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