Targeted disruption of the Mast syndrome gene SPG21 in mice impairs hind limb function and alters axon branching in cultured cortical neurons
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- Soderblom, C., Stadler, J., Jupille, H. et al. Neurogenetics (2010) 11: 369. doi:10.1007/s10048-010-0252-7
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Mast syndrome (SPG21) is a childhood-onset, autosomal recessive, complicated form of hereditary spastic paraplegia (HSP) characterized by dementia, thin corpus callosum, white matter abnormalities, and cerebellar and extrapyramidal signs in addition to spastic paraparesis. A nucleotide insertion resulting in premature truncation of the SPG21 gene product maspardin underlies this disorder, likely leading to loss of protein function. In this study, we generated SPG21−/− knockout mice by homologous recombination as a possible animal model for SPG21. Though SPG21−/− mice appeared normal at birth, within several months they developed gradually progressive hind limb dysfunction. Cerebral cortical neurons cultured from SPG21−/− mice exhibited significantly more axonal branching than neurons from wild-type animals, while comprehensive neuropathological analysis of SPG21−/− mice did not reveal definitive abnormalities. Since alterations in axon branching have been seen in neurons derived from animal models of other forms of HSP as well as motor neuron diseases, this may represent a common cellular pathogenic theme.
KeywordsMaspardinACP33CD4Hereditary spastic paraplegiaRab7
Central nervous system
Diphtheria toxin subunit A
Hereditary spastic paraplegia
Myelin basic protein
Normal goat serum
The hereditary spastic paraplegias (HSPs) are a diverse group of neurological disorders with the cardinal feature of progressive spasticity and weakness of the lower limbs [1–7]. They are classified as “pure” if lower extremity spasticity and weakness occur in isolation or “complicated” if patients exhibit other neurological abnormalities such as cognitive impairment, distal amyotrophy, or white matter abnormalities . The HSPs characteristically result from distal axonal degeneration in the long descending corticospinal tracts and ascending dorsal column fibers, which are among the longest motor and sensory axons, respectively, in the central nervous system (CNS) . More recently, the identification of multiple genetic loci (SPG1-46) and 20 gene products for the HSPs has permitted a molecular genetic classification of these disorders [2–7]. Based on the proteins identified, several additional mechanisms for pathogenesis have been advanced. These include abnormal cell signaling or migration, mitochondrial abnormalities, aberrations of myelination, impaired cholesterol or neurosteroid metabolism, alterations in intracellular ER network morphology, and defects in intracellular trafficking and transport. Most known proteins appear to fall into the latter two categories—including atlastin-1 (SPG3A), spastin (SPG4), NIPA1 (SPG6), strumpellin (SPG8), KIF5A (SPG10), spastizin (SPG15), seipin (SPG17), spartin (SPG20), acidic cluster protein 33 (ACP33)/maspardin (SPG21), and receptor expression enhancing protein 1 (REEP1; SPG31) [2–10]—suggesting that defects in protein and organelle trafficking may underlie distal axonal degeneration.
Mast syndrome (SPG21; MIM 248900) is a childhood-onset, autosomal recessive, complicated HSP characterized by progressive spastic paraparesis in association with dementia, developmental defects, thin corpus callosum, white matter abnormalities, and cerebellar and extrapyramidal signs [11,12]. The early onset of SPG21 indicates that it may have a neurodevelopmental component, though it is also clearly progressive during adulthood [11,12]. All known cases of SPG21 are autosomal recessive, caused by a single nucleotide insertion (601insA) in exon 7 of the SPG21 gene (aliases MASPARDIN, ACP33, MAST, BM-019, and GLO10), resulting in a reading frame shift and subsequent premature termination (fs201-212X213) of the 308-amino acid maspardin protein . Individuals heterozygous for the 601insA mutation do not exhibit any neurological symptoms. Thus, disease pathogenesis likely results from a lack of functional maspardin protein rather than a toxic gain of function, consistent with the autosomal recessive inheritance of Mast syndrome.
In this study, we show that targeted disruption of the SPG21 gene in mice using homologous recombination results in hind limb dysfunction, suggesting that these knockout mice may serve as a model to study some pathological aspects observed in Mast syndrome patients. Although morphological studies of the nervous system did not reveal definitive neuropathologic changes, primary cerebral cortical neurons cultured from SPG21−/− animals showed prominent alterations in axon branching.
Materials and methods
Generation and breeding of knockout mice
SPG21−/− mice were generated commercially (Caliper Life Sciences). For construction of the targeting vector, the mouse chromosome 9 sequence (nt # 65,660,000∼65,740,000) was retrieved from the Ensembl database (http://www.ensembl.org) and used as reference in this project. BAC clone RP24-285H11 was used for generating homologous arms and Southern probes by PCR or the RED cloning/gap-repair method. The 5′ (4.1 kb) and 3′ (5.8 kb) homologous arms were generated by RED cloning/gap repair. They were cloned into the LloxNwCD vector, with fidelity confirmed by restriction digestion and end sequencing. The final vector was obtained by standard molecular cloning methods. Aside from homologous arms, the final vector also contained a LoxP-flanked Neo expression cassette (for positive selection of embryonic stem (ES) cells) and a diphtheria toxin subunit A (DTA) expression cassette (for negative selection of ES cells). Final vector construction was confirmed by both restriction digestion and end sequencing analysis. NotI digestion was used to linearize the final vector for electroporation. 5′ and 3′ external probes were generated by PCR using proofreading TaKaRa LA Taq (Takara Bio) and tested using genomic Southern blot analysis for ES screening. They were cloned into the pCR4.0-TOPO backbone and confirmed by DNA sequencing.
For genotyping, genomic DNA was isolated from tail snips using standard procedures. Gene-specific PCR was carried out in an MJ Research PTC-200 DNA Engine Thermal Cycler (Bio-Rad) using Taq DNA polymerase (Invitrogen) and primers specific for exon 3 of SPG21 (forward: 5′-CGTGGATGACGATGACAGTA-3′), Neo cassette (forward: 5′-GCCAGCTCATTCCTCCCACTCAT-3′), and genomic reverse (5′-AAAACTAAAGGCTAGCCGGG-3′). Reactions were subjected to an initial denaturing step of 2 min at 94°C, followed by 30 cycles of 45 s at 94°C, 90 s at 60°C, and 60 s at 72°C, with a final extension of 72°C for 10 min.
For the beam-walking test, male mice of the indicated ages (2–12 months) were placed at one end of an 80-cm long, 1-cm square wooden rod elevated 30 cm above the bench with wood supports. They were allowed to walk to the goal box at the other end, with the time to traverse recorded. The number of foot slips (one or both hind limbs slipped from the beam) was recorded using a tally counter.
All animal experiments were approved by the NINDS/NIDCD Animal Care and Use Committee. Mice were perfused transcardially with 5% paraformaldehyde (PFA). Brains and spinal cords were isolated and post-fixed in 5% PFA for at least 48 h. Whole brains and large sections of spinal cord were then Golgi-stained by incubation in 3% potassium dichromate in 5% PFA for up to 1 week, followed by two washes in freshly prepared 0.75% silver nitrate and incubation in 0.75% silver nitrate for up to 1 week subsequently. Sections (50 μm thick) were then cut in distilled water on a sliding vibratome and were Nissl-stained subsequently.
Immunocytochemistry of tissue sections
Mice were transcardially perfused with 4% PFA, and skeletal muscles, spinal cords, and brains were isolated, post-fixed in 4% PFA overnight, and cryoprotected sequentially in 15% and 30% sucrose. Brains were cut coronally, and spinal cords and some muscles were cut transversely on a cryostat (14 μm thick sections); the other muscle sections (30 μm thick) were cut longitudinally. Polyclonal rabbit anti-synaptotagmin I antibodies (gift of Dr. Peter Low, Karolinska Institutet) were used to stain all section types, while polyclonal rabbit anti-GluR2/3 (3 μg/ml) (gift of Dr. Katherine Roche, NINDS, NIH), chicken anti-neurofilament-H (Clontech; 1:50,000), and rat anti-myelin basic protein (MBP) (Millipore; 1:300) were applied to brain and spinal cord sections. Additionally, polyclonal rabbit anti-neurofilament-M (Millipore; 1:300) was used to label muscle sections. After washing, each primary antibody was followed by an appropriate Alexa Fluor-conjugated secondary antibody and α-bungarotoxin (Invitrogen) where indicated for muscles. At least six sections were analyzed per animal.
For whole-mount analysis of adult hind limb muscles, mice were transcardially perfused with 4% PFA. Lateral gastrocnemius muscles were isolated, post-fixed in 4% PFA overnight, and partitioned into four longitudinal sections using a scalpel. The tissue was rinsed in phosphate-buffered saline (PBS) and incubated in 0.1 M glycine/PBS at 4°C overnight. Subsequently, tissue was permeabilized in 1% Triton X-100/PBS at room temperature for 8 h. Antibody staining was performed with anti-neurofilament-M antibody in 5% normal goat serum (NGS)/PBS at 4°C for 40 h. Samples were washed 3 × 20 min in PBS and incubated at 4°C overnight using Alexa Fluor-conjugated secondary antibody (1:100) and α-bungarotoxin in 5% NGS in PBS. Muscles were washed four times for 20 min in 5% NGS/PBS, rinsed in PBS, and mounted. Images were obtained with an inverted Zeiss LSM 510 META confocal microscope. Z-stack projections were made from serial scanning every 1 μm to reconstruct the neuromuscular junction (NMJ) using the tool available in the LSM software. Measurements of average cross-sectional areas and number of muscle fibers per NMJ and relative fluorescent intensities of brain and spinal cord markers were performed using ImageJ (NIH).
Neuronal culture and immunofluorescence microscopy
Primary cultures of mouse cerebral cortical neurons were prepared from P0 mice, plated at a density of ∼1.0 × 104/cm2 on coverslips, and maintained and immunostained as described previously [16,17]. Coverslips were mounted using Gel/Mount reagent (Biomeda). Fluorescence images were acquired using Zeiss LSM510 or Zeiss LSM710 confocal microscopes and processed with Adobe Photoshop 7.0 software.
Protein preparation, gel electrophoresis, and immunoblotting
For immunoblotting, proteins were resolved by SDS-PAGE and then electrophoretically transferred to nitrocellulose. After blocking with 5% non-fat milk/0.1% Tween 20/Tris-buffered saline (TBS, pH 7.4), antibodies (1–5 μg/ml) were added for 2.5 h at room temperature. The rabbit polyclonal anti-maspardin, anti-atlastin-1, anti-spastin, and anti-REEP1 antibodies have been described previously [10,14,16]. Rabbit polyclonal antibodies against KIF5A and KIF5B were from Abcam. Mouse polyclonal anti-ALDH16A1 antibodies (B01P) were purchased from Abnova, and goat polyclonal anti-Rab7 antibodies (C-19) were from Santa Cruz Biotechnology. Mouse monoclonal anti-β-actin antibody (clone AC-15; IgG1) was obtained from Sigma-Aldrich. Following several washes in TBS, horseradish peroxidase-conjugated secondary antibodies (1:1000; GE Healthcare Life Sciences) were added for 1.5 h in blocking buffer. Finally, after several washes with 0.1% Tween/TBS, immunoreactive proteins were revealed using Renaissance Enhanced Chemiluminescence Reagent (PerkinElmer Life Sciences). Images were acquired using the Molecular Imager ChemiDoc XRS System and Quantity One software (Bio-Rad).
BDNF treatment of neurons
Cerebral cortical neurons prepared from P0 mice as above were plated at a density of 4–5 × 106 per 10 cm plate and were maintained. At 7 days in vitro, 50 ng/ml BDNF in neurobasal medium was applied to neurons for 0–240 min. At the appropriate time point, cells were washed twice with PBS, then lysed with a buffer consisting of 50 mM Tris–Cl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 2% SDS, 1 mM PMSF, and complete protease inhibitor cocktail (Roche Applied Science) for 10 min on ice. Samples were subjected to SDS-PAGE and then immunoblotted using mouse monoclonal anti-TrkB (1:300; BD Biosciences) and rabbit polyclonal anti-phospho TrkA (Tyr490)/TrkB (Tyr516) (1:1,000; Cell Signaling).
Brains of SPG21−/− knockout mice and SPG21+/+ littermate controls (ages 6 and 15 months; n = 3 for each group) were dissected. RNA was extracted, and microarray analysis using the Affymetrix GeneChip mouse genome 430 2.0 array was performed by the NIH Neuroscience Microarray Consortium (http://np2.ctrl.ucla.edu/np2/home.do) using Affymetrix GeneChip Operating Software v1.4. All RNA samples met rigorous quality control evaluations by the NIH Neuroscience Microarray Consortium that included present call rates, scaling factor, GAPDH ratio, and background analysis.
Protein content determination
Protein concentrations were determined using the Pierce BCA Protein Assay Reagent (Thermo Fisher Scientific) with bovine serum albumin as the standard.
Statistical analyses were performed using Student's t test, assuming unequal variance, with p < 0.05 considered significant.
SPG21−/− mice exhibited defects in hind limb function
Cultured cortical neurons from SPG21−/− mice displayed increased axonal branching
Since differential effects on axon growth and branching have been observed in response to stimulation with different growth factors [21,22], and maspardin has been identified as a negative regulator of CD4 , we examined whether depletion of maspardin affected either phosphorylation of growth factor receptors in response to ligand binding or else the subsequent degradation of these receptors. We focused on BDNF, which binds the TrkB receptor, and examined both TrkB levels and TrkB phosphorylation at Tyr516. We noted no significant differences in any of these studies when studying neurons derived from SPG21−/− and SPG21+/+ mouse cortices over a period of 4 h after stimulation (data not shown).
SPG21−/− mice exhibited no gross neuropathologic abnormalities
We completed a comprehensive pathological characterization of aged SPG21−/− knockouts by investigating peripheral tissues as well as the central nervous system (ORS/VRP Mouse Phenotyping Service, National Institutes of Health, Bethesda, MD). Three SPG21−/− mice with gait disorders and three SPG21+/+ animals, including both males and females, were examined at 18–19 months of age. This examination included serum chemistries, hematology studies, organ/body weights, and comprehensive gross and histopathological analyses. Examination of the CNS and peripheral nerves did not reveal differences between the wild-type and mutant mice (n = 3 of each group). Pathologic changes seen in both groups included degenerative joint disease of the knee and temporomandibular joints, mild degenerative disc disease of the spine, fibro-osseous lesions of the femur and skull, and loss of neurons from the spiral ganglia of the inner ear. These changes, however, can be observed in aged animals of any strain.
Gene chip analysis of mRNA changes in SPG21−/− mice
Differential expression of mRNAs in brains from SPG21−/− compared to SPG21+/+ mice
GenBank accession #
Guanine nucleotide binding protein beta 1
Spermatogenesis associated glutamate-rich protein 7, pseudogene 1
DEAH box polypeptide 30
G-protein coupled receptor, family C, group 5, member b
Arginine and glutamate rich 1
Ring finger and KH domain containing 3
ATP-binding cassette, sub-family C (CFTR/MRP), member 5
Aldehyde dehydrogenase family 18, member A1
Histocompatibility 2, D region locus 1
DNA segment, ERATO Doi 553, expressed, mRNA
Uncoupling protein 2
Neuronal pentraxin receptor
Speckle-type POZ protein
Basic helix-loop-helix family, member e22
Serine protease inhibitor, Kunitz type 2
Kinesin family member 5B
RAD21 homolog (Schizosaccharomyces pombe)
v-raf murine sarcoma 3611 viral oncogene homolog
Lon peptidase 2, peroxisomal
Eukaryotic translation initiation factor 2, subunit 2 (beta)
SH2 domain containing 5
A mutation in the SPG21 gene results in a shift of reading frame and premature stop codon, causing maspardin protein truncation and presumed loss of function that results in the complicated form of HSP known as Mast syndrome (SPG21). Maspardin was first identified as a CD4-binding protein and proposed as a negative regulatory factor involved in CD4-dependent T cell activation . However, maspardin is a ubiquitously expressed protein, including in CD4-negative cells types, and thus likely has other functions. In this study, we generated knockout mice lacking the SPG21 gene encoding maspardin as an animal model for SPG21.
SPG21−/− animals had a clear but slowly progressive defect in motor function, with the hind limbs most affected. Some of the characteristic pathological features of human SPG21 were not clearly seen, however. In particular, we found no clear evidence of white matter disease. Even so, clear effects on axonal branching were seen when neurons were cultured from the cerebral cortex of neonatal SPG21−/− animals, with significantly more branching but no differences in primary axon length. This finding fits broadly with the proposed role of maspardin as a negative regulatory factor for CD4, since maspardin might also act as a negative regulator of other growth factor/cytokine receptors.
Since some growth factors have been described which stimulate branching of axons without increasing the primary axon length , as we described here, the presumptive loss of inhibition might result in positive modulation of growth factor signaling. Alternatively, based on the localization of maspardin to late endosomes and lysosomes [13,14], its co-immunoprecipitation with Rab7 , and the presence of a cluster of acidic amino acids at the N-terminus , maspardin may be involved in protein sorting in the late endosomal/lysosomal pathway. Thus, although we did not detect any changes in TrkB phosphorylation or degradation, it remains possible that ACP33/maspardin may mediate subtle changes in intracellular targeting of this or other growth factor receptors, as suggested previously for it effects on CD4 . Lastly, since growth factors can regulate levels of microtubule-severing proteins in neurons and increase branching , this could account for the observed increase in branching. Importantly, however, we did not see an increase in protein levels of the SPG4 microtubule-severing protein spastin in the SPG21−/− mice. Another known interaction of maspardin is with the aldehyde dehydrogenase ALDH16A1 . Interestingly, our comparative gene chip analysis identified a significant up-regulation in mRNA levels of another aldehyde dehydrogenase, ALDH18A1. However, very little is known about either of these two aldehyde dehydrogenase subtypes, and any pathogenic significance remains speculative.
Thus, a key factor limiting the interpretation of our data reporting an increase in axonal branching in SPG21−/− mice is that the function of maspardin itself is not well understood. Since it is a protein highly conserved throughout evolution, it likely plays a fundamental role within cells. Also, though the presumed catalytic triad of maspardin has some differences from other proteins in the esterase/lipase superfamily, it will be important to confirm that it lacks enzymatic activity. In some aspects, maspardin is reminiscent of a protein known as comparative gene identification-58 (CGI-58), which also has a short, continuous stretch of acidic amino acids at the N-terminus as well as a noncatalytic α/β-hydrolase domain . CGI-58, also designated as α/β-hydrolase domain containing-5 (ABHD-5), is a lipid droplet-associated protein that activates adipose triglyceride lipase and acylates lysophosphatidic acid . Since the SPG17 gene product seipin and the SPG20 gene product spartin are both associated with lipid droplets, and a number of other HSP proteins have been implicated in lipid metabolism [25–29], this pathway also could be affected. Interestingly, some animal models for amyotrophic lateral sclerosis also exhibit increased branching of axons , prefiguring a pathogenic link between some forms of HSP and amyotrophic lateral sclerosis. The identification of other maspardin-interacting proteins will clarify the function of maspardin and facilitate mechanistic investigations of the SPG21−/− mice.
The authors wish to thank James Nagle and Debbie Kauffman (NINDS DNA Sequencing Facility) for DNA sequencing and Dr. Peng-Peng Zhu for generation of the phylogenetic tree. This work was supported by the Intramural Research Program of the National Institute of Neurological Disorders and Stroke, National Institutes of Health [to C.S., J.S., H.J., C.B, and M.C.H.], the National Institute of General Medical Sciences Pharmacology Research Associate (PRAT) Program and the Swedish Research Council [grant numbers 13473, 20587 to O.S.].