Sodium vanadate combined with l-ascorbic acid delays disease progression, enhances motor performance, and ameliorates muscle atrophy and weakness in mice with spinal muscular atrophy
- Huei-Chun Liu1, 2,
- Chen-Hung Ting1Email author,
- Hsin-Lan Wen1,
- Li-Kai Tsai3,
- Hsiu-Mei Hsieh-Li4,
- Hung Li^1 and
- Sue Lin-Chao1Email author
© Liu et al; licensee BioMed Central Ltd. 2013
Received: 24 September 2012
Accepted: 14 February 2013
Published: 14 February 2013
Proximal spinal muscular atrophy (SMA), a neurodegenerative disorder that causes infant mortality, has no effective treatment. Sodium vanadate has shown potential for the treatment of SMA; however, vanadate-induced toxicity in vivo remains an obstacle for its clinical application. We evaluated the therapeutic potential of sodium vanadate combined with a vanadium detoxification agent, L-ascorbic acid, in a SMA mouse model.
Sodium vanadate (200 μM), L-ascorbic acid (400 μM), or sodium vanadate combined with L-ascorbic acid (combined treatment) were applied to motor neuron-like NSC34 cells and fibroblasts derived from a healthy donor and a type II SMA patient to evaluate the cellular viability and the efficacy of each treatment in vitro. For the in vivo studies, sodium vanadate (20 mg/kg once daily) and L-ascorbic acid (40 mg/kg once daily) alone or in combination were orally administered daily on postnatal days 1 to 30. Motor performance, pathological studies, and the effects of each treatment (vehicle, L-ascorbic acid, sodium vanadate, and combined treatment) were assessed and compared on postnatal days (PNDs) 30 and 90. The Kaplan-Meier method was used to evaluate the survival rate, with P < 0.05 indicating significance. For other studies, one-way analysis of variance (ANOVA) and Student's t test for paired variables were used to measure significant differences (P < 0.05) between values.
Combined treatment protected cells against vanadate-induced cell death with decreasing B cell lymphoma 2-associated X protein (Bax) levels. A month of combined treatment in mice with late-onset SMA beginning on postnatal day 1 delayed disease progression, improved motor performance in adulthood, enhanced survival motor neuron (SMN) levels and motor neuron numbers, reduced muscle atrophy, and decreased Bax levels in the spinal cord. Most importantly, combined treatment preserved hepatic and renal function and substantially decreased vanadium accumulation in these organs.
Combined treatment beginning at birth and continuing for 1 month conferred protection against neuromuscular damage in mice with milder types of SMA. Further, these mice exhibited enhanced motor performance in adulthood. Therefore, combined treatment could present a feasible treatment option for patients with late-onset SMA.
KeywordsL-ascorbic acid combined treatment SMA vanadate.
Spinal muscular atrophy (SMA) is an inherited neurodegenerative disease characterized by motor neuron degeneration in the anterior horn of the spinal cord that leads to muscle atrophy and paralysis . SMA is classified into different types based on the age at onset and disease severity. Symptoms of type I SMA manifest before 6 months of age, and patients never achieve the ability to sit. The onset of type II SMA occurs between 6 and 18 months, and patients are never able to stand or walk. Patients with type III SMA present with symptoms after 18 months, and they are able to walk at some point [2–4]. Two survival motor neuron (SMN) genes on chromosome 5q13 have been correlated with SMA: telomeric SMN1 and centromeric SMN2. SMA is caused by deletions or loss-of-function mutations in SMN1 with the retention of SMN2 [5–8], resulting in production of insufficient full-length SMN transcripts. SMN2 primarily transcribes exon 7-excluded mRNA because of a C-to-T transition at position 6 in exon 7 [9, 10] and produces an unstable C-terminally truncated SMN protein. However, patients with SMA present with varying degrees of severity depending on the number of SMN2 copies, a finding that has also been replicated in SMA mouse models [7, 11, 12], indicating that SMN2 could serve as the SMA modifier and is therefore a natural target for SMA therapy [12–16].
Two SMA therapy strategies that target SMN2 to produce more SMN have been investigated: enhancing SMN2 promoter activity and correcting SMN2 alternative splicing. Some compounds have been demonstrated to activate the SMN2 promoter and/or to change the SMN2 alternative splicing pattern, including histone deacetylase inhibitors (sodium butyrate, valproic acid (VPA), trichostatin A, suberoylanilide hydroxamic acid, and LBH589), prolactin, salbutamol, and sodium vanadate (SV) [17–24]. Synthesized antisense oligonucleotides (ASO) have also been shown to effectively reverse the SMN2 splicing pattern in vitro and in vivo, and they have displayed promising efficacy in treating SMA [25–28]. However, many of these compounds are known to be toxic at high doses, and their biosafety for human clinical trials remains to be proven [29, 30].
SV is a candidate compound for SMA therapy in vitro [23, 31]. SV and SV derivatives have been effective in treating diabetes in rodent models [32–34] and are currently in phase II clinical trials . However, high doses or long-term administration of vanadium damages organs and causes reproductive and developmental problems in animals [36–38]. Chelation therapy that combines vanadium compounds with chelating agents capable of binding vanadium in vivo to reduce poisoning has been one approach to reducing vanadium toxicity [39–41]. L-ascorbic acid (L-AA; vitamin C) is a natural vanadium detoxification agent that has been demonstrated to be safe for human use [40, 42, 43]. The interaction between L-AA and SV occurs under physiological conditions and is known to decrease vanadium toxicity [44, 45].
In the present work, the therapeutic potential of SV in combination with L-AA (combined treatment) was investigated in a mouse model of late-onset SMA that was previously used as a preclinical therapeutic testing system for SMA [26, 46]. The results indicate that combining L-AA with SV does not disrupt the ability of SV to increase the production of SMN levels but it eliminates SV-induced cytotoxicity in vitro. Mice with late-onset SMA that received combined treatment on postnatal days (PNDs) 1 to 30 exhibited delayed disease progression and enhanced motor activity in adulthood (PND 90). We also found sustained and elevated SMN levels, increased motor neuron numbers, improved muscle pathology, and reduced Bax levels in the spinal cords of the adult mice. Importantly, vanadium accumulation in the kidneys and livers of these mice was largely reduced, and those organs retained normal function during development and adulthood. Therefore, our study provides a potentially feasible and effective approach to treating patients with late-onset SMA.
Cell culture and chemical treatment
The procedures for culturing NSC34 cells stably expressing SMN2 (SMN2-NSC34) have been described previously . A dermal biopsy obtained from a patient with type II SMA (a 39-year-old woman with three copies of SMN2) was acquired from the Department of Neurology, National Taiwan University Hospital, and primary human dermal fibroblasts (HDFs) were cultured following standard procedures . The protocol for the human study was approved by the Research Ethics Committee of the National Taiwan University Hospital (NTUH-REC no. 201011059RB). Control wild-type (WT) primary HDFs (from a 29-year-old woman) were purchased from Cell Application Inc. (San Diego, CA, USA). Primary HDFs were cultured in fibroblast growth medium (Cell Application Inc.) at 37°C with 5% CO2 in a humidified incubator. The cells were plated 1 day before treatment with 400 μM L-AA (Sigma Aldrich, St Louis, MO, USA), 200 μM SV (Sigma), or 400 μM L-AA and 200 μM SV and harvested at the indicated times.
Cell viability assay
At 1 day before treatment and harvesting, 5 × 105 cells were plated onto six-well culture plates. Following treatment the total cell numbers were measured by the trypan blue exclusion test using the Countess Automated Cell Counter (Invitrogen, Carlsbad, CA, USA). Cell viability was evaluated three times for each condition.
SMA-like mice were previously generated via a homozygous knockout of Smn exon 7 with a transgene of human SMN2 (Smn-/-SMN2+/-) by our laboratory . The mice model of late-onset SMA (Smn-/-SMN2+/+) used in these studies had four copies of SMN2 and mice were generated via an initial breeding with a mouse model of late-onset SMA . Genotyping was performed as described previously . SMA mice were maintained on a 12-h light and 12-h dark schedule in accordance with the principles of laboratory animal care. The mice were supplied with sterile water ad libitum and rodent pellets under the control of the animal facility of the Institute of Molecular Biology, Academia Sinica, Taiwan. All procedures were approved by the Academia Sinica Animal Care and Use Committee, Master Protocol no. RMiIMBLH2008024.
SV and L-AA were dissolved in sterile deionized water. Vehicle (water), SV (20 mg/kg once daily), L-AA (40 mg/kg once daily), or SV (20 mg/kg) combined with L-AA (40 mg/kg) were orally administered on PNDs 1 to 30 using a 24-gauge feeding needle as described previously .
Cell or tissue lysates (20 μg) were prepared for western blot studies as described previously . Primary antibodies used for western blotting included mouse anti-SMN (1:5,000; BD Biosciences, San Diego, CA, USA), mouse anti-β-actin (1:10,000; Sigma), rabbit anti-Bax (1:1,000; Millipore, Temecula, CA, USA), and rabbit anti-caspase 3 (1:1,000; Cell Signaling, Temecula, CA, USA). Secondary antibodies conjugated with horseradish peroxidase (Millipore) were used at a dilution of 1:5,000.
Ear morphology analysis
Mice were examined from PND 50 to PND 96 and their ear integrity was determined. The severity of ear morphology was assigned a score from 0 to 4, with 0 indicating the most severe loss of integrity. A score of 4 indicated normal ear morphology, 3 indicated a red color at the tip, 2 indicated purple and black colorations indicative of necrosis, and 1 indicated loss of half of the external ear. A score of 0 indicated loss of nearly all of the external ear. Scores were plotted and analysis of variance (ANOVA) was applied to determine significance.
Motor functions in the mice were analyzed by a battery of behavioral tests. In the surface-righting assay, each pup was placed in a supine position, and the time to turn over was measured (maximum 30 s) every day on PNDs 1 to 12 . In the geotaxis assay, each mouse was placed on a 30° incline with its head facing the bottom of the incline. Success was judged if the mouse was able to reorient itself 180° within 30 s ; measurements were taken daily on PNDs 1 to 12. Hind limb strength was determined by the tube test. Measurements were taken as a mouse hung by its hind limbs on the lip of a 50-ml tube . The tube test was performed daily on PNDs 1 to 12. Locomotion and exploratory movement were measured in the adult mice by the open-field test. Each mouse was placed alone in the corner of an open-field cage (480 × 480 mm2) made of polyvinyl chloride. The activity of the animal in the open-field cage was detected for 60 minutes using a video imaging system . Motor function was also measured by the accelerating rotarod test. Each mouse was evaluated as the rotation speed increased from 4 to 40 rpm over 5 minutes; the final score was an average of three trials .
Mice (n = 3 in each group) were perfused with 4% paraformaldehyde (PFA). Lumbar spinal cords and tibialis anterior (TA) muscles were excised, fixed overnight, dehydrated using an infiltration machine (Leica Microsystems Nussloch GmbH, Nussloch, Germany), and embedded in paraffin. Lumbar spinal cords (4 μm) were serially sectioned at 50 μm intervals, and muscles (4 μm) were cross-sectioned at the midpoint. The sections were mounted on slides and stained with hematoxylin and eosin (H&E). Images were observed using a Zeiss Axio Observer Z1 microscope (Carl Zeiss, Jena, Germany) with a 10 × objective and analyzed using MetaMorph software (v.7.7.2; Molecular Devices, Sunnyvale, CA, USA). Using this software TA muscle area (> 500 myofibers per mouse) and the mean motor neuron numbers per section (> 10 sections per mouse) were determined. For whole-mount immunostaining, TA muscles were excised and fixed in 4% PFA overnight at 4°C. After three washes in 0.1 M phosphate-buffered saline (PBS)/0.1 M glycine, the muscles were blocked in blocking buffer (3% bovine serum albumin (BSA) and 0.5% Triton-X 100 in 0.1 M PBS), followed by incubation with anti-neurofilament H antibody (diluted to 1:500; Millipore) in blocking solution overnight at 4°C. The following day, the samples were washed in five changes of rinse solution (1% BSA and 0.5% Triton-X 100 in 0.1 M PBS) over a period of 5 h and then incubated with Alexa Fluor 488 donkey anti-rabbit IgG and tetramethylrhodamine-5-(and 6)-isothiocyanate (TRITC)-conjugated α-bungarotoxin (α-BTX) (diluted to 1:1,000; Molecular Probes, Carlsbad, CA, USA). After washing with five changes of rinse solution over a period of 5 h, muscle samples were mounted onto slides using fluorescence mounting solution (Invitrogen). Confocal images were captured using an LSM710 microscope (Carl Zeiss) with a 25 × objective lens. Neuromuscular junction (NMJ) area measurements were made using MetaMorph software. For gem number counts, fixed primary dermal fibroblasts were blocked and incubated with anti-SMN antibody as described previously . Images were obtained using an LSM META 510 laser-scanning confocal microscope (Carl Zeiss), and SMN nuclear localization in fibroblasts was confirmed by 4',6-diamidino-2-phenylindole (DAPI) counterstaining.
Hepatic and renal function test
Blood samples were collected from the facial vein of the mice using a lancet. The samples were then mixed with ethylenediaminetetra-acetic acid (EDTA). The solution was then centrifuged for 30 minutes at 4,700 g. The serum samples from each group of mice were analyzed using a DRI-CHEM clinical chemistry analyzer (FDC 3500; FujiFilm Medical Co, Tokyo, Japan) for glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), blood urea nitrogen (BUN), and creatinine (CRE).
Determination of vanadium
To investigate vanadium levels in tissue, snap-frozen kidney and liver tissues and blood were weighed and homogenized in 3 ml HNO3. The lysates were heated to 200°C within 15 minutes and then maintained at 200°C for 25 minutes using the ultra-high throughput microwave digestion system (MARSXpress; CEM Corporation, Matthews, NC, USA). After cooling, vanadium levels were determined by inductively coupled plasma mass spectrometry (ICP-MS) (X-Series II; Thermo Fisher Scientific Inc., Waltham, MA, USA).
The findings were confirmed by at least three independent experiments. Data were analyzed using Prism 5 software (GraphPad Software Inc., San Diego, CA, USA). Statistical significance was determined by Student's t test or one-way analysis of variance (Tukey's test). P < 0.05 was considered statistically significant.
Effect of L-AA combined with SV on SMN expression closely mimics SV alone
To extend these results to a clinically relevant model, we isolated HDFs from a type II SMA patient. The expression level of SMN protein in fibroblasts obtained from an SMA patient was approximately 59.2% of the amount observed in cells from a healthy donor (Additional file 1). The SMN protein level was restored by SV alone or combined treatment (n = 3, Figure 1I, upper panel, and L) and no change in SMN expression was observed following L-AA treatment alone in the fibroblasts derived from the SMA or healthy donors. These results demonstrate that SMN2-NSC34 cells receiving L-AA in addition to SV had a similar pattern of SMN-production compared to cells receiving only SV. In fibroblasts derived from healthy and SMA donors, both combined treatment and SV treatment alone resulted in increases of SMN expression. Therefore, L-AA has minimal to no negative impact on the efficacy of SV induction of SMN protein expression.
L-AA protects cells against SV-induced cytotoxicity
SV treatment-induced cytotoxicity has been well documented [54, 55]. To determine whether L-AA protects cells against SV-induced cell death, we assessed the viability of SMN2-NSC34 cells as well as WT and SMA patient-derived HDFs following treatment with SV (200 μM) in combination with L-AA (400 μM). We further determined whether L-AA protects SMN2-NSC34 cells and WT and SMA HDFs against SV-induced death by trypan blue exclusion staining (Figure 1E-G). The viability of SV-treated cells significantly decreased from 8 to 24 h in all cell types (n = 3 in each group) (Figure 1E-G, middle; Additional files 2, 3 and 4). By contrast, both NSC34 cells and HDFs that received combined treatment displayed no obvious signs of cell death from 8 to 24 h and exhibited improved viability compared with SV-treated cells as measured by trypan blue staining (Figure 1E-G, right; Additional files 2, 3 and 4). L-AA alone did not affect cell viability (Figure 1E-G, left). In addition, we found that SV-treatment resulted in increased proapoptotic Bax expression compared to L-AA treatment in SMN2-NSC34 cells (1.75 ± 0.11 vs 1.06 ± 0.16, P < 0.05), WT HDFs (1.29 ± 0.02 vs 0.92 ± 0.01, P < 0.001), and SMA HDFs (1.23 ± 0.01 vs 0.90 ± 0.02, P < 0.001) (Figure 1H, I, K, M). In contrast, combined treatment resulted in significantly lower Bax expression compared to SV treatment in SMN2-NSC34 cells (0.90 ± 0.06 vs 1.75 ± 0.11, P < 0.01), WT HDFs (1.05 ± 0.02 vs 1.29 ± 0.02, P < 0.01) and SMA HDFs (1.08 ± 0.04 vs 1.23 ± 0.01, P < 0.05) (Figure 1H, I, K, M). These results demonstrate that L-AA protected both NSC34 cells and HDFs against SV-induced toxicity.
L-AA eliminates SV-induced toxicity in vivo
Combined treatment delays disease progression in mice with late-onset SMA
Early combined treatment improves motor function in mice with late-onset SMA into adulthood
Combined treatment displays motor neuron protective effects and reduces muscle atrophy in mice with late-onset SMA
Combined treatment decreases Bax levels during development of mice with late-onset SMA
Combined treatment does not affect hepatic and renal functions in mice with late-onset SMA
Hepatic and renal function in wild-type (WT) mice and vehicle-treated, sodium vanadate (SV)-treated, L-ascorbic acid (L-AA)-treated, and L-AA+SV-treated spinal muscular atrophy (SMA) mice
54 to 298
17 to 77
8 to 33
0.2 to 0.9
Postnatal day 30
55.67 ± 10.20
35.67 ± 6.67
19.82 ± 0.56
93.69 ± 20.28
30.16 ± 2.64
22.22 ± 0.77
0.23 ± 0.02
129.62 ± 33.65
31.15 ± 2.14
23.98 ± 0.91
0.26 ± 0.02
119.87 ± 28.25
31.27 ± 2.99
22.80 ± 1.01
0.21 ± 0.03
Postnatal day 90
125.40 ± 30.09
57.60 ± 7.63
24.38 ± 2.65
107.05 ± 18.45
42.20 ± 4.19
22.27 ± 0.90
169.54 ± 19.09
37.69 ± 2.96
24.32 ± 0.65
143.50 ± 20.66
39.62 ± 3.85
25.17 ± 1.30
Accumulation of vanadium (μg/g) in the liver, kidneys, and blood of vehicle-treated, sodium vanadate (SV)-treated, L-ascorbic acid (L-AA)-treated, and L-AA+SV-treated spinal muscular atrophy (SMA) mice
Postnatal day 6:
2.11 ± 0.18
3.89 ± 0.60
0.59 ± 0.01 ***
1.37 ± 0.26 *
Postnatal day 30:
0.22 ± 0.02
0.22 ± 0.04
0.07 ± 0.01
Postnatal day 90:
0.08 ± 0.04
0.07 ± 0.02
0.02 ± 0.01
In the present study, we show that L-AA largely decreases vanadium toxicity both in vitro and in vivo and that administration of SV combined with L-AA delays disease progression, improves motor activities and muscle pathology, and protects spinal motor neurons in a mouse model of late-onset SMA.
Early intervention with combined treatment provides long-term efficacy in mice with late-onset SMA
Several reports indicated that SMA mice that received treatment before disease onset exhibited a satisfactory recovery of SMN levels [17, 20, 22, 27, 28, 66], an improvement in SMA symptoms [17, 20, 22, 27, 28, 66], and rescue of the SMA-like phenotype [28, 66]. Based on these results, SMA mice received combined treatment for 1 month beginning on PND 1. The initial results revealed improved SMN levels in the brains and spinal cords of the mice that received combined treatment relative to those of the mice that received L-AA alone (Figure 6A-D). In addition, combined treatment improved the motor performance of the adult mice with late-onset SMA (Figure 5C), most probably because of a protective effect on motor neurons (Figure 6E-H) and an improvement in muscle pathology (Figure 7) due to the recovery of SMN levels. These findings are consistent with the fact that SMN is required for normal development and that early treatment resulting in sufficient SMN levels improves the prognosis in SMA models. While early treatment has been shown to be essential to mitigating disease severity in mice with early-onset SMA, whether early drug intervention is also necessary for mice with late-onset SMA has not been established. Hua et al. reported that early (E15) ASO injection in mice with late-onset SMA  dramatically reduced the disease severity and improved motor function, indicating that early treatment is also beneficial in late-onset SMA. However, most cases of type II and III SMA are not diagnosed in the earlier stages of the disease, making it necessary to evaluate the efficacy of later interventions in the late-onset SMA model. In this study, we did not evaluate whether combined treatment administered to mice with late-onset SMA near or after the onset of symptoms could achieve ideal therapeutic effects. However, there has been some evidence that other drugs such as SV that correct the SMN2 alternative splicing may be therapeutic even when administered later in the disease progression. Some reports have indicated that type II SMA patients that receive VPA for 6 months at disease onset showed significant increases in muscle strength and function [67, 68]. Additionally, some type II and III SMA patients who received salbutamol treatment for 6 months at disease onset also showed an increase of muscle strength . These findings therefore support the possibility that combined L-AA and SV treatment applied at later stages of late-onset SMA may be beneficial and present a promising avenue for further study. However, the efficacy of later interventions with combined treatment should be further investigated. SV treatment (20 mg/kg once daily) alone caused substantially reduced weight gain and mortality before PND 6 in mice (Figure 3). Decreasing the SV dosage to 15 mg/kg prevented lethality but still resulted in a reduced growth rate in juvenile mice (Additional file 5). By contrast, the mice that received combined treatment displayed normal growth rates with no obvious hepatic or renal damage (Figure 3 and Table 1) and reduced vanadium accumulation in the liver and kidneys on PND 30 (Table 2) that decreased to very low levels in the adult mice after drug therapy ended. The effects of SV on the liver or kidneys are of particular concern because of their involvement in the excretory mechanism [43, 44]. In addition, a report indicated that the daily tolerance of vanadium ranges from 10 to 60 μg/day in humans . However, the average basal and normative vanadium requirement has been difficult to ascertain. Data acquired from deprivation studies in animals indicated that 2 to 25 ng/day vanadium often induced no significant clinical effects, and many diets supply approximately 15 to 30 μg of vanadium daily, suggesting that dietary intake of vanadium of approximately 10 μg/day is safe . Moreover, the addition of L-AA dramatically reduces SV-induced toxicity in vitro and in vivo (Figures 1 and 3). L-AA has very low toxicity, and the minimum dietary requirement in humans is generally 40 to 100 mg/day, however, concentrations of up to 100-fold higher have been shown to be within a safe range . Therefore, combined treatment provides a novel and useful strategy for SMA therapy in the near future.
Combined treatment results in reduced Bax levels and attenuated motor neuron death
Motor neuron loss has been found in the lumbar spinal cord in all types of SMA . Inhibition of neuronal apoptosis is one potential strategy for SMA therapy [18, 58, 65]. The proapoptotic protein Bax is involved in neuron death after trophic factor deprivation and during development  and is induced by SV treatment [54, 55]. Abolishing Bax-dependent apoptosis prolongs lifespan in a mouse model of type I SMA , indicating that Bax may play a deleterious role in SMA pathogenesis. Although SV enhances SMN2 expression , the toxic effect of SV on cells (especially NSC34 cells and HDFs) in this study (Figure 1E-G) is a major obstacle to the application of vanadium-related compounds in SMA therapy. L-AA protects cells against SV-induced cell death (Figures 1E-G and 8). The levels of Bax, but not of caspase 3, were significantly downregulated in cells (Figure 1H, I, K, M) and animals (Figure 8) that received combined treatment, indicating that L-AA protects motor neurons from death caused by decreased Bax expression through a Bax-dependent mechanism. However, the Bax level remained higher in the mice with late-onset SMA that received combined treatment than in the WT mice (Figure 8). It is possible that L-AA only functions to eliminate SV-induced Bax levels but fails to reverse SMA pathogenesis. In addition, we attributed the reduced Bax levels observed in the mice that received combined treatment (Figure 8) to SMN induction.
Future perspectives of combined treatment
Vanadate is a small compound that can pass through the blood-brain barrier. It moves through the circulatory system and enters the metabolic pathways . The vanadate derivatives bis(ethylmaltolato)oxovanadium(IV) and bis(maltolato)oxovanadium(IV) are insulin-mimetic agents currently being investigated in phase II clinical trials for type II diabetes . Those drugs may present novel opportunities for SMA therapy in the near future. Although some reports indicated that vanadate is not toxic when administered orally , the effects of vanadium accumulation in organs and the toxic effects of long-term administration of vanadate-based compounds need to be carefully investigated. Also, the optimal timing of combined treatment (that is, when to begin and the duration), in addition to the optimal time for additional courses of drug administration, also need to be established. Furthermore, L-AA appears to be an ideal chelating agent to combine with vanadium compounds. However, other chelating agents that are effective in combination with vanadium compounds are being investigated in a diabetic model. The ingestion of a tea decoction with vanadium results in reduced vanadium accumulation in most tissues , and when administered orally over 14 months, this combination induces long-term glycemic stability without obvious organ toxicity . The development of improved chelating agents with strong antioxidant properties that are readily biodegradable, cost effective, and stable within a wide pH range would boost the safety and efficacy of vanadium for SMA treatment.
Our work demonstrates that early treatment with vanadate combined with L-AA has considerable potential for treating patients with late-onset type II/III SMA. Furthermore, the development of a vanadate derivative and the usage of vanadium compounds in combination with chelating agents are other feasible strategies for SMA therapy.
blood urea nitrogen
glutamate oxaloacetate transaminase
glutamate pyruvate transaminase
human dermal fibroblasts
spinal muscular atrophy
survival motor neuron
We thank the Taiwan Mouse Clinic, which is funded by the National Research Program for Biopharmaceuticals at the National Science Council (NSC) of Taiwan, for technical support with the open-field and rotarod experiments. We are grateful to the Incubation Center at the Genomics Research Center for technical support with the ICP-MS experiments. We would like to thank Ms Su-Ping Lee at the Image Core Facility at the IMB, Academia Sinica, and Dr Shauh-Der Yeh in the Institute of Biomedical Science, Academia Sinica, for technical support. We would also like to thank Dr Neil Cashman for kindly providing the NSC34 cell line. We would like to thank Dr AndreAna Pena at the IMB editorial office for manuscript editing. One of the authors, Dr Hung Li, passed away in 2009. This article is dedicated to his memory. This work was supported by research grants from the NSC (NSC100-2321-B-001-008) and Academia Sinica (AS034006 and AS022323).
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