Transient caloric restriction in early adulthood hastens disease endpoint in male, but not female, Cu/Zn-SOD mutant G93A mice

Long-term caloric restriction (CR) prolongs the lifespan in healthy insects, rodents, and nonhuman primates. We previously reported that long-term CR improves motor performance but hastens clinical onset of disease in an animal model of amyotrophic lateral sclerosis (G93A mice). G93A mice overexpress the mutant human Cu/Zn-SOD gene and show progressive lower motor neuron weakness and increased oxidative stress. To study short-term (15 days) CR in the same animal model, we investigated the effect of transient caloric restriction (TCR) on paw grip endurance, clinical onset, disease progression (time from clinical onset to endpoint), and lifespan. Starting at age 40 days, 32 separately caged G93A mice were randomly divided into two groups: ad libitum (AL, n = 17; 10 females, 7 males) and TCR (n = 15; 6 females, 9 males) with a diet equal to 60% of AL. When the TCR mice lost 30% of their weight they were offered food AL until endpoint, otherwise all TCR mice were provided food AL from age 55 days until endpoint (i.e., range of TCR = 13-15 days). Paw grip endurance started to decrease significantly at age 96 days compared with baseline values for all the groups. TCR males reached clinical onset 5 days sooner than TCR females. Disease progression was 8 days faster in TCR mice than AL mice and 6 days faster in male mice than female mice. The probability of survival was significantly different between the groups, with the TCR males having a faster rate of reaching endpoint than TCR females, AL males, and AL females. We conclude that TCR hastens clinical onset of disease and shortens the lifespan in male, but not female, G93A mice. Moreover, TCR hastens progress of disease but has no effect on paw grip endurance. The female sex is protective against the detrimental effects of short-term CR in G93A mice. Assuming we can extrapolate these results to humans, short-term CR should be avoided in patients with amyotrophic lateral sclerosis, especially men.     

Behavioural and anatomical effects of systemically administered leukemia inhibitory factor in the SOD1(G93A G1H) mouse model of familial amyotrophic lateral sclerosis

We investigated the anatomical and behavioural effects of daily intraperitoneal injection of 25 microg/kg of LIF in the SOD1(G93A G1H) mouse model of familial ALS. We found some subtle beneficial behavioural changes in LIF treated mice. These included later onset of clinical disease in females as determined by clinical scoring; better grip strength in males; and delayed development of motor impairment in males as determined by the rotarod test. However, we found no significant rescue of motoneurons or prolongation of survival as a result of this systemic dose of LIF in these mice.     

Hippocampal ultrastructural changes and apoptotic cell death in rats following endurance training and acute exhaustive exercise

BACKGROUND： Exhaustive exercise can lead to apoptosis of skeletal muscle cells and myocardial cells as a result of pathological changes in the corresponding cellular ultrastructure. It is hypothesized that such changes could also occur in neurons. OBJECTIVE： To observe brain cell apoptosis and ultrastructural changes in hippocampal neurons in rats following endurance training and acute exhaustive exercise. DESIGN, TIME AND SETTING： A randomized, controlled, morphological analysis was performed at the Medical Laboratory Center of Zhengzhou University between July and November 2007. MATERIALS： Forty male, 8-week-old, Sprague Dawley rats were included in this study. METHODS： Endurance training consisted of treadmill running once a day, 6 days a week, for 4. weeks. For acute exhaustive exercise, graded treadmill running was conducted. Rats were exposed to exercise at an increasing speed （10 m/min, increasing to 20 and 36 m/min for moderate- and high-intensity exhaustive exercise, respectively, and then was continued until exhaustion）. A total of 40 rats were evenly distributed into the following 4. groups： Group A rats were not exercised; Group B rats were not trained but sacrificed 24 hours after acute exhaustive treadmill running exercise; Group C-rats were subjected to endurance training and sacrificed immediately after acute exhaustive treadmill running exercise; Group D rats were subjected to endurance training and sacrificed 24 hours after acute exhaustive treadmill running exercise. MAIN OUTCOME MEASURES： Apoptotic cell death was detected by the TUNEL method and hippocampal neuronal ultrastructural change was observed through using transmission electron microscopy. RESULTS： All 40 rats were included in the final analysis. Subsequent to exhaustive exercise, rat cerebral cortex and hippocampal neurons appeared contracted and degenerated. In addition, high amount of lipofuscin was visible in the hippocampal region, Necrotic neurons encased by glial cells appeared in the cerebral cortex and hip     

Effect of physical activity on hippocampal high affinity choline uptake and muscarinic binding: a comparison between young and old F344 rats

Normal aging has been associated with a progressive decline in hippocampal cholinergic function. In the present study, specific markers of hippocampal cholinergic function, high-affinity choline uptake (HACU) and muscarinic quinuclidinylbenzilate (QNB) binding, were shown to be altered by endurance training (6 months of treadmill running, 5 days/week, 30 min/day). HACU and QNB binding were determined in synaptosomes of endurance trained F344 rats and their age-matched sedentary controls. Comparison of synaptosomes of sedentary rats ages 3 months, 12 months and 25 months (distinguished in this paper as young (Y), middle age (MA) and old (O), respectively) showed maximum HACU at 12 months and subsequent reduction in HACU and QNB binding at 25 months (P less than 0.05). This decline at 25 months is consistent with previous reports of an age-related decline in cholinergic function. Endurance trained rats (trained from 6 months to 12 months of age) showed a reduction (P less than 0.02) in HACU and an increase (P less than 0.05) in QNB binding compared to their age-matched sedentary controls whereas endurance trained rats (trained from 19 months to 25 months of age) showed no significant difference in either parameter from their age-matched sedentary controls. From these results, it appears that while both training and normal aging reduce HACU, the reductions may be different in presynaptic mechanism and postsynaptic consequence.     

丁苯酞对SOD1~(G93A)转基因鼠脊髓Ferritin表达的影响

目的探讨丁苯酞对SOD1G 93A转基因鼠的神经保护作用及其可能的机制。方法选取SOD1G 93A阳性小鼠48只,随机分为丁苯酞组(发病早期及终末期)和安慰剂组(发病早期及终末期),每组12只,观察SOD1G 93A转基因鼠的发病时间和生存期,采用免疫组化和蛋白印迹的方法观察SOD1G 93A转基因鼠腰髓铁蛋白(Ferritin)表达。结果丁苯酞组的发病时间为118.0±2.5d,与安慰剂组的109.5±2.2d相比较,P<0.05;生存时间为138.6±2.3d,与安慰剂组的128.1±2.8d相比较,P<0.05;免疫组化显示:发病初期,安慰剂组Ferritin阳性细胞显示胞浆和胞膜周围均匀着色,丁苯酞组Ferritin仅胞膜周围着色,胞核和胞浆着色不明显;终末期,丁苯酞组与安慰剂组相比,二者的Ferritin在胞核和胞浆均有着色;蛋白印迹显示:发病初期,Ferritin在腰髓表达量的相对值,丁苯酞组为0.81±0.23,与安慰剂组的1.57±0.43相比较,P<0.05;终末期,丁苯酞组为1.45±0.45,与安慰剂组的1.73±0.36相比较,P>0.05。结论丁苯酞延迟ALS小鼠的发病时间并延长其生存期,丁苯酞延迟小鼠发病可能与调节铁代谢有关。     

Caloric restriction transiently improves motor performance but hastens clinical onset of disease in the Cu/Zn-superoxide dismutase mutant G93A mouse

Caloric restriction (CR) prolongs lifespan in insects, rodents, and nonhuman primates, a process attributed to a reduction in oxidative stress. Transgenic mice that overexpress the mutant human Cu/Zn-superoxide dismutase (SOD1) gene (G93A mice) are an animal model of amyotrophic lateral sclerosis showing progressively lower motor neuron weakness and increased oxidative stress. We investigated the effect of CR on motor performance, clinical onset, disease progression, and lifespan in G93A mice. Starting at 40 days of age, 14 separately caged G93A mice were randomly divided into two groups: ad libitum (AL; n = 6) and calorie-restricted (CR; n = 8) with a diet equal to 60% of AL. The CR mice (mean +/- SEM: 14.0 +/- 0.7 g) weighed 31% less than the AL mice (20.3 +/- 1.0 g) (P = 0.0002). From 74 to 93 days of age, the CR mice performed better on the rotarod than the AL mice: fall time, P = 0.039; fall speed, P = 0.009. The CR mice had a faster rate of reaching clinical onset than the AL mice (hazard ratio = 4.3, P = 0.0006). The CR and AL mice reached clinical onset of disease at age 99 +/- 1 and 110 +/- 2 days, respectively (P = 0.0003), with no significant difference in disease progression. The CR mice tended to reach endpoint sooner than the AL mice (age-specific death: 125 +/- 3 vs. 133 +/- 3 days, respectively, P = 0.09). We conclude that CR diet transiently improves motor performance but hastens clinical onset of disease in G93A mice. These results suggest that CR diet is not a protective strategy for patients with amyotrophic lateral sclerosis (ALS) and hence is contraindicated.     

Oral Administration of Memantine Prolongs Survival in a Transgenic Mouse Model of Amyotrophic Lateral Sclerosis

Background and purpose

N-methyl-D-aspartate (NMDA)-mediated neurotoxicity and oxidative stress have been implicated in the etiology of amyotrophic lateral sclerosis (ALS). Memantine is a low-affinity, noncompetitive NMDA receptor antagonist that may protect against motor neuron degeneration.

Methods

Thirty transgenic mice expressing the G93A SOD1 mutation were randomly divided into control, low-dose memantine (30 mg/kg/day), and high-dose memantine (90 mg/kg/day) groups, with memantine supplied daily with drinking water beginning at 75 days of age. Body weight, survival, and behavioral performances including a rotarod test, paw grip endurance, and hindlimb extension reflex were assessed in the control and memantine-diet groups.

Results

Clinical symptoms were evident in the G93A transgenic mice by 11 weeks of age. Memantine was tolerated well. Compared to control, mice treated with memantine performed better in the rotarod test and hindlimb extension reflex. Moreover, low-dose memantine treatment significantly prolonged the survival of the transgenic mice relative to control mice (141 vs 134 days, p<0.05).

Conclusions

These findings suggest that memantine, even when administered at the time of symptom onset, has beneficial effects on patients with ALS.     

丁苯酞对FALS模型鼠雄激素受体表达的影响

目的探讨丁苯酞(NBP)对家族性肌萎侧索硬化(FALS)模型hSOD1G93A转基因鼠雄激素受体表达的影响及机制。方法观察NBP干预后不同性别hSOD1G93A转基因小鼠的发病时间及生存期,应用免疫组化的方法观察hSOD1G93A转基因小鼠终末期脊髓前角神经元雄激素受体的表达结果 NBP组雄性hSOD1G93A转基因小鼠生存期为133.52±9.16与安慰剂组120.27±8.98相比,P=0.00;雌性的hSOD1G93A转基因小鼠生存期136.21±11.90与安慰剂组128.13±8.20相比,P=0.04。NBP组雄、雌性存活期相比,P=0.58。NBP组雄性hSOD1G93A转基因小鼠发病时间为110.22±9.19天与安慰剂组101.13±8.90天相比,P=0.00;雌性的hSOD1G93A转基因小鼠发病时间113.04±12.73天,与安慰剂组96.07±4.92天相比,P=0.00。NBP组雄、雌发病时间相比,P=0.46。免疫组化结果显示终末期NBP组SOD1G93A转基因小鼠AR阳性的运动神经元数目5.87±1.598与安慰剂组2.93±1.1、空白对照组3.00±1.134相比,P<0.05;雄:雌残存神经元数量相比为6.75±1.282:4.86±1.345,P=0.02。结论 NBP可以推迟hSOD1G93A转基因小鼠的发病时间,延长生存期,与性别无明显相关;NBP增加hSOD1G93A转基因小鼠脊髓前角运动神经元雄激素受体的表达。     

Comparison of peripheral sudomotor sensitivity to acetylcholine in endurance and non‐endurance trained male subjects

Introduction: We investigated the effect of endurance and non‐endurance training on peripheral sudomotor sensitivity. Methods: The quantitative sudomotor axon reflex test (QSART) was performed. Results: Endurance‐trained subjects (ET, long‐distance runners) had a significantly shorter onset time of sweating, greater sweat volume, increased density of activated sweat glands and sweat gland output per single activated gland, greater volume of transepidermal water loss, and higher skin temperature compared with those in the other 2 groups [non–endurance‐trained group (NET), sedentary control group (CT)]. NET subjects (baseball players) had a tendency to increase in these variables; thus, some values were greater than control subjects. Conclusions: These results suggest that endurance training much more effectively modifies sudomotor sensitivity than non‐endurance training. Muscle Nerve 50 : 407–412, 2014     

The matrix metalloproteinases inhibitor Ro 28-2653 [correction of Ro 26-2853] extends survival in transgenic ALS mice

In amyotrophic lateral sclerosis (ALS), there is increased expression of matrix metalloproteinases (MMPs) and degradation of the extracellular matrix in postmortem spinal cord tissue. We used zymography and in situ zymography to analyze the expression of MMP-2 and MMP-9 in spinal cord tissue from the G93A transgenic mouse model of ALS. Expression of MMP-9 was increased in the spinal cord of G93A mice. For functional analysis of the role of MMPs, we investigated the effects of oral administration of the MMP inhibitor Ro 28-2653 (100 mg/kg), starting at the age of 30 days (n = 19) and on disease onset (starting at the age of 90 days (n = 10)). Treatment with the MMP inhibitor Ro 28-2653 starting at 30 days of age improved motor performance and significantly (P < 0.05) prolonged the survival time of the animals (136 +/- 12 versus 123 +/- 12 days, mean +/- SD), however, administration at disease onset did not significantly improve survival time. Our experiments show that MMPs are expressed in an animal model of ALS and may play a role in the complex pathophysiologic changes. Early pharmacologic inhibition with a synthetic MMP inhibitor extends survival of the animals which suggest a role of MMPs in the early phase of the disease.     

Muscle contractility dysfunction precedes loss of motor unit connectivity in SOD1(G93A) mice

Introduction: Electrophysiological measurements are used in longitudinal clinical studies to provide insight into the progression of amyotrophic lateral sclerosis (ALS) and the relationship between muscle weakness and motor unit (MU) degeneration. Here, we used a similar longitudinal approach in the Cu/Zn superoxide dismutase (SOD1[G93A]) mouse model of ALS. Methods: In vivo muscle contractility and MU connectivity assays were assessed longitudinally in SOD1(G93A) and wild type mice from postnatal days 35 to 119. Results: In SOD1(G93A) males, muscle contractility was reduced by day 35 and preceded MU loss. Muscle contractility and motor unit reduction were delayed in SOD1(G93A) females compared with males, but, just as with males, muscle contractility reduction preceded MU loss. Discussion: The longitudinal contractility and connectivity paradigm employed here provides additional insight into the SOD1(G93A) mouse model and suggests that loss of muscle contractility is an early finding that may precede loss of MUs and motor neuron death. Muscle Nerve 59 :254–262, 2019     

CGP 3466B has no effect on disease course of (G93A) mSOD1 transgenic mice.

BACKGROUND: There is an accumulating body of evidence that apoptosis is involved in the motor neuron death that occurs in ALS, and in the (G93A) mSOD1 transgenic mouse model (mSOD1 mice). CGP 3466B, a tricyclic propargylamine structurally related to (-)-deprenyl, was found to inhibit apoptosis in a wide variety of in vitro and in vivo models. We therefore studied the effect of CGP 3466B in mSOD1 mice. METHODS: As the effect of CGP 3466B was previously reported to have a bell-shaped curve, we performed a dose-ranging study. High-copy G93A mSOD1 mice were treated subcutaneously from the age of 50 days until death with four concentrations of CGP 3466B (0.39 microg kg(-1), 3.9 microg kg(-1), 39 microg kg(-1), and 390 microg kg(-1)). Behavioural tests were performed daily to determine disease onset, disease progression and survival. At the age of 110 days, two mice per group were sacrificed for histopathological analysis of the lumbar ventral horn and for semiquantitative analysis of motor neuron number. RESULTS: We observed no effect on disease onset, disease progression, or survival of the mice. We also did not observe a significant effect on the number of motor neurons due to CGP 3466B. CONCLUSIONS: We conclude that in high-copy G93A mSOD1 mice, chronic subcutaneous treatment with CGP 3466B offers no clinical benefit.     

Mechano-growth factor, an IGF-I splice variant, rescues motoneurons and improves muscle function in SOD1 mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by motoneuron degeneration. Although viral delivery of IGF-I has shown therapeutic efficacy in the SOD1^G93A mouse model of ALS, clinical trials of IGF-I in ALS patients have led to conflicting results. Here we examine the effects of an IGF-I splice variant, mechano-growth factor (MGF) which has previously been shown to have greater neuroprotective effects than IGF-I in a number of models of neurodegeneration. A mammalian expression plasmid containing either MGF or, for comparison, the IGF-I cDNA sequence was delivered to the hindlimb muscles of SOD1^G93A mice at 70 days of age, at symptom onset. Treatment with either IGF-I or MGF resulted in a significant improvement in hindlimb muscle strength, and an increase in motor unit and motoneuron survival. Significantly more motoneurons survived in MGF treated mice.     

Recurrent systemic infections with Streptococcus pneumoniae do not aggravate the course of experimental neurodegenerative diseases

Neurological symptoms of patients suffering from neurodegenerative diseases such as Alzheimer's dementia (AD), Parkinson's disease (PD), or amyotrophic lateral sclerosis (ALS) often worsen during infections. We assessed the disease‐modulating effects of recurrent systemic infections with the most frequent respiratory pathogen, Streptococcus pneumoniae, on the course of AD, PD, and ALS in mouse models of these neurodegenerative diseases [transgenic Tg2576 mice, (Thy1)‐[A30P]αSYN mice, and Tg(SOD1‐G93A) mice]. Mice were repeatedly challenged intraperitoneally with live S. pneumoniae type 3 and treated with ceftriaxone for 3 days. Infection caused an increase of interleukin‐6 concentrations in brain homogenates. The clinical status of (Thy1)‐[A30P]αSYN mice and Tg(SOD1‐G93A) mice was monitored by repeated assessment with a clinical score. Motor performance was controlled by the tightrope test and the rotarod test. In Tg2576 mice, spatial memory and learning deficits were assessed in the Morris water maze. In none of the three mouse models onset or course of the disease as evaluated by the clinical tests was affected by the recurrent systemic infections performed. Levels of α‐synuclein in brains of (Thy1)‐[A30P]αSYN mice did not differ between infected animals and control animals. Plaque sizes and concentrations of Aβ 1–40 and Aβ 1–42 were not significantly different in brains of infected and uninfected Tg2576 mice. In conclusion, onset and course of disease in mouse models of three common neurodegenerative disorders were not influenced by repeated systemic infections with S. pneumoniae, indicating that the effect of moderately severe acute infections on the course of neurodegenerative diseases may be less pronounced than suspected. © 2009 Wiley‐Liss, Inc.     

The effects of endurance training in persons with a hereditary myosin myopathy

OBJECTIVE: To evaluate muscle performance and its consequences in eight individuals with a hereditary myopathy and the effects of an 8-week endurance training program. MATERIAL AND METHODS: Handgrip, muscle strength and endurance and oxygen consumption by breath-by-breath analysis during a stepless bicycle ergonometer test were evaluated. Walking, balance test and activities of daily living (ADL) were assessed, and a questionnaire for activity level and perceived symptoms was used. The design was a before-after trial in comparison with data from a control population, bicycling at 70% of maximal workload, 30 min/day, 5 days/week for 8 weeks. RESULTS: The subjects were weaker than age-matched controls. After training, the peak watt increased by almost 20% (P < 0.05). Muscle strength (flexion/extension) and isometric endurance (40% of maximum at 60 degrees ) did not change significantly. The average self-selected walking speed increased significantly (P < 0.05) from 1.25 to 1.45 m/s. Compliance was excellent and no serious adverse events occurred. CONCLUSION: Endurance training seems to function for this myopathy.     

The influence of metallothionein treatment and treadmill running exercise on disease onset and survival in SOD1G93A amyotrophic lateral sclerosis mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, characterised by the degeneration of motor neurons innervating skeletal muscle. The mechanisms underlying neurodegeneration in ALS are not yet fully elucidated, and with current therapeutics only able to extend lifespan by a matter of months there is a clear need for novel therapies to increase lifespan and patient quality of life. Here, we evaluated whether moderate‐intensity treadmill exercise and/or treatment with metallothionein‐2 (MT2), a neuroprotective protein, could improve survival, behavioural or neuropathological outcomes in SOD1^G93A familial ALS mice. Six‐week‐old female SOD1^G93A mice were allocated to one of four treatment groups: MT2 injection, i.m.; moderate treadmill exercise; neither MT2 nor exercise; or both MT2 and exercise. MT2‐treated mice survived around 3% longer than vehicle‐treated mice, with this mild effect reaching statistical significance in Cox proportional hazards analysis once adjusted for potential confounders. Mixed model body weight trajectories over time indicated that MT2‐treated mice, with or without exercise, reached maximum body weight at a later age, suggesting a delay in disease onset of around 4% compared to saline‐treated mice. Exercise alone did not significantly increase survival or delay disease onset, and neither exercise nor MT2 substantially ameliorated gait abnormalities or muscle strength loss. We conclude that neither exercise nor MT2 treatment was detrimental in female SOD1^G93A mice, and further study could determine whether the mild effect of peripheral MT2 administration on disease onset and survival could be improved via direct administration of MT2 to the central nervous system.     

MicroRNA expression in animal models of amyotrophic lateral sclerosis and potential therapeutic approaches

A review of recent animal models of amyotrophic lateral sclerosis showed a large number of mi RNAs had altered levels of expression in the brain and spinal cord,motor neurons of spinal cord and brainstem,and hypoglossal,facial,and red motor nuclei and were mostly upregulated.Among the mi RNAs found to be upregulated in two of the studies were mi R-21,mi R-155,mi R-125 b,mi R-146 a,mi R-124,mi R-9,and mi R-19 b,while those downregulated in two of the studies included mi R-146 a,mi R-29,mi R-9,and mi R-125 b.A change of direction in mi RNA expression occurred in some tissues when compared(e.g.,mi R-29 b-3 p in cerebellum and spinal cord of wobbler mice at 40 days),or at different disease stages(e.g.,mi R-200 a in spinal cord of SOD1(G93 A)mice at 95 days vs.108 and 112 days).In the animal models,suppression of mi R-129-5 p resulted in increased lifespan,improved muscle strength,reduced neuromuscular junction degeneration,and tended to improve motor neuron survival in the SOD1(G93 A)mouse model.Suppression of mi R-155 was also associated with increased lifespan,while lowering of mi R-29 a tended to improve lifespan in males and increase muscle strength in SOD1(G93 A)mice.Overexpression of members of mi R-17~92 cluster improved motor neuron survival in SOD1(G93 A)mice.Treatment with an artificial mi RNA designed to target h SOD1 increased lifespan and improved muscle strength in SOD1(G93 A)animals.Further studies with animal models of amyotrophic lateral sclerosis are warranted to validate these findings and identify specific mi RNAs whose suppression or directed against h SOD1 results in increased lifespan,improved muscle strength,reduced neuromuscular junction degeneration,and improved motor neuron survival in SOD1(G93 A)animals.     

Decline in Daily Running Distance Presages Disease Onset in a Mouse Model of ALS

Amyotrophic lateral sclerosis (ALS) is characterized by progressive degeneration of lower motor neurons resulting in paralysis and death. Epidemiological and clinical findings suggest that a decline in athletic performance may presage the clinical onset of ALS, but this possibility has not been tested in an animal model. By placing running wheels in each mouse’s cage to measure their exercise activity, we show that presymptomatic G93A SOD1 ALS mice are more active runners (15–20 km/day) than control mice (7–9 km/day). The ALS mice then exhibit a sharp decline in daily running distance 10–20 days prior to the onset of clinical disease. Within the group of ALS mice, there were no significant correlations between cumulative lifetime running distance and age at clinical disease onset or age at death, suggesting that amount of exercise did not affect the course of the disease process. Our data show that presymptomatic ALS mice have a propensity for running long distances, and then dramatically reduce the amount they run prior to the appearance of clinical symptoms. The monitoring of voluntary running distance may provide a valuable biomarker to evaluate the efficacy of potential therapeutic interventions for ALS in preclinical studies. Daniel A. Bruestle and Roy G. Cutler contributed equally to this work.     

Diapocynin and apocynin administration fails to significantly extend survival in G93A SOD1 ALS mice

NADPH oxidase has recently been identified as a promising new therapeutic target in ALS. Genetic deletion of NADPH oxidase (Nox2) in the transgenic SOD1^G93A mutant mouse model of ALS was reported to increase survival remarkably by 97 days. Furthermore, apocynin, a widely used inhibitor of NADPH oxidase, was observed to dramatically extend the survival of the SOD1^G93A ALS mice even longer to 113 days (Harraz et al. J Clin Invest 118: 474, 2008). Diapocynin, the covalent dimer of apocynin, has been reported to be a more potent inhibitor of NADPH oxidase. We compared the protection of diapocynin to apocynin in primary cultures of SOD1^G93A-expressing motor neurons against nitric oxide-mediated death. Diapocynin, 10 μM, provided significantly greater protection compared to apocynin, 200 μM, at the lowest statistically significant concentrations. However, administration of diapocynin starting at 21 days of age in the SOD1^G93A-ALS mouse model did not extend lifespan. Repeated parallel experiments with apocynin failed to yield protection greater than a 5-day life extension in multiple trials conducted at two separate institutions. The maximum protection observed was an 8-day extension in survival when diapocynin was administered at 100 days of age at disease onset. HPLC with selective ion monitoring by mass spectrometry revealed that both apocynin and diapocynin accumulated in the brain and spinal cord tissue to low micromolar concentrations. Diapocynin was also detected in the CNS of apocynin-treated mice. The failure to achieve significant protection with either apocynin or diapocynin raises questions about the utility for treating ALS patients.     

Therapeutic immunization with a glatiramer acetate derivative does not alter survival in G93A and G37R SOD1 mouse models of familial ALS

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. The cause of motor neuron degeneration remains largely unknown, and there is no potent treatment. Overexpression of various human mutant superoxide dismutase-1 (SOD1) genes in mice and rats recapitulates some of the clinical and pathological characteristics of sporadic and familial ALS. Glatiramer acetate (GA) is an approved drug for the treatment of multiple sclerosis and neuroprotective properties in some neurodegenerative conditions. A recent report suggested that GA immunization could delay disease progression in some, but not all, G93A SOD1 transgenic mouse models of amyotrophic lateral sclerosis (ALS). Moreover, it has been theorized that derivatives of GA could enhance immunogenicity and positively affect disease outcomes. The purpose of our study was to assess the neuroprotective efficacy of TV-5010, a high molecular weight GA, in three different SOD1 mutant mouse models. We used large numbers of two SOD1 transgenic mouse strains overexpressing the G93A mutation, B6SJL-TgN[SOD1-G93A]1Gur and B6.Cg-Tg(SOD1-G93A)1Gur/J, and the SOD1 mutant mouse overexpressing G37R (line 29). Regardless of the frequency of injections and the dose, treatment with TV-5010 was ineffective at altering either disease onset or survival in both SOD1 G93A mutants used and in the SOD1 G37R transgenic mice; in multiple studies, disease was accelerated. These studies suggest that, at a range of dosing regimens and carrier used, TV-5010 immunization was ineffective in delaying disease in multiple preclinical therapeutic models for ALS. The biological response in animals, and ultimate clinical translation, will ultimately be dependent on careful and appropriate dose, route and carrier paradigms.