Imaging Flow Cytometry Analysis to Identify Differences of Survival Motor Neuron Protein Expression in Patients With Spinal Muscular Atrophy

BackgroundSpinal muscular atrophy is a neurodegenerative disorder caused by the deficient expression of survival motor neuron protein in motor neurons. A major goal of disease-modifying therapy is to increase survival motor neuron expression. Changes in survival motor neuron protein expression can be monitored via peripheral blood cells in patients; therefore we tested the sensitivity and utility of imaging flow cytometry for this purpose.MethodsAfter the immortalization of peripheral blood lymphocytes from a human healthy control subject and two patients with spinal muscular atrophy type 1 with two and three copies of SMN2 gene, respectively, we used imaging flow cytometry analysis to identify significant differences in survival motor neuron expression. A bright detail intensity analysis was used to investigate differences in the cellular localization of survival motor neuron protein.ResultsSurvival motor neuron expression was significantly decreased in cells derived from patients with spinal muscular atrophy relative to those derived from a healthy control subject. Moreover, survival motor neuron expression correlated with the clinical severity of spinal muscular atrophy according to SMN2 copy number. The cellular accumulation of survival motor neuron protein was also significantly decreased in cells derived from patients with spinal muscular atrophy relative to those derived from a healthy control subject.ConclusionsThe benefits of imaging flow cytometry for peripheral blood analysis include its capacities for analyzing heterogeneous cell populations; visualizing cell morphology; and evaluating the accumulation, localization, and expression of a target protein. Imaging flow cytometry analysis should be implemented in future studies to optimize its application as a tool for spinal muscular atrophy clinical trials.     

脊髓性肌萎缩症的快速基因诊断研究

目的研究我国近端型脊髓性肌萎缩症（ＳＭＡ）患者的运动神经元生存基因（ＳＭＮ）外显子的缺失情况,探讨其快速基因诊断的可行性和临床应用价值。方法应用ＰＣＲ－酶切法检测２６例确诊的ＳＭＡ患者及２０名正常对照ＳＭＮ基因的第７、８号外显子的缺失情况。结果在２６例及２５例患者中,分别发现缺失了端粒ＳＭＮ基因（ＳＭＮ１）的第７和８号外显子,缺失率达１００％（２６／２６）和９６％（２５／２６）,而正常对照及患者的家系成员均未发现外显子缺失。结论应用ＰＣＲ－酶切法检测ＳＭＮ１基因缺失从而进行ＳＭＡ患者的基因诊断,具有准确、简便和快速的优点。     

The Genetics of Spinal Muscular Atrophy: Progress and Challenges

Spinal muscular atrophies (SMAs) are a group of inherited disorders characterized by motor neuron loss in the spinal cord and lower brainstem, muscle weakness, and atrophy. The clinical and genetic phenotypes incorporate a wide spectrum that is differentiated based on age of onset, pattern of muscle involvement, and inheritance pattern. Over the past several years, rapid advances in genetic technology have accelerated the identification of causative genes and provided important advances in understanding the molecular and biological basis of SMA and insights into the selective vulnerability of the motor neuron. Common pathophysiological themes include defects in RNA metabolism and splicing, axonal transport, and motor neuron development and connectivity. Together these have revealed potential novel treatment strategies, and extensive efforts are being undertaken towards expedited therapeutics. While a number of promising therapies for SMA are emerging, defining therapeutic windows and developing sensitive and relevant biomarkers are critical to facilitate potential success in clinical trials. This review incorporates an overview of the clinical manifestations and genetics of SMA, and describes recent advances in the understanding of mechanisms of disease pathogenesis and development of novel treatment strategies.

Electronic supplementary material

The online version of this article (doi:10.1007/s13311-014-0314-x) contains supplementary material, which is available to authorized users.     

Failure of lower motor neuron radial outgrowth precedes retrograde degeneration in a feline model of spinal muscular atrophy

Feline spinal muscular atrophy (SMA) is a fully penetrant, autosomal recessive lower motor neuron disease in domestic cats that clinically resembles human SMA Type III. A whole genome linkage scan identified a ～140-kb deletion that abrogates expression of LIX1, a novel SMA candidate gene of unknown function. To characterize the progression of feline SMA, we assessed pathological changes in muscle and spinal cord from 3 days of age to beyond onset of clinical signs. Electromyographic (EMG) analysis indicating denervation occurred between 10 and 12 weeks, with the first neurological signs occurring at the same time. Compound motor action potential (CMAP) amplitudes were significantly reduced in the soleus and extensor carpi radialis muscles at 8-11 weeks. Quadriceps femoris muscle fibers from affected cats appeared smaller at 10 weeks; by 12 weeks atrophic fibers were more prevalent than in age-matched controls. In affected cats, significant loss of L5 ventral root axons was observed at 12 weeks. By 21 weeks of age, affected cats had 40% fewer L5 motor axons than normal. There was no significant difference in total L5 soma number, even at 21 weeks; thus degeneration begins distal to the cell body and proceeds retrogradely. Morphometric analysis of L5 ventral roots and horns revealed that 4 weeks prior to axon loss, motor axons in affected cats failed to undergo radial enlargement, suggesting a role for the putative disease gene LIX1 in radial growth of axons.     

Nutritional Status and Nutrient Intake Challenges in Children With Spinal Muscular Atrophy

BackgroundNutrition is recognized as a core component of multidisciplinary care for patients with spinal muscular atrophy, but specific nutritional challenges in this population are not well described. We aimed to describe the nutritional status and nutrient intake in children with spinal muscular atrophy.MethodsWe performed a retrospective medical record review of prospectively collected data from children with spinal muscular atrophy followed at a multidisciplinary clinic at a tertiary referral center. We collected data including clinical parameters; anthropometrics, including weight, height, and body mass index (BMI); and 24-hour dietary intake records in all children followed in the clinic. Available data were found in records from the dietitian as part of a standard evaluation process, and additional clinical data were acquired from patient medical records. Subjects were classified based on spinal muscular atrophy type, and nutritional intake data were compared with dietary reference intakes for gender and age. Z-scores were calculated for weight for age (WAZ), height for age, and BMI (BMIZ) using the World Health Organization AnthroPlus software with appropriate World Health Organization reference growth standards. Subjects were classified as malnourished if their WAZ was <−2 or >+2. Anthropometric measurements were obtained at first visit and at a follow-up visit at an average of a 3-year interval between the clinic visits. A decline of more than 0.5 WAZ over this period was defined a priori as significant nutritional deterioration.ResultsWe analyzed data from 60 subjects, 26 (43%) female, with median age 5.5 years (interquartile range 2 years to 12 years). The cohort consisted of children with spinal muscular atrophy type 1 (28 %), type 2 (45 %), and type 3 (27 %). At the first clinic visit, nine (15%) patients were malnourished. Thirteen (23%) subjects had a significant decline in WAZ from −0.35 (−1.31 to 0.58) to −1.04 (−2.15 to 0.02) at follow-up after approximately 3 years. A third of these subjects were already malnourished at first visit. A significant decline in BMIZ was noted in 47% of the cohort, and the prevalence of severe malnutrition (BMIZ < −3) increased from 2% to 17% after 3 years. In children receiving specialized enteral nutrition via a feeding tube, overfeeding was recorded in 29% and underfeeding was recorded in 35%. Suboptimal vitamin D intake was recorded in 35% of patients with enteral feeding device.ConclusionsMalnutrition was prevalent in children with spinal muscular atrophy, and nearly half the cohort demonstrated nutritional deterioration over time. Energy, protein, and vitamin D intakes were inadequate in a majority of the cohort. Underfeeding was highly prevalent, but overfeeding was also present in a third of the enterally fed cohort. Future studies describing optimal nutrient requirements and body composition variables in this group are required.     

慢性脊髓性肌萎缩症的临床和肌活检

文章报道２４例慢性脊髓性肌萎缩症，其中婴儿型、少年型、成年型慢性近端型分别为５例、２例及１３例，面肩肱型２例，远端型和肩腓型各１例。本病主要临床表现为肌无力、肌萎缩和不同程度的肌束震颤，锥体束和周围神经一般不受累。各型肌萎缩的部位不同。３例患者伴有ＣＰＫ浓度增高。除２例肌电图正常外，其余表现为失神经性改变。光镜提示神经原性萎缩。电镜下见肌原纤维数量减少，Ｚ线变粗或波浪状以及线粒体和内质网肿胀。     

Decremental Responses to Repetitive Nerve Stimulation in X-Linked Bulbospinal Muscular Atrophy

Background and Purpose

X-linked bulbospinal muscular atrophy (X-BSMA) is characterized by bulbar and spinal muscular weakness and fasciculations. Although X-BSMA is a motor neuronopathy, there are several reports of myasthenic symptoms or decremental responses to repetitive nerve stimulation (RNS). We report the results of applying the RNS test to 15 patients among 41 with genetically confirmed X-BSMA; these 15 patients complained of fatigue, ease of becoming tired, or early muscular exhaustion.

Methods

The 3-Hz RNS test was performed on the trapezius, nasalis, orbicularis oculi, flexor carpi ulnaris, and abductor digiti quinti muscles. A decrement greater than 10% was considered abnormal. Additionally, a pharmacologic response to neostigmine was identified in three patients.

Results

A significant decrement was observed in 67% of patients, and was most common in the trapezius muscle (nine cases). The decrement of the trapezius muscle response ranged from 15.9% to 36.9%. The decrement was inversely correlated with the amplitude of compound muscle action potentials at rest. Neostigmine injection markedly improved the decrement in three patients, who showed noticeable decremental responses to 3-Hz RNS.

Conclusions

This study shows that myasthenic symptoms and abnormal decremental responses to low-rate RNS are common in X-BSMA.     

Health-Related Quality of Life in Children and Adolescents With Spinal Muscular Atrophy in the Czech Republic

BackgroundSpinal muscular atrophy is a rare hereditary neuromuscular disorder (with a prevalence of 1 per 30,000) that greatly debilitates patients and, in most cases, shortens their life expectancy. Although there is no causal therapy, improvements in symptomatic therapy have extended patients' life expectancy and increased their quality of life. Unfortunately, the advancements in care vary from country to country. To improve the care for children with spinal muscular atrophy in the Czech Republic, we created a survey to obtain the baseline information about their quality of life and compared the data with equivalent data from the United States.MethodsWe used the Pediatric Quality of Life Inventory 3.0 Neuromuscular Measurement Model, which is a health-related quality of life questionnaire specific to children with neuromuscular disorders. The survey was conducted on 35 children with genetically proven spinal muscular atrophy and their parents.ResultsCompared with the US data, the Czech data generally show a lower quality of life, mainly in the family resources part. The greatest score was achieved in the section about communication. Altogether, the parents' scores are lower than those of the children.ConclusionIn the Czech Republic, patients with spinal muscular atrophy and, especially their parents, have a significantly lower quality of life compared with US patients, mostly because of economic factors and a lack of social support. Our results reveal areas toward which improvement should be directed. The need for family support through social care as well as civic, patient, or organizational support is accentuated.     

Telomeric Region of the Spinal Muscular Atrophy Locus Is Susceptible to Structural Variations

BackgroundMost patients with spinal muscular atrophy lack the survival motor neuron 1 gene (SMN1) in the telomeric region of the spinal muscular atrophy locus on chromosome 5q13. On the other hand, the copy number of SMN2, a centromeric homolog of SMN1, is increased in many of these patients. This study aimed to clarify the mechanism underlying these structural variations.MethodsWe determined the copy numbers of telomeric and centromeric genes in the spinal muscular atrophy locus of 86 patients and 22 control subjects using multiplex ligation-dependent probe amplification analysis. Then, we chose 74 patients lacking SMN1 exons 7 and 8, and compared their dataset with that of 22 control subjects retaining SMN1 exons 7 and 8.ResultsThe SMN2 copy number was shown to vary widely and to correlate with the disease severity of the patients. Interestingly, telomeric NAIP and telomeric GTF2H2 showed similar tendencies. We also noted positive correlations among the copy number of SMN2 and the telomeric genes of the spinal muscular atrophy locus. However, the copy numbers of centromeric NAIP and centromeric GTF2H2 were stable among the patients, with both approximating a value of two.ConclusionOur findings suggested that the telomeric region of the spinal muscular atrophy locus appears to be susceptible to structural variation, whereas the centromeric region is stable. Moreover, according to our results, new SMN2 copies may be generated in the telomeric region of the spinal muscular atrophy locus, supporting the SMN1-to-SMN2 gene conversion theory.     

Emerging concepts underlying selective neuromuscular dysfunction in infantile-onset spinal muscular atrophy

Infantile-onset spinal muscular atrophy is the quintessential example of a disorder characterized by a predominantly neurodegenerative phenotype that nevertheless stems from perturbations in a housekeeping protein.Resulting from low levels of the Survival of Motor Neuron(SMN)protein,spinal muscular atrophy manifests mainly as a lower motor neuron disease.Why this is so and whether other cell types contribute to the classic spinal muscular atrophy phenotype continue to be the subject of intense investigation and are only now gaining appreciation.Yet,what is emerging is sometimes as puzzling as it is instructive,arguing for a careful re-examination of recent study outcomes,raising questions about established dogma in the field and making the case for a greater focus on milder spinal muscular atrophy models as tools to identify key mechanisms driving selective neuromuscular dysfunction in the disease.This review examines the evidence for novel molecular and cellular mechanisms that have recently been implicated in spinal muscular atrophy,highlights breakthroughs,points out caveats and poses questions that ought to serve as the basis of new investigations to better understand and treat this and other more common neurodegenerative disorders.     

Neurodevelopmental abnormalities in neurosphere-derived neural stem cells from SMN-depleted mice

Spinal muscular atrophy (SMA) is a genetic disorder caused by depletion of survival motor neuron (SMN) protein and characterized by degeneration of alpha-motor neurons in the spinal cord. We investigated the morphology and differentiation of neurosphere-derived neural stem cells (NSCs) generated from the brains of a hypomorphic series of SMA mice. Neurospheres from the Smn(-/-);SMN2 mice, which represent a model of very severe SMA, produced NSCs with increased proliferation during growth and differentiation. These cells produced fewer Tuj1-positive neuronal cells, which displayed morphological alterations and had fewer and shorter neurites. The decrease in the number of Tuj1-positive cells was not a result of enhanced apoptosis but was accompanied by an increase in the number of nestin-positive cells. These results provide insight into the most severe model of SMA, in which SMN is nearly completely depleted, and suggest that SMN has a role in neurodevelopment as well as in neuromaintenance. Our work raises the possibility that SMN depletion affects neurodevelopment and neuromaintenance to varying extents, leading to SMA pathogenesis.     

Distribution of neuronal apoptosis inhibitory protein-like immunoreactivity in the rat central nervous system

We have recently shown that spinal muscular atrophy (SMA), an autosomal recessive disorder characterized by motor neuron loss, is associated with deletion of a gene that encodes the neuronal apoptosis inhibitory protein (NAIP). In the present study, we have examined the distribution of NAIP-like immunoreactivity (NAIP-LI) in the rat central nervous system (CNS) by using an affinity-purified polyclonal antibody against NAIP. In the forebrain, immunoreactive neurons were detected in the cortex, the hippocampus (pyramidal cells, dentate granule cells, and interneurons), the striatum (cholinergic interneurons), the basal forebrain (ventral pallidum, medial septal nucleus, and diagonal band), the thalamus (lateral and ventral nuclei), the habenula, the globus pallidus, and the entopenduncular nucleus. In the midbrain, NAIP-LI was located primarily within neurons of the red nucleus, the substantia nigra pars compacta, the oculomotor nucleus, and the trochlear nucleus. In the brainstem, neurons containing NAIP-LI were observed in cranial nerve nuclei (trigeminal, facial, vestibular, cochlear, vagus, and hypoglossal nerves) and in relay nuclei (pontine, olivary, lateral reticular, cuneate, gracile nucleus, and locus coeruleus). In the cerebellum, NAIP-LI was found within both Purkinje and nuclear cells (interposed and lateral nuclei). Finally, within the spinal cord, NAIP-LI was detected in Clarke's column and in motor neurons. Taken together, these results indicate that NAIP-LI is distributed broadly in the CNS. However, high levels of NAIP-LI were restricted to those neuronal populations that have been reported to degenerate in SMA. This anatomical correspondence provides additional evidence for NAIP involvement in the neurodegeneration observed in acute SMA. J. Comp. Neurol. 382:247-259, 1997. © 1997 Wiley-Liss, Inc.     

SMA valiant trial: A prospective,double‐blind,placebo‐controlled trial of valproic acid in ambulatory adults with spinal muscular atrophy

Introduction: An open‐label trial suggested that valproic acid (VPA) improved strength in adults with spinal muscular atrophy (SMA). We report a 12‐month, double‐blind, cross‐over study of VPA in ambulatory SMA adults. Methods: There were 33 subjects, aged 20–55 years, included in this investigation. After baseline assessment, subjects were randomized to receive VPA (10–20 mg/kg/day) or placebo. At 6 months, patients were switched to the other group. Assessments were performed at 3, 6, and 12 months. The primary outcome was the 6‐month change in maximum voluntary isometric contraction testing with pulmonary, electrophysiological, and functional secondary outcomes. Results: Thirty subjects completed the study. VPA was well tolerated, and compliance was good. There was no change in primary or secondary outcomes at 6 or 12 months. Conclusions: VPA did not improve strength or function in SMA adults. The outcomes used are feasible and reliable and can be employed in future trials in SMA adults. Muscle Nerve 49 : 187–192, 2014     

Rasch analysis of clinical outcome measures in spinal muscular atrophy

Introduction: Trial design for SMA depends on meaningful rating scales to assess outcomes. In this study Rasch methodology was applied to 9 motor scales in spinal muscular atrophy (SMA). Methods: Data from all 3 SMA types were provided by research groups for 9 commonly used scales. Rasch methodology assessed the ordering of response option thresholds, tests of fit, spread of item locations, residual correlations, and person separation index. Results: Each scale had good reliability. However, several issues impacting scale validity were identified, including the extent that items defined clinically meaningful constructs and how well each scale measured performance across the SMA spectrum. Conclusions: The sensitivity and potential utility of each SMA scale as outcome measures for trials could be improved by establishing clear definitions of what is measured, reconsidering items that misfit and items whose response categories have reversed thresholds, and adding new items at the extremes of scale ranges. Muscle Nerve 49 :422–430, 2014     

The Effect of Phenobarbital on the Multiple Neuronal Activity in the Hippocampus of El Mice

Abstract: Phenobarbital (PB) (5, 25 and 50 mg/kg), which was administered intraperitoneally to evaluate the effect of it on El mice, inhibited convulsive seizures dose-dependently. On the other hand, microelectrodes were implanted continually into the hippocampus of El mice, after which changes in the neuronal activity caused by a seizure were studied, as well as the effect of PB (5, 25 and 50 mg/kg) on the neuronal activity. The neuronal activity increased during the convulsive seizure when compared to the neuronal activity as the mice moved about freely. Further, after the convulsive seizure, five or six groups of bursts were seen periodically. PB reduced the neuronal activity dose-dependently judging by the number of bursts in the initial group of bursts.     

Advances in SMA research: Review of gene deletions

The term spinal muscular atrophy (SMA) is used to encompass a group of inherited disorders in which the striking pathological feature is loss of the cell bodies of alpha motor neurons in the anterior horn cell of the spinal cord and, in some cases, of the bulbar motor nuclei. Although the pathological features of these disorders have been well characterized, the nature of the primary underlying biochemical abnormality remains to be determined. In the 1990s genetic linkage was established for the childhood onset recessive forms of SMA (types I, II and III) to markers mapping to the chromosomal region 5q11.2–13.3. Physical maps of the region were then constructed, several candidate genes isolated and in 1995 deletions in two genes, the survival motor neuron (SMN) gene and the neuronal apoptosis inhibitory protein (NAIP) gene, were identified in significant numbers of patients. Already the impact of the characterization of these deletions is being seen in clinical practice in terms of aiding diagnosis in symptomatic cases and in prenatal diagnosis. As discussed in this review however, several questions remain unresolved. It is unclear whether deletions in one or both of these genes, or indeed in other, as yet unidentified, genes are important in generating the SMA phenotype. The function of the protein product of the SMN gene is unknown. The NAIP gene encodes a protein which inhibits apoptosis in a mammalian cell line: is it disruption of this function which is relevant in SMA? What underlies the variation in disease severity evident both between and within families? Resolution of such issues is of crucial importance if the identification of these deleted gene sequences is to lead to the development of rational therapies for motor neuron diseases.