Induced pluripotent stem cells from a spinal muscular atrophy patient.

Spinal muscular atrophy is one of the most common inherited forms of neurological disease leading to infant mortality. Patients have selective loss of lower motor neurons resulting in muscle weakness, paralysis and often death. Although patient fibroblasts have been used extensively to study spinal muscular atrophy, motor neurons have a unique anatomy and physiology which may underlie their vulnerability to the disease process. Here we report the generation of induced pluripotent stem cells from skin fibroblast samples taken from a child with spinal muscular atrophy. These cells expanded robustly in culture, maintained the disease genotype and generated motor neurons that showed selective deficits compared to those derived from the child's unaffected mother. This is the first study to show that human induced pluripotent stem cells can be used to model the specific pathology seen in a genetically inherited disease. As such, it represents a promising resource to study disease mechanisms, screen new drug compounds and develop new therapies.

Restoration of SMN to Emx-1 expressing cortical neurons is not sufficient to provide benefit to a severe mouse model of Spinal Muscular Atrophy.

Spinal Muscular Atrophy (SMA), an autosomal recessive neuromuscular disorder, is a leading genetic cause of infant mortality. SMA is caused by the homozygous loss of Survival Motor Neuron-1 (SMN1). However, low, but essential, levels of SMN protein are produced by a nearly identical copy gene called SMN2. Detailed analysis of neuromuscular junctions in SMA mice has revealed a selective vulnerability in a subset of muscle targets, suggesting that while SMN is reduced uniformly, the functional deficits manifest sporadically. Additionally, in severe SMA models, it is becoming increasing apparent that SMA is not restricted solely to motor neurons. Rather, additional tissues including the heart, vasculature, and the pancreas contribute to the complete SMA-associated pathology. Recently, transgenic models have been utilized to examine the tissue-specific requirements of SMN, including selective depletion and restoration of SMN in motor neurons. To determine whether the cortical neuronal populations expressing the Emx-1 promoter are involved in SMA pathology, we generated a novel SMA mouse model in which SMN expression was specifically induced in Emx-1 expressing cortical neurons utilizing an Emx-1-Cre transgene. While SMN expression was robust in the central nervous system as expected, SMA mice did not live longer. Weight and time-to-right motor function were not significantly improved.

Delivery of recombinant follistatin lessens disease severity in a mouse model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is the most common genetic cause of infant mortality. SMA is caused by loss of functional survival motor neuron 1 (SMN1), resulting in death of spinal motor neurons. Current therapeutic research focuses on modulating the expression of a partially functioning copy gene, SMN2, which is retained in SMA patients. However, a treatment strategy that improves the SMA phenotype by slowing or reversing the skeletal muscle atrophy may also be beneficial. Myostatin, a member of the TGF-beta super-family, is a potent negative regulator of skeletal muscle mass. Follistatin is a natural antagonist of myostatin, and over-expression of follistatin in mouse muscle leads to profound increases in skeletal muscle mass. To determine whether enhanced muscle mass impacts SMA, we administered recombinant follistatin to an SMA mouse model. Treated animals exhibited increased mass in several muscle groups, elevation in the number and cross-sectional area of ventral horn cells, gross motor function improvement and mean lifespan extension by 30%, by preventing some of the early deaths, when compared with control animals. SMN protein levels in spinal cord and muscle were unchanged in follistatin-treated SMA mice, suggesting that follistatin exerts its effect in an SMN-independent manner. Reversing muscle atrophy associated with SMA may represent an unexploited therapeutic target for the treatment of SMA.

The spinal muscular atrophy mouse model, SMAΔ7, displays altered axonal transport without global neurofilament alterations.

Spinal muscular atrophy (SMA) is a neurodegenerative disease resulting from decreased levels of survival motor neuron 1 (SMN1) protein. Reduced SMN1 levels are linked to pathology at neuromuscular junctions (NMJs), which includes decreased vesicle density and organization, decreased quantal release, increased endplate potential duration, and neurofilament (NF) accumulations. This work presents a first study towards defining molecular alterations that may lead to the development of NMJ pathology in SMA. Fast, anterograde transport of synaptic vesicle 2 (SV2-c) and synaptotagmin (Syt1) proteins was reduced 2 days prior to the observed decrease in synaptic vesicle density. Moreover, reduced accumulation of SV2-c or Syt1 was not due to reduced protein expression or reduced kinesin activity. Dynein levels were reduced at times that are consistent with NF accumulations at NMJs. Furthermore, NF distribution, from cell body to sciatic nerve, appeared normal in SMA∆7 mice. Taken together, these results suggest that reduced axonal transport may provide a mechanistic explanation for reduced synaptic vesicle density and concomitant synaptic transmission defects, while providing evidence that suggests NF accumulations result from local NMJ alterations to NFs.

Effect of diet on the survival and phenotype of a mouse model for spinal muscular atrophy.

Proximal spinal muscular atrophy (SMA) is a leading genetic cause of infant death. Patients with SMA lose alpha-motor neurons in the ventral horn of the spinal cord which leads to skeletal muscle weakness and atrophy. SMA is the result of reduction in Survival Motor Neuron (SMN) expression. Transgenic mouse models of SMA have been generated and are extremely useful in understanding the mechanisms of motor neuron degeneration in SMA and in developing new therapeutic candidates for SMA patients. Several research groups have reported varying average lifespans of SMNDelta7 SMA mice (SMN2(+/+);SMNDelta7(+/+);mSmn(-/-)), the most commonly used mouse model for preclinical therapeutic candidate testing. One environmental factor that varied between research groups was maternal diet. In this study, we compared the effects of two different commercially available rodent chows (PicoLab20 Mouse diet and Harlan-Teklad 22/5 diet) on the survival and motor phenotype of the SMNDelta7 mouse model of SMA. Specifically, the PicoLab20 diet significantly extends the average lifespan of the SMNDelta7 SMA mice by approximately 25% and improved the motor phenotype as compared to the Harlan diet. These findings indicate that maternal diet alone can have considerable impact on the SMA phenotype.

The Wallerian degeneration slow (Wld(s)) gene does not attenuate disease in a mouse model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a severe neuromuscular disease characterized by loss of spinal alpha-motor neurons, resulting in the paralysis of skeletal muscle. SMA is caused by deficiency of survival motor neuron (SMN) protein levels. Recent evidence has highlighted an axon-specific role for SMN protein, raising the possibility that axon degeneration may be an early event in SMA pathogenesis. The Wallerian degeneration slow (Wld(s)) gene is a spontaneous dominant mutation in mice that delays axon degeneration by approximately 2-3 weeks. We set out to examine the effect of Wld(s) on the phenotype of a mouse model of SMA. We found that Wld(s) does not alter the SMA phenotype, indicating that Wallerian degeneration does not directly contribute to the pathogenesis of SMA development.

Breast Cancer Subtype Influences the Accuracy of Predicting Pathologic Response by Imaging and Clinical Breast Exam After Neoadjuvant Chemotherapy.

BACKGROUND/AIM: Clinical response evaluation after neoadjuvant chemotherapy (NACT) for breast cancer could include various imaging methods, as well as clinical breast exam (CBE). We assessed the accuracy of CBE and imaging to predict pathologic response after NACT administration according to breast cancer subtype. PATIENTS AND METHODS: This retrospective cohort study included 84 patients with records of NACT and subsequent primary breast surgery from 2003-2013. Patients were divided into 4 breast cancer subtypes according to hormone receptor (HR) status and human epidermal growth factor receptor-2 (HER2) status. Negative predictive value (NPV), false-negative rate (FNR), false-positive rate (FPR) and positive predictive value (PPV) were calculated for CBE and imaging post-NACT and prior to breast cancer surgery. RESULTS: NPV, FNR, FPR and PPV varied by breast cancer subtype and clinical response evaluation method. Imaging resulted in a higher NPV and a lower FNR than CBE among the entire cohort. There was a lower FPR with CBE. Clinical response evaluation by CBE was highly accurate for predicting pathologic residual disease in HR+ tumors (CBE PPV: 95.5% in HR+HER2-, 100.0% in HR+HER2+). In triple-negative breast cancer (TNBC), the imaging NPV was 100% and the imaging FNR was 0%. CONCLUSION: The use of imaging in HR+ tumors post-NACT may provide little to no additional value that is not already garnered by performance of a CBE. For TNBC, imaging may play a critical role in the prediction of pathologic complete response (pCR) post-NACT.

Transgenic inactivation of murine myostatin does not decrease the severity of disease in a model of Spinal Muscular Atrophy.

Spinal Muscular Atrophy (SMA) is a devastating neurodegenerative disease and is a leading genetic cause of infantile death. SMA is caused by the homozygous loss of Survival Motor Neuron-1 (SMN1). The presence of a nearly identical copy gene called SMN2 has led to the development of several strategies that are designed to elevate SMN levels, and it is clear that SMN2 is an important modifier gene. However, the possibility exists that SMN-independent strategies to lessen the severity of the SMA phenotype could provide insight into disease development as well as aid in the identification of potential therapeutic targets. Muscle enhancement has been considered an interesting target for a variety of neurodegenerative diseases, including SMA. Previously we have shown in SMA mice that delivery of recombinant follistatin resulted in an extension in survival and a general lessening of disease severity. Follistatin is known to functionally block myostatin (MSTN), a potent inhibitor of muscle development. However, follistatin is a multifaceted protein involved in a variety of cellular pathways. To determine whether MSTN inhibition was the primary pathway associated with the previously reported follistatin results, we generated an animal model of SMA in which Mstn was genetically inactivated. In this report we characterize the novel SMA/Mstn model and demonstrate that Mstn inactivation does not significantly enhance muscle development in neonatal animals, nor does it result in an amelioration of the SMA phenotype.

Delivery of therapeutic agents through intracerebroventricular (ICV) and intravenous (IV) injection in mice.

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most often results in failure of potential therapeutics designed for neurodegenerative disorders. Neurodegenerative diseases such as Spinal Muscular Atrophy (SMA), in which the lower motor neurons are affected, can benefit greatly from introducing the therapeutic agents into the CNS. The purpose of this video is to demonstrate two different injection paradigms to deliver therapeutic materials into neonatal mice soon after birth. One of these methods is injecting directly into cerebral lateral ventricles (Intracerebroventricular) which results in delivery of materials into the CNS through the cerebrospinal fluid. The second method is a temporal vein injection (intravenous) that can introduce different therapeutics into the circulatory system, leading to systemic delivery including the CNS. Widespread transduction of the CNS is achievable if an appropriate viral vector and viral serotype is utilized. Visualization and utilization of the temporal vein for injection is feasible up to postnatal day 6. However, if the delivered material is intended to reach the CNS, these injections should take place while the blood brain barrier is more permeable due to its immature status, preferably prior to postnatal day 2. The fully developed blood brain barrier greatly limits the effectiveness of intravenous delivery. Both delivery systems are simple and effective once the surgical aptitude is achieved. They do not require any extensive surgical devices and can be performed by a single person. However, these techniques are not without challenges. The small size of postnatal day 2 pups and the subsequent small target areas can make the injections difficult to perform and initially challenging to replicate.