Objective To report results of intrathecal nusinersen in children with later-onset spinal muscular atrophy (SMA). Methods Analyses included children from a phase 1b/2a study (ISIS-396443-CS2; NCT01703988) who first received nusinersen during that study and were eligible to continue treatment in the extension study (ISIS-396443-CS12; NCT02052791). The phase 1b/2a study was a 253-day, ascending dose (3, 6, 9, 12 mg), multiple-dose, open-label, multicenter study that enrolled children with SMA aged 2-15 years. The extension study was a 715-day, single-dose level (12 mg) study. Time between studies varied by participant (196-413 days). Assessments included the Hammersmith Functional Motor Scale-Expanded (HFMSE), Upper Limb Module (ULM), 6-Minute Walk Test (6MWT), compound muscle action potential (CMAP), and quantitative multipoint incremental motor unit number estimation. Safety also was assessed. Results Twenty-eight children were included (SMA type II, n = 11; SMA type III, n = 17). Mean HFMSE scores, ULM scores, and 6MWT distances improved by the day 1,150 visit (HFMSE: SMA type II, + 10.8 points; SMA type III, + 1.8 points; ULM: SMA type II, + 4.0 points; 6MWT: SMA type III, + 92.0 meters). Mean CMAP values remained relatively stable. No children discontinued treatment due to adverse events. Conclusions Nusinersen treatment over similar to 3 years resulted in motor function improvements and disease activity stabilization not observed in natural history cohorts. These results document the long-term benefit of nusinersen in later-onset SMA, including SMA type III.

Spinal muscular atrophy (SMA), traditionally described as a predominantly childhood form of motor neurone disease, is the leading genetic cause of infant mortality. Although motor neurones are undoubtedly the primary affected cell type, the severe infantile form of SMA (Type I SMA) is now widely recognised to represent a multisystem disorder where a variety of organs and systems in the body are also affected. Here, we report that the spleen is disproportionately small in the Taiwanese' murine model of severe SMA (Smn(-/-);SMN2(tg/0)), correlated to low levels of cell proliferation and increased cell death. Spleen lacks its distinctive red appearance and presents with a degenerated capsule and a disorganised fibrotic architecture. Histologically distinct white pulp failed to form and this was reflected in an almost complete absence of B lymphocytes necessary for normal immune function. In addition, megakaryoctyes persisted in the red pulp. However, the vascular density remained unchanged in SMA spleen. Assessment of the spleen in SMA patients with the infantile form of the disease indicated a range of pathologies. We conclude that development of the spleen fails to occur normally in SMA mouse models and human patients. Thus, further analysis of immune function is likely to be required to fully understand the full extent of systemic disease pathology in SMA.

Mutations in ATP1A3 cause Alternating Hemiplegia of Childhood (AHC) by disrupting function of the neuronal Na+/K+ ATPase. Published studies to date indicate 2 recurrent mutations, D801N and E815K, and a more severe phenotype in the E815K cohort. We performed mutation analysis and retrospective genotype-phenotype correlations in all eligible patients with AHC enrolled in the US AHC Foundation registry from 1997-2012. Clinical data were abstracted from standardized caregivers’ questionnaires and medical records and confirmed by expert clinicians. We identified ATP1A3 mutations by Sanger and whole genome sequencing, and compared phenotypes within and between 4 groups of subjects, those with D801N, E815K, other ATP1A3 or no ATP1A3 mutations. We identified heterozygous ATP1A3 mutations in 154 of 187 (82%) AHC patients. Of 34 unique mutations, 31 (91%) are missense, and 16 (47%) had not been previously reported. Concordant with prior studies, more than 2/3 of all mutations are clustered in exons 17 and 18. Of 143 simplex occurrences, 58 had D801N (40%), 38 had E815K (26%) and 11 had G937R (8%) mutations. Patients with an E815K mutation demonstrate an earlier age of onset, more severe motor impairment and a higher prevalence of status epilepticus. This study further expands the number and spectrum of ATP1A3 mutations associated with AHC and confirms a more deleterious effect of the E815K mutation on selected neurologic outcomes. However, the complexity of the disorder and the extensive phenotypic variability among subgroups merits caution and emphasizes the need for further studies.

Kisspeptin, through its receptor, GPR54, regulates gonadotropin releasing hormone (GnRH) mRNA. However, the relationship between mRNA and protein is controversial and confounded by the lack of a method for analyzing protein levels from tissue sections. GnRH mRNA is markedly suppressed in GPR54GnRH knockout (KO) mice. We determined whether reduced mRNA dictated less protein expression in the neurons. We examined two features: axon diameter and somal size. We hypothesized that the absence of GPR54 would produce decreased GnRH levels reflected by the thickness of the immunostained axons and the size of the cell bodies. Sections from global and conditional GPR54 KO mice were stained using the ABC immunoperoxidase method and compared to sections from wild type controls. We first learned that the optimum staining conditions for the KO mice grossly overstained the WT. When conditions were adjusted to be in the linear part of the titration curve, obvious differences in protein levels emerged. Axons containing GnRH were imaged at a magnification of 200x, then skeletonized to 1 µm width, enabling calculation of total axon length in the field. The axon area divided by the skeletonized length would then be equivalent to the axon diameter. Axon volume was calculated. For cells, somal width was determined. Our results demonstrate that the GnRH+ axons in GPR54 KO mice are approximately three times thinner than WT axons and somal diameter was proportionately reduced. The results stress the importance of primary antibody titrations.Funded by NIH grants R01HD370246, U01HD66435 and U01HD66432 to SR and GEH

OBJECTIVE:Spinal muscular atrophy (SMA) is the number 1 genetic killer of young children. It is caused by mutation or deletion of the survival motor neuron 1 (SMN1) gene. Although SMA is primarily a motor neuron disease, metabolism abnormalities such as metabolic acidosis, abnormal fatty acid metabolism, hyperlipidemia, and hyperglycemia have been reported in SMA patients. We thus initiated an in-depth analysis of glucose metabolism in SMA.METHODS:Glucose metabolism and pancreas development were investigated in the Smn(2B/-) intermediate SMA mouse model and type I SMA patients.RESULTS:Here, we demonstrate in an SMA mouse model a dramatic cell fate imbalance within pancreatic islets, with a predominance of glucagon-producing α cells at the expense of insulin-producing β cells. These SMA mice display fasting hyperglycemia, hyperglucagonemia, and glucose resistance. We demonstrate similar abnormalities in pancreatic islets from deceased children with the severe infantile form of SMA in association with supportive evidence of glucose intolerance in at least a subset of such children.INTERPRETATION:Our results indicate that defects in glucose metabolism may play an important contributory role in SMA pathogenesis.

Alternating hemiplegia of childhood (AHC) is a rare, severe neurodevelopmental syndrome characterized by recurrent hemiplegic episodes and distinct neurological manifestations. AHC is usually a sporadic disorder and has unknown etiology. We used exome sequencing of seven patients with AHC and their unaffected parents to identify de novo nonsynonymous mutations in ATP1A3 in all seven individuals. In a subsequent sequence analysis of ATP1A3 in 98 other patients with AHC, we found that ATP1A3 mutations were likely to be responsible for at least 74% of the cases; we also identified one inherited mutation in a case of familial AHC. Notably, most AHC cases are caused by one of seven recurrent ATP1A3 mutations, one of which was observed in 36 patients. Unlike ATP1A3 mutations that cause rapid-onset dystonia-parkinsonism, AHC-causing mutations in this gene caused consistent reductions in ATPase activity without affecting the level of protein expression. This work identifies de novo ATP1A3 mutations as the primary cause of AHC and offers insight into disease pathophysiology by expanding the spectrum of phenotypes associated with mutations in ATP1A3.

Objective: To examine the pathology of the motor unit in spinal muscular atrophy (SMA). Background SMA is an inherited motor neuron disease, which often causes early mortality. Although there is currently no disease-modify treatment to offer patients, several drugs show promising effects in SMA mouse models in which the pathology of the motor unit has been well defined. In mice, there are early abnormalities of motor neuron (MN) terminals prior to MN loss with differential susceptibility of certain MN groups. Limited prior studies of human SMA pathology have not characterized early changes of MNs, their axons, or terminals. Design/Methods: At autopsy, spinal cord, nerve roots, phrenic nerve, and skeletal muscle were collected from 14 SMA cases and analyzed with confocal and electron microscopy methods. Results: Vast numbers of small, round myofibers were evident in the psoas, paraspinal, and intercostal muscles, but myofiber size was relatively preserved in diaphragm muscle. Phrenic nerve showed an average of 1830±177 axons/nerve in SMA cases, while ventral roots exhibited a severe reduction in axonal density. Active axonal degeneration was present in ventral roots and phrenic nerve where 3.7±0.45% of axons showed a compacted myelin appearance. Neuromuscular junctions (NMJs) in the diaphragm were often well innervated, but many axon terminals displayed neurofilament aggregation and others were very thin. EM analysis confirmed neurofilament aggregation and reduced density of synaptic vesicles in SMA diaphragm NMJs. Conclusions: These studies show that motor units are differentially affected in human SMA, with phrenic nerve motor neurons innervating the diaphragm less affected than those innervating the paraspinal, intercostal, or proximal limb muscles. The diaphragm may therefore provide important insights into early SMA disease pathology. In this muscle, most NMJs were innervated but structurally abnormal. These studies raise the possibility that the distal motor neuron terminal and axon are the earliest sites of disease pathogenesis in human SMA. Supported by: The SMA Foundation and by R01NS062869 (CJS) from NINDS and by R01-HD054599 (KJS) from NICHD. Disclosure: Dr. Crowder has nothing to disclose. Dr. Polley has nothing to disclose. Dr. Kong has nothing to disclose. Dr. Van Meerbeke has nothing to disclose. Dr. Murphy has nothing to disclose. Dr. Swoboda has received research support from BioMarin Pharmaceuticals and Orphamed, Dr. Crawford has nothing to disclose. Dr. Sumner has nothing to disclose.

Spinal muscular atrophy, the most prevalent hereditary motor neuron disease, is caused by mutations in the survival motor neuron (SMN) 1 gene. A significant reduction in the encoded SMN protein leads to the degeneration of motor neurons. However, the molecular events leading to this process are not well understood. The present study uses a previously developed neuronal cell culture model of spinal muscular atrophy for a multiplex transcriptome analysis. Furthermore, gene expression analysis was performed on in vitro cell cultures, as well as tissue samples of spinal muscular atrophy patients and transgenic mice. RNA and subsequent Western blot protein analyses suggest that low SMN levels are associated with significantly lower alpha-synuclein expression. Examination of two genes related to vesicular transport showed a similar though less dramatic decrease in expression. The 140-amino acid protein alpha-synuclein, dominant mutations of which have previously been associated with an autosomal dominant form of Parkinson's disease, is strongly expressed in select neurons of the brain. Although not well understood, the physiologic functions of alpha-synuclein have been linked to synaptic vesicular neurotransmitter release and neuroprotection, suggesting a possible contribution to Smn-deficient motor neuron pathology. Furthermore, alpha-synuclein may be a genetic modifier or biomarker of spinal muscular atrophy.

Spinal Muscular Atrophy (SMA) is the most common neurodegenerative disease in live births, and is caused by the homozygous deletion of the SMN1 gene and modulated by the amount of protein product of the SMN2 gene. Higher primates are the only animals to carry both the SMN1 and SMN2 genes; all other organisms have the single copy of SMN1 and its deletion causes an embryonic lethal condition. Chick embryos are ideally situated for (1) detection of distinct stages of development, (2) manipulation via electroporation of an interfering RNA (RNAi) construct into the one copy of SMN in the developing chick, and (3) analysis of the resulting deficiency within a short period of time (48 hours) after RNAi introduction. We show that electroporation alone or electroporation of the GFP reporter construct do not cause phenotypic abnormalities which are present with the electroporation of the SMN/GFP RNAi, and that the presence of the SMN/GFP RNAi reduces the amount of SMN protein in the chick as demonstrated by Western Blot and immunohistochemistry. Creating this new model of a common neurodegenerative disease allows us to modulate the severity, and thus more closely mimic the clinical presentation of the disease, by targeting the microinjection/electroporation to earlier or later stages of development; test drug and other interventions; and determine some of the earliest embryonic manifestations of SMA. 1RO1HD054599‐01 to KJS

Parkinson's Disease (PD) affects more than 1 million Americans and 50,000 new cases are diagnosed in the US each year. The vast majority of PD cases have been shown to be idiopathic and caused by environmental toxins; therefore creating an animal model to test both whether a given toxin is causative of the disease or not, and what drugs can be administered to ameliorate the effects of those toxins would advance prevention and treatment of PD in humans. The remaining 10% of the human cases of PD are caused by genetic factors. Drosophila melanogaster provides us with an excellent model organism for both idiopathic and genetic disease modeling, but we are faced with the challenge of comparing different studies and their different reports of dopaminergic neuronal loss (the diagnostic hallmark of PD in humans). We have developed an optimized protocol of fixation, embedding, and immunohistochemistry in the fly brain to demonstrate those neurons. This protocol combines a weak cross-linking fixative containing an anti-oxidant to provide protection to the tissue, microwave-enhanced penetration of the fixative into the fly brain, thick agarose sectioning with a vibratome, and immunostaining using an antibody specific to the Drosophila ortholog of a dopaminergic epitope to identify these neurons.

Parkinson's disease (PD) affects >1 million Americans and is marked by the loss of dopaminergic neurons in the substantia nigra. PD has been linked to two causative factors: genetic risks (hereditary PD) and environmental toxins (idiopathic PD). In recent years, considerable effort has been devoted to the development of a Drosophila model of human PD that might be useful for examining the cellular mechanisms of PD pathology by genetic screening. In 2000, Feany and Bender reported a Drosophila model of PD in which transgenic flies expressing human mutant α-synuclein exhibited shortened life spans, dopaminergic losses, Parkinsonian behaviors, and Lewy bodies in surviving dopaminergic neurons. Since then, a number of studies have been published that validate the model or build on it; conversely, a number report an inability to replicate the results and suggest that most protocols for dopaminergic histology underreport the actual numbers of dopaminergic neurons in the insect brain. Here we report the optimization of dopaminergic histology in Drosophila and identification of new dopaminergic neurons, show the remarkable dendritic complexity of these neurons, and provide an updated count of these neurons in adult brains. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Fatty acid utilization is initiated by fatty acid-CoA ligase, which converts free fatty acids into fatty acyl-CoA esters. We have cloned previously the human long-chain fatty acid-CoA ligase 4 (FACL4), which is a central enzyme in controlling the free arachidonic acid level in cells and thereby regulating eicosanoid production. We report here the expression of this gene in tissues, particularly in different parts of the brain. We found that FACL4 encoded a 75 kDa enzyme and that there was a modified translation product expressed in the brain. FACL4 was expressed in early stages of development with a significant amount of FACL4 mRNA detected in an E7 mouse embryo. In addition, FACL4 was highly expressed in both adult and newborn mouse brain especially in the granule cells of the dentate gyrus and the pyramidal cell layer of CA1 in hippocampus, and the granular cell layer and Purkinje cells of the cerebellum.

Background and Aims: Interleukin 6 (IL-6) is a pro-inflammatory cytokine which is predominately produced by human immunocytes, fibroblasts and endothelial cells.However, increased mncosal IL-6 level had been detected in several gastrointestinal diseases.It is still unknown if IL-6 could be produced by gastric epithelial ceils.We aimed to determine if the gastric epithelial cell could produce IL-6 after inflammatory cytokine TNF-cx and IL-113 stimulation and to study their signal pathway.Methods: Gastric epithelial cell line MKN-28, a well differentiated gastric adenocarcinoma cell line 2x105/ml was cultured in a 96 well culture plate, with 10% FCS RPMI-1640 and stimulated with different doses of TNF-ct, IL-I[3 and protein kinase inhibitors.Cell culture supernatant IL-6 level was determined by ELISA.Cell viability was tested by routine MT'f methods.Results: Two, 8, 24 or 36 hr after TNF-ct and IL-1[~ stimulation, MKN-28 cell produced an significant increase of IL-6 in the culture media (7.6 +_0.6, 21.9 +_ 1.6, 26.7 +_ 6.1, 33.9 +_ 1.2 ng/ml and 6.97 + 2.1, 19.8 + 1.27, 27.2 -+ 2.4, 32.1 + 2.1 ng/ml respectively), and was about 3-12 fold over media control.This increase was also showed a doserelated manner.The protein tyrosine kinase (PTK) inhibitor herbimycin, at the doses of 0.1, 1, 10 ~M dose-dependently reduced TNF-ct and IL-I[~ induced IL-6 expression by 8.55%, 24.9%, 56.6% and 23.08%, 26.7%, 40.5% respectively.Dexamethasone mimic this effect by reducing TNF-ct and IL-I~ induced IL-6 expression in a dose related manner.Protein kinase (PK) C inhibitors GF109203X and PKA inhibitor H8 had no effect on TNF-ct and IL-I~ induced IL-6 production.Conclusions: These results indicated that in addition to the previously described cell systems, gastric epithelial cell could also induce IL-6 production and this expression involved PTK activation and was sensitive to dexamethasone, but not PKC and PKA activation.