Stepper-Based Training Improves Monocyte-Platelet Aggregation and Thrombin Generation in Nonambulatory Hemiplegic Patients.

PURPOSE: Nonambulatory stroke patients are extremely sedentary, but most available data concerning exercise training in stroke patients are related to ambulatory patients. This study aimed to investigate the efficacy of stepper-based exercise training on cardiopulmonary fitness, monocyte subtypes, and associated monocyte-platelet aggregates (MPA) and thrombin generation (TrG) in nonambulatory hemiplegic patients with ischemic stroke. METHOD: Thirty-eight patients were randomized into exercise training (ET, n = 20) and usual care (UC, n = 18) groups. The ET underwent supervised exercise training (60% peak work rate) using a recumbent stepper for two to four sessions per week and 36 sessions in total. In addition, 12 healthy participants were enrolled as healthy controls. Monocyte characteristics, MPA, and plasma TrG kinetics were determined before and after intervention by flow cytometry and calibrated automated thrombogram® (CAT). RESULTS: Seventeen and 15 patients completed the protocol in the ET and UC groups. Peak V̇O2 improved in ET (15.7 ± 4.8 vs 18.9 ± 5.3 mL·min-1·kg-1, +20%), so did the phase angle of the hemiplegic limbs. The counts of total MPA and MPA associated with three monocyte subtypes, alongside CD42b expression all declined in ET with subtypes 2 and 1 being the most prominent. Macrophage inflammatory protein 1ß (MIP-1 ß) level also declined. The TrG kinetics was attenuated after ET by delaying initiation and reducing the rising slope and peak of thrombin production. In UC, no difference was revealed in the pre-post comparison. CONCLUSIONS: Stepper-based ET is feasible in nonambulatory hemiplegic patients and is effective in improving aerobic fitness. Moreover, it decreases heteroaggregation of monocytes with platelets, especially in monocyte subtypes 2 and 1. Thrombin generation was also attenuated. Hence, stepper-based ET may be incorporated in the rehabilitation of nonambulatory hemiplegic patients.

Comparative analysis of alternating hemiplegia of childhood and rapid-onset dystonia-parkinsonism ATP1A3 mutations reveals functional deficits, which do not correlate with disease severity.

Heterozygous mutations in the ATP1A3 gene, coding for an alpha subunit isoform (α3) of Na+/K+-ATPase, are the primary genetic cause for rapid-onset dystonia-parkinsonism (RDP) and alternating hemiplegia of childhood (AHC). Recently, cerebellar ataxia, areflexia, pes cavus, optic atrophy and sensorineural hearing loss (CAPOS), early infantile epileptic encephalopathy (EIEE), childhood rapid onset ataxia (CROA) and relapsing encephalopathy with rapid onset ataxia (RECA) extend the clinical spectrum of ATP1A3 related disorders. AHC and RDP demonstrate distinct clinical features, with AHC symptoms being generally more severe compared to RDP. Currently, it is largely unknown what determines the disease severity, and whether severity is linked to the degree of functional impairment of the α3 subunit. Here we compared the effect of twelve different RDP and AHC specific mutations on the expression and function of the α3 Na+/K+-ATPase in transfected HEK cells and oocytes. All studied mutations led to functional impairment of the pump, as reflected by lower survival rate and reduced pump current. No difference in the extent of impairment, nor in the expression level, was found between the two phenotypes, suggesting that these measures of pump dysfunction do not exclusively determine the disease severity.

Neuronal modeling of alternating hemiplegia of childhood reveals transcriptional compensation and replicates a trigger-induced phenotype.

Alternating hemiplegia of childhood (AHC) is a rare neurodevelopmental disease caused by heterozygous de novo missense mutations in the ATP1A3 gene that encodes the neuronal specific α3 subunit of the Na,K-ATPase (NKA) pump. Mechanisms underlying patient episodes including environmental triggers remain poorly understood, and there are no empirically proven treatments for AHC. In this study, we generated patient-specific induced pluripotent stem cells (iPSCs) and isogenic controls for the E815K ATP1A3 mutation that causes the most phenotypically severe form of AHC. Using an in vitro iPSC-derived cortical neuron disease model, we found elevated levels of ATP1A3 mRNA in AHC lines compared to controls, without significant perturbations in protein expression. Microelectrode array analyses demonstrated that in cortical neuronal cultures, ATP1A3+/E815K iPSC-derived neurons displayed less overall activity than neurons differentiated from isogenic mutation-corrected and unrelated control cell lines. However, induction of cellular stress by elevated temperature revealed a hyperactivity phenotype following heat stress in ATP1A3+/E815K neurons compared to control lines. Treatment with flunarizine, a drug commonly used to prevent AHC episodes, did not impact this stress-triggered phenotype. These findings support the use of iPSC-derived neuronal cultures for studying complex neurodevelopmental conditions such as AHC and provide a platform for mechanistic discovery in a human disease model.

Factors in the disease severity of ATP1A3 mutations: Impairment, misfolding, and allele competition.

Dominant mutations of ATP1A3, a neuronal Na,K-ATPase α subunit isoform, cause neurological disorders with an exceptionally wide range of severity. Several new mutations and their phenotypes are reported here (p.Asp366His, p.Asp742Tyr, p.Asp743His, p.Leu924Pro, and a VUS, p.Arg463Cys). Mutations associated with mild or severe phenotypes [rapid-onset dystonia-parkinsonism (RDP), alternating hemiplegia of childhood (AHC), or early infantile epileptic encephalopathy (EIEE)] were expressed in HEK-293 cells. Paradoxically, the severity of human symptoms did not correlate with whether there was enough residual activity to support cell survival. We hypothesized that distinct cellular consequences may result not only from pump inactivation but also from protein misfolding. Biosynthesis was investigated in four tetracycline-inducible isogenic cell lines representing different human phenotypes. Two cell biological complications were found. First, there was impaired trafficking of αß complex to Golgi apparatus and plasma membrane, as well as changes in cell morphology, for two mutations that produced microcephaly or regions of brain atrophy in patients. Second, there was competition between exogenous mutant ATP1A3 (α3) and endogenous ATP1A1 (α1) so that their sum was constant. This predicts that in patients, the ratio of normal to mutant ATP1A3 proteins will vary when misfolding occurs. At the two extremes, the results suggest that a heterozygous mutation that only impairs Na,K-ATPase activity will produce relatively mild disease, while one that activates the unfolded protein response could produce severe disease and may result in death of neurons independently of ion pump inactivation.

[Hypoglycemic Hemiplegia].

Hypoglycemia may cause acute hemiplegia. The most common diffusion-weighted MR imaging finding in patients with hypoglycemic hemiplegia is the hyperintense lesion involving the internal capsule, mimicking acute ischemic stroke. Thus, in patients with acute onset hemiplegia, it is important to differentiate hypoglycemia on arrival by immediate blood glucose measurement. It has recently been shown that hypoglycemic brain injury start in large white matter tracts such as internal capsule and spread throughout the whole brain, including the gray matter. However, it is still unclear why focal signs such as hemiplegia develope in metabolic disorders affecting the whole brain.

Digital clubbing: forms, associations and pathophysiology.

Among proposed mechanisms to explain digital clubbing, the release of cytokines, specifically vascular endothelial growth factor and platelet-derived growth factor, from aggregated platelets and megakaryocytes has emerged as the most likely explanation. This review describes these and other contributory processes.

α-synuclein assemblies sequester neuronal α3-Na+/K+-ATPase and impair Na+ gradient.

Extracellular α-synuclein (α-syn) assemblies can be up-taken by neurons; however, their interaction with the plasma membrane and proteins has not been studied specifically. Here we demonstrate that α-syn assemblies form clusters within the plasma membrane of neurons. Using a proteomic-based approach, we identify the α3-subunit of Na+/K+-ATPase (NKA) as a cell surface partner of α-syn assemblies. The interaction strength depended on the state of α-syn, fibrils being the strongest, oligomers weak, and monomers none. Mutations within the neuron-specific α3-subunit are linked to rapid-onset dystonia Parkinsonism (RDP) and alternating hemiplegia of childhood (AHC). We show that freely diffusing α3-NKA are trapped within α-syn clusters resulting in α3-NKA redistribution and formation of larger nanoclusters. This creates regions within the plasma membrane with reduced local densities of α3-NKA, thereby decreasing the efficiency of Na+ extrusion following stimulus. Thus, interactions of α3-NKA with extracellular α-syn assemblies reduce its pumping activity as its mutations in RDP/AHC.

A randomized comparison of energy consumption when using different canes, inpatients after stroke.

OBJECTIVES: To investigate the differences in oxygen consumption associated with gait in hemiplegic patients according to the type of cane they use. DESIGN: A randomized crossover design. SETTING: University hospital-based rehabilitation center, Korea. SUBJECTS: Thirty consecutive patients (mean ± SD age, 56.3 ± 3.2 years) with chronic stroke, 17 (56.7%) males and 13 (43.3%) females. INTERVENTIONS: At approximately the same time of day for three consecutive days, each participant completed a walk with one of three randomly assigned types of canes: a single-point cane, a quad cane, and a hemi-walker. MAIN OUTCOME MEASURE: Energy expenditure (O2 rate, mL/kg/min), energy cost (O2 cost, mL/kg/m), and heart rate (HR) via a portable gas analyzer, a 10-meter walk test (10MWT), and a 6-minute walk test (6MWT). RESULTS: Energy expenditure, gait endurance, and gait velocity for a single-point cane were higher (p<0.001 or p=0.005) than for any other type of cane. Energy cost (0.5 ± 0.2 mL/kg/m vs. 0.6 ± 0.2 mL/kg/m vs. 0.6 ± 0.2 ml/kg/m, respectively, p=0.001) was lower for the single-point cane, except for HR (p ≥ 0.05) after the Bonferroni correction (0.05/5=0.01). CONCLUSIONS: A single-point cane requires less oxygen use at a given speed, or permits greater speed for the same oxygen consumption.

Energy cost of obstacle crossing in stroke patients.

OBJECTIVE: The purpose of this study was to analyze the effects of clearing and skirting obstacles during the gait on the energetic cost of walking (ECW) of patients with chronic hemiplegia. The hypothesis was that hemiplegia would have a greatest increase in the ECW than in the healthy group. DESIGN: Fifteen healthy subjects and 17 patients with chronic hemiplegia completed two 6-min walking sessions: one with obstacles and the other without obstacles. During both sessions, the patients were equipped with a portable gas analyzer to measure oxygen uptake (V˙o2). Gait velocity and ECW were calculated. RESULTS: In both groups, gait velocity was lower in the with-obstacles condition and the ECW was significantly higher. V˙o2 was greater in the with-obstacles condition for the healthy group, whereas it remained unchanged for the group with hemiplegia. CONCLUSIONS: Results demonstrated that the addition of obstacles during gait increased the ECW and decreased mean walking speed in both the healthy subjects and the patients with hemiplegia. More interestingly, the authors found differences in adaptation strategies between the healthy subjects and the patients with hemiplegia. During the with-obstacles condition, the oxygen uptakes of the healthy subjects increased and mean walking speed decreased, whereas, in the subjects with hemiplegia, only mean walking speed decreased.

Alternating Hemiplegia of Childhood mutations have a differential effect on Na(+),K(+)-ATPase activity and ouabain binding.

De novo mutations in ATP1A3, the gene encoding the α3-subunit of Na(+),K(+)-ATPase, are associated with the neurodevelopmental disorder Alternating Hemiplegia of Childhood (AHC). The aim of this study was to determine the functional consequences of six ATP1A3 mutations (S137Y, D220N, I274N, D801N, E815K, and G947R) associated with AHC. Wild type and mutant Na(+),K(+)-ATPases were expressed in Sf9 insect cells using the baculovirus expression system. Ouabain binding, ATPase activity, and phosphorylation were absent in mutants I274N, E815K and G947R. Mutants S137Y and D801N were able to bind ouabain, although these mutants lacked ATPase activity, phosphorylation, and the K(+)/ouabain antagonism indicative of modifications in the cation binding site. Mutant D220N showed similar ouabain binding, ATPase activity, and phosphorylation to wild type Na(+),K(+)-ATPase. Functional impairment of Na(+),K(+)-ATPase in mutants S137Y, I274N, D801N, E815K, and G947R might explain why patients having these mutations suffer from AHC. Moreover, mutant D801N is able to bind ouabain, whereas mutant E815K shows a complete loss of function, possibly explaining the different phenotypes for these mutations.

Functional studies and proteomics in platelets and fibroblasts reveal a lysosomal defect with increased cathepsin-dependent apoptosis in ATP1A3 defective alternating hemiplegia of childhood.

Alternating hemiplegia of childhood (AHC) is a rare syndrome with repeated hemiplegic episodes, paroxysmal events and global neurological impairment. Recently, heterozygous de novo ATP1A3 missense mutations have been identified in AHC patients, but the underlying pathogenesis mechanism remains unknown. Mutation analysis of ATP1A3 in 9 unrelated AHC cases revealed mostly D801N or E815K variants. As platelets represent a good cellular model to study defects in neuropathologies, morphological and functional experiments were performed in these subjects. Platelets from the AHC patients presented with structural and functional abnormalities of granules positive for the lysosomal marker CD63. Similar structural granule abnormalities were detected in patients' fibroblasts. Proteomic analysis of platelets and fibroblasts showed a total of 93 differentially expressed proteins in AHC mainly involved in metabolism. Interestingly, 7 of these proteins were detected in both cell types, including the lysosomal protein cathepsin. AHC fibroblasts revealed significantly increased levels of activated cathepsin B, which induces a stronger activation of apoptosis. Our study is the first to link ATP1A3 defects in AHC to a platelet and fibroblast lysosomal defect with evidence of increased apoptosis. Further studies are needed to define how this lysosomal defect is related to decreased ATPase activity. Biological Significance Only recently, the genetic cause of AHC was identified as heterozygous ATP1A3 mutations, but the underlying pathophysiological mechanism still remains unknown. By performing functional, morphological and proteomic studies in AHC patients we found a structural and functional granule defect in AHC platelets and fibroblasts that was specifically found in granules positive for the lysosomal marker CD63. In particular, proteomics identified several differentially expressed proteins in fibroblasts and platelets from AHC cases that are predicted to have an important role in cell function and maintenance, a pathway typically attributed to lysosomes. The lysosomal protein cathepsin was found to be differentially expressed in both platelets and fibroblasts of AHC patients, inducing a stronger activation of mainly the intrinsic apoptosis. Despite the precise mechanism for the increased lysosomal cathepsin B-dependent apoptosis detected in AHC in relation to impaired ATP1A3 deserves further studies, we could here show some evidence for a defective regulation of apoptosis in AHC, a disease that still has no biochemical or neuroradiological parameters for diagnosis.

Altered autophagy gene expression and persistent atrophy suggest impaired remodeling in chronic hemiplegic human skeletal muscle.

INTRODUCTION: Upper motor neuron lesions after stroke are a major cause of disability. We aimed to determine whether skeletal muscles from these patients display typical molecular signatures of inflammation, growth arrest, and atrophy. METHODS: Muscle biopsies were analyzed for morphological, histochemical, ultrastructural, and molecular features indicative of changes in gene expression involved in muscle atrophy. RESULTS: Chronic hemiplegia resulted in ~9.5% atrophy, fiber type shifts, and histochemical and ultrastructural signs of impaired remodeling. TNF and TWEAK expressions were unaltered, but MSTN mRNA was lower (-73%, P < 0.05) in paretic tibialis anterior vs. age-matched controls. The expression of autophagy-related genes (BCN-1, LC3, and GABARAPL1) was lower in paretic tibialis anterior (-81%, -48%, and -60%, respectively, P < 0.01) and soleus (-85%, -54%, and -60% respectively, P < 0.01) compared with old controls. CONCLUSIONS: Persistent atrophy in chronic spastic hemiplegia may be associated with impaired remodeling partly due to altered autophagy gene expression.

Dopaminergic D2-like agonists produce yawning in the myelin mutant taiep and Sprague-Dawley rats.

Systemic administration of D2-like dopaminergic-receptor agonists increases yawning behavior. However, only a few studies have been done in animals with pathological conditions. The taiep rat is a myelin mutant with an initial hypomyelination followed by progressive demyelination, being the brainstem one of the most affected areas. In our experiments, we analyzed the effects of systemic administration of the D2-family agonists and antagonists on yawning behavior, and correlated them with the lipid myelin content in the brainstem and other areas in the central nervous system (CNS) in 8 month old male taiep and Sprague-Dawley rats. Subjects were maintained under standard conditions in Plexiglas cages with a 12:12 light-dark cycle, lights on at 0700 and free access to rodent pellets and tap water. Drugs were freshly prepared injected ip at 0800 and subjects were observed for 60 min. When antagonists were used it was administered 15 min before the agonist. Sprague-Dawley and taiep rats significantly increased their yawning frequency after systemic injection of (-)-quinpirole hydrochloride, R(+)-7-Hydroxy-2-(dipropylamino)tetralin hydrobromide (7-OH-DPAT) or trans-(±)-3,4,4a,10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano [4,3-b]-1,4-oxazin-9-ol hydrochloride ((±)-PD 128,907). Among D2-like agonists used higher effects are obtained with (-)-quinpirole. The effects caused by (-)-quinpirole can be reduced by (-)-sulpiride; and yawning caused by 7-OH-DPAT was decreased by tiapride only in taiep rats. In Sprague-Dawley only (-)-sulpiride is able to decrease (-)-quinpirole-caused yawning. In conclusion, dopaminergic D2-like agonists are still able to cause yawning despite the severe myelin loss in taiep rats. Similarly, patients with various CNS illnesses that affect myelin, such as stroke or multiple sclerosis, are able to yawn suggesting that trigger neurons are still able to command this innate behavior.

Role of SDF1/CXCR4 interaction in experimental hemiplegic models with neural cell transplantation.

Much attention has been focused on neural cell transplantation because of its promising clinical applications. We have reported that embryonic stem (ES) cell derived neural stem/progenitor cell transplantation significantly improved motor functions in a hemiplegic mouse model. It is important to understand the molecular mechanisms governing neural regeneration of the damaged motor cortex after the transplantation. Recent investigations disclosed that chemokines participated in the regulation of migration and maturation of neural cell grafts. In this review, we summarize the involvement of inflammatory chemokines including stromal cell derived factor 1 (SDF1) in neural regeneration after ES cell derived neural stem/progenitor cell transplantation in mouse stroke models.

Alternating hemiplegia of childhood: metabolic studies in the largest European series of patients.

Alternating hemiplegia of childhood (AHC) is a rare disorder with diagnosis based on clinical criteria, as no laboratory, neuroradiological or genetic markers are currently available. The pathogenic mechanisms are still an enigma. Some hypotheses have been proposed such as hemiplegic migraine variant, epileptic mechanism, channelopathy and mitochondrial disorder, but none of these has been confirmed. Our aim was to analyze the results of metabolic studies performed on a series of 157 European patients who fulfilled diagnostic criteria for AHC. We tried to find a common metabolic abnormality, related with AHC. We did not find significant abnormalities in basic metabolic screening, at different ages. Neurotransmitters in cerebrospinal fluid (n = 26) were normal in all of the patients. Mitochondrial respiratory chain enzyme activities were analyzed in 19 muscle biopsies; in 4 cases, different MRC enzyme deficiencies were demonstrated, ranging from mild-unspecific deficiencies to more profound and probably primary defects. Although we did not find specific metabolic markers in our series, some metabolic disorders such as pyruvate dehydrogenase deficiency, MELAS, cerebral glucose transporter defect and neurotransmitter deficiency can exhibit symptoms similar to those of AHC and need to be ruled out before a diagnosis of AHC can be established. Further studies including high-throughput diagnostic technologies seem necessary to elucidate the etiology of this severe and enigmatic disorder.

A new case of idiopathic hemiplegia hemiconvulsion syndrome.

We report a new case of infantile idiopathic hemiconvulsion-hemiplegia syndrome (HH). A prolonged right-sided febrile convulsion was followed 4 days later, by right hemiconvulsive status epilepticus, documented by video-electroencephalogram (EEG) recording. The child developed an ipsilateral hemiplegia, partially improved during the first month of follow-up. Sequential cerebral magnetic resonance imaging (MRI) and proton magnetic resonance spectroscopy (1H-MRS) at 6, 15, 30 days of follow-up showed a cytotoxic edema in the left hemisphere and a subsequent necrosis. At 1-year of follow-up, we performed MRI control because of febrile convulsion lasting few minutes that confirmed a non-progressive left hemisphere atrophy. After 2 years, the patient was seizure-free, with a mild right hemiplegia and language skills deficit. We discuss the unclear pathogenesis of HH through sequential neuroradiological evaluation.

An update on peptide drugs for voltage-gated calcium channels.

Voltage-gated calcium channels are one of the major ion channels distributed in the human central nervous system, and mediate an influx of extracellular Ca(2+) in response to membrane depolarization. Calcium channels are of particular interest in a broad range of cellular functions including cell proliferation and differentiation, gene expression, neurite outgrowth, transmitter and hormone release, and brain plasticity. The dysfunction of calcium channels is related to a variety of clinical disorders such as migraine, hemiplegia, and epilepsy. Therefore, calcium channels have gained great pharmaceutical interest as a privileged target class for the treatment of a wide range of human diseases. This review will examine the known marketed peptide drugs for calcium channels and address the development of some important patented peptide molecules targeting calcium channels.