Dysfunction of proprioceptive sensory synapses is a pathogenic event and therapeutic target in mice and humans with spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by a varying degree of severity that correlates with the reduction of SMN protein levels. Motor neuron degeneration and skeletal muscle atrophy are hallmarks of SMA, but it is unknown whether other mechanisms contribute to the spectrum of clinical phenotypes. Here, through a combination of physiological and morphological studies in mouse models and SMA patients, we identify dysfunction and loss of proprioceptive sensory synapses as key signatures of SMA pathology. We demonstrate that SMA patients exhibit impaired proprioception, and their proprioceptive sensory synapses are dysfunctional as measured by the neurophysiological test of the Hoffmann reflex (H-reflex). We further show that loss of excitatory afferent synapses and altered potassium channel expression in SMA motor neurons are conserved pathogenic events found in both severely affected patients and mouse models. Lastly, we report that improved motor function and fatigability in ambulatory SMA patients and mouse models treated with SMN-inducing drugs correlate with increased function of sensory-motor circuits that can be accurately captured by the H-reflex assay. Thus, sensory synaptic dysfunction is a clinically relevant event in SMA, and the H-reflex is a suitable assay to monitor disease progression and treatment efficacy of motor circuit pathology.

GLUT-1 deficiency syndrome caused by haploinsufficiency of the blood-brain barrier hexose carrier.

The high metabolic requirements of the mammalian central nervous system require specialized structures for the facilitated transport of nutrients across the blood-brain barrier. Stereospecific high-capacity carriers, including those that recognize glucose, are key components of this barrier, which also protects the brain against noxious substances. Facilitated glucose transport in vertebrates is catalyzed by a family of carriers consisting of at least five functional isoforms with distinct tissue distributions, subcellular localizations and transport kinetics. Several of these transporters are expressed in the mammalian brain. GLUT-1, whose sequence was originally deduced from cDNAs cloned from human hepatoma and rat brain, is present at high levels in primate erythrocytes and brain endothelial cells. GLUT1 has been cloned and positionally mapped to the short arm of chromosome 1 (1p35-p31.3; refs 6-8). Despite substantial metabolic requirements of the central nervous system, no genetic disease caused by dysfunctional blood-brain barrier transport has been identified. Several years ago, we described two patients with infantile seizures, delayed development and acquired microcephaly who have normal circulating blood glucose, low-to-normal cerebrospinal fluid (CSF) lactate, but persistent hypoglycorrachia (low CSF glucose) and diminished transport of hexose into isolated red blood cells (RBC). These symptoms suggested the existence of a defect in glucose transport across the blood brain barrier. We now report two distinct classes of mutations as the molecular basis for the functional defect of glucose transport: hemizygosity of GLUT1 and nonsense mutations resulting in truncation of the GLUT-1 protein.

Fatal infantile cardioencephalomyopathy with COX deficiency and mutations in SCO2, a COX assembly gene.

Mammalian cytochrome c oxidase (COX) catalyses the transfer of reducing equivalents from cytochrome c to molecular oxygen and pumps protons across the inner mitochondrial membrane. Mitochondrial DNA (mtDNA) encodes three COX subunits (I-III) and nuclear DNA (nDNA) encodes ten. In addition, ancillary proteins are required for the correct assembly and function of COX (refs 2, 3, 4, 5, 6). Although pathogenic mutations in mtDNA-encoded COX subunits have been described, no mutations in the nDNA-encoded subunits have been uncovered in any mendelian-inherited COX deficiency disorder. In yeast, two related COX assembly genes, SCO1 and SCO2 (for synthesis of cytochrome c oxidase), enable subunits I and II to be incorporated into the holoprotein. Here we have identified mutations in the human homologue, SCO2, in three unrelated infants with a newly recognized fatal cardioencephalomyopathy and COX deficiency. Immunohistochemical studies implied that the enzymatic deficiency, which was most severe in cardiac and skeletal muscle, was due to the loss of mtDNA-encoded COX subunits. The clinical phenotype caused by mutations in human SCO2 differs from that caused by mutations in SURF1, the only other known COX assembly gene associated with a human disease, Leigh syndrome.

Hypoalaninemia: a concomitant of ketotic hypoglycemia.

The cause of of ketotic hypoglycemia, the commonest form of hypoglycemia in childhood, is not known. The present study was undertaken to determine whether the primary defect in this condition is a deficiency of gluconeogenic precursor(s) or an abnormality in the hepatic gluconeogenic enzyme system. Plasma glucose, alanine, and insulin and blood beta-hydroxybutyrate (beta-OHB), pyruvate, and lactate levels were determined in eight ketotic hypoglycemic children and seven agematched controls maintained on a normal diet and after being fed a provocative hypocaloric low-carbohydrate diet (1200 kcal/1.73 m(2), 15% carbohydrate, 17% protein, and 68% fat). On a normal diet, overnight fasting plasma alanine (211+/-10 muM) and glucose (68+/-4 mg/100 ml) were significantly lower and blood beta-OHB (1.22+/-0.37 mM) significantly higher in ketotic hypoglycemic children than in controls (alanine, 315+/-15 muM; glucose, 81+/-3 mg/100 ml; beta-OHB, 0.18+/-0.08 mM). All ketotic hypoglycemic children developed symptomatic hypoglycemia (33+/-3 mg/100 ml) and ketosis (beta-OHB, 3.70+/-0.32 mM) 8-16 hr after starting the provocative diet and these changes were associated with a further decline in plasma alanine (155+/-17 muM). Normal children, even after 36 hr on this diet, maintained higher plasma glucose (48+/-2 mg/100 ml) and alanine (225+/-5 muM) and lower beta-OHB levels (2.56+/-0.44 mM). Intravenous infusions of alanine (250 mg/kg) uniformly restored the hypoglycemic plasma glucose levels of ketotic hypoglycemic children to normal. Cortisone acetate (300 mg/m(2)), given orally in three divided doses during feeding of the provocative diet, produced a 3- to 4-fold increase in plasma alanine within 4-6 hr after beginning steroid therapy and completely prevented the development of hypoglycemia and ketosis. Quantitative amino acid profiles were performed and demonstrated that alanine was the only gluconeogenic amino acid which differed significantly between the two groups. Plasma insulin and blood lactate and pyruvate levels did not differ significantly between normal and ketotic hypoglycemic children under all conditions examined. These results support the hypothesis that a deficiency in gluconeogenic precursor (e.g., alanine) rather than a defect in the hepatic gluconeogenic enzyme apparatus represents the most likely factor in the pathogenesis of ketotic hypoglycemia.

Poor facial affect recognition among boys with duchenne muscular dystrophy.

Children with Duchenne or Becker muscular dystrophy (MD) have delayed language and poor social skills and some meet criteria for Pervasive Developmental Disorder, yet they are identified by molecular, rather than behavioral, characteristics. To determine whether comprehension of facial affect is compromised in boys with MD, children were given a matching-to-sample test with four types of visual recognition (Object, Face, Affect, and Situation matching) developed by Lucci and Fein. Within-group analyses on 50 boys with MD found decreased Affect matching relative to the other matching conditions. Between-group comparisons on 20 sibling pairs found the boys with Duchenne performed more poorly only on the Affect-matching condition. Thus, mildly impaired facial affect recognition may be part of the phenotype associated with Duchenne or Becker MD.

Disparity in expression of protein kinase C alpha in human glioma versus glioma-derived primary cell lines: therapeutic implications.

Intracellular signal transduction by the protein kinase C (PKC) family of enzymes plays a critical role in carcinogenesis and cellular growth regulation. Recent studies have suggested that the PKC isoform alpha may be a critical target for antiglioma therapy in humans (G. H. Baltuch et al., Can. J. Neurol. Sci., 22: 264-271, 1995). We studied the expression and subcellular distribution of the PKC alpha isoform in human high- and low-grade gliomas and also in glioma-derived cell lines with immunoblot analyses. Cell lines derived from high-grade gliomas expressed higher levels of PKC alpha than did cell lines derived from low-grade gliomas. In glioblastoma-derived cell lines, PKC alpha was mainly expressed in the soluble (cytosolic) fraction, indicating an inactive state of the enzyme. When analyzed in freshly frozen samples from human gliomas, the expression of PKC alpha was at similar levels in high- and low-grade tumors and was also similar to the levels in normal brain tissue controls. The PKC partial antagonist bryostatin 1, currently undergoing Phase II testing in patients with malignant gliomas, was capable of specifically down-regulating PKC alpha in vitro in glioblastoma-derived cell lines. However, this was not associated with significant growth inhibition. We conclude that the observed overexpression of PKC alpha in glioblastoma-derived cell lines may be an artifact of in vitro growth. Furthermore, we conclude that expression of PKC alpha in glioma-derived cell lines is not essential for cellular growth in vitro because down-regulation of PKC alpha following treatment with bryostatin 1 was not associated with growth inhibition.

Mutational analysis of GLUT1 (SLC2A1) in Glut-1 deficiency syndrome.

Fifteen children presenting with infantile seizures, acquired microcephaly, and developmental delay were found to have novel heterozygous mutations in the GLUT1 (SLC2A1). We refer to this condition as the Glut-1 Deficiency Syndrome (Glut-1 DS). The encoded protein (Glut-1), which has 12 transmembrane domains, is the major glucose transporter in the mammalian blood-brain barrier. The presence of GLUT1 mutations correlates with reduced cerebrospinal fluid glucose concentrations (hypoglycorrhachia) and reduced erythrocyte glucose transporter activities in the patients. We used Florescence in situ hybridization, PCR, single-stranded DNA conformational polymorphism, and DNA sequencing to identify novel GLUT1 mutations in 15 patients. These abnormalities include one large-scale deletion (hemizygosity), five missense mutations (S66F, R126L, E146K, K256V, R333W), three deletions (266delC, 267A>T; 904delA; 1086delG), three insertions (368-369 insTCCTGCCCACCACGCTCACCACG, 741-742insC, 888-889insG), three splice site mutations (197+1G>A; 1151+1G>T; 857T>G, 858G>A, 858+1del10), and one nonsense mutation (R330X). In addition, six silent mutations were identified in exons 2, 4, 5, 9, and 10. The K256V missense mutation involved the maternally derived allele in the patient and one allele in his mother. A spontaneous R126L missense mutation also was present in the paternally derived allele of the patient. The apparent pathogenicity of these mutations is discussed in relation to the functional domains of Glut-1.

Acetylcholinesterase activity in patients with torsion dystonia. Measurement in erythrocyte membranes.

Anticholinergic therapy provides symptomatic relief in many patients with dystonia. The mechanism underlying this therapeutic action is poorly understood; however, one possibility is that the degradation of acetylcholine is perturbed in these conditions. To investigate this possibility, acetylcholinesterase activity was measured in erythrocyte membranes from healthy volunteers and patients with torsion dystonia. Enzyme activities in erythrocytes from 14 patients with adult-onset, childhood-onset idiopathic, and childhood-onset familial dystonias did not differ significantly from activities measured in erythrocyte membranes from 17 healthy volunteers. Moreover, when blood samples from several members of a family with dominant inheritance of dystonia were assayed simultaneously, similar enzyme activities were found in the affected and unaffected individuals. The data suggest that a generalized acetylcholinesterase deficiency is not involved in the pathogenesis of torsion dystonia.

Steroid-responsive encephalomyelitis in childhood.

The syndrome of parainfectious encephalomyelitis evolves from an antecedent infection. Several etiologic agents have been associated with this complication, although the pathogenesis in each instance may prove to be more uniform. Considerable evidence suggests that the syndrome is mediated immunologically. The seven cases reported here were clinically similar, although the infectious etiologies were diverse. Leptospirosis antedated the neurologic syndrome in two cases, and a "viral" illness preceded the other five cases. The evolution of the syndrome was slowly progressive in each case, and six patients had prominent involvement of rhombencephalic structures. The progressive course was reversed rapidly with eventual full recovery in each instance after initiation of corticosteroid therapy. Our experience with these cases coupled with a review of the literature suggests that corticosteroid therapy should be considered in the subacute or chronic cases of parainfectious encephalomyelitis.

Poor verbal working memory across intellectual level in boys with Duchenne dystrophy.

OBJECTIVE: To determine whether all boys with Duchenne muscular dystrophy (DMD) have a similar verbal and memory profile of skills, or whether only a subset is affected, and to determine whether the weak areas in their profile are substantially different from a control group. METHODS: Performance of patients with DMD on neuropsychological tests of verbal and memory skills was examined in two ways. Standardized test scores for 80 boys with DMD (estimated IQ range, 70 to 160) were ranked individually from worst to best, and the individual rankings were compared across the group using Friedman rank analysis. Additionally, performance of 41 boys with DMD was compared with that of their sibling control subjects of similar age and estimated IQ using multivariate analysis of variance. RESULTS: Individual cognitive profiles were significantly similar among the subjects with DMD, such that for most subjects digit span, story recall, and comprehension were the tests on which each performed most poorly. This finding remained true regardless of whether they were of high or low intellectual function. In contrast, no significant cognitive profile was found among their sibling control subjects, and when compared with their siblings, the DMD group scored significantly more poorly on digit span, comprehension, and story recall, but not on other verbal and memory measures. CONCLUSIONS: Boys with DMD have a specific cognitive profile, regardless of their general level of cognitive function. Specifically, boys with DMD performed more poorly on tests requiring attention to complex verbal information than they did on other verbal or memory measures. The possibility that the missing dystrophin brain products may contribute to selective cognitive processing is considered.

Adult Reye's syndrome: a review with new evidence for a generalized defect in intramitochondrial enzyme processing.

A 42-year-old woman developed a flu-like illness and died 8 days later with Reye's syndrome (RS). There are 26 other cases of adult-onset RS reported. Biochemical, immunologic, and molecular studies of liver, brain, and skeletal muscle revealed a non-uniform decrease in several mitochondrial residual enzyme activities in liver and brain. Pyruvate carboxylase activity was negligible. Cross-reacting material was present in normal abundance in isolated liver mitochondria for several enzymes that had reduced catalytic activity including pyruvate carboxylase. Subunit II (encoded by mitochondrial DNA) and subunit IV (encoded by nuclear DNA) of cytochrome c oxidase also were present in normal abundance with normal electrophoretic mobility. These observations, combined with pertinent findings of other investigators, allow us to speculate that the intramitochondrial matrix chemical environment is disturbed by preceding pathophysiologic events resulting in a lowered ATP/ADP ratio. The lowered intramitochondrial energetic state interferes with the refolding and assembly of imported mitochondrial proteins, causing a loss of the catalytic efficiency of these enzymes. This explains the selective vulnerability of mitochondria in RS and the non-uniform, disproportionate loss of enzyme activity.

Normal fibroblast mitochondrial malic enzyme activity in Friedreich's ataxia.

Mitochondrial and cytoplasmic malic isoenzymes were assayed fluorometrically in digitonin-fractionated fibroblasts from three patients with Friedreich's ataxia (FA). Normal activity was found, failing to verify an earlier report of reduced fibroblast mitochondrial malic enzyme activity in FA. The previously reported disturbance in fibroblast mitochondrial malic enzyme may be an epiphenomenon in patients with the FA phenotype. Further studies are necessary to define the primary genetic defect in this inherited disorder.

Congenital myasthenic syndrome caused by low-expressor fast-channel AChR delta subunit mutation.

OBJECTIVE: To determine the molecular basis of a disabling congenital myasthenic syndrome (CMS) observed in two related and one unrelated Arab kinship. BACKGROUND: CMS can arise from defects in presynaptic, synaptic basal lamina-associated, or postsynaptic proteins. Most CMS are postsynaptic, and most reside in the AChR epsilon subunit; only two mutations have been reported in the AChR delta subunit to date. METHODS: Cytochemistry, electron microscopy, alpha-bungarotoxin binding studies, microelectrode and patch-clamp recordings, mutation analysis, mutagenesis, and expression studies in human embryonic kidney cells were employed. RESULTS: Endplate studies showed AChR deficiency, fast decaying, low-amplitude endplate currents, and abnormally brief channel opening events. Mutation analysis revealed a novel homozygous missense mutation (deltaP250Q) of the penultimate proline in the first transmembrane domain (TMD1) of the AChR delta subunit. Expression studies indicate that deltaP250Q (1) hinders delta/alpha subunit association during early AChR assembly; (2) hinders opening of the doubly occupied closed receptor (A(2)R); and (3) speeds the dissociation of acetylcholine from A(2)R. Mutagenesis studies indicate that deltaP250L also has fast-channel effects, whereas epsilon P245L and epsilon P245Q, identical mutations of the corresponding proline in the epsilon subunit, have mild slow-channel effects. CONCLUSIONS: deltaP250Q represents the third mutation observed in the AChR delta subunit. The severe phenotype caused by deltaP250Q is attributed to endplate AChR deficiency, fast decay of the synaptic response, and lack of compensatory factors. That the penultimate prolines in TMD1 of the delta and epsilon subunits exert a reciprocal regulatory effect on the length of the channel opening bursts reveals an unexpected functional asymmetry between the two subunits.

Potentially fatal cardiac dysrhythmia and hyperkalemic periodic paralysis.

An 11-year-old boy was evaluated for mild periodic muscular weakness exacerbated on separate occasions by disopyramide phosphate and procainamide. He and his mother both had bidirectional ventricular tachydysrhythmia (BVT), short stature, microcephaly, and clinodactyly. The mother, but not the child, had lingual myotonia. The two antiarrhythmic drugs worsened the muscular weakness without benefiting the cardiac dysrhythmia. Potassium loading produced skeletal muscle weakness and transient conversion of the BVT to normal sinus rhythm. Hypokalemia aggravated the BVT without causing weakness. Acetazolamide had no effect. The patient suffered a nonfatal cardiac arrest after several days of increased carbohydrate intake. Imipramine controlled the dysrhythmia without inducing weakness. Periodic paralysis should be considered as the diagnosis in children with BVT, a potentially fatal condition.

Cerebral hyperemia, stroke, and transfusion in sickle cell disease.

To investigate cerebral hemodynamics in sickle cell disease (SCD), we used the 133Xenon inhalation technique of quantifying cerebral blood flow (CBF) in 67 patients. Clinical examinations and cerebral magnetic resonance imaging also were performed in all patients. Compared with age-matched healthy controls, CBF was elevated by 68% in patients, and inversely related to hematocrit. An experimental index of cerebral blood volume, pr4, was also elevated in the patients in a similar manner. Cerebral blood volume was positively correlated to CBF in SCD patients but not in controls. History of stroke and current neurologic symptoms were associated with lower flow and higher cerebral blood volume. Transfusion therapy reduced the hyperemia, the reduction being greater than expected by hematocrit elevation alone. These findings document a vasodilatory hyperemia in SCD. This dilatation may be a risk factor for ischemic distal-field infarctions, as visualized by MRI, due to a limitation of cerebrovascular reserve capacity.

Familial cerebellar ataxia with muscle coenzyme Q10 deficiency.

OBJECTIVE: To describe a clinical syndrome of cerebellar ataxia associated with muscle coenzyme Q10 (CoQ10) deficiency. BACKGROUND: Muscle CoQ10 deficiency has been reported only in a few patients with a mitochondrial encephalomyopathy characterized by 1) recurrent myoglobinuria; 2) brain involvement (seizures, ataxia, mental retardation), and 3) ragged-red fibers and lipid storage in the muscle biopsy. METHODS: Having found decreased CoQ10 levels in muscle from a patient with unclassified familial cerebellar ataxia, the authors measured CoQ10 in muscle biopsies from other patients in whom cerebellar ataxia could not be attributed to known genetic causes. RESULTS: The authors found muscle CoQ10 deficiency (26 to 35% of normal) in six patients with cerebellar ataxia, pyramidal signs, and seizures. All six patients responded to CoQ10 supplementation; strength increased, ataxia improved, and seizures became less frequent. CONCLUSIONS: Primary CoQ10 deficiency is a potentially important cause of familial ataxia and should be considered in the differential diagnosis of this condition because CoQ10 administration seems to improve the clinical picture.

Cardiac size and function during adrenocorticotropic hormone-induced systolic systemic hypertension in infants.

The effects of adrenocorticotropic hormone (ACTH) on systolic blood pressure, and echocardiographic indexes of heart size and function were investigated in 14 infants. After 25 days (range 13 to 46) of treatment with ACTH, systolic blood pressure increased from 93 +/- 9 to 118 +/- 20 mm Hg (p < 0.001; mean +/- 1 SD). Systolic hypertension (systolic blood pressure greater than the 95th percentile for age) developed in 10 of 14 infants and was associated with an increase in left ventricular (LV) shortening fraction from 41 +/- 5% to 52 +/- 8% (p < 0.001). Myocardial hypertrophy and an increase in echocardiographic indexes of myocardial contractility were observed also. To assess the temporal relation between the onset of systolic hypertension and these cardiac changes, data from 8 infants with serial echocardiograms and blood pressure determinations were examined. After a mean 14 days (range 8 to 18) of ACTH, LV shortening fraction increased from 39 +/- 6% to 53 +/- 8% (p < 0.01), whereas systolic blood pressure remained normal in 7 of 8 infants. In addition, a decrease in LV end-systolic dimension was observed during this early phase. This report documents myocardial changes in individual patients, which occur before and during the development of systolic hypertension.

Cytochrome c oxidase-associated Leigh syndrome: phenotypic features and pathogenetic speculations.

Fourteen new cases of cytochrome oxidase (COX)-associated Leigh syndrome (LS) are combined with 20 reported cases to describe the clinical, laboratory, and radiological features of this devastating metabolic condition. Three clinical stages are identified. Most patients have normal neurological development during the first 8-12 months (stage I). Somatic complaints are common, including chronic diarrhea, recurrent vomiting, anorexia, and decelerating body and head growth. The second stage evolves during late infancy and early childhood when motor regression becomes evident. Eye signs, altered breathing patterns, pyramidal, extrapyramidal, and cerebellar signs emerge and sudden clinical deterioration occurs during intercurrent infectious or metabolic stress. The last stage may extend from 2 to 10 years and is manifested by extreme hypotonia, swallowing difficulties and undernutrition. Feeding assistance is necessary and seizures may occur. The CSF lactate concentration is consistently elevated and MRI abnormalities are seen in the subcortical structures. COX deficiency affects most tissues, but is not always generalized. For example, 3 patients with a cardiomyopathy had normal COX activity in cultured skin fibroblasts. Nearly normal amounts of cross-reacting material are present by ELISA and immunoblot analyses. Parental consanguinity has been found in several families, the hereditary pattern is recessive and males are affected more commonly (2:1). The biomolecular abnormality causing COX deficiency in LS is unknown, but the available evidence implicates a nuclear-encoded protein that affects the structure or the stability of the holoenzyme complex.

Neonatal presentations of mitochondrial metabolic disorders.

Because of the high energy requirements of the growing neonate, disorders of mitochondrial metabolism caused by defects in fatty acid oxidation, pyruvate metabolism, and the respiratory chain may often present in the neonatal period. Common neonatal presentations are hypotonia, lethargy, feeding and respiratory difficulties, failure to thrive, psychomotor delay, seizures, and vomiting. Laboratory clues include alterations in the levels of lactate, pyruvate (and the lactate/pyruvate ratio), glucose, and ketone bodies. Diagnosis usually depends on specific enzyme assays or on molecular genetic analysis. Without treatment, most infants die in the first few days or months of life. In the last decade, there have been significant advances in the understanding of the molecular basis of these disorders. This review discusses the major subgroups of mitochondrial disorders, focusing on defects of pyruvate oxidation, the Krebs cycle, and the respiratory chain. Disorders caused by respiratory chain defects may involve nuclear DNA, mitochondrial DNA, or intergenomic signaling. Recognition and early diagnosis of these conditions are important in the genetic counseling of these families.