Hemodynamic evidence linking cognitive deficits in clinically isolated syndrome to regional brain inflammation.

BACKGROUND AND PURPOSE: To investigate the relation between hemodynamic measurements and memory function in patients with clinically isolated syndrome (CIS). METHODS: Forty CIS patients were administered tests of verbal short-term/working memory and passage learning. Using dynamic susceptibility contrast MRI cerebral blood volume (CBV), cerebral blood flow and mean transit time values were estimated in 20 cerebral regions of interest, placed in normal appearing white matter (NAWM) and normal appearing deep gray matter structures, bilaterally. RESULTS: CIS patients showed significantly impaired scores on working memory and secondary verbal memory that correlated inversely with elevated CBV values in the left frontal and periventricular NAWM, thalamus, right caudate and corpus callosum. CONCLUSIONS: Verbal memory in CIS correlates inversely with elevated CBV values of brain structures involved in memory. As these hemodynamic changes, detected in CIS, are indicative of inflammation, the observed cognitive disturbances may relate to widespread brain inflammatory processes that prevail in early multiple sclerosis.

Benign multiple sclerosis in Crete.

Our objective was to study multiple sclerosis on Crete, an island of 0.6 million inhabitants sharing a similar genetic background and the same environment. Case ascertainment was achieved using the MS Epidemiology Program Project of Crete. The diagnosis and classification of multiple sclerosis were made by established clinical and magnetic resonance imaging criteria, and disease evolution was assessed by periodic evaluations. Thorough clinical and laboratory evaluations were conducted; a detailed history, including a questionnaire of 36 items, was taken. Data obtained were analysed for possible interaction with disease prognosis. We identified 587 cases of multiple sclerosis (F:M = 1.6), >90% of which were of Cretan origin from both parental lines. Age at onset was 31.5 +/- 10.3 years (mean +/- SD) and disease duration 12.7 +/- 9.1 years. About 84.6% had relapsing remitting multiple sclerosis, 9.4% primary progressive multiple sclerosis and 6% clinically isolated syndrome. Nearly 40% of our multiple sclerosis patients with disease duration >10 years (mean = 16.2 +/- 5.3 years) remained with no or mild disability (Expanded Disability Status Scale [EDSS] < or =3). Also, about 30% of patients with relapsing remitting multiple sclerosis showed benign disease evolution (EDSS < or =3) more than 20 years (mean = 24.0 +/- 3.3) after onset. Factors predisposing to benign multiple sclerosis included younger age at onset, shorter disease duration and a lower number of relapses. We conclude that a substantial proportion of patients with multiple sclerosis from Crete follow a rather benign disease course, and this may relate to the genetic background of the population and/or to environmental factors.

Abnormal glutamate metabolism in an adult-onset degenerative neurological disorder.

In patients with recessive, adult-onset olivopontocerebellar degeneration associated with a partial deficiency of glutamate dehydrogenase, the concentration of glutamate in plasma was significantly higher than that in controls. Plasma alpha-ketoglutarate was significantly lower. Oral administration of monosodium glutamate resulted in excessive accumulation of this amino acid in plasma and lack of increase in the ratio of plasma lactate to pyruvate in the glutamate dehydrogenase-deficient patients. Decreased glutamate catabolism may result in an excess of glutamate in the nervous system and cause neuronal degeneration.

Patients with horizontal gaze palsy and progressive scoliosis due to ROBO3 E319K mutation have both uncrossed and crossed central nervous system pathways and perform normally on neuropsychological testing.

BACKGROUND: Horizontal gaze palsy and progressive scoliosis (HGPPS) is caused by mutations of the ROBO3 gene, which encodes a receptor associated with axonal guidance during development. Although there is evidence for uncrossed cuneatal and corticospinal tracts in HGPPS, it is unclear whether other central nervous system pathways are involved. OBJECTIVE: To study two patients with HGPPS homozygotic for the ROBO3 E319K mutation using a variety of neurophysiological and neuropsychological tests. METHODS: A battery of neuropsychological tests was applied to assess various cognitive and perceptual functions. The corticospinal, somatosensory and auditory pathways were evaluated using appropriate neurophysiological tests. To access motor pathways to the neck muscles, electromyographic recordings were obtained from the sternocleidomastoideus and splenius capitis muscle during active head rotation. RESULTS: Both patients performed normally on manual dexterity, complex sensory and visuospatial functions, reading and general intelligence tests. Motor evoked potentials in both patients showed uncrossed corticospinal tracts for the extremities, although in one patient, electromyography indicated pyramidal tract crossing for the neck muscles. Although somatosensory evoked potentials showed uncrossed somatosensory fibres subserving proprioception and light touch, right median nerve somatosensory evoked potential in one patient indicated a partial lemniscal crossing. Sympathetic skin response and blink reflex showed a midline crossing of the spinothalamic and quintothalamic tracts. Brain stem auditory evoked potentials indicated a lack of crossing in the level of the trapezoid body. CONCLUSIONS: Our patients with the ROBO3 E319Kappa mutation show normal perceptual and cognitive functions and have both crossed and uncrossed motor, sensory and auditory pathways.

Central nervous system involvement as the presenting manifestation of autoimmune rheumatic diseases: an observational study using the American College of Rheumatology nomenclature for neuropsychiatric lupus.

OBJECTIVE: We sought to describe CNS involvement as initial presentation of autoimmune rheumatic diseases using a standardized nomenclature. PATIENTS AND METHODS: A 6-year observational study (1999-2005) was conducted in the University Hospital of Heraklion Crete, a regional referral secondary/tertiary care academic center. Patients presenting with new neurological symptoms of acute/subacute onset underwent clinical and laboratory screening for systemic autoimmune disorders. The diagnosis of an autoimmune rheumatic disorder was based upon the American College of Rheumatology (ACR) classification criteria, whereas for primary antiphospholipid syndrome (PAPS) we used the Sapporo preliminary criteria. In order to describe the neurological syndromes we used the ACR nomenclature for neuropsychiatric lupus. RESULTS: During this period fourteen patients (ten females and four males) were recorded. Eight patients had systemic lupus erythematosus (SLE), four had primary APS and the remaining two had systemic vasculitis. Four out of the eight SLE patients had secondary APS. Two of them presented with movement disorder (chorea). The other two and all four patients with primary APS presented with cerebrovascular disease (CVD). These six patients comprised the 5.7% of young adults under < 45 years old with cerebrovascular accident admitted over the 6-year period. CONCLUSION: SLE and APS either primary or secondary to SLE were the most common underlying systemic autoimmune rheumatic diseases, in patients presenting with a neurological event of acute onset. Young adults (< 45 years old) with CVD should undertake screening for SLE/APS.

Cloning and characterization of a glutamate transporter cDNA from human cerebellum.

The glutamate/aspartate transporters are essential for the elimination and recycling of synaptic glutamate released from nerve endings during neurotransmission. Evidence suggests that these processes are altered in ischemia and neuronal degenerations linked to excitotoxicity. We screened a cDNA library constructed from human cerebellar mRNA, and isolated a cDNA that shows an 88.5% and a 98.7% sequence similarity at the nucleotide and amino acid level, respectively, with a rat brain specific Na(+)-dependent glutamate/aspartate transporter. The human cDNA is expressed in brain and it may prove useful in the study of human neurodegenerations linked to glutamate dysfunction.

Molecular cloning of human brain glutamate/aspartate transporter II.

Glutamate transporters are membrane-bound proteins which are localized in glial cells and/or pre-synaptic glutamatergic nerve endings and are essential for the removal and termination of action of synaptic glutamate. Several cDNAs encoding glutamate transporters have been isolated from mammalian tissues, including human cerebellum. Here, we screened cDNA libraries derived from human brain stem and cerebellum, and isolated a novel cDNA that encodes for a glutamate transporter. This cDNA predicts a protein which contains 565 amino acids and is homologous to a rat brain Na(+)-dependent glutamate/aspartate transporter. The new cDNA is expressed in brain and is structurally distinct from the previously reported human glutamate transporter cDNA.

Normal bone marrow hematopoietic stem cell reserves and normal stromal cell function support the use of autologous stem cell transplantation in patients with multiple sclerosis.

Bone marrow (BM) stem cell reserves and function and stromal cell hematopoiesis supporting capacity were evaluated in 15 patients with multiple sclerosis (MS) and 61 normal controls using flow cytometry, clonogenic assays, long-term BM cultures (LTBMCs) and enzyme-linked immunosorbent assays. MS patients displayed normal CD34+ cell numbers but a low frequency of colony-forming cells (CFCs) in both BM mononuclear and purified CD34+ cell fractions, compared to controls. Patients had increased proportions of activated BM CD3+/HLA-DR+ and CD3+/CD38+ T cells that correlated inversely with CFC numbers. Patient BM CD3+ T cells inhibited colony formation by normal CD34+ cells and patient CFC numbers increased significantly following immunomagnetic removal of T cells from BMMCs, suggesting that activated T cells may be involved in the defective clonogenic potential of hematopoietic progenitors. Patient BM stromal cells displayed normal hematopoiesis supporting capacity indicated by the CFC number in the nonadherent cell fraction of LTBMCs recharged with normal CD34+ cells. Culture supernatants displayed normal stromal derived factor-1 and stem cell factor/kit ligand but increased flt-3 ligand levels. These findings provide support for the use of autologous stem cell transplantation in MS patients. The low clonogenic potential of BM hematopoietic progenitors probably reflects the presence of activated T cells rather than an intrinsic defect.

Linkage disequilibrium between the expanded (CAG)n repeat and an allele of the adjacent (CCG)n repeat in Huntington's disease patients of Greek origin.

Huntington's disease (HD) is associated with an expanded unstable (CAG)n repeat in the IT15 gene. This repeat was investigated in 44 HD patients and 59 of their relatives at risk who were members of 29 unrelated families from various parts of Greece. Abnormal elongation of the (CAG)n repeat ranging from 39 to 95 trinucleotide units was found in all but one of the 44 HD patients tested with 70% of these patients showing 42-47 repeats. The size of the expanded sequence correlated inversely with the age at disease onset (r = 0.77, p < 0.00001, n = 43). In a single sporadic case, de novo expansion of the (CAG)n repeat was detected. Twenty-four of 59 asymptomatic family members at risk showed expansion of the (CAG)n repeat in the HD range (39-56 trinucleotide units) while three had intermediate alleles (36-37 repeats). Evaluation of the adjacent polymorphic (CCG)n repeat showed a strong linkage disequilibrium between the 7-unit (CCG)n repeat allele and the HD mutation, with 51% of normal and 93% of HD chromosomes showing this allele (chi 2 = 15.55, p < 0.0001, n - 260). These data on HD patients of Greek origin are consistent with the thesis that the (CAG)n expansion is the primary gene defect of the disease and that this mutation occurred primarily on chromosomes with the (CCG)7 repeat haplotype.

Oculogyric crises and parkinsonism. A case of recent onset.

Oculogyric crisis in association with postencephalitic parkinsonism has been reported only following encephalitis lethargica (Economo's disease). Generally, it has been assumed that this phenomenon would fade away with the demise of these postencephalitic patients. Therefore, we report a case of recent onset of persistent oculogyric crisis with parkinsonism in a 35-year-old man following an apparent attack of encephalitis in 1972.

Thiamine deficiency: selective impairment of the cerebellar serotonergic system.

To explore the role of thiamine deficiency in synaptic transmission, the high-affinity uptake and release systems for putative neurotransmitters were studied in synaptosomal preparations isolated from the telencephalon, hypothalamus, and cerebellum of rats made thiamine deficient by diet or pyrithiamine. There was significant decrease in the uptake of serotonin by the synaptosomal preparations of the cerebellum. Although thiamine and its phosphorylated forms added in vitro did not restore the decreased serotonin uptake, the administration of the vitamin in vivo resulted in a significant reversibility of the inhibition of serotonin uptake, coinciding with dramatic clinical improvement. The study supports the possibility of an important serotonergic innervation of the cerebellum and suggests a selective involvement of this system in the pathogenesis of some of the neurologic manifestations of thiamine deficiency.

Trichinosis: severe myopathic involvement with recovery.

A case of clinically severe trichinosis following ingestion of commercially prepared pickled pigs' feet is reported. An unusual finding on muscle biopsy was a larval count of 4,046 per gram of muscle, the second highest density of muscle invasion recorded to date. Survival has not been reported previously in patients with greater than 1,000 trichinae per gram of muscle. Complete symptomatic recovery followed combined corticosteroid and thiabendazole administration.

X-linked adrenoleukodystrophy presenting as neurologically pure familial spastic paraparesis.

The familial spastic paraplegias (FSPs) are heterogeneous neurologic disorders that are known to occur clinically as "pure" or "complicated" forms. Although some of the complicated FSPs have been linked to specific metabolic defects, the pure forms of this disorder remain idiopathic and are considered to be primary system degenerations. We report the case of a 28-year-old man who has evidenced a neurologically pure spastic paraparesis since age twenty-five. Consistent with this diagnosis were the findings of magnetic resonance imaging, which revealed atrophy of the thoracic spinal cord without evidence for white matter disease in the cerebrum, cerebellum, or brainstem. His 32-year-old brother has also evidenced progressive spastic paraparesis since age 30, but his case is confounded by a severe head injury at age 24 that caused a skull fracture and a focal demyelinating lesion of the right frontal lobe. Both patients have had hypogonadism, requiring treatment with testosterone, since age 20. Measurement of plasma levels of very long-chain fatty acids (VLCFA) revealed that both brothers had concentrations diagnostic of adrenoleukodystrophy; their mother had plasma VLCFA levels in the heterozygous range. We conclude that neurologically pure FSP can be an early manifestation of adrenoleukodystrophy and that levels of plasma VLCFA should be determined for all cases of FSP in which X-linked inheritance appears tenable. These considerations may have bearing on the ongoing linkage studies for these disorders.

Alterations in uptake and metabolism of aspartate and glutamate in brain of thiamine deficient animals.

The high affinity uptake systems of aspartate, glutamate, glycine and taurine were studied in synaptosomal preparations isolated from the cerebellum, medulla-pons and the telencephalon of rats made thiamine deficient (TD) by diet or pyrithiamine (PT). There was a significant enhancement in the uptake of asparate/glutamate (probably transported by the same carrier) by the synaptosomal preparations of the cerebellum only, in both groups of thiamine-deficient animals as compared to controls. This was due to an increase in the number of uptake sites and not to an alteration of the binding affinity. Aspartate levels decreased significantly in all three brain areas of PT-treated animals and this change was greatest in the medulla-pons and the cerebellum and least in the cortex. Glutamate and serine levels were significantly decreased only in the medulla-pons whilst the concentration of glutamine was significantly increased in the three brain regions studied. The changes in both uptake and levels of amino acids in TD rats were reversed by thiamine therapy. Though the uptake studies do not discriminate between an altered aspartergic or glutamergic system, the changes in the levels of aspartate in the cerebellum suggest that the aspartergic system is involved. Since earlier studies showed a selective impairment in the high affinity uptake of serotonin by cerebellar synaptosomes, thiamine deficiency could impair cerebellar function by inducing an imbalance in its neurotransmitter systems.

Effect of thiamine deficiency on brain serotonin turnover.

Serotonin turnover has been investigated in regional brain areas of rats made thiamine deficient by pyrithiamine (PT). Following intracisternal injection of [14C]5-hydroxytryptamine ([14C]5-HT), a marked increase in the accumulation of [14C]5-hydroxyindoleacetic acid ([14C]5-HIAA) was found in the medulla-pons, hypothalamus and cerebral cortex. [14C]5-HT levels were normal in all of the brain areas except the cerebral cortex which had an increase of 58%. The ratio of [14C]5-HIAA/[14C]5-HT was significantly increased in every brain region of PT-treated rats except the cerebral cortex. Part of this increase in [14C]5-HIAA was shown to be due to impairment of active transport of this 5-HT metabolite out of the brain. However, increased 5-HT synthesis in the cerebellum, hypothalamus, striatum, hippocampus and cerebral cortex was demonstrated by measurement of 5-HT accumulation after inhibition of brain monoamine oxidase. PT-induced increase in endogenous 5-HIAA in the medulla-pons occurred simultaneously with the onset of neurological signs and both parameters were reversible by thiamine administration. These results suggest that acute thiamine deficiency, induced by PT, both increases brain 5-HT synthesis and impairs 5-HIAA efflux from the brain. There is a close correlation between neurological manifestations and changes in brain 5-HT metabolism in acute thiamine deficiency.

Neuron-specific human glutamate transporter: molecular cloning, characterization and expression in human brain.

A cDNA encoding a neuron-specific glutamate/aspartate transporter was isolated from human brain cDNA libraries and characterized. The new cDNA, designated human glutamate transporter III, is structurally distinct from two previously described brain specific glutamate transporters. This human cDNA is 90% and 95% homologous at nucleotide and amino acid level, respectively, with a previously reported rabbit glutamate/aspartate transporter. Northern blot analysis of human tissues revealed that the mRNA of this transporter is expressed in brain, liver, muscle, ovary, testis and in retinoblastoma cell lines. In situ hybridization in human brain sections showed that the mRNA is densely expressed in substantia nigra, red nucleus, hippocampus, and in cerebral cortical layers. Southern blot analysis revealed that the gene encoding this mRNA exists as a single copy in the human genome.

L-glutamate binding sites of normal and atrophic human cerebellum.

The binding kinetics, pharmacologic properties, ontogeny and localization of L-glutamate binding sites were studied in membrane preparations and sections of normal and olivopontocerebellar atrophy (OPCA) human cerebellum. One binding component was found with a Kd value in the order of 150 x 10(-9) M. No significant changes of Kd values were observed with age, whereas the highest Bmax value was observed at the age of 1 year. L-Aspartate, ibotenate, quisqualate and L-homocysteic acid were potent inhibitors of L-[3H]glutamate binding. Quantitative densitometric measurements indicated the presence of L-glutamate sites in both the molecular and granule cell layer. In OPCA cerebella a very significant decrease of L-[3H]glutamate specific binding (Bmax) was observed, whereas Kd values were found unchanged. The pharmacologic properties of L-[3H]glutamate binding sites of OPCA cerebellar tissues were similar to those of normal cerebellum. [3H]quinuclidinyl benzylate binding, expressed in fmol/mg protein, did not show significant differences between normal and OPCA cerebella.

Immunohistochemical localization of the neuron-specific glutamate transporter EAAC1 (EAAT3) in rat brain and spinal cord revealed by a novel monoclonal antibody.

Neuronal regulation of glutamate homeostasis is mediated by high-affinity sodium-dependent and highly hydrophobic plasma membrane glycoproteins which maintain low levels of glutamate at central synapses. To further elucidate the molecular mechanisms that regulate glutamate metabolism and glutamate flux at central synapses, a monoclonal antibody was produced to a synthetic peptide corresponding to amino acid residues 161-177 of the deduced sequence of the human neuron-specific glutamate transporter III (EAAC1). Immunoblot analysis of human and rat brain total homogenates and isolated synaptosomes from frontal cortex revealed that the antibody immunoreacted with a protein band of apparent Mr approximately 70 kDa. Deglycosylation of immunoprecipitates obtained using the monoclonal antibody yielded a protein with a lower apparent Mr (approximately 65 kDa). These results are consistent with the molecular size of the human EAAC1 predicted from the cloned cDNA. Analysis of the transfected COS-1 cells by immunocytochemistry confirmed that the monoclonal antibody is specific for the neuron-specific glutamate transporter. Immunocytochemical studies of rat cerebral cortex, hippocampus, cerebellum, substantia nigra and spinal cord revealed intense labeling of neuronal somata, dendrites, fine-caliber fibers and puncta. Double-label immunofluorescence using antibody to glial fibrillary acidic protein as a marker for astrocytes demonstrated that astrocytes were not co-labeled for EAAC1. The localization of EAAC1 immunoreactivity in dendrites and particularly in cell somata suggests that this transporter may function in the regulation of other aspects of glutamate metabolism in addition to terminating the action of synaptically released glutamate at central synapses.

Glutamate transport and metabolism in dopaminergic neurons of substantia nigra: implications for the pathogenesis of Parkinson's disease.

Parkinson's disease (PD) is associated with degeneration of the pigmented dopaminergic neurons located in the ventral mesencephalon. Although the mechanisms by which these neurons degenerate in PD are poorly understood, indirect evidence suggests involvement of glutamatergic mechanisms in the pathogenesis of this disorder. Glutamate, the major excitatory transmitter in the mammalian central nervous system, is known to be neurotoxic when present in excess at the synapses. Two major mechanisms protect neurons from glutamate-induced toxicity: (a) removal of synaptic glutamate via a high affinity uptake carried out by cytoplasmic membrane proteins known as excitatory amino acid transporters (EAAT); and (b) metabolism and recycling of glutamate by synaptic astrocytes via glutamine synthetase, an ATP-requiring reaction. However, when extra-cellular glutamate levels are high (0.5-1.0 mM), glutamate metabolism may be shifted toward the ATP-generating oxidative deamination (glutamate dehydrogenase)-TCA cycle pathway. We have cloned and characterized two human glutamate dehydrogenases (GDH), one of which is nerve tissue specific. This isoenzyme requires ADP for its activity and it may become functional when cellular energy charge is low. We have also cloned three human glutamate transporters. One of these (EAAT3) is neuron specific. In situ hybridization studies using human brain revealed that the pigmented dopaminergic neurons, which degenerate in PD, express EAAT3 at high levels. Primary nerve tissue cultures derived from rat ventral mesencephalon were established and studied for their ability to metabolize glutamate. Results showed that mature cultures expressing high levels of GDH activity were capable of rapidly utilizing glutamate added to the medium at high concentrations (1-1.2 mM). This was associated with little release of aspartate and alanine into the medium. In contrast, immature cultures expressing low GDH activity utilized glutamate at lower rates while releasing substantial amounts of aspartate and alanine into the medium. These data suggest that immature mesencephalic cells metabolize a substantial fraction of the glutamate they take up from the medium via the transamination pathway, compared to mature mesencephalic cultures. Immunocytochemical studies on these cultures revealed that dopaminergic neurons (identified by their tyrosine hydroxylase content) showed intense staining for GDH. Furthermore, inhibition of GDH expression by antisense oligonucleotides was toxic to cultured mesencephalic neurons, with dopaminergic neurons being affected at the early stages of this inhibition. Hence, the dense expression by dopaminergic neurons of proteins involved in the transport and metabolism of glutamate may serve particular biological needs intrinsic to these cells. Further studies are required to test whether these properties render these neurons vulnerable to excitotoxic mechanisms or to abnormalities of glutamate metabolism.

Relationship of inferior olive-climbing fibers to p,p'-DDT-induced myoclonus in rats.

The effects of 3-acetylpyridine (3-AP), which destroys the inferior olive, and harmaline, which stimulates inferior olive-climbing fiber activity, on DDT-induced myoclonus, wee studied in rats. 3-AP shortened and harmaline delayed the time of onset of myoclonus after intragastric administration of DDT. 3-AP also counteracted the antimyoclonic action of L-5-hydroxytryptophan plus chlorimipramine, clonazepam and phenoxybenzamine in this animal model. The results suggest that these antimyoclonic agents require an intact olivocerebellar pathway for their action.