Pam Fredman

A procedure for the quantitative isolation of brain gangliosides

In a systematic study of the optimal conditions for the quantitative isolation of gangliosides from brain tissue and their further purification the yield of gangliosides obtained by extraction of the tissue twice with twenty volumes of chloroform/methanol/water (4 : 8 : 3, v/v) was larger than that obtained with all other solvents tested, including tetrahydrofuran/phosphate buffer. The gangliosides were separated from other lipids by phase partition, water was added to the total lipid extract to give a final chloroform/methanol/water volume ratio of 4 : 8 : 5.6. Isolation of gangliosides from the total lipid extract with the aid of anion-exchange resins was not practical as a routine procedure on a large scale. The crude gangliosides extract was freed from low molecular weight contaminants by dialysis against water. This method was superior to the purification on gel filtration media or on anion-exchange resins, which required large columns with selective losses of gangliosides as a result. The present method has been applied to human brain, and the concentration and distribution of gangliosides in the human forebrain in infancy and old age are given.

Cerebrospinal fluid in the diagnosis of multiple sclerosis: a consensus report.

The Committee of the European Concerted Action for Multiple Sclerosis (Charcot Foundation) organised five workshops to discuss CSF analytical standards in the diagnosis of multiple sclerosis. This consensus report from 12 European countries summarises the results of those workshops. It is hoped that neurologists will confer with their colleagues in clinical chemistry to arrange the best possible local practice. The most sensitive method for the detection of oligoclonal immunoglobulin bands is isoelectric focusing. The same amounts of IgG in parallel CSF and serum samples are used and oligoclonal bands are revealed with IgG specific antibody staining. All laboratories performing isoelectric focusing should check their technique at least annually using “blind” standards for the five different CSF and serum patterns. Quantitative measurements of IgG production in the CNS are less sensitive than isoelectric focusing. The preferred method for detection of blood-CSF barrier dysfunction is the albumin quotient. The CSF albumin or total protein concentrations are less satisfactory. These results must be interpreted with reference to the age of the patient and the local method of determination. Cells should be counted. The normal value is no more than 4 cells/microliters. Among evolving optional tests, measurement of the combined local synthesis of antibodies against measles, rubella, and/or varicella zoster could represent a significant advance if it offers higher specificity (not sensitivity) for identifying chronic rather than acute inflammation. Other tests that may have useful correlations with clinical indices include those for oligoclonal free light chains, IgM, IgA, or myelin basic protein concentrations.

Structural membrane alterations in Alzheimer brains found to be associated with regional disease development; increased density of gangliosides GM1 and GM2 and loss of cholesterol in detergent- resistant membrane domains

The formation of neurotoxic beta‐amyloid fibrils in Alzheimers disease (AD) is suggested to involve membrane rafts and to be promoted, in vitro, by enriched concentrations of gangliosides, particularly GM1, and the cholesterol therein. In our study, the presence of rafts and their content of the major membrane lipids and gangliosides in the temporal cortex, reflecting late stages of AD pathology, and the frontal cortex, presenting earlier stages, has been investigated. Whole tissue and isolated detergent‐resistant membrane fractions (DRMs) were analysed from 10 AD and 10 age‐matched control autopsy brains. DRMs from the frontal cortex of AD brains contained a significantly higher concentration (µmol/µmol glycerophospholipids), of ganglioside GM1 (22.3 ± 4.6 compared to 10.3 ± 6.4, p < 0.001) and GM2 (2.5 ± 1.0 compared to 0.55 ± 0.3, p < 0.001). Similar increases of these gangliosides were also seen in DRMs from the temporal cortex of AD brains, which, in addition, comprised significantly lower proportions of DRMs. Moreover, these remaining rafts were depleted in cholesterol (from 1.5 ± 0.2 to 0.6 ± 0.3 µmol/µmol glycerophospholipids, p < 0.001). In summary, we found an increased proportion of GM1 and GM2 in DRMs, and accelerating plaque formation at an early stage, which may gradually lead to membrane raft disruptions and thereby affect cellular functions associated with the presence of such membrane domains.

Dynamic association of human insulin receptor with lipid rafts in cells lacking caveolae

Cholesterol‐sphingolipid rich plasma membrane domains, known as rafts, have emerged as important regulators of signal transduction. The adipocyte insulin receptor (IR) is localized to and signals via caveolae that are formed by polymerization of caveolins. Caveolin binds to IR and stimulates signalling. We report that, in liver‐derived cells lacking caveolae, autophosphorylation of the endogenous IR is dependent on raft lipids, being compromised by acute cyclodextrin‐mediated cholesterol depletion or by antibody clustering of glycosphingolipids. Moreover, we provide evidence that IR becomes recruited to detergent‐resistant domains upon ligand binding and that clustering of GM2 ganglioside inhibits IR signalling apparently by excluding the ligand‐bound IR from these domains. Our results indicate that, in cells derived from liver, an important insulin target tissue, caveolae are not required for insulin signalling. Rather, the dynamic recruitment of the ligand‐bound IR into rafts may serve to regulate interactions in the initiation of the IR signalling cascade.

Blood-brain barrier disturbance in patients with Alzheimer's disease is related to vascular factors

To investigate the blood‐brain barrier (BBB function), serum and cerebro‐spinal fluid (CSF) from 118 patients with Alzheimers disease (AD) and 50 healthy controls was analyzed with regard to albumin concentrations. For the AD group, clinical vascular factors were also recorded. The CSF/serum albumin ratio was used as a measure of BBB function. When compared with controls, the AD group showed a higher mean albumin ratio, indicating a decline in BBB function. However, no significant reduction in BBB function was found in AD patients without vascular factors. These findings support the hypothesis that BBB disturbance is related to vascular factors, coexisting with AD. In the control group, no significant correlation between age and albumin ratio was found, which suggests that aging per se is not primarily associated with a decline in BBB function. The present study provides evidence in favour of the conclusion that a reduced BBB function is related neither to AD nor to aging, but to clinical vascular factors.

A population study of apoE genotype at the age of 85: relation to dementia, cerebrovascular disease, and mortality

OBJECTIVES To study the association of apoE genotypes with dementia and cerebrovascular disorders in a population based sample of 85 year old people. METHODS A representative sample of 85 year old people (303 non-demented, 109 demented) were given a neuropsychiatric and a medical examination and head CT. The apoE isoforms were determined. Dementia was diagnosed according to DSM-III-R. RESULTS At the age of 85, carriers of the apoE ε4 allele had an increased odds ratio (OR) for dementia (1.9; p<0.01) and its subtypes Alzheimer’s disease (1.9; p<0.05) and vascular dementia (2.0; p<0.05). Among those categorised as having vascular dementia, the apoE ε4 allele was associated with mixed Alzheimer’s disease-multi-infarct dementia (OR 6.5; p<0.05), but not with pure multi-infarct dementia (OR 1.5; NS). Only carriers of the apoE ε4 allele who also had ischaemic white matter lesions on CT of the head had an increased OR for dementia (OR 6.1; p=0.00003), and its main subtypes Alzheimer’s disease (OR 6.8; p=0.002) and vascular dementia (OR 5.6; p=0.0007), whereas carriers of the apoE ε4 allele without white matter lesions had an OR for dementia of 1.0 (OR for Alzheimer’s disease 1.8; NS and for vascular dementia 0.6; NS) and non-carriers of the apoE ε4 allele with white matter lesions had an OR for dementia of 2.2; NS (OR for Alzheimer’s disease 2.7; NS and for vascular dementia 1.6; NS). The apoE allele variants were not related to mortality or incidence of dementia between the ages of 85 and 88. The ε2 allele was related to a higher prevalence of stroke or transient ischaemic attack at the age of 85 (OR 2.1; p<0.05) and a higher incidence of multi-infarct dementia during the follow up (OR 2.9; p<0.05). CONCLUSIONS Neither the apoE ε4 allele nor white matter lesions are sufficient risk factors by themselves for dementia at very old ages, whereas possession of both these entities increases the risk for Alzheimer’s disease and vascular dementia substantially.

Separation of gangliosides on a new type of anion-exchange resin

A new anion exchange resin, Spherosil-DEAE-Dextran, consisting of porous glass beads covered with cross-linked DEAE-Dextran, was used in the separation of gangliosides. The gangliosides were eluted from the resin with a discontinuous gradient of potassium acetate in methanol, separating the gangliosides quantitatively into mono-, di, tri-, tetra- and pentasialoganglioside fractions. The new resin was found to have higher binding capacity, to show less unspecific adsorption and to give a better separation of the higher oligosialogangliosides than did DEA-Spherosil, QAE-Sephadex, DEAE-Sephadex and DEAE-Sepharose. Anion exchange chromatography improved discrimination between closely allied gangliosides as well as quantification and identification of individual gangliosides, especially the minor ones. The new procedure was used in the separation of the gangliosides in human infant forebrain and cerebellum.

White matter changes in normal pressure hydrocephalus and Binswanger disease: Specificity, predictive value and correlations to axonal degeneration and demyelination

Objectives– To analyse the diagnostic and prognostic value of periventricular hyperintensity (PVH) and deep white matter hyperintensity (DWMH) magnetic resonance imaging (MRI) changes and their relation to symptoms and cerebrospinal fluid (CSF) markers of demyelination (sulphatide) and axonal degeneration [neurofilament triplet protein (NFL)] in a large series of patients with normal pressure hydrocephalus (NPH) and Binswanger disease (BD). Materials and methods– PVH and DWMH were determined by a semi‐automatic segmentation method on T2‐weighted images in 29 patients with NPH and 17 patients with BD. CSF analyses, psychometric testing and quantification of balance, gait and continence were performed in all patients and also postoperatively in NPH patients. Results– No MRI variable could identify NPH or BD patients. Abundant PVH and DWMH preoperatively correlated with improvement in gait, balance and psychometric performance after shunt surgery (P < 0.05). CSF sulphatide correlated positively with the amount of DWMH (P < 0.05) while NFL was correlated to both PVH and DWMH (P < 0.05). Abundant PVH correlated with poor psychometric performance while DWMH correlated with gait disturbance (P < 0.05). Postoperative reduction in PVH correlated with improvement in gait, balance and psychometric performance. Conclusion– In spite of a refined quantification method, NPH and BD patients exhibited similar MRI changes. MRI had a predictive value in NPH patients. DWMH might relate to demyelination and PVH to neuronal axonal dysfunction. NPH and BD share the major part of symptoms and MRI changes, indicating a common pathophysiological pattern, and we raise the question of how to treat BD patients.

Cerebrospinal fluid apolipoprotein E is reduced in Alzheimer's disease

Apolipoprotein E (ApoE) has been implicated in the pathogenesis of Alzheimers disease (AD). ApoE is synthesized within the brain and has been suggested to be involved in the re-utilization of membrane lipids during neuronal repair and remyelination after injury. Spherical ApoE-containing lipoprotein particles are found in the cerebrospinal fluid (CSF). To study further the pathogenetic role of ApoE in degenerative brain disorders, we analysed ApoE in CSF. A significant (p < 0.001) reduction of CSF ApoE (1.5 +/- 1.2 ng ml-1) was found in AD compared with controls (5.0 +/- 2.7 ng ml-1). A less pronounced reduction was also found in frontal lobe dementia (3.1 +/- 1.5 ng ml-1; p < 0.05). These findings support the hypothesis that ApoE is involved in the pathogenesis of degenerative brain disorders such as AD. An increased reutilization of ApoE-lipid complexes in the brain, as part of a generalized repair process, may explain the low CSF ApoE in AD. Alternatively, the reduction of CSF ApoE may be caused by absorption of ApoE to senile plaques and neurofibrillary tangles.

Characterization of new gangliosides of the lactotetraose series in murine xenografts of a human glioma cell line

The major mono–and disialogangliosides of the extensively characterized established human glioma line D54MG were isolated and purified from subcutaneous solid xenografts grown in athymic (nu/nu) mice. Structural determination showed that they belonged to the lactotetraosylceramide series. The sialyllactotetraosylceramide contained 90% N–glycolyl– and 10% N–acetylneuraminic acid linked in an α2–3 linkage (IV3NeuGc–LcOse4Cer, IV3NeuAc–LcOse4Cer). The disialogangliosides had a previously undescribed type of structure with sialic acids linked to the terminal galactose in an α2–3 linkage and to N–acetylglucosamine in an α2–6 linkage. Not only did species with NeuAc or NeuGc occur, but also species with mixtures of the two sialic acids, e.g. NeuAC and NeuGc. The schematic structures of the new disialogangliosides are