P. A. Bolhuis

Concentrations of amyloid-β protein in cerebrospinal fluid increase with age in patients free from neurodegenerative disease

Cerebral deposition of amyloid-beta protein (A beta) is central to the pathogenesis of Alzheimers disease (AD). Increasing age is one of the few definitively established risk factors for this disease. The concentration of A beta was measured in cerebrospinal fluid (CSF) with a sensitive enzyme-linked immunosorbent assay in 18 adult neurological patients free from neurodegenerative disease. CSF A beta increased with age, yielding a significant correlation of 0.84. This observation suggests that increased levels of A beta in CSF may be an index of age-related changes in the processing of the amyloid-beta precursor protein resulting in an increased risk for AD.

Cerebrospinal Fluid Markers of Alzheimer's Disease

Objective: To review studies on cerebrospinal fluid (CSF) in patients with Alzheimers disease (AD) in order to answer the question whether CSF contains a specific marker which can be used to support a clinical diagnosis of AD.

Effects of 2,5-hexanedione on calpain-mediated degradation of human neurofilaments in vitro

2,5-Hexanedione (2,5-HD), the neurotoxic metabolite of n-hexane, can structurally modify neurofilaments (NF) by pyrrole adduct formation and subsequent covalent cross-linking. 2,5-HD also induces accumulations of NF within the pre-terminal axon. We examined whether exposure of NF to 2,5-HD affected NF degradation. Two different models were used: (1) NF-enriched cytoskeletons isolated from human sciatic nerve were incubated with 2,5-HD in vitro and (2) differentiated human neuroblastoma cells (SK-N-SH) were exposed to 2, 5-HD in culture prior to isolation of cytoskeletal proteins. The cytoskeletal preparations were subsequently incubated with calpain II. The amount of NF-H and NF-L remaining after proteolysis was determined by SDS-PAGE and quantitative immunoblotting. NF-M proteolysis could not be quantified. Incubation of sciatic nerve cytoskeletal preparations with 2,5-HD resulted in cross-linking of all three NF proteins into high molecular weight (HMW) material with a range of molecular weights. Proteolysis of the NF-H and NF-L polypeptides was not affected by 2,5-HD-exposure. Degradation of the HMW material containing NF-H or NF-L was retarded when comparing with degradation of the NF-H and NF-L polypeptides, respectively, from control samples, but not as compared to the corresponding NF polypeptides from 2,5-HD-treated samples. Exposure of SK-N-SH cells to 2,5-HD also resulted in considerable cross-linking of NF. No differences were found between the proteolytic rates of NF-L and NF-H from exposed cells as compared with those subunits from control cells. Moreover, degradation of cross-linked NF-H was not different from monomeric NF-H. In conclusion, whether 2,5-HD affects calpain-mediated degradation of cross-linked NF proteins will depend on which model better reflects NF cross-linking as occurring in 2, 5-HD-induced axonopathy. However, with both models it was demonstrated that exposure of NF proteins to 2,5-HD without subsequent cross-linking is not adequate to inhibit NF proteolysis in vitro by added calpain.

Evidence against increased oxidative stress in fibroblasts from patients with non-superoxide-dismutase-1 mutant familial amyotrophic lateral sclerosis

Fibroblasts were cultured from 5 unrelated familial amyotrophic lateral sclerosis (FALS) patients and from healthy control subjects. In parallel, fibroblasts were examined for signs of abnormal oxidative stress by study of reactive oxygen species metabolism and, concurrently, leukocyte DNA from the same patients was examined for superoxide dismutase 1 (SOD1) mutations. The endogenous production of reactive oxygen species was assessed by following the menadione-induced reduction of oxidized cytochrome o, added to the medium. FALS and control fibroblasts exhibited the same rate of metabolism. Also levels of thiobarbturic-acid-reactive species (TBARS), a marker of lipid peroxidation, were similar in fibroblasts from either group. The search for SOD1 mutations by linkage study and cycle sequencing proved negative. We did not find evidence for SOD1 mutations by either method of study. Our results provide no evidence for increased oxidative stress in fibroblasts from non-SOD1 mutant FALS.

Sensitivity to 2,5-Hexanedione of Neurofilaments in Neuroblastoma Cell Line SK-N-SH Increases During Differentiation

The effect of 2,5-hexanedione (2,5-HD) on the distribution of the neurofilamental (NF) proteins and vimentin was examined in human neuroblastoma cell line SK-N-SH with immunocytochemical methods. Retinoic acid (10 μM) induced differentiation into neuronal cells resulting in the outgrowth of processes and synthesis of NF proteins in the majority of the cells. A minority (4%) differentiated as large fibroblasts. Cells were exposed to 0–10 mM 2,5-HD for 3 days. In neuronal cells a concentration-dependent accumulation of NF proteins was detected as a spherical structure in the perikaryon. Neurofilaments in differentiated SK-N-SH cells were more susceptible to 2,5-HD than NF in undifferentiated cells, as the effects were observed at much lower 2,5-HD concentrations. In contrast, no accumulation of vimentin was detected in the fibroblastic cells.

Muscle cell cultures in Menkes' disease: Copper accumulation in myotubes

SummaryWe present64Cu uptake studies in cultured muscle cells from a one-year-old patient with Menkes disease. The cultured muscle cells from the patient showed a five-fold higher64Cu uptake than control muscle cells. Copper uptake in muscle cells was of the same magnitude as that found in fibroblasts from the patient and also from other Menkes patients. The copper content of a muscle biopsy from the patient was twice that of a control biopsy. The enhanced uptake is probably copper specific, since zinc uptake was unaltered in both muscle cells and fibroblasts from the patient.Cytochrome c oxidase in the muscle of the patient was reduced to one-third of the value for controls, which is in agreement with the hypothesis that in Menkes disease copper accumulates in a biologically non-active form. However, in cultured muscle cells and fibroblasts from the patient the cytochrome c oxidase activity was in the normal range, probably because of the relatively large amount of copper already available in the culture medium.

Metallothionein in Menkes' disease: Induction in cultured muscle cells

Menkes disease is an inherited disturbance of copper metabolism. Addition of copper to the medium of cultured fibroblasts and lymphoblasts from patients with Menkes disease results in an increased induction of metallothionein. We investigated the metallothionein induction in response to copper and zinc in muscle cells (myoblasts and myotubes). Metallothionein synthesis was analyzed by gel electrophoresis of labeled proteins and metallothionein synthesis in muscle cells was compared with the synthesis in fibroblasts. The induction by copper was higher both in muscle cells and in fibroblasts from the Menkes patient compared to the control cells. Hybrid myotubes obtained by fusion of control myoblasts and Menkes myoblasts render a system in which complementation can be studied. Metallothionein synthesis in hybrid myotubes occurred at a level intermediate between the synthesis in Menkes and control myotubes. The abnormal accumulation of copper-induced metallothionein was only partially corrected by fusion with normal cells. Metallothionein induction by zinc was similar in Menkes and control fibroblasts. Combination of copper and zinc yielded no differences in additional metallothionein synthesis for Menkes cells and control fibroblasts. Therefore, metallothionein induction in Menkes disease can primarily be accounted for by copper rather than by zinc.

Normal platelet aggregation in myotonic dystrophy

In 18 patients with myotonic dystrophy, spontaneous aggregation and platelet aggregation induced by thrombin, adenosine diphosphate and epinephrine were compared with normal aggregation patterns. In 17 of the 18 patients the results were not significantly different from normal. In 1 patient spontaneous aggregation and hypersensitive platelets were found. These results are in disagreement with earlier reports on a specific hypersensitivity to epinephrine in myotonic dystrophy. Neither the clinical data (myotonia, paresis) nor the laboratory data (creatine kinase, myoglobin, immunoglobulin G) were correlated with the platelet aggregations.

Mitochondrial Function and Biogenesis in Cultured Mammalian Cells without Functional Respiratory Chains

Long-term impairment of mitochondrial gene expression is possible in cultured mammalian cells grown under specific conditions. This allowed us to study the expression and the function of nuclearly coded mitochondrial proteins in cells that contain no mitochondrial gene products and that are therefore depleted of functional oxidative phosphorylation systems. These cells appeared to synthesise nuclearly coded mitochondrial proteins at near-normal rates. The proteins are imported into the mitochondria, implying that a mitochondrial membrane potential can still be generated. We conclude that mitochondrial gene expression does not influence the expression of nuclear mitochondrial genes, at least not under the culture conditions used.