Antidiabetics, statins and the risk of amyotrophic lateral sclerosis.

BACKGROUND: Medications that are used for treatment of metabolic disorders have been suggested to be associated with the development of amyotrophic lateral sclerosis (ALS). METHODS: To examine the associations of antidiabetics and statins with the subsequent risk of ALS we conducted a population-based nested case-control study of 2475 Swedish residents diagnosed with ALS during July 2006 to December 2013 and 12 375 population controls (five for each ALS case). We extracted information on filled prescriptions of antidiabetics and statins for both cases and controls from the Swedish Prescribed Drug Register during the years before ALS diagnosis. Conditional logistic regression was used to calculate odds ratios (ORs) for the associations of these medications with ALS risk. RESULTS: Patients with ALS were less likely to have been prescribed with antidiabetics compared with controls [OR, 0.76; 95% confidence intervals (CI), 0.65-0.90]. Conversely, statins were not associated with ALS risk overall (OR, 1.08; 95% CI, 0.98-1.19), although a positive association was noted among women (OR, 1.28; 95% CI, 1.10-1.48). The latter association was mostly explained by ALS cases being more likely to have a first prescription of statins during the year before diagnosis compared with controls (OR, 2.54; 95% CI, 1.84-3.49). CONCLUSIONS: The inverse association of antidiabetics with ALS is consistent with the previously reported inverse association between type 2 diabetes and ALS risk. The increase in prescription of statins during the year before ALS diagnosis deserves attention because it might reflect an acceleration of the course of ALS due to statin use.

Detection of BMAA in the human central nervous system.

Amyotrophic lateral sclerosis (ALS) is an extremely devastating neurodegenerative disease with an obscure etiology. The amino acid ß-N-methylamino-l-alanine (BMAA) produced by globally widespread phytoplankton has been implicated in the etiology of human motor neuron diseases [corrected]. BMAA was recently proven to be present in Baltic Sea food webs, ranging from plankton to larger Baltic Sea organisms, some serving as important food items (fish) for humans. To test whether exposure to BMAA in a Baltic Sea setting is reflected in humans, blood and cerebrospinal fluid (CSF) from individuals suffering from ALS were analyzed, together with sex- and age-matched individuals not inflicted with ALS. Ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and multiple reaction monitoring (MRM), in conjunction with diagnostic transitions revealed BMAA in three (12%) of the totally 25 Swedish individuals tested, with no preference for those suffering from ALS. The three BMAA-positive samples were all retrieved from the CSF, while BMAA was not detected in the blood. The data show that BMAA, potentially originating from Baltic Sea phytoplankton, may reach the human central nervous system, but does not lend support to the notion that BMAA is resident specifically in ALS-patients. However, while dietary exposure to BMAA may be intermittent and, if so, difficult to detect, our data provide the first demonstration of BMAA in the central nervous system of human individuals ante mortem quantified with UHPLC-MS/MS, and therefore calls for extended research efforts.

Founder effect in spinal and bulbar muscular atrophy (SBMA) in Scandinavia.

We haplotyped 13 Finnish, 10 Swedish, 12 Danish and 2 Norwegian SBMA (spinal and bulbar muscular atrophy, Kennedy disease) families with a total of 45 patients and 7 carriers for 17 microsatellite markers spanning a 25.2 cM region around the androgen receptor gene on chromosome Xq11-q12 in search of a genetic founder effect. In addition, the haplotypes of 50 Finnish, 20 Danish and 22 Swedish control males were examined. All the Scandinavian SBMA families shared the same 18 repeat allele for the intragenic GGC repeat, which was present in only 24% of the controls. Linkage disequilibrium was also seen for the closest microsatellite markers. In addition, extended haplotypes of the Finnish, Swedish and Danish SBMA families revealed country-specific common founder haplotypes, which over time became gradually shortened by recombinations. No common haplotype was found among the controls. The data suggest that the SBMA mutation was introduced into western Finland 20 generations ago. Haplotype analysis implies a common ancestor for the majority of Scandinavian SBMA patients.

Multiple founder effects in spinal and bulbar muscular atrophy (SBMA, Kennedy disease) around the world.

SBMA (spinal and bulbar muscular atrophy), also called Kennedy disease, is an X-chromosomal recessive adult-onset neurodegenerative disorder caused by death of the spinal and bulbar motor neurones and dorsal root ganglia. Patients may also show signs of partial androgen insensitivity. SBMA is caused by a CAG repeat expansion in the first exon of the androgen receptor (AR) gene on the X-chromosome. Our previous study suggested that all the Nordic patients with SBMA originated from an ancient Nordic founder mutation, but the new intragenic SNP marker ARd12 revealed that the Danish patients derive their disease chromosome from another ancestor. In search of relationships between patients from different countries, we haplotyped altogether 123 SBMA families from different parts of the world for two intragenic markers and 16 microsatellites spanning 25 cM around the AR gene. The fact that different SBMA founder haplotypes were found in patients from around the world implies that the CAG repeat expansion mutation has not been a unique event. No expansion-prone haplotype could be detected. Trinucleotide diseases often show correlation between the repeat length and the severity and earlier onset of the disease. The longer the repeat, the more severe the symptoms are and the onset of the disease is earlier. A negative correlation between the CAG repeat length and the age of onset was found in the 95 SBMA patients with defined ages at onset.

Myelin breakdown and elimination in the posterior funiculus of the adult cat after dorsal rhizotomy: a light and electron microscopic qualitative and quantitative study.

Adult cats were subjected to unilateral dorsal L6, L7, and S1 rhizotomy. After survival times of 1-1,552 days the degeneration of axons and myelin sheaths and the elimination of degenerating myelin was studied qualitatively and quantitatively with light and electron microscopy and in Marchi-stained sections in the posterior funiculus in T12-L2. Degeneration was first observed as swollen or shrunken nerve fibers. Somewhat later there was an increased occurrence of collapsed myelin sheaths. The latter lost their myelin periods and appeared to be transformed into myelin bodies. The occurrence of myelin bodies coincided temporally with the presence of many Marchi-positive bodies. Later, an increasing number of intracellularly located lipid droplets occurred, paralleled by the occurrence of a great number of Marchi-positive granules and crystalline structures. Profiles of collapsed myelin sheaths, myelin bodies, and lipid droplets were frequently seen in the cytoplasm of microglial cells. Later, astrocytes and perivascular cells became filled with numerous lipid droplets. The findings suggest that microglial cells take up collapsed myelin sheaths and within these cells the sheaths become transformed into myelin bodies and subsequently into lipid droplets. These two products of myelin disintegration appear to correspond to the Marchi-positive structures seen during the degeneration process. The lipid droplets appear to be transported to astrocytes and perivascular cells.

Spontaneous phagocytosis of C-type synaptic terminals by spinal alpha-motoneurons in newborn kittens. An electron microscopic study.

The cell bodies and proximal dendrites of spinal alpha-motoneurons were studied electron microscopically with the aid of serial sections during the first postnatal week in the cat. The observations suggested that some of the synaptic terminals of the so-called C-type on the cell bodies and dendrites are phagocytosed by the motoneurons during the first few days after birth. This finding is discussed in relation to the earlier demonstrated postnatal loss of synaptic terminals on the motoneurons after birth and differences demonstrated between different functional types of spinal motoneurons with respect to the number and distribution of C-type terminals in the adult cat.

An ultrastructural study of serially sectioned Renshaw cells. I. Architecture of the cell body, axon hillock, initial axon segment and proximal dendrites.

Seventeen neurons which were postsynaptic to axon collateral boutons of intracellularly HRP-stained triceps surae alpha-motoneurons were studied ultrastructurally. All 17 neurons were situated in lamina VII, ventro-medially to the main motor nuclei. This and other facts support the assumption that the observed neurons are morphological correlates to the physiologically defined Renshaw cells. The contours of the cell bodies, as observed in the midnucleolus plane, were elongated. The axons originated either from the cell bodies or from dendrites. The number of dendrites of each neuron varied between 3 and 7. The appearance of the presumed Renshaw cells was also compared with that of a larger sample of neurons from the ventral part of lamina VII which was studied light microscopically in semithin sections. It was suggested that the Renshaw cells belong to the larger and more elongated neurons in the area.

An ultrastructural study of serially sectioned Renshaw cells. II. Synaptic types.

Boutons and synaptic contacts on 17 presumed Renshaw cells were studied ultrastructurally. All 17 neurons were postsynaptic to axon collateral boutons of intracellularly HRP-stained triceps and surae alpha-motoneurons and located in lamina VII, ventromedially to the main motor nuclei. The boutons and synaptic contacts could be classified into two main categories on the basis of synaptic vesicles: S-type boutons with spherical synaptic vesicles and F-type boutons with flattened vesicles, the alpha-motoraxon collateral boutons falling into the S-category. In addition, some S-type boutons containing neurofilaments and some being apposed by small presynaptic boutons were observed. The results are discussed to earlier observations on the synaptology of central neurons, particularly spinal alpha-motoneurons.

An ultrastructural study of the synaptic contacts of alpha-motoneurone axon collaterals. I. Contacts in lamina IX and with identified alpha-motoneurone dendrites in lamina VII.

Horseradish peroxidase (HRP) was injected intracellularly in triceps surae alpha-motoneurones. The axons and axon collaterals of these neurones were traced light and electron microscopically. Synaptic boutons of collaterals, in Rexed's lamina IX or in synaptic contact with HRP-stained motoneurone dendrites in lamina VII, were studied ultrastructurally. The boutons exhibited spherical synaptic vesicles and made synaptic contacts of two different types with HRP-stained alpha-motoneurone dendrites in lamina IX and VII, dendrites and cell bodies of large neurones in lamina IX, dendrites of unknown origin in lamina IX and with one cell body of a medium size neurone in lamina IX. The observations are discussed in relation to earlier qualitative and quantitative studies on the synaptology of cat spinal alpha-motoneurones.

An ultrastructural study of the synaptic contacts of alpha 1-motoneuron axon collaterals. II. Contacts in lamina VII.

Horseradish peroxidase (HRP) was injected intracellularly in triceps surae alpha-motoneurons. The axons and axon collaterals of these neurons were traced light and electron microscopically. Synaptic boutons of collaterals in the ventral part of Rexed's lamina VII were studied ultrastructurally. The boutons exhibited spherical synaptic vesicles and made synaptic contacts with cell bodies and proximal dendrites of neurons assumed to be Renshaw cells and with dendrites of unknown origin. The observations are discussed in relation to earlier qualitative and quantitative studies on the other known synaptic contacts of the alpha-motor axons, both in the central and peripheral nervous system.

Cytotoxic activity in the plasma of amyotrophic lateral sclerosis (ALS) patients against normal erythrocytes. Quantitative determinations.

Erythrocytes from healthy controls were incubated during 5.5 h under physiological conditions in own plasma, and plasma from blood type-matched ALS patients and diseased controls. Free haemoglobin (Hb) was repeatedly measured spectrophotometrically as a measure of haemolysis. Using a standardized procedure, the interexperimental variation in Hb release during incubation in own control plasma was kept very low in a large series of incubations (n = 77). This indicated that the condition of the erythrocytes had been constant. Parallel incubations in plasma from ALS patients (n = 20) rendered a clear-cut increase in Hb release, which in all cases exceeded the confidence interval of the controls. There was no correlation between Hb release in the ALS group and age, sex, type of disease or duration of symptoms. The cytotoxic effect of ALS plasma was abolished by heating to 56 degrees C. In a material of incubations in plasma of diseased controls (traumatic spinal cord disease, Guillain-Barré syndrome), the haemolysis was slightly larger than in control plasma, but in all cases smaller than in ALS plasma.

Further studies on the occurrence of serum autoantibodies against a membrane bound AChE fraction in ALS/MND patients and controls.

Autoantibodies of the IgG and IgA types against a membrane bound AChE fraction were studied in the serum of ALS/MND patients, controls with neurological or autoimmune diseases, and normal controls. About 70% of ALS and progressive spinal muscle atrophy (PMA) patients had antibodies of both Ig types (titre > 1/81), compared to about 12% of neurologically diseased controls and about 8% of the other controls. A higher proportion of individuals having autoantibodies was found in the older age group in both patients and controls.

Pitfalls in the evaluation of isometric strength (TQNE) data in ALS.

The phase of rapid reduction of isometric strength in single muscle groups was identified and its slope was calculated in patients studied with the TQNE method. This parameter was studied in the extremities and in respiration in 4 ALS patients and 4 PMA (progressive spinal muscle atrophy) patients. The slopes of the strength reduction in these muscle groups and their mean values exceeded the slopes of the corresponding megascores; this difference was larger in the extremities than in respiration. It is concluded that repeated spirometry gives a good expression of reduction of muscle strength ir ALS, and is well suited for monitoring treatment effects.

Abnormal tissue distribution of lead in amyotrophic lateral sclerosis.

The lead content of cerebrospinal fluid (CSF) was found to be significantly elevated in 12 patients with amyotrophic lateral sclerosis, when compared to 28 control subjects having non-degenerative neurological disorders. The difference could not be explained as being merely secondary to blood-CSF barrier damage. A hypothetical model of the pathogenesis of the disease is advanced and the results are discussed in relation to this model.

Further studies on the erythrocyte uptake of lead in vitro in amyotrophic lateral sclerosis (ALS) patients and controls. Abnormal erythrocyte fragility in ALS.

Following our earlier observations on increased plasma concentrations of lead in amyotrophic lateral sclerosis (ALS), the erythrocyte uptake of lead from plasma has been studied in vitro. Whole-blood from ALS patients and controls was incubated after the addition of lead (0.6 mumol/l whole-blood) and plasma lead concentrations were repeatedly determined. Incubation was continued until haemolysis developed. Fairly stable plasma lead concentrations were established at, on the average, 0.5-0.6 mumol/l after 10-30 min and persisted throughout the incubation with no significant difference between ALS- and control samples. Unexpectedly, it was also observed that haemolysis occurred significantly earlier in the ALS- than in the control samples. Plateau levels in plasma lead concentration of the same order as in the present experiments have been observed both in ALS- and control samples in previous experiments with the same technique, where the lead dose added was twice as high, and these plateau levels were about 10 times higher than those observed in vivo in ALS patients and controls. It is therefore suggested that the final plasma lead concentrations in vivo is established by factors other than the erythrocyte uptake and it is improbable that the differences between ALS patients and controls in plasma lead concentration are associated with differences in the degree of lead uptake by the red cells. The increased plasma lead concentrations in ALS patients may instead be caused by increased fragility of the erythrocytes, as manifested by the earlier occurrence of haemolysis in the present experiments. The observation of increased red cell fragility is, however, also of interest as a possible manifestation of a generalized membrane defect.

Serum protein binding of lead in vitro in amyotrophic lateral sclerosis patients and controls.

The binding of lead to serum proteins was studied in vitro in ALS-patients and controls. Serum was incubated with 210Pb, the proteins were separated by isoelectric focusing and the radioactivity in the different protein fractions was determined by liquid scintillation counting. The activity was concentrated in the orosomucoid (acid alpha1-glycoprotein) fraction both in ALS-patients and controls under the present experimental conditions and the capacity of this protein to bind lead was demonstrated in a control experiment. The findings are discussed in relation to our earlier observations on increased concentrations of lead in cerebrospinal fluid and plasma in ALS-patients and the hypothetical role of the retrograde axonal transport in motoneurons in the pathogenesis of ALS. The present observations do not lend support to the idea that the increased plasma concentrations of lead in the disease would be related to qualitative differences in serum protein binding.

Immunoglobulins from patients with amyotrophic lateral sclerosis affect human erythrocyte acetylcholinesterase.

Human erythrocyte acetylcholinesterase (AChE) solubilized with Triton X-100 and obtained as a complex with micelles containing Triton and membrane phospholipids was incubated with immunoglobulins (Igs) from patients with amyotrophic lateral sclerosis (ALS) and from normal individuals. The temperature dependence of the AChE activity was determined. Biphasic (broken) Arrhenius plots were obtained with control Igs with the break point at 32.8 +/- 0.3 degrees C (SD, n = 18) indicating that the enzyme changes its conformation at this temperature. With ALS-Igs monophasic (linear) plots were observed in 14 cases and a biphasic in one case. ALS-Igs prevent the conformational change occurring at the break point temperature. The activation energy at physiological temperature increased by 60% from 2.4 to 3.8 kcal/mol (10.0-15.9 kJ/mol) which implies that ALS-Igs inhibit AChE. Thus, ALS-patients have autoantibodies that change the normal behaviour of erythrocyte AChE and which bind to the enzyme molecule or/and to phospholipids associated with the enzyme. At least part of the autoantibodies should be directed against the enzyme molecule, since a change in the Arrhenius plot was also observed in a control experiment with AChE which probably had micelles without any phospholipids. This enzyme was isolated by affinity chromatography and was washed with a buffer containing Triton X-100 before desorption from the affinity column, a treatment known to remove all phospholipids from erythrocyte AChE.

Abnormal distribution of lead in amyotrophic lateral sclerosis--reestimation of lead in the cerebrospinal fluid.

The lead concentration in CSF was determined by flameless atomic absorption spectrophotometry in 16 ALS patients and 22 control cases. The mean values were 0.69 +/- 0.55 (ALS) and 0.41 +/- 0.37 (controls), P < 0.01. This confirms our earlier findings of raised CSF lead levels in ALS but the present values are lower than previously reported for both ALS patients and controls.

Selective vulnerability of alpha motor neurons in ALS: relation to autoantibodies toward acetylcholinesterase (AChE) in ALS patients.

The degenerative process in amyotrophic lateral sclerosis (ALS) concerns primarily alpha motor neurons in the spinal cord and brain stem, and neurons forming descending pathways to the cord, especially in the pyramidal tract. Some degeneration of large peripheral sensory nerve fibers often occurs too, but preganglionic autonomic neurons and gamma motor neurons are most often spared in the disease. The vulnerability of alpha motor neurons compared to other types of neurons in ALS is discussed in relation to retrograde axoplasmic transport from peripheral blood of foreign noxious macromolecules, interneuronal transport of such molecules, and neuronal surface structure properties relevant to uptake for retrograde axoplasmic transport. Certain differences in these aspects between alpha motor neurons and other neuronal types exist. Some differences concern the neuronal turnover of acetylcholinesterase (AChE), which could be of special interest in view of the recent demonstration of regular occurrence of autoantibodies towards this enzyme in ALS patients.

Myelin breakdown in the posterior funiculus of the kitten after dorsal rhizotomy. A qualitative and quantitative light and electron microscopic study.

Morphological aspects of myelin breakdown in the posterior funiculus during Wallerian degeneration were studied in kittens subjected to lumbosacral dorsal rhizotomies 6-8 days after birth. The first sign of myelin breakdown was characterized by swollen or shrunken nerve fibers. Shortly thereafter there was an increased occurrence of collapsed myelin sheaths and later of rounded myelin bodies. Myelin was clearly seen in microglial cells. Correlative observations on Marchi-stained material indicted the simultaneous and frequent appearance of Marchi-positive bodies (MPB:s) and myelin bodies. Due to the rapidity of the degeneration process in the kitten, the increase in the occurrence of Marchi-positive granules (MPG:s) seemed to start concomitantly with increased occurrence of MPB:s. However, the frequent occurrence of MPG:s outlasted that for MPB:s. The findings indicate that the MPB:s may be the counterpart to myelin bodies and the MPG:s to lipid droplets. Microglial cells may be responsible for the primary uptake of degenerating myelin and the subsequent transformation of myelin bodies to lipid droplets. The much faster breakdown of myelin and elimination of lipid material in the degenerating posterior funiculus of the kitten, as compared to the adult, seemed to be due not only to the lower myelin content in the kitten, but also to a higher density of microglial and a greater efficiency in the myelin breakdown process in the degenerating posterior funiculus of the kitten.