Mutations in the vesicular trafficking protein annexin A11 are associated with amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder. We screened 751 familial ALS patient whole-exome sequences and identified six mutations including p.D40G in the ANXA11 gene in 13 individuals. The p.D40G mutation was absent from 70,000 control whole-exome sequences. This mutation segregated with disease in two kindreds and was present in another two unrelated cases (P = 0.0102), and all mutation carriers shared a common founder haplotype. Annexin A11-positive protein aggregates were abundant in spinal cord motor neurons and hippocampal neuronal axons in an ALS patient carrying the p.D40G mutation. Transfected human embryonic kidney cells expressing ANXA11 with the p.D40G mutation and other N-terminal mutations showed altered binding to calcyclin, and the p.R235Q mutant protein formed insoluble aggregates. We conclude that mutations in ANXA11 are associated with ALS and implicate defective intracellular protein trafficking in disease pathogenesis.

Exome sequencing in amyotrophic lateral sclerosis identifies risk genes and pathways.

Amyotrophic lateral sclerosis (ALS) is a devastating neurological disease with no effective treatment. We report the results of a moderate-scale sequencing study aimed at increasing the number of genes known to contribute to predisposition for ALS. We performed whole-exome sequencing of 2869 ALS patients and 6405 controls. Several known ALS genes were found to be associated, and TBK1 (the gene encoding TANK-binding kinase 1) was identified as an ALS gene. TBK1 is known to bind to and phosphorylate a number of proteins involved in innate immunity and autophagy, including optineurin (OPTN) and p62 (SQSTM1/sequestosome), both of which have also been implicated in ALS. These observations reveal a key role of the autophagic pathway in ALS and suggest specific targets for therapeutic intervention.

The C9ORF72 expansion mutation is a common cause of ALS+/-FTD in Europe and has a single founder.

A massive hexanucleotide repeat expansion mutation (HREM) in C9ORF72 has recently been linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we describe the frequency, origin and stability of this mutation in ALS+/-FTD from five European cohorts (total n=1347). Single-nucleotide polymorphisms defining the risk haplotype in linked kindreds were genotyped in cases (n=434) and controls (n=856). Haplotypes were analysed using PLINK and aged using DMLE+. In a London clinic cohort, the HREM was the most common mutation in familial ALS+/-FTD: C9ORF72 29/112 (26%), SOD1 27/112 (24%), TARDBP 1/112 (1%) and FUS 4/112 (4%) and detected in 13/216 (6%) of unselected sporadic ALS cases but was rare in controls (3/856, 0.3%). HREM prevalence was high for familial ALS+/-FTD throughout Europe: Belgium 19/22 (86%), Sweden 30/41 (73%), the Netherlands 10/27 (37%) and Italy 4/20 (20%). The HREM did not affect the age at onset or survival of ALS patients. Haplotype analysis identified a common founder in all 137 HREM carriers that arose around 6300 years ago. The haplotype from which the HREM arose is intrinsically unstable with an increased number of repeats (average 8, compared with 2 for controls, P<10(-8)). We conclude that the HREM has a single founder and is the most common mutation in familial and sporadic ALS in Europe.

Primary lateral sclerosis as a phenotypic manifestation of familial ALS.

Primary lateral sclerosis (PLS) is a diagnosis of exclusion in patients with progressive spinobulbar spasticity and could be part of the clinical spectrum of ALS. Unlike ALS, which is familial in 5 to 10% of the cases, PLS has been described as a sporadic disorder in adults. The authors report two patients with PLS from unrelated SOD1-negative familial ALS families. These observations provide further evidence that PLS can be linked pathophysiologically to ALS.

Euthanasia and physician-assisted suicide among patients with amyotrophic lateral sclerosis in the Netherlands.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a disease that causes progressive paralysis leading to respiratory failure. Patients with ALS may consider physician-assisted suicide. However, it is not known how many patients, if given the option, would actually decide to end their lives by physician-assisted suicide or euthanasia nor at what stage of the disease they would choose to do so. METHODS: We identified physicians of 279 patients in the Netherlands with a diagnosis of ALS who died between 1994 and 1999. Physicians were asked to fill out a validated questionnaire about the end-of-life decisions that were made. Of 241 eligible physicians, 203 returned the questionnaire (84 percent). RESULTS: Of the 203 patients, 35 (17 percent) chose euthanasia and died that way. An additional six patients (3 percent) died as a result of physician-assisted suicide. Patients to whom religion was important were less likely to have died as a result of euthanasia or physician-assisted suicide. The choice of euthanasia or physician-assisted suicide was not associated with any particular characteristics of the disease or of the patient's care, nor was it associated with income or educational level. Disability before death was significantly more severe in patients who died as a result of euthanasia than among those who died in other ways. Physician-assisted suicide appeared to occur somewhat earlier in the course of the disease than did euthanasia. An additional 48 patients (24 percent) received palliative treatment, which probably shortened their lives. CONCLUSIONS: In the Netherlands, we found that one in five patients with ALS died as a result of euthanasia or physician-assisted suicide.

Expression of different metallothionein messenger ribonucleic acids in motor cortex, spinal cord and liver from patients with amyotrophic lateral sclerosis.

In earlier studies of sporadic amyotrophic lateral sclerosis (ALS), a disease of unknown etiology, the amount of metallothioneins (MTs), a group of small (6-7 kDa) metal-binding proteins, appeared higher in liver, kidney and spinal cord from patients than from non-neurologic controls. Immunohistochemically, the expression of MT in the central nervous system appeared limited to glia. Since the highly conserved MTs isotypes share antigenic epitopes, they could not be distinguished by immunological methods. It thus proved necessary to estimate the expression of each individual MT messenger ribonucleic acid (mRNA) by performing reverse transcriptase polymerase chain reaction (RT-PCR)-mediated analysis of tissue samples. Tissues selected included liver, motor cortex and cervical cord at C6; MT mRNAs analyzed included MT1A, 1B, 1E, 1F, 1G, 2A, and 3. Also, special care was taken to avoid interference by amplification of the 6 MT pseudogenes. Except of MT3, already known as brain-specific, and MT1B which was not expressed in any tissue, mRNA levels of the other MT genes tended to be higher in ALS than in control liver samples, but the differences did not attain statistical significance. In the nervous system, the diverse MT genes were expressed over a greater range in ALS than in controls, but exhibited no change in a consistent direction. At the motor cortex, changes seemed to be less pronounced than at C6. MT3 was expressed in the motor cortex and the cord. The results provide no evidence for either the induction of a specific MT repertoire, or for the inability of glia to express any MT gene in ALS. Because the semi-quantitative RT-PCR technique does not permit detailed comparisons between the subtypes of MT expressed in the various tissues, the question whether a single inductor may be held responsible for the elevation of MT in the ALS liver and nervous system remains open. In conclusion, ALS tissue remains capable of expressing all the major MT genes. MT, present in protoplasmic glia, arises locally and is not secondary to increases of hepatic or renal MT. Because MT3 is also expressed by the normal and ALS spinal cord, it is a central nervous system-specific and not only a brain-specific protein. Thus, the excess of MT in ALS liver seems to be an effect of slower catabolism rather than faster synthesis of protein.

Enhanced superoxide dismutase-2 immunoreactivity of astrocytes and occasional neurons in amyotrophic lateral sclerosis.

The recent discovery of missense mutations in the superoxide dismutase (SOD)-1 gene as a cause of familial amyotrophic lateral sclerosis (ALS) and the ensuing description of transgenic SOD-1 mutant mouse models have focussed scientific interest on free radical scavenging mechanisms in all other familial (FALS) and sporadic (SALS) forms of the disease. We have compared the presence of intracellular cytosolic copper-zinc SOD-1 and mitochondrial manganese SOD-2 in the CNS from FALS and SALS patients and from non-neurological controls by immunohistochemical assessment, in the knowledge that no SOD-1 mutations have been found in any of 18 Dutch ALS pedigrees. ALS specimens from the motor cortex and the spinal cord presented enhanced SOD-2 immunoreactivity, especially of astrocytes and occasionally of neurons. Astrocyte staining appeared to be increased at the cerebral cortical and the spinal cervical and lumbar levels, but was only slightly increased in the thoracic anterior horns and not at all in the brain stem. This indicates that, by the time of death, the disease had burnt out in the brain stem and thoracic cord. Increased staining of neurons was limited to the small lateral and dorsal nuclei of the spinal cord. FALS and SALS cases exhibited the same staining patterns. SOD-1 immunoreactivity did not differ between disease and control specimens. SOD-1 and -2 staining was normal in the ALS cortical, brain stem and spinal motoneurons. This suggests that SALS and non-SOD-1 mutant FALS are not accompanied by loss of SOD-1 or -2 protein. An enzyme-linked immunosorbent assay revealed no differences in SOD-1 and SOD-2 levels between ALS patients and controls. Our major finding of locally increased SOD-2 immunoreactivity of astrocytes in FALS and SALS specimens, probably reflects reactive fibrillary and protoplasmatic gliosis in areas of ongoing degeneration but may also result from an attempt at compensation for free radical injury.

Percutaneous endoscopic gastrostomy in patients with amyotrophic lateral sclerosis and impaired pulmonary function.

Amyotrophic lateral sclerosis is a rapidly progressive disease of unknown etiology resulting in tetraparalysis, dysarthria, dysphagia, and ultimately death from respiratory insufficiency. In the course of the disease, recurrent episodes of aspiration, pneumonia, dehydration, and malnutrition may necessitate nasoenteral tube placement, an inconvenient and unattractive arrangement in patients with dribbling and impaired swallowing. A percutaneous endoscopic gastrostomy seemed a better, though potentially hazardous, alternative in view of the often severely restricted pulmonary function of these patients. Therefore, we prospectively investigated the use of percutaneous endoscopic gastrostomy in 68 consecutive patients with amyotrophic lateral sclerosis. Minimum required pulmonary function was defined as forced vital capacity (FVC) of 1 L or more and CO2 gas exchange capability as pCO2 of 45 mm Hg or less. The methodology of insertion was adapted to facilitate the early removal of gastric air. Fifty-five patients (median FVC, 1.7 L; pCO2, 40 mm Hg) were eligible for the gastrostomy procedure, and 13 patients (median FVC, 0.8 L; pCO2, 47 mm Hg) were not. Despite the fact that modification of the method of insertion rendered the procedure more difficult, the success rate was 89% (49/55); it was 96% (49/51) when failures related to distorted anatomy were excluded. The procedure-related mortality rate was 1.8% and the 24-hour in-hospital mortality rate was 3.6%, mainly related to respiratory insufficiency. The 30-day out-of-hospital mortality rate was 11.5%. Major complications (3.6%) consisted of a spontaneously draining cutaneous abscess in 2 cases. Peristomal redness was present in 6 cases, and 5 patients required analgesics for wound pain.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular basis of an adult form of Sandhoff disease: substitution of glutamine for arginine at position 505 of the beta-chain of beta-hexosaminidase results in a labile enzyme.

Sandhoff disease is a lysosomal storage disorder characterized by accumulation of GM2 ganglioside due to mutations in the beta-chain of beta-hexosaminidase. Hexosaminidase activity is negligible in infantile Sandhoff disease whereas residual activity is present in juvenile and adult forms. Here we report the molecular basis of the first described adult form of Sandhoff disease. Southern analysis of chromosomal DNA indicated the absence of chromosomal deletions in the gene encoding the beta-chain. Northern analysis of RNA from cultured fibroblasts demonstrated that at least one of the beta-chain alleles was transcribed into normal-length mRNA. Sequence analysis of the entire cDNA prepared from poly-adenylated RNA showed that only one point mutation was present, consisting of a G-->A transition at nucleotide position 1514. This mutation changes the electric charge at amino acid position 505 by substitution of glutamine for arginine in a highly conserved part of the beta-chain, present even in the slime mold Dictyostelium discoideum. The nucleotide transition generated a new restriction site for DdeI, which was present in only one of the alleles of the patient. Reverse transcription of mRNA followed by restriction with DdeI resulted in complete digestion at the mutation site, demonstrating that the second allele was of an mRNA-negative type. Transfection of COS cells with a cDNA construct containing the mutation but otherwise the normal sequence resulted in the expression of a labile form of beta-hexosaminidase. These results show that the patient's is a genetic compound, and that the lability of beta-hexosaminidase found in this form of Sandhoff disease is based on a single nucleotide transition.

Tissue fixation methods alter the immunohistochemical demonstrability of neurofilament proteins, synaptophysin, and glial fibrillary acidic protein in human cerebellum.

This study has examined the effect of postmortem autolysis, type, and duration of fixation on neurofilament, synaptophysin, and glial fibrillary acidic protein (GFAP) antigen decay as demonstrated by immunohistochemistry, using a streptavidin-biotin peroxidase method. The system used consisted of 5 normal cerebellar cortices. Time intervals, temperature, mode of fixation and storage, and staining technique were well controlled. Anti-neurofilament antibodies comprised SMI-31, MNF, and BF-10 against phosphorylated epitopes, and SMI-32 against a non-phosphorylated epitope. Bouin's and B5 fixative, and Sensofix gave best results, whereas formaldehyde and paraformaldehyde fixation gave much lower immunoreactivity. Phosphorylated neurofilament epitopes were less affected by aldehydes than unphosphorylated epitopes. GFAP staining was most consistent after Bouin fixation while the monoclonal antibody was much more sensitive to the fixative used than the polyclonal one. Aspecific background staining increased considerably after a postmortem interval of 24 hours. Synaptophysin immunoreactivity, as demonstrated by SY-38, proved very sensitive to prolonged fixation and was of poor quality following formaldehyde and paraformaldehyde fixation. Knowledge of antigen decay due to postmortem artifacts is essential for the correct evaluation of immunoperoxidase studies of autolyzed tissues that have been fixed and stored in different modes and for variable time interval.

Effects of midline medullary lesions on velocity storage and the vestibulo-ocular reflex.

1. Crossing fibers were sectioned at the midline of the medulla caudal to the abducens nucleus in four cynomolgus monkeys. In two animals the lesions caused the time constant of horizontal and vertical per- and post-rotatory nystagmus to fall to 5-8 s. The slow rise in optokinetic nystagmus (OKN), as well as optokinetic after-nystagmus (OKAN) and cross-coupling of horizontal to vertical OKN and OKAN were abolished. Steady state velocities could not be maintained during off-vertical axis rotation (OVAR). Pitch and yaw nystagmus were affected similarly. We conclude that the ability to store activity related to slow phase eye velocity, i.e., "velocity storage", was lost in these monkeys for nystagmus about any axis. Velocity storage was partially affected by a small midline lesion in the same region in a third animal. There was no effect of a more superficial midline section in a fourth monkey, and it served as a control. 2. The gain (eye velocity/head velocity) of the vestibulo-ocular reflex (VOR) was unaffected by the midline lesions. Saccades were normal, as was the ability to hold the eyes in eccentric gaze positions. The gain of the fast component of OKN increased in one monkey to compensate for the loss of the slow component. 3. One animal was tested for its ability to adapt the gain of the VOR due to visual-vestibular mismatch after lesion. Average changes in gain in response to wearing magnifying (2.2x) and reducing (0.5x) lenses, were +35% and -30%, respectively. This is within the range of normal monkeys. Thus, a midline lesion that abolished velocity storage did not alter that animal's ability to adapt the gain of the VOR. 4. Lesions that reduced or abolished velocity storage interrupted crossing fibers in the rostral medulla, caudal to the abducens nuclei. Cells that contributed axons to this portion of the crossing fibers are most likely located in central portions of the medial vestibular nucleus (MVN) and/or in rostral portion of the descending vestibular nucleus (DVN). The implication is that velocity storage arises from neurons in MVN and DVN whose axons cross the midline.

Immunohistochemical characterization of the inflammatory infiltrate in amyotrophic lateral sclerosis.

In order to test the hypothesis that the immune system plays a role in the pathogenesis of amyotrophic lateral sclerosis (ALS), the cellular composition of the spinal cord inflammatory infiltrate was analysed in eight cases of sporadic ALS by a panel of monoclonal antibodies. The majority of the many diffusely scattered lymphocytes seen in the anterior and lateral corticospinal tracts and anterior horns belonged to the suppressor/cytotoxicity T-cell subset and were admixed with variable numbers of macrophages. Helper-inducer T-cells were rare and B-cells were conspicuously absent. Compared to controls, ALS specimens exhibited an increase in major histocompatibility complex (MHC) products or human leucocyte antigens (HLA) in the corticospinal tracts and anterior horns. HLA-ABC antigens were expressed in the honeycomb pattern of the glial matrix of the spinal cord, and HLA-DR antigens were strongly expressed by large dendritic cells. In addition, macrophages and endothelial cells were labelled by HLA-DR. These findings suggest that an autoimmune process or infectious agent may play a role in ALS.

Compensation for labyrinthine defects examined by use of a tilting room.

Visual compensation for vestibular deficiency was demonstrated by stabilometry inside a laterally tilting room. Labyrinthless patients showed a predominance of vision over the somatosensory system for a rather long time: in contrast to the controls, the labyrinthless subjects experienced the tilting room often as stationary and the stationary stabilometer as tilting, which resulted in vertical postural instability.