Corticobasal and ataxia syndromes widen the spectrum of C9ORF72 hexanucleotide expansion disease.

Recently, a hexanucleotide (GGGGCC) repeat expansion in the first intron of C9ORF72 was reported as the cause of chromosome 9p21-linked frontotemporal dementia-amyotrophic lateral sclerosis (FTD-ALS). We here report the prevalence of the expansion in a hospital-based cohort and associated clinical features indicating a wider clinical spectrum of C9ORF72 disease than previously described. We studied 280 patients previously screened for mutations in genes involved in early onset autosomal dominant inherited dementia disorders. A repeat-primed polymerase chain reaction amplification assay was used to identify pathogenic GGGGCC expansions. As a potential modifier, confirmed cases were further investigated for abnormal CAG expansions in ATXN2. A pathogenic GGGGCC expansion was identified in a total of 14 probands. Three of these presented with atypical clinical features and were previously diagnosed with clinical olivopontocerebellar degeneration (OPCD), atypical Parkinsonian syndrome (APS) and a corticobasal syndrome (CBS). Further, the pathogenic expansion was identified in six FTD patients, four patients with FTD-ALS and one ALS patient. All confirmed cases had normal ATXN2 repeat sizes. Our study widens the clinical spectrum of C9ORF72 related disease and confirms the hexanucleotide expansion as a prevalent cause of FTD-ALS disorders. There was no indication of a modifying effect of the ATXN2 gene.

Molecular chaperons, amyloid and preamyloid lesions in the BRI2 gene-related dementias: a morphological study.

Molecular chaperons or amyloid-associated proteins (AAPs) are deposited in vascular and parenchymal amyloid lesions in Alzheimer's disease (AD) and other amyloidoses. AAPs, such as apolipoprotein E (ApoE) or apolipoprotein J (ApoJ) have been strongly implicated in the pathogenesis of AD in vitro and in vivo. Furthermore the possession of the ApoE in4 allele is a well-studied risk factor for AD. In view of the similarities between AD and both familial British dementia (FBD) and familial Danish dementia (FDD), we investigated the presence of AAPs in these two diseases to understand better their role in the general process of amyloidogenesis. Immunohistochemistry for ApoE, ApoJ, serum amyloid P (SAP), alpha-1-antichymotrypsin, cystatin C, heparan sulphate proteoglycans, such as agrin, perlecan, syndecans, glypican-1 and for heparan sulphate glycosaminoglycan (HS GAG) side chains was carried out together with immunohistochemical preparations specific to the amyloid subunits. Significant or extensive staining for ApoE, ApoJ, agrin, glypican-1 and HS GAG side chains was found in both amyloid (fibrillar) and preamyloid (nonfibrillar) deposits in FBD and FDD. The remaining AAPs, including SAP, were predominantly found in amyloid lesions. Only very weak staining was present in a small proportion of the amyloid lesions using perlecan immunohistochemistry. These findings suggest that the deposition patterns of AAPs in FBD and FDD are mostly similar to those in AD. The presence of AAPs in the preamyloid lesions supports the notion that chaperon molecules may play a role in the early steps of fibrillogenesis.

Genetic alterations of the BRI2 gene: familial British and Danish dementias.

Classic arguments sustaining the importance of amyloid in the pathogenesis of dementia are usually centered on amyloid beta (Abeta) and its role in neuronal loss characteristic of Alzheimer disease, the most common form of human cerebral amyloidosis. Two non-Abeta cerebral amyloidoses, familial British and Danish dementias, share many aspects of Alzheimer disease, including the presence of neurofibrillary tangles, parenchymal pre-amyloid and amyloid deposits, cerebral amyloid angiopathy, and a widespread inflammatory response. Both early-onset conditions are linked to specific mutations in the BRI2 gene, causing the generation of longer-than-normal protein products and the release of 2 de novo created peptides ABri and ADan, the main components of amyloid fibrils in these inherited dementias. Although the molecular mechanisms and signal transduction pathways elicited by the amyloid deposits and their relation to cognitive impairment remain to be clarified, new evidence indicates that, independent of the differences in their primary structures, Abeta, ABri, and ADan subunits are able to form morphologically compatible ion-channel-like structures and elicit single ion-channel currents in reconstituted lipid membranes. These findings reaffirm the notion that non-Abeta amyloidosis constitute suitable alternative models to study the role of amyloid deposition in the mechanism of neuronal cell death.

Chronic lithium treatment with or without haloperidol fails to affect the morphology of the rat cerebellum.

We used unbiased stereological principles to determine whether long-term administration of lithium at human therapeutic levels, with or without haloperidol, affects the number or sizes of cerebellar Purkinje cells or the volume of histological layers in the rat cerebellum. Twenty-eight rats were randomly divided into three groups, receiving either no treatment, lithium, or lithium combined with haloperidol. The serum lithium levels ranged from 0.50 to 0.77 mmol/l. Haloperidol was given at a daily dose of 1 mg/kg. After 30 weeks of treatment, the animals were killed and the cerebelli were histologically prepared. No statistically significant differences were observed between the groups with respect to the cerebellar measures.

Chromosome 13 dementia syndromes as models of neurodegeneration.

Two hereditary conditions, familial British dementia (FBD) and familial Danish dementia (FDD), are associated with amyloid deposition in the central nervous system and neurodegeneration. The two amyloid proteins, ABri and ADan, are degradation products of the same precursor molecule BriPP bearing different genetic defects, namely a Stop-to-Arg mutation in FBD and a ten-nucleotide duplication-insertion immediately before the stop codon in FDD. Both de novo created amyloid peptides have the same length (34 amino acids) and the same post-translational modification (pyroglutamate) at their N-terminus. Neurofibrillary tangles containing the classical paired helical filaments as well as neuritic components in many instances co-localize with the amyloid deposits. In both disorders, the pattern of hyperphosphorylated tau immunoreactivity is almost indistinguishable from that seen in Alzheimer's disease. These issues argue for the primary importance of the amyloid deposits in the mechanism(s) of neuronal cell loss. We propose FBD and FDD, the chromosome 13 dementia syndromes, as models to study the molecular basis of neurofibrillary degeneration, cell death and amyloid formation in the brain.

A decamer duplication in the 3' region of the BRI gene originates an amyloid peptide that is associated with dementia in a Danish kindred.

Familial Danish dementia (FDD), also known as heredopathia ophthalmo-oto-encephalica, is an autosomal dominant disorder characterized by cataracts, deafness, progressive ataxia, and dementia. Neuropathological findings include severe widespread cerebral amyloid angiopathy, hippocampal plaques, and neurofibrillary tangles, similar to Alzheimer's disease. N-terminal sequence analysis of isolated leptomeningeal amyloid fibrils revealed homology to ABri, the peptide originated by a point mutation at the stop codon of gene BRI in familial British dementia. Molecular genetic analysis of the BRI gene in the Danish kindred showed a different defect, namely the presence of a 10-nt duplication (795-796insTTTAATTTGT) between codons 265 and 266, one codon before the normal stop codon 267. The decamer duplication mutation produces a frame-shift in the BRI sequence generating a larger-than-normal precursor protein, of which the amyloid subunit (designated ADan) comprises the last 34 C-terminal amino acids. This de novo-created amyloidogenic peptide, associated with a genetic defect in the Danish kindred, stresses the importance of amyloid formation as a causative factor in neurodegeneration and dementia.

Selective degeneration of dorsal root ganglia and dorsal nerve roots in methyl mercury-intoxicated rats: a stereological study.

The components of the nervous system of rats that are most critically affected by methyl mercury are still a matter of debate. A recent stereological study of rats with typical symptoms resulting from methyl mercury intoxication demonstrated that the morphology of cerebellar granule cells and Purkinje cells were unchanged at the light microscopic level, even though there was pronounced degeneration of myelinated axons in dorsal nerve root nerves. In the present study, unbiased stereological methods were used to quantify morphological changes in the dorsal root ganglion, and dorsal and ventral nerve roots of the rats used in the previous study. The rats were treated with methyl mercury (2 mg daily/kg, per os) for a 19-day period that was followed by a 32-day period without treatment. The means of the total numbers of A-cell and B-cell perikarya in the dorsal root ganglion of the intoxicated rats were reduced by 60% and 24%, respectively. The mean volume of A-cell perikarya in rats of the experimental group was reduced by 22%, whereas the mean volume of B-cell perikarya was the same in the two groups. In the experimental group, the total number of myelinated axons in the dorsal nerve roots was reduced by 60%, whereas no difference was found in the ventral nerve roots. The areas of axon and myelin sheath, dorsal and ventral nerve roots were not affected. This study demonstrates that extensive loss of dorsal root ganglion cells and myelinated axons in dorsal nerve roots precedes light microscopical changes in the ventral nerve roots and the cerebellum of rats intoxicated with methyl mercury.

The effect of chronic lithium treatment on the calibre of axons and nerve fibres in the rat sural nerve.

Cases of peripheral neuropathy have been reported in humans receiving lithium therapy. However, no previous studies have addressed the question of whether chronic lithium treatment causes morphological changes in the peripheral nervous system in experimental animals. The present study used stereological methods to determine whether long-term administration of lithium affected the calibre of the axons or of the nerve fibres in the rat sural nerve. Twenty-two rats were divided into 2 groups and given either no treatment or lithium, with serum levels averaging from 0.5 to 0.7 mmol/l. After 30 weeks of treatment, the animals were killed and the sural nerve was isolated at the level of the knee and was removed. The cross-sectional area of axons and of nerve fibres was estimated by point counting. Compared with the controls, a strong tendency towards a reduced nerve fibre area in the lithium-treated animals was found, with a between-group difference of 1.79 microns 2 (P = 0.06). For the axon area, the difference was 0.73 micron 2 (P = 0.20).

Redistribution of axoplasm in the motor root in experimental diabetes.

Early experimental diabetes is associated with a reduction in axonal caliber. To elucidate the changes of nerve caliber further, we investigated the proximal and distal regions of the anterior root of rats after 5 weeks of streptozocin-induced diabetes. After vascular perfusion fixation, the fifth lumbar motor root was excised and two 3-mm segments were isolated, one at the level of the spinal cord and one at the dorsal root ganglion. The areas of myelinated fibers and their axons were measured by point counting. Axons from diabetic mice were enlarged proximally as compared to the controls, and reduced distally. It has been hypothesized that the reduction in axonal caliber is caused by an impairment of axonal transport of structural proteins rather than by osmotic shrinkage. Our findings indicate a redistribution of axoplasm in the nerves of the diabetic mice and support the hypothesis that changes in the axonal transport of neurofilaments are responsible for the structural changes seen in early diabetes. Similar changes could also play a role in the development of neuropathy in man.

Structural preservation of cerebellar granule cells following neurointoxication with methyl mercury: a stereological study of the rat cerebellum.

Methyl mercury intoxication causes ataxia. Structural changes of cerebellar and peripheral nerve tissues have been described. However, it is still unclear whether the ataxia is of cerebellar or peripheral origin. To clarify this question further, the effects of methyl mercury intoxication on the numbers of granule and Purkinje cells and the volume of Purkinje cell perikarya have been evaluated with stereological methods. Rats were intoxicated with methyl mercury, at a dose of 2 mg/kg per day for 19 successive days, and the analysis was carried out 2.5 or 4.5 weeks later. The total numbers of cerebellar granule cells and Purkinje cells were estimated using an optical fractionator and the mean volume of the Purkinje cells was estimated by the vertical rotator technique. The volumes of the granular cell layer, the molecular layer and the white matter were estimated using the Cavalieri principle. The intoxicated animals developed hindlimb incoordination when held by the tail. Although pronounced axonal degeneration occurred in the peripheral nervous system, no changes were found in cerebellar cell numbers or cell sizes in either of the test groups. The absence of detectable light microscopic changes in the cerebellum indicates that the peripheral nervous system is affected prior to the cerebellum in rats intoxicated with organic mercury.

Effect of chronic lithium treatment with or without haloperidol on number and sizes of neurons in rat neocortex.

The present study used stereological methods to determine whether long-term administration of lithium, with or without haloperidol, affects the number and average volume of neocortical neurons. Twenty-five rats were divided into three groups and given no treatment, lithium, or lithium combined with haloperidol. Serum lithium levels ranged from 0.5 to 0.8 mmol/l. Haloperidol was injected intraperitoneally at a daily dose of 1 mg/kg. After 30 weeks of treatment, the animals were killed and the brains were prepared. Neocortical volume, density of neurons, total number of neurons and mean volume of neurons were estimated. As no differences were found between the groups, the present study provides no evidence for quantitative morphological changes in the cerebral cortex due to long-term 'therapeutic' levels of lithium, with or without haloperidol.

Number and volume of rat dorsal root ganglion cells in acrylamide intoxication.

Acrylamide intoxication induces a filamentous neuropathy with breakdown of distal axons and chromatolytic reaction of dorsal root ganglion cells. To obtain quantitative information about the perikaryal alterations neurons of the fifth lumbar dorsal root ganglion of rats were examined with stereological techniques following intoxication with a total dose of 500 mg acrylamide. Number, mean volume and distribution of neuron volume were estimated for each of the two cell subpopulations using optical disectors, the four-way-nucleator and systematic sampling techniques. In intoxicated rats perikaryal volume of A-cells was significantly reduced by 28%, from 63,200 micron3 (CV = 0.16) to 45,500 micron3 (CV = 0.19), whereas the volume of B-cells was unchanged. Numbers of A- and B-cells were preserved. The finding of a selective atrophy of A-cell perikaryal volume is in accordance with previous observations of predominant alterations of large myelinated sensory fibres and most likely reflects an attack on the perikaryal neurofilaments abundant in this cell type.

The volume of Purkinje cells decreases in the cerebellum of acrylamide-intoxicated rats, but no cells are lost.

The effects of acrylamide intoxication on the numbers of granule and Purkinje cells and the volume of Purkinje cell perikarya have been evaluated with stereological methods. The analysis was carried out in the cerebella of rats that had received a dose of 33.3 mg/kg acrylamide, twice a week, for 7.5 weeks. The total numbers of cerebellar granule and Purkinje cells were estimated using the optical fractionator and the mean volume of the Purkinje cell perikarya was estimated with the vertical rotator technique. The volumes of the molecular layer, the granular cell layer and the white matter were estimated using the Cavalieri principle. The mean weight of the cerebellum of the intoxicated rats was 7% lower than that of the control rats (2P = 0.001). The numbers of the Purkinje cells and granule cells were the same in both groups, but the mean volume of the perikarya of the Purkinje cells in the intoxicated rats was 10.5% less than that of the control group (2P = 0.004). The volume of the granular cell layer was reduced by 15% (2P = 0.006) but there were no differences in the volumes of the molecular layer and the white matter in the intoxicated and control animals.

The number and mean volume of neurons in the cerebral cortex of rats intoxicated with acrylamide.

Acrylamide was given in an accumulated dose of 500 mg/kg to rats by intraperitoneal injection in two different dosing schedules: 50 mg/kg twice a week for 5 weeks and 33.3 mg/kg twice a week for 7.5 weeks. The effect of acrylamide intoxication on the neurons in the cerebral cortex of the rat was studied using unbiased stereological methods. A reduction of brain weight of 8% was seen in both the intoxicated groups. The volume of neocortex was significantly decreased in the experimental groups, but the density of neurons was increased resulting in an unchanged total number of neurons. The mean volume of neurons in neocortex was significantly decreased in both acrylamide intoxicated groups. There was no difference between the two different intoxication schedules. The possibility that acrylamide causes neuronal death and the effect of eventual differential cellular sensitivity is discussed.

No effect of growth hormone treatment on axonal transport of slow component a in diabetic and nondiabetic rats.

A decreased axonal transport of slow component a (SCa), i.e., neurofilaments, is an early event in experimental diabetes as well as hypothyroidism, and common to these metabolic derangements are decreased levels of serum insulin-like growth factor I (IGF-I). To evaluate the possible connection between these facts, we investigated the effect of growth hormone (GH), which stimulates IGF-I production, on axonal transport of SCa in diabetic and nondiabetic rats. Serum concentrations of IGF-I fell from about 1500 micrograms/L in controls to about 600 micrograms/L in diabetics. GH treatment (100 mu/rat twice daily) normalized IGF-I for the first week of diabetes, after which the level decreased to the level of the untreated diabetics. The SCa transport velocity was found to be decreased in the diabetic nerves as previously reported [0.91 +/- 0.07 = mm/day, n = 9; (mean +/- SD) versus 1.01 +/- 0.09 mm/day, n = 8, in controls, 2 p less than 0.05). No changes were seen for the GH-treated groups (1.03 +/- 0.06 mm/day, (n = 11) in GH-treated controls). The lack of effect of GH treatment can be due to blockage of IGF-I synthesis or the decreased level of thyroid hormone, triiodothyronine (T3), in the diabetic rats.

Stereological estimates of nuclear volume and other quantitative variables in supratentorial brain tumors. Practical technique and use in prognostic evaluation.

The use of morphometry and modern stereology in malignancy grading of brain tumors is only poorly investigated. The aim of this study was to present these quantitative methods. A retrospective feasibility study of 46 patients with supratentorial brain tumors was carried out to demonstrate the practical technique. The continuous variables were correlated with the subjective, qualitative WHO classification of brain tumors, and the prognostic value of the parameters was assessed. Well differentiated astrocytomas (n = 14) had smaller estimates of the volume-weighted mean nuclear volume and mean nuclear profile area, than those of anaplastic astrocytomas (n = 13) (2p = 3.1.10(-3) and 2p = 4.8.10(-3), respectively). No differences were seen between the latter type of tumor and glioblastomas (n = 19). The nuclear index was of the same magnitude in all three tumor types, whereas the mitotic index was significantly increased in glioblastomas (2p = 0.01). Three-dimensional, shape-independent estimates of macroscopical tumor volume were not different in anaplastic astrocytomas and glioblastomas (2p = 0.39). Histological type of tumor and mitotic index were of significant prognostic value (2p = 8.2.10(-6) and 2p approximately 0.05, respectively). Age above the median and short duration of symptoms were significantly associated with short survival (2p = 0.01). Further investigations of larger series of patients are needed to define the clinical usefulness of these objective, reproducible, and quantitative techniques in the prognostic evaluation of primary brain tumors.

The total number of neurons in the human neocortex unbiasedly estimated using optical disectors.

An efficient method is presented for obtaining, in under 4 h, an unbiased estimate of the total number of neurons in the human neocortex, with a coefficient of error on the estimate of approximately 5%. The novel sampling scheme used in this study is unbiased and was designed so that only a small amount of neocortical grey matter had to be removed. Hence, the majority of the cerebral grey matter and all the internal grey matter was left intact for further resampling and analysis. Each cerebral hemisphere was subdivided into the four major neocortical regions, sliced coronally at 7-mm intervals and the volume of the neocortex determined using Cavalieri's principle. Uniform sampling of neocortex was performed in the hemisphere followed by regional subsampling with a varying sampling fraction being taken from each region. Neuronal density estimates were made in thick plastic sections using optical disectors. Shrinkage estimates were made in parallel with the number estimates and found to be negligible. The total number of neocortical neurons in the right hemisphere of five normal 80-year-old men was found to be 13.7 x 10(9) with an inter-individual coefficient of variation of 12%.

Increased thermal stability of collagen after 2,5-hexanedione intoxication in rats.

The present study examined the ability of 2,5-hexanedione (2,5-HD) to cross-link the fibrillary protein, collagen, in vivo. The thermal stability of collagen measured as the isometric contraction-relaxation force was studied in tail tendons from rats, receiving 2,5-HD intraperitoneally for 2 or 6 weeks (1 g/kg/week, divided into 3 doses). The maximum contraction force was significantly increased after 6 weeks of intoxication as compared to controls, indicating an increase in covalent cross-linking between collagen molecules. Likewise, in vitro incubation of rat tail tendons in 2,5-HD for 24 hr induced an increase in thermal isometric contraction force.

The effect of 2,5-hexanedione on newly formed collagen in granulation tissue.

The mechanical strength of newly formed collagen in granulation tissue was investigated by implantation of cellulose sponges into rats intoxicated with 2,5-hexanedione (2,5-HD) and control rats. Intoxication with 2,5-HD caused a significant decrease in mechanical strength after 20 days of implantation. No differences were found between 2,5-HD intoxicated and control groups in the amount of collagen formed in the sponges. The mechanical strength of granulation tissue depends on formation and cross-linking of the newly formed collagen fibrils. 2,5-HD is known to react with epsilon-amino groups of lysine and it is therefore able to interfere with both fibril aggregation and cross-linking of the collagen. On the other hand, 2,5-HD induces the formation of cross-links in intact tendon collagen. Therefore, we stress that it is essential to distinguish between intact and newly formed fibrils when evaluating the effect of 2,5-HD on fibillary proteins.

Diabetes decreases Na+-K+ pump concentration in skeletal muscles, heart ventricular muscle, and peripheral nerves of rat.

Na+-K+-ATPase or the Na+-K+ pump is essential for some specific properties of muscle and nerve tissue such as contractility and excitability. Previous studies have shown conflicting variations in Na+-K+-ATPase activity or Na+-K+ pump concentration of muscle cells in experimental diabetes. Our study demonstrates that early untreated diabetes in rats induced by injection of streptozocin is associated with decreases in [3H]ouabain binding-site concentration of 24-48% in various skeletal muscles and 16% in peripheral nerves as well as a decrease in K+-dependent 3-O-methylfluorescein phosphatase activity of 21% in the heart ventricle. These effects could be prevented by insulin treatment. They probably represent a decrease in the concentration of Na+-K+ pumps. There was no evidence for more than one population of Na+-K+ pumps in intact samples of skeletal muscle and nerves from normal, diabetic, and insulin-treated animals. The decrease in Na+-K+ pump concentration in nerve cells may be due to atrophy of the axons. In skeletal muscles, myocardium, and peripheral nerves, the observed decrease in Na+-K+ pump concentration may be important for the pathophysiology of diabetes. We emphasize that quantification of Na+-K+-ATPase or the Na+-K+ pump in muscle and nerve tissue from diabetic animals should preferably be performed with either intact samples or crude homogenates of whole tissue.