Julian R. Thorpe

Clinical and neuropathologic variation in neuronal intermediate filament inclusion disease.

Background: Recently described neuronal intermediate filament inclusion disease (NIFID) shows considerable clinical heterogeneity. Objective: To assess the spectrum of the clinical and neuropathological features in 10 NIFID cases. Methods: Retrospective chart and comprehensive neuropathological review of these NIFID cases was conducted. Results: The mean age at onset was 40.8 (range 23 to 56) years, mean disease duration was 4.5 (range 2.7 to 13) years, and mean age at death was 45.3 (range 28 to 61) years. The most common presenting symptoms were behavioral and personality changes in 7 of 10 cases and, less often, memory loss, cognitive impairment, language deficits, and motor weakness. Extrapyramidal features were present in 8 of 10 patients. Language impairment, perseveration, executive dysfunction, hyperreflexia, and primitive reflexes were frequent signs, whereas a minority had buccofacial apraxia, supranuclear ophthalmoplegia, upper motor neuron disease (MND), and limb dystonia. Frontotemporal and caudate atrophy were common. Histologic changes were extensive in many cortical areas, deep gray matter, cerebellum, and spinal cord. The hallmark lesions of NIFID were unique neuronal IF inclusions detected most robustly by antibodies to neurofilament triplet proteins and α-internexin. Conclusion: NIFID is a neuropathologically distinct, clinically heterogeneous variant of frontotemporal dementia (FTD) that may include parkinsonism or MND. Neuronal IF inclusions are the neuropathological signatures of NIFID that distinguish it from all other FTD variants including FTD with MND and FTD tauopathies.

Nitric oxide donors decrease the function and survival of human pancreatic islets.

Nitric oxide (NO) has been proposed as a possible mediator of beta-cell damage in human IDDM. This hypothesis is based on in vitro studies with rodent pancreatic islets. In the present study we examined whether human beta-cells are affected by NO. In view of species differences in beta-cell sensitivity to damaging agents, rat islets were investigated in parallel. Isolated islets were exposed for 90 min to different concentrations of three chemically unrelated NO donors, SIN-1, GSNO or RBS. At the end of this incubation, human insulin release was mostly similar in control and NO-treated islets but, 48 h later, islet retrieval, islet DNA and insulin content, and glucose-induced insulin release were markedly lower in islets exposed to NO donors. Rat islets were already inhibited during the initial 90 min; 48 h later their loss in beta-cell function was similar to that in human islets. Nicotinamide or succinic acid monomethyl ester partially protected against SIN-1 induced islet cell loss, but not against the functional inhibition of human pancreatic islets. Exposure of human or rat islets to RBS was associated with significant DNA strand breakage, as judged by the comet assay (single cell gel electrophoresis) and by ultrastructural signs of cell damage. DNA damage was more severe in rat islet cells exposed to similar amounts of RBS. It is concluded that NO donors can damage human pancreatic islets, an effect paralleled by induction of nuclear DNA strand breaks.

A central role for dityrosine crosslinking of Amyloid-β in Alzheimer's disease.

BackgroundAlzheimer’s disease (AD) is characterized by the deposition of insoluble amyloid plaques in the neuropil composed of highly stable, self-assembled Amyloid-beta (Aβ) fibrils. Copper has been implicated to play a role in Alzheimer’s disease. Dimers of Aβ have been isolated from AD brain and have been shown to be neurotoxic.ResultsWe have investigated the formation of dityrosine cross-links in Aβ42 formed by covalent ortho-ortho coupling of two tyrosine residues under conditions of oxidative stress with elevated copper and shown that dityrosine can be formed in vitro in Aβ oligomers and fibrils and that these links further stabilize the fibrils. Dityrosine crosslinking was present in internalized Aβ in cell cultures treated with oligomeric Aβ42 using a specific antibody for dityrosine by immunogold labeling transmission electron microscopy. Results also revealed the prevalence of dityrosine crosslinks in amyloid plaques in brain tissue and in cerebrospinal fluid from AD patients.ConclusionsAβ dimers may be stabilized by dityrosine crosslinking. These results indicate that dityrosine cross-links may play an important role in the pathogenesis of Alzheimer’s disease and can be generated by reactive oxygen species catalyzed by Cu2+ ions. The observation of increased Aβ and dityrosine in CSF from AD patients suggests that this could be used as a potential biomarker of oxidative stress in AD.

Superoxide, nitric oxide, peroxynitrite and cytokine combinations all cause functional impairment and morphological changes in rat islets of Langerhans and insulin secreting cell lines, but dictate cell death by different mechanisms

We have shown that nitric oxide treatment for 30–90 min causes inhibition of insulin secretion, DNA damage and disturbs sub-cellular organization in rat and human islets of Langerhans and HIT-T15 cells. Here rat islets and beta-cell lines were treated with various free radical generating systems S-nitrosoglutathione (nitric oxide), xanthine oxidase plus hypoxanthine (reactive oxygen species), 3-morpholinosydnonimine (nitric oxide, super-oxide, peroxynitrite, hydrogen peroxide) and peroxynitrite and their effects over 4 h to 3 days compared with those of the cytokine combination interleukin-1β, tumour necrosis factor-α and interferon-γ. End points examined were de novo protein synthesis, cellular reducing capacity, morphological changes and apoptosis by acridine orange cytochemistry, DNA gel electrophoresis and electron microscopy. Treatment (24–72 h) with nitric oxide, superoxide, peroxynitrite or combined cytokines differentially decreased redox function and inhibited protein synthesis in rat islets of Langerhans and in insulin-containing cell lines; cytokine effects were arginine and nitric oxide dependent. Peroxynitrite gave rare apoptosis in HIT-T15 cells and superoxide gave none in any cell type, but caused the most beta cell-specific damage in islets. S-nitroso-glutathione was the most effective agent at causing DNA laddering or chromatin margination characteristic of apoptotic cell death in insulin-containing cells. Cytokine-induced apoptosis was observed specifically in islet beta cells, combined cytokine effects on islet function and death most resembled those of the mixed radical donor SIN-1.

A Preferentially Segregated Recycling Vesicle Pool of Limited Size Supports Neurotransmission in Native Central Synapses

Summary At small central synapses, efficient turnover of vesicles is crucial for stimulus-driven transmission, but how the structure of this recycling pool relates to its functional role remains unclear. Here we characterize the organizational principles of functional vesicles at native hippocampal synapses with nanoscale resolution using fluorescent dye labeling and electron microscopy. We show that the recycling pool broadly scales with the magnitude of the total vesicle pool, but its average size is small (∼45 vesicles), highly variable, and regulated by CDK5/calcineurin activity. Spatial analysis demonstrates that recycling vesicles are preferentially arranged near the active zone and this segregation is abolished by actin stabilization, slowing the rate of activity-driven exocytosis. Our approach reveals a similarly biased recycling pool distribution at synapses in visual cortex activated by sensory stimulation in vivo. We suggest that in small native central synapses, efficient release of a limited pool of vesicles relies on their favored spatial positioning within the terminal.

The effect of cytochalasin B on the rate of growth and ultrastructure of wheat coleoptiles and maize roots

Cytochalasin B (CB) inhibits the elongation growth of maize roots, and that of wheat coleoptile segments incubated in indolyl-3-acetic acid, by over 30% after a lag period of about 60 min. This long lag is not due to poor tissue penetration by the inhibitor, but seems to reflect a property of the process inhibited by CB. The only visible ultrastructural change accompanying growth inhibition is the accumulation of secretory vesicles in the vicinity of dictyosomes, which occurs between 90 and 300 min. However, a massive accumulation of vesicles is seen after 120 min in root cap cells which possess very active dictyosomes. The results indicate that CB does not inhibit elongation growth by interfering with cytoplasmic streaming. Instead, they indicate that the drug acts to inhibit the secretion of cell wall components at some stage after vesicle production, but prior to their transport.

A comprehensive assessment of the SOD1G93A low-copy transgenic mouse, which models human amyotrophic lateral sclerosis.

SUMMARY Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder that results in the death of motor neurons in the brain and spinal cord. The disorder generally strikes in mid-life, relentlessly leading to paralysis and death, typically 3–5 years after diagnosis. No effective treatments are available. Up to 10% of ALS is familial, usually autosomal dominant. Several causative genes are known and, of these, mutant superoxide dismutase 1 (SOD1) is by far the most frequently found, accounting for up to 20% of familial ALS. A range of human mutant SOD1 transgenic mouse strains has been produced, and these largely successfully model the human disease. Of these, the most widely used is the SOD1 mouse, which expresses a human SOD1 transgene with a causative G93A mutation. This mouse model is excellent for many purposes but carries up to 25 copies of the transgene and produces a great excess of SOD1 protein, which might affect our interpretation of disease processes. A variant of this strain carries a deletion of the transgene array such that the copy number is dropped to eight to ten mutant SOD1 genes. This ‘deleted’ ‘low-copy’ mouse undergoes a slower course of disease, over many months. Here we have carried out a comprehensive analysis of phenotype, including nerve and muscle physiology and histology, to add to our knowledge of this ‘deleted’ strain and give baseline data for future studies. We find differences in phenotype that arise from genetic background and sex, and we quantify the loss of nerve and muscle function over time. The slowly progressive pathology observed in this mouse strain could provide us with a more appropriate model for studying early-stage pathological processes in ALS and aid the development of therapies for early-stage treatments.

Utilizing the Peptidyl-Prolyl Cis-Trans Isomerase Pin1 as a Probe of Its Phosphorylated Target Proteins: Examples of Binding to Nuclear Proteins in a Human Kidney Cell Line and to Tau in Alzheimer's Diseased Brain

The human parvulin Pin1 is a member of the peptidyl–prolyl cis–trans isomerase group of proteins, which modulate the assembly, folding, activity, and transport of essential cellular proteins. Pin1 is a mitotic regulator interacting with a range of proteins that are phosphorylated before cell division. In addition, an involvement of Pin1 in the tau-related neurodegenerative brain disorders has recently been shown. In this context, Pin1 becomes depleted from the nucleus in Alzheimers disease (AD) neurons when it is redirected to the large amounts of hyperphosphorylated tau associated with the neurofibrillary tangles. This depletion from the nucleus may ultimately contribute to neuron cell death. Recently we have devised a novel methodology in which exogenous Pin1 is used as a TEM probe for its target proteins. Here we extend this methodology to provide further evidence that Pin1 binds at enhanced levels to mitotic nuclear proteins and to hyperphosphorylated tau in AD brain. We suggest that exogenous Pin1 labeling can be used to elucidate the phosphorylation status of its target proteins in general and could specifically provide important insights into the development of tau-related neurodegenerative brain disorders.

Optimization and investigation of the use of 2,2-dimethoxypropane as a dehydration agent for plant tissues in transmission electron microscopy.

After obtaining initially inconsistent results using 2,2-dimethoxypropane (DMP) to dehydrate pea root and stem segments for transmission electron microscopy, it was found that consistent total dehydration of 3-mm pea root and 1- and 3-mm pea stem lengths was only obtained after 2 15 min exposure to DMP, i.e., much longer than originally recommended (L. L. Muller and T. J. Jacks, 1975, J. Histochem. Cytochem., 23, 107). Satisfactory dehydration of a variety of plant tissues was achieved after 2 15 min dehydration in DMP, and no differences in cell ultrastructure were observed between ethanol and DMP-dehydrated samples following three different fixation procedures (glutaraldehyde/OsO4, acrolein/OsO4, and KMnO4). The value of DMP in the retention of cell contents during dehydration was assessed in procedures for the localization of (1) rat liver peroxisomal soluble catalase using 3,3-diaminobenzidine and (2) Na+ K+ and Cl by precipitation techniques in Suaeda maritima leaf segments.

Immunoreactivity to Lys63-linked polyubiquitin is a feature of neurodegeneration

The major human neurodegenerative diseases are characterised by ubiquitin-positive intraneuronal inclusions, however the precise nature of the ubiquitin modifications in these structures is unclear. Using a monoclonal antibody specific for Lys63-linked polyubiquitin we have performed the first immunohistochemical analysis of linkage-specific ubiquitination in vivo associated with neurodegeneration. Immunoreactivity was detected within the pathological lesions of Alzheimers, Huntingtons and Parkinsons disease brains, although staining of Lewy bodies in the substantia nigra in Parkinsons disease was rare, indicating a selective involvement of Lys63-linked polyubiquitin in inclusion biogenesis in this disorder. Immunoreactivity was also a feature in neurons of proteasome-depleted mice, suggesting a proteasomal contribution to the degradation of Lys63-linked polyubiquitinated proteins in vivo.