Flow velocity and pulsatility of the ocular circulation in chronic intracranial hypertension.

OBJECTIVES: The aim of this study is to examine the relationship of ocular blood flow velocities and pulsatility to intracranial pressure (ICP). METHODS: We measured arterial and venous flow velocities using color Doppler imaging (CDI) and calculated resistance indices for the central retinal (CRA) and the ophthalmic (OA) arteries from 16 patients (32 eyes) with chronic intracranial hypertension (ICH) and varying degrees of ICP and papilledema. The results were compared with normal data from 16 age-matched, healthy subjects. RESULTS: Arterial flow velocities were significantly decreased for the aggregate subject group compared with controls. A corresponding rise in arterial resistance with increasing ICP in the mild-moderate range was noted. Unexpectedly, with more severe elevations of ICP these trends reversed. CONCLUSIONS: In mild-moderate increased cerebrospinal fluid (CSF) pressure, a reduction of flow velocities may result because of increased vascular resistance. Paradoxically, in more severe chronic ICH, we hypothesize that local autoregulatory vascular changes and/or diversion of cerebral blood flow into the ophthalmic circulation may normalize these parameters. This phenomenon may partially underlie the relative sparing of visual function early in the course of pseudotumor cerebri (PTC), regardless of actual ICP levels.

Beta-amyloid peptide expression is sufficient for myotube death: implications for human inclusion body myopathy.

Inclusion body myositis (sIBM) is the most common disorder of skeletal muscle in aged humans. It shares biochemical features with Alzheimer's disease, including congophilic deposits, which are immunoreactive for beta-amyloid peptide (Abeta) and C'-terminal betaAPP epitopes. However, the etiology of myofiber loss and the role of intracellular Abeta in IBM is unknown. Here we report correlative evidence for apoptotic cell death in myofibers of IBM patients that exhibit pronounced Abeta deposition. HSV-1-mediated gene transfer of Abeta(42) into cultured C2C12 myotubes resulted in a 12.6-fold increase in dUTP-labeled and condensed nuclei over nonexpressing myotubes (P < 0.05). The C'-terminal betaAPP domain C99 also induced myotube apoptosis, but to a significantly lesser extent than Abeta. Apoptosis specific to Abeta-expressing myotubes was also demonstrated through DNA fragmentation, decreased mitochondrial function and the loss of membrane phospholipid polarity. Myotubes laden with Abeta(42), but not other transgene products, developed cytoplasmic inclusions consisting of fibrillar material. Furthermore, injection of normal mouse gastrocnemius muscle with HSV-encoding Abeta cDNA resulted in TUNEL-positive myofibers with pyknotic nuclei. We conclude that Abeta is sufficient to induce apoptosis in myofibers both in vivo and in vitro and suggest it may contribute to myofiber loss and muscle dysfunction in patients with IBM.

Acetylcholinesterase inhibition in dementia with Lewy bodies: results of a prospective pilot trial.

OBJECTIVE: Dementia with Lewy bodies (DLB) is the second commonest form of dementia. The response to acetylcholinesterase inhibition (AChEI) could be greater in DLB than in Alzheimer's disease (AD) because cholineacetyl-transferase levels are more reduced in the former. This preliminary trial seeks to compare performances in cognitive tasks before and after tacrine administration in DLB and AD subjects. METHODS: Six DLB and 6 AD patients were enrolled in an open, nonrandomized, intervention trial using 80 mg/day tacrine. Patients met ADRDA or DLB consortium criteria for probable diseases. Subjects were matched for Mini Mental State Examination (MMSE) score, age and sex. Mattis Dementia Rating Scale (DRS), Controlled Oral Word Association Test (FAS) and Boston Naming tests were administered at baseline and at 6 months into treatment. RESULTS: AD and DLB groups did not differ in initial mean total DRS scores. In the primary analysis, both groups declined during the course of treatment (-7.3 +/- 4.2 and -16.8 +/- 39.2 DRS points, respectively). Due to the large variability in DLB posttreatment scores, this group was divided post hoc into responders (DLBr) and nonresponders (DLBnr). The DLBr group outperformed the DLBnr group at baseline (p < 0.05) and, notably, in follow-up DRS test scores (p < 0.001). Two-way MANOVA comparing both DLB subgroups with either the entire AD cohort or similarly stratified AD subgroups showed a significant interaction (F = 7.6; p < 0.015), attributed mostly to declines in DLBnr group scores (p < 0.01). Surprisingly, on DRS memory subscale and FAS tests, there were significant improvements in DLBr scores (p < 0.02). A baseline MMSE (or DRS memory) score >/=15 predicted a positive response to tacrine in DLB. Acceleration of parkinsonism occurred in all DLB subjects. CONCLUSION: Results from a primary analysis of the therapeutic effect of 80 mg/day tacrine in DLB and AD were negative. However, post hoc analysis showed that mild to moderate DLB responds favorably to AChEI relative to AD through stabilization of global cognitive decline and improvements in specific cognitive areas. These results could be useful in the planning of a more definitive study.

Enhancer function and novel DNA binding protein activity in the near upstream betaAPP gene promoter.

The role of betaAPP gene transcription and promoter regulation in modifying amyloid beta-peptide (Abeta) levels is not well understood. Increased production of Abeta or changes in Abeta42/Abeta40 ratio by fibroblasts occurs in the presence of mutant presenilin or betaAPP alleles in familial Alzheimer's disease subjects. Both betaAPP mRNA and Abeta levels are increased in trisomy 21. The APP gene promoter is in a class of housekeeping genes and contains two putative consensus sites for the binding of transcription factor AP1. Electrophoretic mobility shift (EMSA) and DNase protection assays using human fibroblast and HeLa nuclear extract identified specific protein binding with novel Sp1-like properties to both a near-upstream and a downstream domain of the betaAPP promoter. The upstream binding activity was localized to a putative AP1 consensus site and its immediate 5'-adjacent GC-rich element. However, c-Jun antibody and competition experiments had no effect on binding to this domain. A series of 5'-deleted betaAPP promoter-reporter gene transfections in HeLa and fibroblast cells showed that the domain-containing region, n.t. -383 to -348, exerts a 2.9-fold activating influence on basal pbetaAPP-reporter transcription. When subcloned to test enhancer function, the 5'-GC element/'AP1 site' tandem construct conferred four-fold greater activity than either element alone and two-fold greater than the more 3'-situated HSE consensus sequence. Phorbol ester treatment had no effect in these reporter assays. This element shares homology and binding properties with a domain immediately 5' to the downstream E-box/USF element. An interaction model involving both domains and looping of interjacent DNA is proposed. We conclude that this newly described binding protein-enhancer complex is required for full betaAPP promoter activation.

Expression of ryanodine receptors in human embryonic kidney (HEK293) cells.

It has been shown previously that mobilization of caffeine-sensitive intracellular calcium (Ca2+i) stores increased the release of amyloid beta-peptide (Abeta) from transfected human embryonic kidney cells (HEK293) [Querfurth, Jiang, Geiger and Selkoe (1997) J. Neurochem. 69, 1580-1591]. The present study was to test the hypothesis that the caffeine/Abeta responses were due to interactions with specific subtypes of ryanodine receptors (RyR) using [3H]ryanodine receptor binding, epifluorescence imaging of Ca2+i, immunocytofluorescence, immunoprecipitation and PCR techniques. [3H]Ryanodine bound to a single class of high-affinity caffeine-sensitive sites (Kd=9.9+/-1.6 nM, Bmax=25+/-4 fmol/mg of protein). RyRs were immuno-decorated in a punctate reticulo-linear pattern. Results from SDS/PAGE and reverse transcriptase-PCR demonstrated endogenous expression of type 1 (skeletal) and type 2 (cardiac) RyRs. HEK293 cell RyRs were functionally active, because (i) [Ca2+]i increased 2.8-fold over baseline following applications of 5-15 mM caffeine, (ii) repetitive spiked increases in [Ca2+]i were observed, and (iii) evidence for a use-dependent block was obtained. Some of these findings were extended to include HeLa and human fibroblast cell lines, suggesting a broader applicability to cells of epithelioid lineage. Implications for the processing of the beta-amyloid precursor protein in Alzheimer's disease and for calcium channel research using transfected HEK293 cells are discussed.

Caffeine stimulates amyloid beta-peptide release from beta-amyloid precursor protein-transfected HEK293 cells.

Extracellular amyloid beta-peptide (A beta) deposition is a pathological feature of Alzheimer's disease and the aging brain. Intracellular A beta accumulation is observed in the human muscle disease, inclusion body myositis. A beta has been reported to be toxic to neurons through disruption of normal calcium homeostasis. The pathogenic role of A beta in inclusion body myositis is not as clear. Elevation of intracellular calcium following application of calcium ionophore increases the generation of A beta from its precursor protein (betaAPP). A receptor-based mechanism for the increase in A beta production has not been reported to our knowledge. Here, we use caffeine to stimulate ryanodine receptor (RYR)-regulated intracellular calcium release channels and show that internal calcium stores also participate in the genesis of A beta. In cultured HEK293 cells transfected with betaAPP cDNA, caffeine (5-10 mM) significantly increased the release of A beta fourfold compared with control. These actions of caffeine were saturable, modulated by ryanodine, and inhibited by the RYR antagonists ruthenium red and procaine. The calcium reuptake inhibitors thapsigargin and cyclopiazonic acid potentiated caffeine-stimulated A beta release. NH4Cl and monensin, agents that alter acidic gradients in intracellular vesicles, abolished both the caffeine and ionophore effects. Immunocytochemical studies showed some correspondence between the distribution patterns of RYR and cellular betaAPP immunoreactivities. The relevance of these findings to Alzheimer's disease and inclusion body myositis is discussed.

Beta APP mRNA transcription is increased in cultured fibroblasts from the familial Alzheimer's disease-1 family.

Familial (autosomal dominant) Alzheimer's disease (FAD) is a genetically heterogeneous disorder. Mutations in exons 16 and 17 of the amyloid beta-protein precursor (beta PP) gene currently account for less than 2% of FAD kindreds. No known defect in beta PP quantity, structure, or processing accounts for disease-associated beta-amyloid deposition in the majority of early-onset FAD kindreds. Only two out of a sample of 48 pedigrees (particularly the early onset FAD 4 kindred) contributed noticeably to evidence of linkage at the D21S16/13 and S1/S11 loci in the chromosomal region 21q21 [75]. Many early onset FAD pedigrees (including the FAD 1 and FAD 4 kindreds) show strong evidence of linkage to markers in the chromosome 14q24.3 region. Patients with trisomy 21 (Down's syndrome, DS) virtually always develop a histopathological phenotype indistinguishable from FAD, presumably on the basis of increased beta PP gene dosage and transcription. Whereas no beta PP gene duplication has been found in FAD, other mechanisms that augment beta PP production by effects at the transcriptional level could explain some FAD cases. Here, we report that cultured fibroblasts from affected members of the FAD 1 pedigree show a approximately 1.9 fold increase (P = 0.007) in beta PP mRNA levels compared to unaffected members when the cells are grown under stressed conditions in 0.5% serum. The elevated levels of beta PP mRNA in cells cultured in 0.5% serum also cosegregate with haplotypes in the 14q24.3 region when analyzed by linkage methods (LOD score = 3.26 at theta = 0.001). This is the chromosomal region to which FAD in this family has previously been mapped. As expected, fibroblasts from patients with DS used as a control show a similar beta PP mRNA increase. Fibroblasts from the FAD 4 pedigree did not show this defect under the conditions utilized here. beta PP and A beta protein levels were determined quantitatively after metabolic labeling and immunoprecipitation and found to increase 2.0 and 2.5 fold, respectively, in the fibroblasts from affected FAD 1 members. Finally, transient transfections of a beta PP promoter/chloramphenicol acetyl transferase reporter gene construct demonstrated a approximately 3-4 fold increase in beta PP promoter activity in affected fibroblasts from the FAD 1 but not the FAD 4 pedigree. Taken together, these data raise the possibility that an increase in beta PP transcription may underlie the AD phenotype in at least some of the chromosome 14-linked FAD families.

Calcium ionophore increases amyloid beta peptide production by cultured cells.

Amyloid beta peptide (A beta) is released into the media of a variety of cells in culture during normal metabolism. The discovery of several missense mutations within or flanking the A beta region of the beta amyloid precursor protein (beta APP) in familial Alzheimer's disease provides strong evidence for a role of altered processing of beta APP in the pathogenesis of this disorder. The cellular mechanisms that regulate the relative utilization of the secretory pathway, which causes beta APP to be cleaved within the A beta domain, and the alternative proteolytic pathway, which produces intact A beta, are unknown. It is hypothesized that a number of neurodegenerative diseases, including Alzheimer's disease, are characterized by abnormal calcium metabolism. We investigated the effect of disordered calcium homeostasis on A beta production in human kidney 293 cells transfected with beta APP cDNA. A beta immunoprecipitated from the conditioned media of cells was compared to immunoprecipitated full-length and secreted forms of beta APP in both metabolic labeling and pulse-chase labeling paradigms. The calcium ionophore A23187 consistently increased the production of A beta approximately 3-fold. This effect was dependent on the presence of extracellular calcium in intact cells. Caffeine also increased A beta production, possibly through release of calcium from intracellular stores. The increase in A beta was cAMP-independent, and it was not mediated by a protein kinase C-dependent pathway, as treatment with phorbol esters decreased A beta levels. The effects of the ionophore on beta APP maturation and phosphorylation were also established. We conclude that elevation of intracellular calcium levels has an important effect on beta APP maturation and proteolytic processing and substantially enhances the production and release of the amyloidogenic A beta peptide.

Sodium channels in normal and regenerated feline ventral spinal roots.

Regenerated and remyelinated nerve fibers have shorter internodes and thus more nodes than normal mature fibers. This requires either a decrease in the number of sodium channels per node or an increase in the number of channels per fiber or both. The purpose of this investigation was to determine what happens to sodium channel number, as estimated by 3H-saxitonin (STX) binding, in regenerated fibers and to relate this to nodal number. Five adult cats underwent cryoaxotomy of ventral root levels L5, L6, L7, and S1 on the left side. After regeneration for 16-45 weeks, binding parameters were determined. On the right (control) side, binding was consistent with that in unoperated animals (b = 1.3, Bmax = 10.2 +/- 0.4 fmol/mg wet, Kd = 0.6 +/- 0.1 nM). However, the regenerated nerves showed a 3.5-fold increase in maximal binding (b = 1.3, Bmax = 36.1 +/- 0.5, Kd = 0.45 +/- 0.4). Computer-aided histologic analysis of the regenerated roots revealed a decrease in fiber size; a significant decrease in internodal length for fibers in a given size class; and a 1.35-fold increase in total fibers per root. These factors account for a 2.36-fold increase in nodes per milligram (wet). The number of STX binding sites per regenerated node was calculated to be 1.95 X 10(6) (1.31, 3.07, 95% confidence limits), whereas it was 1.26 X 10(6) (0.78, 2.02) for the control roots. The difference was not significant (p greater than 0.05). It is concluded that, in regeneration, the increase in nodal number is accompanied by an increase in sodium channels, so that the number of channels per node is normal or slightly increased. There is a marked increase in channels per fiber and an even greater increase in channels per anterior horn cell. The implications of these data for nodal reorganization in remyelination are discussed.