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The Research Objective: of this study was to test whether variation in mitochondrial composition is associated with "selective vulnerability" in Alzheimer brain. The term "selective vulnerability" refers to the loss of relatively vulnerable brain cells and the sparing of relatively resistant brain cells in disorders in which a genetic defect or environmental agent acts on both types of cells. The mechanisms underlying selective vulnerability are largely unknown, but mitochondria may be involved; the composition of mitochondria varies among different types of neurons, and mitochondria have an important role in cell death. Alzheimer's Disease (AD) is one of a number of neurodegenerative disorders in which both selective vulnerability and abnormalities of mitochondria occur., Methods: We examined by immunohistochemistry the cellular distribution of a mitochondrial constituent (the alpha-ketoglutarate dehydrogenase complex, KGDHC) known to be deficient in AD, in relation to the known selective vulnerability of neurons in areas 21 and 22 of the temporal lobe in this neurodegenerative disorder., Results: In normal human brain, cortical layers III and V contain neurons intensely immunoreactive for KGDHC, compared to other cells in these areas. The KGDHC-enriched cells are lost in AD (p < 0.001). In layer III, the loss of KGDHC-enriched cells is proportional to total loss of neurons, as determined by immunoreactivity to neuron specific enolase (NSE). In layer V, a higher proportion of the KGDHC-enriched neurons are lost than of other (NSE positive) neurons (p < 0.001)., Significance: Variations in mitochondrial composition may be one of the factors determining which cells die first when different types of cells are exposed to the same stress.

Frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17) is a neurological disorder associated with tau pathology.Tau deposits in FTDP-17 brains consist of polymerized filaments of hyperphosphorylated tau, the morphology of which is determined by the nature of the tau gene mutation observed in each case. A number of mutations associated with FTDP-17 have been identified in the 5' splice site of exon 10 and in exons 9-13 of the tau gene. The exon 10 5' splice site mutations disrupt alternative splicing and thus alter the ratio of 4R and 3R Tau isoforms. The majority of Tau missense mutations decrease its ability to bind tubulin and promote microtubule assembly. The extent of reduction varies depending on the site and nature of the mutation. Some Tau missense mutations also have a direct effect on the rate and the extent of tau filament formation. In the presence of polymerization-inducing agents such as heparin or arachidonic acid, mutant tau forms polymers more efficiently than wild type tau in vitro. Tau mutations affect polymerization at both nucleation and elongation phases. One mutation (R406W) is also known to alter the susceptibility of tau to phosphorylation. Expression of mutant tau in cultured cells changes the cytoskeletal integrity of CHO and COS-7 cells, but none of the tau transfected cells display tau filament inclusions. These findings suggest involvement of at least two mechanisms in the pathogenesis of FTDP-17.

The degradation of different isoforms of human recombinant tau (R-tau; T39, T40, and T44) and fetal tau (F-tau) by cathepsin D (CD) was investigated. Gel electrophoresis and Coomassie Blue staining of different R-tau species digested at pH 3.5 showed very little differences in CD susceptibility. Immunoblotting analyses revealed that amino and carboxy termini of tau were cleaved before other regions. F-tau was most vulnerable to proteolysis at both termini. Digestion of R-tau with 0.01 unit of CD/ml at pH 3.5 resulted in cleavage between Phe8-Glu9, Met419-Val420, Thr427-Leu428-Ala429, and Leu436-Ala437 as determined by amino acid sequencing and mass spectroscopy (numbering of amino acids was based on T40). With higher concentrations of CD (1 unit/ml), additional sites of digestion were detected between amino acids 34-161, 200-257, and 267-358. The cleavage sites at amino acids 34-161 and 267-358 were observed at pH 3.5, whereas that at amino acids 200-257 was detected at pH 7.0. Our results suggest that CD cleavage of tau could generate tau fragments with intact microtubule binding domains, which could have a role in the pathogenesis of paired helical filaments (PHFs) in Alzheimer's disease. Such proteolysis might also contribute to the changes of PHF phenotype observed in intracellular and extracellular tangles.

To explore the utility of cultured skin fibroblasts in investigating diseases of the nervous system in which constituents characteristic of neurons are involved, sensitive immunochemical methods were used to test for the presence in skin fibroblasts of low amounts of proteins normally used as neuronal markers. The presence of each of the neurofilament triplet proteins and of neuron-specific enolase was demonstrated by immunoblotting and by immunocytochemistry, and of an 86-kDa synapsin-like material by immunoblotting. These observations agree with previous suggestions that readily available cultured fibroblasts may be useful in investigations of disorders in which molecules are involved which are typically associated with neurons in vivo, such as Alzheimer's disease.

Molecular cloning and cDNA sequencing have indicated that the fibril-forming, amyloidogenic beta/A4 peptide of cerebrovasculature and plaque core in AD is encoded as part of a larger precursor, amyloid precursor protein (APP). A panel of antibodies directed against synthetic peptides, which correspond to distinct domains of this putative APP molecule (i.e., amino acid residues 45-62, 587-596, 597-606, 597-638 [beta/A4 peptide], 638-658 and 653-661), were used to probe immunohistochemically serial sections of formalin-fixed, paraffin-embedded Alzheimer's disease (AD) brains for the presence of APP and/or its derivatives. Histochemical staining of adjacent sections with Bielschowsky's silver impregnation and with Congo red or thioflavin S-staining techniques was also done to identify the structures with amyloid deposition. All these antibodies exhibited intense immunoreactivity with amyloidotic cerebral vessels, including meningeal and parenchymal. This observation indicates that the amyloidotic vasculature of AD brain contains, in addition to the fibril-forming beta/A4 protein, nonamyloidogenic APP and/or its derivatives. More importantly, this APP immunoreactivity colocalized with angiopathic amyloid, which is characterized by phenol-resistant, birefringent congophilia. Parallel analyses with a dual SABC/silver impregnation procedure further confirmed that APP and/or its derivatives, including the amyloidogenic beta/A4, colocalized with argentophilic amyloid in the cerebrovasculature of AD.

Of three human neuroblastoma lines tested, IMR32K (and IMR32 parental line) was the only cell line that, after its exposure to a differentiation medium, consistently developed materials recognized immunocytochemically by a panel of antibodies against paired helical filaments (PHF). Ultrastructurally, these cells accumulated, at their perikarya and neuritic extensions, spatially discrete arrays of fibrils, which occasionally occurred in twisted pairs. When these fibrillar structures appeared as paired helices, they exhibited dimensions and configurations reminiscent of PHF found in affected Alzheimer neurons, although less compact. Immunoelectron microscope examinations of the fibrillar structures in these neuroblastoma cells with one of these anti-PHF immunoprobes revealed that only subsets of fibrillar structures that appeared thickened or aggregated to form bundles were selectively immunolabeled. Cultures of these immortal neuroblastoma lines may provide a convenient model for studying aspects of PHF formation that are hard to examine in Alzheimer brain obtained at autopsy.

Sodium butyrate has been shown to inhibit the growth and induce the differentiation of F-98 rat glioma cells. In agreement with the morphological changes, we have found that mRNAs for fibronectin and collagen in these cells could be reversibly induced by butyrate. While Ki-ras mRNA levels remained relatively unchanged, mRNAs for fos and sis increased significantly during the course of butyrate induced differentiation. c-fos induction can be detected 30 min after butyrate addition, a peak level (greater than 20 fold) was reached at 2 h, with a subsequent gradual decline. c-sis induction was detectable 24 h after butyrate exposure, at which time the cells have assumed morphological transition. Interestingly, the sis mRNA induction was not reversible upon butyrate withdrawal. The sis mRNA half-life increased from 40 min in the untreated cells to 100 min in the butyrate induced cells indicating that the increase in the stability of sis mRNA contributed, at least in part, to the elevated levels of sis expression. These findings demonstrate a coordinated induction of fibronectin and collagen genes in the butyrate-treated F-98 cells. In addition, fos and sis transcripts were differentially induced; a rapid and transient induction of fos followed by an irreversible induction of sis at a later stage of differentiation.

The widely used intracerebral tumor implantation method by freehand injection into parietal or hippocampal areas of the rat brain has proven inadequate for reliable experimental therapeutic studies. Problems include poor intracerebral growth yields and significant rates of spread to extracranial tissues, lungs, and spinal cord. Major variables have been examined experimentally on a model using nitrosourea-induced nervous system tumor cell lines in sygeneic rats. A rapid stereotaxic method greatly improved the consistency of tumor placement. The optimal site was found to be the caudate nucleus. The production of a spheroid intracerebral growth was further facilitated by the use of 1% agar in the cell suspension medium and by an injection volume of 10 mu1 containing at least 10(4) cells. Further improvements involved injection technique and flushing of the operative field. These modifications have resulted in a 99% to 100% yield of intracerebral growth, with a marked reduction in the number and size of extracranial extensions and with distant metastasis rates of 0% to 5%. These results have continually improved with further experience. The method is satisfactory for radiation and chemotherapeutic trials in which survival time as an index of tumor size may be used an an end point.

The effect of butyrate on cellular morphologic features and morphometric parameters was examined in vitro with clonal glioma cells of the inbred CD Fischer rat. Untreated cells were multipolar with a high nuclear:cytoplasmic ratio, contained few attenuated rough endoplasmic reticular (RER) and cytoskeletal components in the cytoplasm, and were devoid of junctional complexes. Following exposure to butyrate, the glioma cells appeared epithelioid, and each was characterized by a polygonal cell body with a substantially reduced nuclear:cytoplasmic ratio. In contrast to the coarsely clumped nuclear chromatin of untreated glioma cells, the nuclei of the butyrate-exposed cells consisted of finely dispersed chromatin. The cytoplasm of treated cells contained numerous distended RER packed with proteinaceous material and had an elaborate cyto-skeletal network. Butyrate also induced the development of junctional devices between apposed surface membranes of adjacent cells. The acquisition of these phenotypic traits more characteristic of normal glial cells makes butyrate a potent, naturally occurring reverse transformation agent.

Frog esophageal mucosa contains peptic glands which are innervated by cholinergic neurons. When incubated in a medium containing 1.5 mM CaCl2, pepsinogen release from esophageal mucosa was increased by a high potassium concentration (55 mM KCl), 1,1-dimethyl-4-phenylpiperazinium (DMPP) or bethanechol. Whereas the response to bethanechol remained little changed, the response to high KCl concentrations or DMPP was abolished in the absence of Ca2+. The stimulatory effects of high KCl concentrations and DMPP were also eliminated by the presence of atropine or somatostatin. Furthermore, pepsinogen release in response to bethanechol was dose-dependently inhibited by somatostatin. Frog esophagus was found to contain somatostatin-like immunoreactivity, with a higher density at the end adjacent to the stomach. Chromatography of mucosa extract on Sephadex G-50 revealed a single peak of somatostatin-like immunoreactivity that coeluted with somatostatin-14. Immunohistochemical staining of the mucosa with peroxidase antiperoxidase technique demonstrated the presence of two varieties of somatostatin-like immunoreactivity-containing cells, one individually dispersed within the intercalated septa and the other in groups within the interlobular septa of the peptic glands. These results seem to indicate that somatostatin or somatostatin-like immunoreactivity may play a modulatory role in neurally mediated pepsinogen secretion in the frog esophagus.