Multiple system atrophy/progressive supranuclear palsy: alpha-Synuclein, synphilin, tau, and APOE.

Article abstract-Alpha synuclein, tau, synphilin, and APOE genotypes were analyzed in patients with multiple system atrophy (MSA) and progressive supranuclear palsy (PSP) and controls. The predisposing effect of the tau insertion polymorphism to the development of PSP is confirmed. However, no effect of alpha-synuclein, synphilin, or APOE variability on the development of PSP, or of tau, alpha-synuclein, APOE, or synphilin gene variability on the development of MSA, are demonstrated.

Cell-specific expression of beta-amyloid precursor protein isoform mRNAs and proteins in neurons and astrocytes.

The abnormal accumulation of beta-amyloid (A beta) in senile plaques appears to be a central pathological process in Alzheimer's disease. A beta is formed by proteolysis of beta-amyloid precursor protein (APP) with several isoforms generated by alternative splicing of exons 7, 8 and 15. A semi-quantitative reverse transcription (RT)-polymerase chain reaction (PCR) analysis showed that APP695 mRNA lacking exon 7 and 8 was most abundant in primary cultures of rat neurons, while APP770 and APP751 representing, respectively, the full length and exon 8 lacking isoforms predominated in cultured astroglial cells. Antisera AP-2 and AP-4 were produced by immunizing rabbits with keyhole limpet haemocyanin coupled with synthetic peptides representing KPI region APP301-316 and A beta region APP670-686 of APP770, respectively. These polyclonal antisera were purified against the corresponding peptide using affinity chromatography. Western blot analysis of homogenates of relatively enriched neuronal and astroglial cultures showed that these antibodies discretely stained bands of proteins in a cell-specific manner. Dot-blot analysis using AP-2, AP-4 and 22C11 antibodies indicated that, in comparison with neurons, cultured astrocytes contained 3-fold greater KPI-containing APP isoform proteins. The amount of total APP proteins, which include both KPI-containing and KPI-lacking APP isoforms, was approximately 90% higher in astrocytes than in neurons. Consistent with these in vitro findings in cultured astrocytes, in fimbria-fornix lesioned rat hippocampus, labelling with AP-2 antibody, which specifically reacts with KPI-containing APP proteins, was mainly observed in glial fibrillary acidic protein-positive reactive astrocytes in vivo. The results showed that APP isoforms are expressed in a cell type-specific manner in the brain and, since deposition of A beta is closely associated with the expression of KPI-containing APP isoforms, provide further evidence for the involvement of astrocytes in plaque biogenesis.

Presenilins and early-onset familial Alzheimer's disease.

Thirty-seven missense mutations and a splice-site mutation in the presenilin gene PS1 on chromosome 14 and two missense mutations PS2 on chromosome 1 co-segregate with early-onset familial Alzheimer's disease (AD). The presenilins belong to a family of conserved integral membrane proteins which include Caenorhabditis elegans SPE4 and SEL12 and the rat apoptosis-linked gene, ALG3. This review summarizes the genetics of presenilins in AD and indicators of putative function based on cellular localization and the functions of non-human homologues. Findings to date suggest an important role of presenilins in beta-amyloid (A beta) production: in vitro and in vivo studies have shown that presenilin mutations are associated with relatively increased production of the longer, and highly fibrillogenic A beta 42(43) peptide, and a marked elevation in the number of A beta 42-immunoreactive plaques in the brains of individuals with familial AD who carry PS1 and PS2 mutations. There is growing evidence that the deposition of A beta 42(43) could in some cases be an early and key event in the AD pathogenic cascade. The genetic and molecular biological data discussed in this review describe mechanisms by which presenilin mutations could lead to the development of AD. Also, mutant presenilins may be more proapoptotic. It is argued that the understanding of the processes by which presenilin mutations lead to the development of AD will help in devising a coherent framework for therapeutic strategies.

beta-Amyloid-mediated inhibition of redox activity (MTT reduction) is not an indicator of astroglial degeneration.

The reduction in 3-[4,5-dimethylthiazol]-2,5-diphenyltetrazolium bromide (MTT) to a coloured formazan compound by cultured cells has been extensively used as an in vitro model for understanding neurobiological mechanisms involved in amyloid beta-protein (A beta-mediated cell death. In primary cultures of astrocytes, very low concentrations of aggregated A beta 1-4C, but not A beta 4C-1, produced a significant inhibition in the reduction of the dye MTT. This inhibitory response was rapid and persisted as long as A beta 1-4C was present in the culture medium. Such a severe reduction in cell redox activity for days failed to cause death of astroglial cells, measured in terms of trypan blue uptake and lactate dehydrogenase release. Interleukin-1 beta (IL-1 beta), which is known to attenuate excitotoxic neurodegeneration, had no effect on A beta 1-4C-induced inhibition of MTT reduction. These results suggest that even though inhibition of MTT reduction represents an early indicator of the A beta 1-4C mediated cell injury, without other corroborating evidence, it should not be used as a measure of cell death.

Preparation and purification of antisera against different regions or isoforms of beta-amyloid precursor protein.

We describe a procedure for the production and peptide affinity purification of polyclonal antisera against synthetic peptides representing different domains of beta-amyloid precursor protein (APP). Rabbits were immunised with keyhole limpet haemocyanin coupled to synthetic peptides representing the amino-terminal APP18-32, Kunitz-type protease inhibitor (KPI) region APP301-316, the A beta region APP670-686, and the carboxy-terminal APP756-770 of APP770 for the production of antisera anti-AP-1, anti-AP-2, anti-AP-4 and anti-AP-5, respectively. Each antiserum was purified to specific antibody using the respective cognate peptides immobilised on affinity columns as ligand, using the 1-ethyl-3-(dimethylaminopropyl)carbodiimide-diaminodipropylamine method. Purified antibodies of these four antisera were highly specific and in enzyme-linked immunosorbent assays (ELISA) reacted only to the corresponding peptide. These purified antisera have been used in Western blot, immunohistochemical and immunoprecipitation techniques to facilitate the understanding of the regulation of APP and amyloid beta-protein (A beta). The A beta is formed by proteolysis of APP, and its deposition leading to the formation of senile plaques in the brain is considered to be a key step in the pathogenesis of Alzheimer's disease.

Polymorphism and chromosomal localization of the GI-form of human glutathione peroxidase (GPX2) on 14q24.1 by in situ hybridization.

We have isolated a 3.3-kb DNA containing the two exons and a 2.6-kb intron of the human CPX2 gene. This gene encodes the intestinal isoenzyme of glutathione peroxidase, GSHPx-GI. Consistent signals were detected at 14q24.1 when this DNA was used as a probe for fluorescence in situ hybridization on metaphase chromosomes. Based on either single-stranded conformation polymorphism or sequencing analysis, two sites containing DNA polymorphism were found in the intron: the 5' end had a single A/T alteration, and the 3' end had a microsatellite of TC repeats.

Genetic studies in Alzheimer's disease with an NACP/alpha-synuclein polymorphism.

The non-Abeta component of Alzheimer's disease amyloid (NAC) is copurified with amyloid from the brain tissue of Alzheimer's disease victims and is immunohistochemically localized to amyloid fibrils. NAC is a hydrophobic peptide fragment from the NAC precursor protein (NACP/alpha-synuclein) that is localized to presynaptic terminals. We used a polymorphic dinucleotide repeat sequence in a genomic clone of NACP for genetic association and linkage studies. Screening of Alzheimer's disease families failed to establish linkage between NACP and Alzheimer's disease. Nevertheless, one of the NACP polymorphisms (NACP allele 2) was shown to have significant association with healthy elderly control individuals with apolipoprotein E risk. This may indicate a possible protective function of the allele.