Long-lasting acetylcholinesterase splice variations in anticholinesterase-treated Alzheimer's disease patients.

Protein levels of different acetylcholinesterase (AChE) splice variants were explored by a combination of immunoblot techniques, using two different antibodies, directed against the C-terminus of the AChE-R splice variant or the core domain common to all variants. Both AChE-R and AChE-S splice variants as well as several heavier AChE complexes were detected in brain homogenates from the parietal cortex of patients with or without Alzheimer's disease (AD) as well as the cerebrospinal fluid (CSF) of AD patients, compatible with the assumption that CSF AChEs might originate from CNS neurons. Long-term changes in the composition of CSF AChE variants were further pursued in AD patients treated with rivastigmine (n = 11) or tacrine (n = 17) in comparison to untreated AD patients (n = 5). In untreated patients, AChE-R was markedly reduced as compared with the baseline level (37%), whereas the medium size AChE-S complex was increased by 32%. Intriguingly, tacrine produced a general and profound up-regulation of all detected AChE variants (up to 117%), whereas rivastigmine treatment caused a mild and selective up-regulation of AChE-R ( approximately 10%, p < 0.05). Moreover, the change in the ratio of AChE-R to AChE-S (R/S-ratio) strongly and positively correlated with sustained cognition at 12 months (p < 0.0001). Thus, evaluation of changes in the composition of CSF AChE variants may yield important information referring to the therapeutic efficacy and/or development of drug tolerance in AD patients treated with anti-cholinesterases.

Development of human antibody fragments directed towards synaptic acetylcholinesterase using a semi-synthetic phage display library.

Current Alzheimer's disease therapies suppress acetylcholine hydrolysis by inhibiting acetylcholinesterase (AChE) at cholinergic synapses. However, anticholinesterases promote alternative splicing changing the composition of brain AChE variants. To study this phenomenon we developed monoclonal antibodies to acetylcholinesterase synaptic peptide (ASP), a synthetic peptide with the C-terminal sequence unique to the human synaptic variant AChE-S. Screening of a phage display human antibody library allowed the isolation of single-chain Fv (scFv) antibodies that were highly specific for ASP, and displayed closely related third complementarity determining regions of the variable heavy chain domain (V(H)-CDR3). BIAcore analysis demonstrated dissociation constants at the micromolar range: 1.6 x 10(-6) and 2.0 x 10(-6) M for ASP and the complete AChE-S protein, respectively. The anti-ASP antibodies provide a novel tool for studying the synaptic AChE-S variant, the expression of which is altered in ageing and dementia.

Excess "read-through" acetylcholinesterase attenuates but the "synaptic" variant intensifies neurodeterioration correlates.

Acute stress increases the risk for neurodegeneration, but the molecular signals regulating the shift from transient stress responses to progressive disease are not yet known. The "read-through" variant of acetylcholinesterase (AChE-R) accumulates in the mammalian brain under acute stress. Therefore, markers of neurodeterioration were examined in transgenic mice overexpressing either AChE-R or the "synaptic" AChE variant, AChE-S. Several observations demonstrate that excess AChE-R attenuates, whereas AChE-S intensifies, neurodeterioration. In the somatosensory cortex, AChE-S transgenics, but not AChE-R or control FVB/N mice, displayed a high density of curled neuronal processes indicative of hyperexcitation. In the hippocampus, AChE-S and control mice, but not AChE-R transgenics, presented progressive accumulation of clustered, heat shock protein 70-immunopositive neuronal fragments and displayed a high incidence of reactive astrocytes. Our findings suggest that AChE-R serves as a modulator that may play a role in preventing the shift from transient, acute stress to progressive neurological disease.

The ecto-5'-nucleotidase subunits in dimers are not linked by disulfide bridges but by non-covalent bonds.

It has long been considered that ecto-5'-nucleotidase (eNT) dimers consist of subunits linked by disulfide bonds. Hydrophilic (6.7S) and amphiphilic (4.0S) dimers were separated by sedimentation analysis of eNT purified from bull seminal plasma. Hydrophilic (4. 2S) and amphiphilic (2.6S) eNT monomers were obtained after reduction of disulfide bonds in dimers. The amphiphilic eNT dimers or monomers were converted into their hydrophilic variants with phosphatidylinositol-specific phospholipase C. SDS-PAGE plus Western blot showed 68 kDa subunits, regardless of the addition of beta-mercaptoethanol to the SDS mixture. Active eNT monomers were obtained by addition of 1 M guanidinium chloride (Gdn) to dimers, and unfolded subunits by addition of 4 M Gdn. The results unambiguously demonstrate that the subunits in eNT dimers are not linked by disulfide bridges, but by non-covalent bonds, and that dissociation precedes inactivation and unfolding.

Biochemical properties of 5'-nucleotidase from mouse skeletal muscle.

Ecto-5'-nucleotidase (eNT) from mouse muscle has been purified after extraction with detergent followed by chromatography on concanavalin A- and AMP-Sepharose. Three fractions were recovered: UF was NT non-retained in immobilised AMP; F-I was bound enzyme eluted with beta-glycerophosphate, and F-II was bound NT released with AMP. eNT was 80000-fold purified in F-II, this fraction showing proteins of 74, 68 and 51 kDa after immunoblotting. NT in UF migrated at 6.7S after centrifugation in sucrose gradients with Triton X-100, the peak being split into two of 6.7S and 4.4S in gradients with Brij 96. Ecto-NT in F-I or F-II migrated at 5.8S in Triton X-100-, or 4.4S in Brij 96-containing gradients. The hydrodynamic behaviour, concentration in Triton X-114, binding to phenyl-agarose, and sensitivity to phosphatidylinositol-specific phospholipase C revealed that enzyme forms in F-I or F-II were amphiphilic dimers with linked phosphatidylinositol residues, whilst most of NT forms in UF were hydrophilic dimers. A zinc/protein molar ratio of 2.2 was determined for eNT in F-II. NT activity was decreased in assays made in imidazole buffer, and was partly restored with 10 microM Zn2+ or 100 microM Mn2+. In assays with Tris buffer, NT showed a Km for AMP of 12 microM, and was competitively inhibited by ATP or ADP.

Differential interaction of the monoclonal antibody AE-1 with acetylcholinesterase oligomers and monomers from rabbit muscle microsomes, human brain and fetal bovine serum.

The monoclonal antibody AE-1 raised against acetylcholinesterase (AChE) from human erythrocytes (HE) is shown to react with active asymmetric and tetrameric AChE components from rabbit muscle microsomes (RMM), and with tetrameric forms from human brain (HB) or fetal bovine serum (FBS). However, it failed to bind to AChE monomers from RMM or HB. The results of Western blot revealed that the determinant for AE-1 consisted of a conformational domain, not a primary sequence region, in the AChE subunit. The antibody recognized HE monomers and FBS dimers, but not FBS monomers. The formation of labile immunocomplexes between AE-1 and AChE subunits may explain the lack of interaction between the antibody and the monomers from non-erythrocyte sources.

Conversion of acetylcholinesterase hydrophilic tetramers into amphiphilic dimers and monomers.

Exposure of purified hydrophilic tetramers of acetylcholinesterase (AChE) from fetal bovine serum to various guanidinium chloride (Gdn) concentrations led to inactive tetramers (2 M Gdn) and dimers (6 M Gdn). The native tetramers were almost fully monomerized by reduction, a minor fraction of the released monomers remaining active. Sedimentation analysis and hydrophobic chromatography showed that the modified tetramers, dimers and monomers had amphiphilic properties. Intrinsic fluorescence spectra and binding of the amphiphilic probe, 1-anilino-8-naphthalene sulfonate (ANS), revealed that AchE subunit in the modified tetramers were in a 'molten globule' structure, the dimers in a denatured stated, and the inactive monomers in a 'native-like' structure. These data show that AChE subunits possess a flexible conformation, which may be important for generating a full set of molecular forms. In addition, the behavior of the active monomers with amphiphiles may explain the interactions of type II AChE forms with membranes.