Gerd Multhaup

Neuronal origin of a cerebral amyloid: neurofibrillary tangles of Alzheimer's disease contain the same protein as the amyloid of plaque cores and blood vessels.

The protein component of Alzheimers disease amyloid [neurofibrillary tangles (NFT), amyloid plaque core and congophilic angiopathy] is an aggregated polypeptide with a subunit mass of 4 kd (the A4 monomer). Based on the degree of N‐terminal heterogeneity, the amyloid is first deposited in the neuron, and later in the extracellular space. Using antisera raised against synthetic peptides, we show that the N terminus of A4 (residues 1‐11) contains an epitope for neurofibrillary tangles, and the inner region of the molecule (residues 11‐23) contains an epitope for plaque cores and vascular amyloid. The non‐protein component of the amyloid (aluminum silicate) may form the basis for the deposition or amplification (possible self‐replication) of the aggregated amyloid protein. The amyloid of Alzheimers disease is similar in subunit size, composition but not sequence to the scrapie‐associated fibril and its constituent polypeptides. The sequence and composition of NFT are not homologous to those of any of the known components of normal neurofilaments.

Alzheimer's disease amyloidogenic glycoprotein: expression pattern in rat brain suggests a role in cell contact.

The cloned cDNA encoding the rat cognate of the human A4 amyloid precursor protein was isolated from a rat brain library. The predicted primary structure of the 695‐amino acid‐long protein displays 97% identity to its human homologue shown previously to resemble an integral membrane protein. The protein was detected in rodent brain and muscle by Western blot analysis. Using in situ hybridization and immunocytochemistry on rat brain sections, we discovered that rat amyloidogenic glycoprotein (rAG) and its mRNA are ubiquitously and abundantly expressed in neurons indicating a neuronal original for the amyloid deposits observed in humans with Alzheimers disease (AD). The protein appears in patches on or near the plasma membranes of neurons suggesting a role for this protein in cell contact. Highest expression was seen in rat brain regions where amyloid is deposited in AD but also in areas which do not contain deposits in AD. Since amyloid deposits are rarely observed in rat brain, we conclude that high expression of AG is not the sole cause of amyloidosis.

Identification, transmembrane orientation and biogenesis of the amyloid A4 precursor of Alzheimer's disease.

The precursor of the Alzheimers disease‐specific amyloid A4 protein is an integral, glycosylated membrane protein which spans the bilayer once. The carboxy‐terminal domain of 47 residues was located at the cytoplasmic site of the membrane. The three domains following the transient signal sequence of 17 residues face the opposite side of the membrane. The C‐terminal 100 residues of the precursor comprising the amyloid A4 part and the cytoplasmic domain have a high tendency to aggregate, and proteinase K treatment results in peptides of the size of amyloid A4. This finding suggests that there is a precursor‐product relationship between precursor and amyloid A4 and we conclude that besides proteolytic cleavage other events such as post‐translational modification and membrane injury are primary events that precede the release of the small aggregating amyloid A4 subunit.

Localization of the putative precursor of Alzheimer's disease-specific amyloid at nuclear envelopes of adult human muscle.

Cloning and sequence analysis revealed the putative amyloid A4 precursor (pre‐A4) of Alzheimers disease to have characteristics of a membrane‐spanning glycoprotein. In addition to brain, pre‐A4 mRNA was found in adult human muscle and other tissues. We demonstrate by in situ hybridization that pre‐A4 mRNA is present in adult human muscle, in cultured human myoblasts and myotubes. Immunofluorescence with antipeptide antibodies shows the putative pre‐A4 protein to be expressed in adult human muscle and associated with some but not all nuclear envelopes. Despite high levels of a single 3.5‐kb pre‐A4 mRNA species in cultured myoblasts and myotubes, the presence of putative pre‐A4 protein could not be detected by immunofluorescence. This suggests that putative pre‐A4 protein is stabilized and therefore functioning in the innervated muscle tissue but not in developing, i.e. non‐innervated cultured muscle cells. The selective localization of the protein on distinct nuclear envelopes could reflect an interaction with motor endplates.

Proteolytic Fragments of Alzheimer’s Disease‐Associated Presenilin 1 Are Present in Synaptic Organelles and Growth Cone Membranes of Rat Brain

Abstract: Previous studies have demonstrated the molecular linkage of three causative genes for early‐onset Alzheimer’s disease: the presenilin 1 gene on chromosome 14, the presenilin 2 gene on chromosome 1, and the amyloid precursor protein gene on chromosome 21. In the present study, we have investigated the distributions of the ∼20‐kDa C‐terminal and ∼30‐kDa N‐terminal fragments of presenilin 1 and the amyloid precursor protein in rat brain and compared them with the distribution of several marker proteins. The fragments of presenilin 1 are present in synaptic plasma membranes, neurite growth cone membranes, and small synaptic vesicles of rat brain. Both proteolytic fragments are coenriched in the corresponding tissue fractions. Based on this observation, it seems likely that N‐ and C‐terminal presenilin 1 fragments form a functional unit while remaining associated. In contrast to a predominant subcellular localization of presenilin 1 to the endoplasmic reticulum and Golgi apparatus in different cell lines, our results indicate that rat brain presenilin 1 fragments exit from these biosynthetic compartments to reach synaptic organelles in neurons.

Stage-specific proteophosphoglycan from Leishmania mexicana amastigotes - Structural characterization of novel mono-, di-, and triphosphorylated phosphodiester-linked oligosaccharides

Intracellular amastigotes of the protozoan parasite Leishmania mexicana secrete a macromolecular proteophosphoglycan (aPPG) into the phagolysosome of their host cell, the mammalian macrophage. The structures of aPPG glycans were analyzed by a combination of high pH anion exchange high pressure liquid chromatography, gas chromatography-mass spectrometry, enzymatic digestions, electrospray-mass spectrometry as well as1H and 31P NMR spectroscopy. Some glycans are identical to oligosaccharides known from Leishmania mexicana promastigote lipophosphoglycan and secreted acid phosphatase. However, the majority of the aPPG glycans represent amastigote stage-specific and novel structures. These include neutral glycans ([Glcβ1–3]1–2Galβ1–4Man, Galβ1–3Galβ1–4Man, Galβ1–3Glcβ1–3Galβ1–4Man), several monophosphorylated glycans containing the conserved phosphodisaccharide backbone (R-3-[PO4-6-Gal]β1–4Man) but carrying stage-specific modifications (R = Galβ1-, [Glcβ1–3]1–2Glcβ1-), and monophosphorylated aPPG tri- and tetrasaccharides that are uniquely phosphorylated on the terminal hexose (PO4-6-Glcβ1–3Galβ1–4Man, PO4-6-Glcβ1–3Glcβ1–3Galβ1–4Man, PO4-6-Galβ1–3Glcβ1–3Galβ1–4Man). In addition aPPG contains highly unusual di- and triphosphorylated glycans whose major species are PO4-6-Glcβ1–3Glcβ1–3[PO4-6-Gal]β1–4Man, PO4-6-Galβ1–3Glcβ1–3[PO4-6-Gal]β1–4Man, PO4-6-Galβ1–3Glcβ1–3Glcβ1–3[PO4-6-Gal]β1–4Man, PO4-6-Glcβ1–3[PO4-6-Glc]β1–3[PO4-6-Gal]β1–4Man, PO4-6-Galβ1–3[PO4-6-Glc]β1–3Glcβ1–3[PO4-6-Gal]β1–4Man, and PO4-6-Glcβ1–3[PO4-6-Glc]β1–3Glcβ1–3[PO4-6-Gal]β1–4Man. These glycans are linked together by the conserved phosphodiester R-Manα1-PO4-6-Gal-R or the novel phosphodiester R-Manα1-PO4-6-Glc-R and are connected to Ser(P) of the protein backbone most likely via the linkage R-Manα1-PO4-Ser. The variety of stage-specific glycan structures in Leishmania mexicana aPPG suggests the presence of developmentally regulated amastigote glycosyltransferases which may be potential anti-parasite drug targets.

Role of Copper and Other Transition Metal Ions in the Pathogenesis of Parkinson’s Disease, Prion Diseases, Familial Amyotrophic Lateral Sclerosis, and Alzheimer’s Disease

During the last few years, there has been growing evidence based on experimental data that Cu and other transition metal ions may have important roles in the pathogenesis of a series of hereditary and sporadic disorders of the central nervous system (CNS). It is suggested that metal ions such as Cu exert toxicity during electron-transfer reactions and also play a structural role by changing the conformation of proteins upon specific binding. In contrast, Zn is assumed to play a purely structural role because it exists exclusively in one oxidation state. According to presently accumulating knowledge, Cu complexes are especially sensitive to redox reactions (in the form of “free”) -Cu ions in the Fenton-type reaction). On the other hand, copper is an essential element—being a cofactor of detoxifying enzymes such as of Cu/Zn-superoxide dismutase (SOD). According to the high-affinity binding of Cu to the native amyloid precursor protein (APP) and the prion protein (PrP), Cu is suggested to exert conformation stabilizing functions, to act as a redox active detoxifying cofactor, and is involved in metabolic transport (APP) or absorption-secretory (PrP and APP) functions. Currently, there are no data to confirm the hypothesis that an enhanced exogeneous exposure to Cu (or other transition metal ions) may accelerate the progression of neurodegenerative diseases or even to show a significant association with increased metal-ion levels in general, or what may occur with a higher frequency in response to exogenous exposure. In contrast, a causal relationship has been shown for neurodegenerative symptoms and chronic inhalatory exposure of Mn because of its unique ability to cross the blood-brain barrier via the olfactory epithelium.