Helmut Romig

A loss of function mutation of presenilin-2 interferes with amyloid beta-peptide production and notch signaling.

Presenilin-1 (PS1) facilitates γ-secretase cleavage of the β-amyloid precursor protein and the intramembraneous cleavage of Notch1. Although Alzheimer’s disease-associated mutations in the homologous presenilin (PS2) gene elevate amyloid β-peptide (Aβ42) production like PS1 mutations, here we demonstrate that a gene ablation of PS2 (unlike that of PS1) in mice does not result in a severe phenotype resembling that of Notch-ablated animals. To investigate the amyloidogenic function of PS2 more directly, we mutagenized a conserved aspartate at position 366 to alanine, because the corresponding residue of PS1 is known to be required for its amyloidogenic function. Cells expressing the PS2 D366A mutation exhibit significant deficits in proteolytic processing of β-amyloid precursor protein indicating a defect in γ-secretase activity. The reduced γ-secretase activity results in the almost complete inhibition of Aβ and p3 production in cells stably expressing PS2 D366A, whereas cells overexpressing the wild-type PS2 cDNA produce robust levels of Aβ and p3. Using highly sensitive in vivo assays, we demonstrate that the PS2 D366A mutation not only blocks γ-secretase activity but also inactivates PS2 activity in Notch signaling by inhibiting the proteolytic release of the cytoplasmic Notch1 domain. These data suggest that PS2 is functionally involved in Aβ production and Notch signaling by facilitating similar proteolytic cleavages.

Glycine 384 is required for presenilin-1 function and is conserved in bacterial polytopic aspartyl proteases.

Endoproteolysis of β-amyloid precursor protein (βAPP) and Notch requires conserved aspartate residues in presenilins 1 and 2 (PS1 and PS2). Although PS1 and PS2 have therefore been proposed to be aspartyl proteases, no homology to other aspartyl proteases has been found. Here we identify homology between the presenilin active site and polytopic aspartyl proteases of bacterial origin, thus supporting the hypothesis that presenilins are novel aspartyl proteases.

Expression of Alzheimer's disease-associated presenilin-1 is controlled by proteolytic degradation and complex formation.

Numerous mutations causing early onset Alzheimer’s disease have been identified in the presenilin (PS) genes, particularly the PS1 gene. Like the mutations identified within the β-amyloid precursor protein gene, PS mutations cause the increased generation of a highly neurotoxic variant of amyloid β-peptide. PS proteins are proteolytically processed to an N-terminal ∼30-kDa (NTF) and a C-terminal ∼20-kDa fragment (CTF20) that form a heterodimeric complex. We demonstrate that this complex is resistant to proteolytic degradation, whereas the full-length precursor is rapidly degraded. Degradation of the PS1 holoprotein is sensitive to inhibitors of the proteasome. Formation of a heterodimeric complex is required for the stability of both PS1 fragments, since fragments that do not co-immunoprecipitate with the PS complex are rapidly degraded by the proteasome. Mutant PS fragments not incorporated into the heterodimeric complex lose their pathological activity in abnormal amyloid β-peptide generation even after inhibition of their proteolytic degradation. The PS1 heterodimeric complex can be attacked by proteinases of the caspase superfamily that generate an ∼10-kDa proteolytic fragment (CTF10) from CTF20. CTF10 is rapidly degraded most likely by a calpain-like cysteine proteinase. From these data we conclude that PS1 metabolism is highly controlled by multiple proteolytic activities indicating that subtle changes in fragment generation/degradation might be important for Alzheimer’s disease-associated pathology.

Presenilin-1 differentially facilitates endoproteolysis of the β-amyloid precursor protein and Notch

Mutations in the presenilin-1 (PS1) gene are associated with Alzheimer’s disease and cause increased secretion of the neurotoxic amyloid-β peptide (Aβ). Critical intramembraneous aspartates at residues 257 and 385 are required for the function of PS1 protein. Here we investigate the biological function of a naturally occurring PS1 splice variant (PS1 Δexon 8), which lacks the critical aspartate 257. Cell lines that stably express PS1 Δexon 8 or a PS1 protein in which aspartate residue 257 is mutated secrete significant levels of Aβ, whereas Aβ generation is severely reduced in cells transfected with PS1 containing a mutation of aspartate 385. In contrast, endoproteolytic processing of Notch is almost completely inhibited in cell lines expressing any of the PS1 variants that lack one of the critical aspartates. These data indicate that PS1 may differentially facilitate γ-secretase-mediated generation of Aβ and endoproteolysis of Notch.

The Biological and Pathological Function of the Presenilin-1 ΔExon 9 Mutation Is Independent of Its Defect to Undergo Proteolytic Processing

The two homologous presenilins are key factors for the generation of amyloid β-peptide (Aβ), since Alzheimer’s disease (AD)-associated mutations enhance the production of the pathologically relevant 42-amino acid Aβ (Aβ42), and a gene knockout of presenilin-1 (PS1) significantly inhibits total Aβ production. Presenilins undergo proteolytic processing within the domain encoded by exon 9, a process that may be closely related to their biological and pathological activity. An AD-associated mutation within the PS1 gene deletes exon 9 (PS1Δexon9) due to a splicing error and results in the accumulation of the uncleaved full-length protein. We now demonstrate the unexpected finding that the pathological activity of PS1Δexon9 is independent of its lack to undergo proteolytic processing, but is rather due to a point mutation (S290C) occurring at the aberrant exon 8/10 splice junction. Mutagenizing the cysteine residue at position 290 to the original serine residue completely inhibits the pathological activity in regard to the elevated production of Aβ42. Like PS1Δexon9, the resulting presenilin variant (PS1Δexon9 C290S) accumulates as an uncleaved protein and fully replaces endogenous presenilin fragments. Moreover, PS1Δexon9 C290S exhibits a significantly increased biological activity in a highly sensitive in vivo assay as compared with the AD-associated mutation. Therefore not only the increased Aβ42 production but also the decreased biological function of PS1Δexon9 is due to a point mutation and independent of the lack of proteolytic processing.

Elevated vulnerability to oxidative stress-induced cell death and activation of caspase-3 by the Swedish amyloid precursor protein mutation.

The Swedish double mutation (KM670/671NL) of amyloid precursor protein (APPsw) is associated with early‐onset familial Alzheimers disease (FAD) and results in from three‐ to sixfold increased β‐amyloid production. The goal of the present study was to elucidate the effects of APPsw on mechanisms of apoptotic cell death. Therefore, PC12 cells were stably transfected with human APPsw. Here we report that the vulnerability of APPsw‐bearing PC12 cells to undergo apoptotic cell death was significantly enhanced after exposure to hydrogen peroxide compared to human wild‐type APP‐bearing cells, empty vector‐transfected cells, and parent untransfected cells. In addition, we have analyzed the potential influence of several mechanisms that can interfere with the execution of the apoptotic cell death program: the inhibition of cell death by the use of caspase inhibitors and the reduction of oxidative stress by the use of (±)‐α‐tocopherol (vitamin E). Interestingly, oxidative stress‐induced cell death was significantly attenuated in APPsw PC12 cells by pretreatment with caspase‐3 inhibitors but not with caspase‐1 inhibitors. In parallel, caspase‐3 activity was markedly elevated in APPsw PC12 after stimulation with hydrogen peroxide for 6 hr, whereas caspase‐1 activity was unaltered. In addition, oxidative stress‐induced cell death could be reduced after pretreatment of APPsw cells with (±)‐α‐tocopherol. The protective potency of (±)‐α‐tocopherol was even greater than that of caspase‐3 inhibitors. Our findings further emphasize the role of mutations in the amyloid precursor protein in apoptotic cell death and may provide the fundamental basis for further efforts to elucidate the underlying processes caused by FAD‐related mutations. J. Neurosci. Res. 64:183–192, 2001.

Dependence of cerebrospinal fluid Tau protein levels on apolipoprotein E4 allele frequency in patients with Alzheimer's disease

Consistent pathological hallmarks of Alzheimers disease (AD) are the formation of brain amyloid and neurofibrillary tangles (NFTs). Levels of the major protein component of NFTs, the microtubule associated protein Tau, were shown to be increased in cerebrospinal fluid (CSF) of AD patients as compared to age-matched controls. The presence of apolipoprotein E-epsilon 4 allele (APOE4) is a risk factor for sporadic and familial late-onset AD. ApoE may interact with the binding of Tau to microtubules and Tau phosphorylation in an isoform-specific manner. We investigated whether direct evidence of an isoform-specific interaction of apoE and Tau can be demonstrated in the CSF of live AD patients. We measured the apoE genotype and CSF levels of Tau in 19 patients with probable AD and 12 age-matched control subjects. We found that CSF levels of Tau increase with increasing APOE allele frequency (Spearman rank correlation, zeta = 2.71, P = 0.007). This finding may be in agreement with reports of a lesser binding of apoE4 to Tau, compared to apoE2 and apoE3, resulting in higher levels of unbound Tau in CSF.

Selective CB2 receptor agonists. Part 2: Structure–activity relationship studies and optimization of proline-based compounds

Through a ligand-based pharmacophore model (S)-proline based compounds were identified as potent cannabinoid receptor 2 (CB2) agonists with high selectivity over the cannabinoid receptor 1 (CB1). Structure-activity relationship investigations for this compound class lead to oxo-proline compounds 21 and 22 which combine an impressive CB1 selectivity profile with good pharmacokinetic properties. In a streptozotocin induced diabetic neuropathy model, 22 demonstrated a dose-dependent reversal of mechanical hyperalgesia.

Selective CB2 receptor agonists. Part 3: The optimization of a piperidine-based series that demonstrated efficacy in an in vivo neuropathic pain model

A novel class of potent cannabinoid receptor 2 (CB2) agonists based on a (S)-piperidine scaffold was identified using ligand-based pharmacophore models. Optimization of solubility and metabolic stability led to the identification of several potent CB2 agonists (e.g., 30) that displayed selectivity over cannabinoid receptor 1 (CB1) and acceptable drug like properties. In rats, compound 30 demonstrated a favorable pharmacokinetic profile and efficacy in a Streptozotocin-induced diabetic neuropathy model, with full reversal of mechanical hyperalgesia.