Rupert Sandbrink

APP Gene Family Alternative Splicing Generates Functionally Related Isoformsa

The Alzheimers βA4‐amyloid protein precursor (APP) and the APP‐like proteins (APLPs) are transmembrane glycoproteins with a similar modular domain structure. APP exists in 8 isoforms generated by alternative splicing of exons 7, 8, and 15, of which the L‐APP mRNAs lacking exon 15 are ubiquitously expressed in rat tissues but not in neurons. Rat APLP2, the nearest relative of APP, is similarly expressed in 4 different isoforms due to alternative splicing of inserts encoding a Kunitz protease inhibitor domain (KPI, homologous to exon 7 of APP) and a divergent region of 12 amino acids on the NH2‐terminal side of the transmembrane domain (12 aa exon). KPI‐APLP2 transcripts are highly expressed in neurons, in contrast to KPI‐APPs, while L‐APLP2 mRNA isoforms lacking the 12 aa exon are predominantly expressed in non‐neuronal rat tissues, similar to L‐APPs. Further examination of the divergent domains in APP and APLP2 harboring the similarly alternatively spliced APP exon 15 and the 12 aa exon of APLP2 revealed some structural similarities of the amino acid sequences and the predicted secondary structures. In both L‐APLP2 and L‐APP, a putative xylosyl‐transferase recognition site for chondroitin sulfate glycosaminoglycan attachment is present that is interrupted in APP and APLP2 isoforms expressing APP exon 15 or the 12 aa exon of APLP2. Thus, a related function of the divergent domains and the corresponding alternatively spliced APP and APLP2 isoforms in regulation of the binding properties of the ectodomain is suggested. Additionally, β‐secretase cleavage of APP might be sterically hindered selectively in proteoglycan L‐APP but not in APP lacking the proteoglycan attachment site. Neurons which have a uniquely low portion of L‐APP and high content of APP might therefore be especially susceptible to βA4‐protein liberation. This could explain the selective vulnerability of neurons that is observed in Alzheimers disease.

Cerebral changes and cerebrospinal fluid beta-amyloid in Alzheimer's disease : a study with quantitative magnetic resonance imaging

Pathological and biochemical studies indicate that β-amyloid (βA4) deposition is a hallmark in the pathogenesis of Alzheimers disease (AD).1–4 Neuroimaging studies demonstrate that the respective cerebral changes primarily strike the temporal lobe and the amygdala-hippocampus complex and may be reliably assessed using quantitative magnetic resonance imaging (MRI).5,6 Therefore one may expect that reduced βA4-levels are significantly correlated with measures of the temporal lobe rather than global cerebral atrophy in AD patients. To test this hypothesis in a clinical study, cerebrospinal fluid concentrations of total β A4 and its major C-terminal variations β A4 1–40 and β A4 1–42 were compared with cerebral changes as assessed by quantitative magnetic resonance imaging (MRI). Significantly (P < 0.05) reduced β A4 1–40 and β A4 1–42 levels were found in the AD patients (17 female; six male; AD/NINCDS-ADRDA-criteria)7 in comparison to the patients with major depression (seven female; two male; DSM-III-R).8 Within the AD group, βA4 and β xA4 1–42 levels were significantly correlated with the volume of the temporal lobes (r = 0.46 and r = 0.48, respectively) but none of the other volumetric measures. These findings indicate that changes in cerebral β A4 levels contribute to temporal lobe atrophy in AD and support the possibility that βA4 is central to the etiology of AD.

Transforming growth factor β mediates increase of mature transmembrane amyloid precursor protein in microglial cells

By using the immortalized microglial cell line BV‐2, we show that the high expression of theβA4 amyloid precursor protein (APP), its biogenesis and metabolism is modulated by TGF β, a cytokine with immunosuppressive activity, and by the microglia‐stimulating agent LPS. TGFβ induces accumulation of cellular mature APP, the putative precursor of the amyloid subunit of Alzheimers disease. LPS leads to an increase in cellular immature, non‐amyloidogenic APP and secretion of also non‐amyloidogenic APP fragments. We also demonstrate a functional involvement of ECM molecules in the regulation of microglial APP expression at mRNA and protein level by TGFβ and LPS.

Complete nucleotide and deduced amino acid sequence of rat amyloid protein precusor-like protein 2 (APLP2APPH): Two amino acids length difference to human and murine homologues

We present the cDNA sequence of the rat amyloid precursor-like protein 2 (APLP2) comprising the complete coding sequence of 765 amino acid residues. By alternative splicing of two exons, transcripts encoding for 753, 709 and 697 amino acids are also generated. The derived amino acid sequence displays a sequence identity to human APLP2 of approx. 92% and to murine CDEI binding protein of approx. 95%, but differs from both by a deletion of 2 amino acids and an insertion of 4 amino acids within the acidic domain.

Expression of the APP Gene Family in Brain Cells, Brain Development and Aging

The Alzheimers beta A4-amyloid protein precursor (APP) and the APP-like proteins (APLPs) are transmembrane glycoproteins with a similar modular domain structure. Alternatively spliced exons found in both genes comprise a Kunitz protease inhibitor domain encoding exon, and another exon within the divergent regions adjacent to the transmembrane domain, i.e. exon 15 of the APP gene and an exon encoding 12 residues in APLP2. Omission of the latter exons in L-APP and L-APLP2 isoforms, respectively, generates a functional recognition sequence for xylosyltransferase-mediated addition of glycosaminoglycans and proteoglycan formation. In this paper, we summarize our analyses of the regulated expression of these alternatively spliced exons in APP and APLP2 in primary cultured rat brain cells, rat brain development and aging. In conjunction with additional data for the human brain, these data provide important clues for understanding the functional significance of alternative splicing and glycosylation in APP biology. On the basis of recent results showing a higher amyloidogenicity of exon 15 encoding APP than L-APP isoforms, we further discuss the potential significance of the low levels of L-APP in neurons for the susceptibility of the brain towards Alzheimers disease.

Regulation of APP Expression, Biogenesis and Metabolism by Extracellular Matrix and Cytokinesa

We have identified and characterized the ligand binding properties of the Alzheimers disease (AD) βA4 amyloid protein precursor (APP), mapped the APP ligand binding sites and analyzed the regulation of APP expression, biogenesis and metabolism by components of the extracellular matrix (ECM) and cytokines.

Expression of L‐APP mRNA in Brain Cellsa

Several reports addressed the issue of how the alternative splicing of exon 7 and 8 in the APP pre‐mRNA is regulated in different tissues. Of special interest here was the potential involvement of exon 7 containing APP splice isoforms, since this exon codes for a serine protease inhibitor and is therefore of putative relevance for amyloidogenic catabolism of the precursor protein. The recent identification of a third alternative splice site in close proximity to the βA4‐amyloid portion in the APP gene which may also increase APP amyloidogenicity, allowed us to investigate its regulation in cells of the central nervous system. With our assay, we were able to resolve six different APP isoforms of the eight potential isoforms which can be generated from the three alternatively spliced exons 7, 8, and 15. We demonstrate here that, in addition to rat brain microglia cells, astrocyte‐enriched cultures also skip the novel alternative 3′‐splice site in front of exon 15, generating L‐APP mRNA. Neurons are the only cells in the central nervous system which seem to use the 3′‐splice site of intron 14 nearly 100%. Interestingly, this very 3′‐splice site is the only one present in the APP gene that completely matches the consensus sequence for the branchpoint sequence proposed for introns. We would therefore suggest that neurons lack a specific splicing factor which inhibits the use of the rather strong 3′‐splice site in front of exon 15. It remains to be shown whether this is also the case for neurons in Alzheimers disease.

APP Expression in Primary Neuronal Cell Cultures fromP6 Mice during in vitro Differentiation

Primary neuronal cell cultures from P6 mice were investigated in order to study amyloid protein precursor (APP) gene expression in differentiating neurons. Cerebellar granule cells which strongly express APP 695 allowed the identification of three distinct isoforms of neuronal APP 695. The high-molecular-weight form of APP 695 is sialylated. The expression pattern of neuronal APP 695 changes during in vitro differentiation. Sialylated forms become more abundant upon longer cultivation time. The secreted forms of sialylated, neuronal APP 695 are shown to comigrate with APP isolated from cerebrospinal fluid. We suggest that the different sialylation states of APP 695 may reflect the modulation of cell-cell and cell-substrate interactions during in vitro differentiation and regeneration.

Interaction of the Presenilins with the Amyloid Precursor Protein (APP).

The genes encoding presenilin-1 (PS1) and presenilin-2 (PS2) were identified as the genes that harbour mutations that cause more than 60% of early onset familial Alzheimers disease cases (FAD) (1-3). So far, more than 40 missense mutations have been described for presenilin-1 and two have been found in the gene coding for presenilin-2 (reviewed in refs. 4 and 5). Carriers of mutated presenilin genes develop in their brain neuropathological changes characteristic of Alzheimers disease including the deposition of amyloid Aβ peptide. The latter is released from its cognate amyloid precursor protein (APP) by a two-step proteolytic conversion: first, proteolysis of APP by β-secretase, which releases the N-terminus of Aβ, and second, conversion of the remaining fragment by γ-secretase, which cleaves within the predicted transmembrane region of APP. This releases the C-terminus of Aβ, which may end either at position 40 or, to a lesser extent, at position 42 (reviewed in ref. 6). The latter species, Aβ(1-42), is more prone to aggregation and deposition than Aβ(1-40) and is produced at higher levels in the brains and primary fibroblasts of FAD patients carrying PS missense mutations (7). The same result was obtained when cultured cells transfected with mutated PS1 orPS2, or transgenic mice harboring missense PS1 were analyzed for the production of Aβ(1-42): in every case increased amounts of the longer Aβ(1-42) species were observed (8-10). The mechanisms by which mutations in the PS genes affect the proteolytic processing of APP by γ-secretase have not been resolved in detail. There are two possibilities by which the normal processing of APP may be disturbed: either mutations in the presenilins affect APP metabolism in an indirect way by modulation of proteases or interaction with proteins involved in APP intracellular routing, or presenilins may modulate APP processing directly through physical interactions with APP. Such a direct interaction between presenilins and APP was first demonstrated by us for PS2 (11). Later on, formation of stable complexes with APP was reported not only for PS2 but also for PS1 (12,13,13a).