Gopal Thinakaran

Familial Alzheimer's Disease–Linked Presenilin 1 Variants Elevate Aβ1–42/1–40 Ratio In Vitro and In Vivo

Mutations in the presenilin 1 (PS1) and presenilin 2 genes cosegregate with the majority of early-onset familial Alzheimers disease (FAD) pedigrees. We now document that the Abeta1-42(43)/Abeta1-40 ratio in the conditioned media of independent N2a cell lines expressing three FAD-linked PS1 variants is uniformly elevated relative to cells expressing similar levels of wild-type PS1. Similarly, the Abeta1-42(43)/Abeta1-40 ratio is elevated in the brains of young transgenic animals coexpressing a chimeric amyloid precursor protein (APP) and an FAD-linked PS1 variant compared with brains of transgenic mice expressing APP alone or transgenic mice coexpressing wild-type human PS1 and APP. These studies provide compelling support for the view that one mechanism by which these mutant PS1 cause AD is by increasing the extracellular concentration of Abeta peptides terminating at 42(43), species that foster Abeta deposition.

Endoproteolysis of Presenilin 1 and Accumulation of Processed Derivatives In Vivo

The majority of early-onset cases of familial Alzheimers disease (FAD) are linked to mutations in two related genes, PS1 and PS2, located on chromosome 14 and 1, respectively. Using two highly specific antibodies against nonoverlapping epitopes of the PS1-encoded polypeptide, termed presenilin 1 (PS1), we document that the preponderant PS1-related species that accumulate in cultured mammalian cells, and in the brains of rodents, primates, and humans are approximately 27-28 kDa N-terminal and approximately 16-17 kDa C-terminal derivatives. Notably, a FAD-linked PS1 variant that lacks exon 9 is not subject to endoproteolytic cleavage. In brains of transgenic mice expressing human PS1, approximately 17 kDa and approximately 27 kDa PS1 derivatives accumulate to saturable levels, and at approximately 1:1 stoichiometry, independent of transgene-derived mRNA. We conclude that PS1 is subject to endoproteolytic processing in vivo.

Effects of PS1 Deficiency on Membrane Protein Trafficking in Neurons

We have examined the trafficking and metabolism of the beta-amyloid precursor protein (APP), an APP homolog (APLP1), and TrkB in neurons that lack PS1. We report that PS1-deficient neurons fail to secrete Abeta, and that the rate of appearance of soluble APP derivatives in the conditioned medium is increased. Remarkably, carboxyl-terminal fragments (CTFs) derived from APP and APLP1 accumulate in PS1-deficient neurons. Hence, PS1 plays a role in promoting intramembrane cleavage and/or degradation of membrane-bound CTFs. Moreover, the maturation of TrkB and BDNF-inducible TrkB autophosphorylation is severely compromised in neurons lacking PS1. We conclude that PS1 plays an essential role in modulating trafficking and metabolism of a selected set of membrane and secretory proteins in neurons.

Evidence that levels of presenilins (PS1 and PS2) are coordinately regulated by competition for limiting cellular factors.

Mutations in two related genes, PS1and PS2, account for the majority of early onset cases of familial Alzheimer’s disease. PS1 and PS2 are homologous polytopic membrane proteins that are processed endoproteolytically into two fragments in vivo. In the present report we examine the fate of endogenous PS1 and PS2 after overexpression of human PS1 or PS2 in mouse N2a neuroblastoma cell lines and human PS1 in transgenic mice. Remarkably, in N2a cell lines and in brains of transgenic mice expressing human PS1, accumulation of human PS1 derivatives is accompanied by a compensatory, and highly selective, decrease in the steady-state levels of murine PS1 and PS2 derivatives. Similarly, the levels of murine PS1 derivatives are diminished in cultured cells overexpressing human PS2. To define the minimal sequence requirements for “replacement” we expressed familial Alzheimer’s disease-linked and experimental deletion variants of PS1. These studies revealed that compromised accumulation of murine PS1 and PS2 derivatives resulting from overexpression of human PS1 occurs in a manner independent of endoproteolytic cleavage. Our results are consistent with a model in which the abundance of PS1 and PS2 fragments is regulated coordinately by competition for limiting cellular factor(s).

Generation of APLP2 KO Mice and Early Postnatal Lethality in APLP2/APP Double KO Mice

Amyloid precursor protein (APP) is a member of a larger gene family including amyloid precursor-like proteins (APLP), APLP2 and APLP1. To examine the function of APLP2 in vivo, we generated APLP2 knockout (KO) mice. They are of normal size, fertile, and appear healthy up to 22 months of age. We observed no impaired axonal outgrowth of olfactory sensory neurons following bulbectomy, suggesting against an important role for APLP2 alone in this process. Because APLP2 and APP are highly homologous and may serve similar functions in vivo, we generated mice with targeted APLP2 and APP alleles. Approximately 80% of double KO mice die within the first week after birth, suggesting that APLP2 and APP are required for early postnatal development. The surviving approximately 20% of double KO mice are 20-30% reduced in weight and show difficulty in righting, ataxia, spinning behavior, and a head tilt, suggesting a deficit in balance and/or strength. Adult double KO mice mate poorly, despite apparent normal ovarian and testicular development. Otherwise, double KO mice appear healthy up to 13 months of age. We conclude, that APLP2 and APP can substitute for each other functionally.

COMPARATIVE EVALUATION OF SYNAPTOPHYSIN-BASED METHODS FOR QUANTIFICATION OF SYNAPSES

SummaryDevelopment, ageing, and a variety of neurological disorders are characterized by selective alterations in specific populations of nerve cells which are, in turn, associated with changes in the numbers of synapses in the target fields of these neurons. To begin to delineate the significance of changes in synapses in development, ageing, and disease, it is first essential to quantify the number of synapses in defined regions of the CNS. In the past, investigators have used EM methods to assess synapse numbers or density, but these approaches are costly, labour intensive, and technically difficult, particularly in autopsy material. To begin to define reliable strategies useful for studies of both animals and humans, we used three techniques to measure synaptophysin-immunoreactivity in rat brain. The levels of synaptophysin protein were determined by Western blots of five hippocampal subregions; the intensity of synaptophysin-immunoreactivity in dentate gyrus and stratum oriens was determined by optical densitometry of immunocytochemically stained sections; and the total number of synaptophysin-immunoreactivity presynaptic boutons in dentate gyrus and stratum oriens was assessed by unbiased stereology. Each approach has advantages and disadvantages. Western blotting is the least time-consuming of the three methods and allows simultaneous processing of multiple samples. In systematically sampled histological sections, both densitometry and stereology allow precise definition of the region of interest, and the sterological optical disector method allows quantitation of the numbers of synaptophysin-immunoreactive boutons. Stereology was the only method that clearly demonstrated greater synaptophysin-immunoreactivity in the dentate gyrus as compared to stratum oriens. The use of systematic sampling and the disector technique offer a high degree of anatomical resolution (lacking in Western blot methods) and has quantitative advantage over the greyscale-based density approach. Thus, at present, stereology is the most useful method for estimating synaptic numbers in defined regions of the brain.

Endoproteolytic Processing and Stabilization of Wild-type and Mutant Presenilin

Presenilin 1 (PS1), mutated in pedigrees of early-onset familial Alzheimer’s disease, is a polytopic integral membrane protein that is endoproteolytically cleaved into 27-kDa N-terminal and 17-kDa C-terminal fragments. Although these fragments are the principal PS1 species found in normal mammalian brain, the role of endoproteolysis in the maturation of PS1 has been unclear. The present study, which uses stably transfected mouse neuroblastoma N2a cells, demonstrates that full-length polypeptides, derived from either wild-type or A246E FAD-mutant human (hu) PS1, are relatively short-lived (t½ 1.5 h) proteins that give rise to the N- and C-terminal PS1 fragments, which are more stable (t½ ∼ 24 h). N-terminal fragments, generated artificially by engineering a stop codon at amino acid 306 (PS1–306) of wild-type huPS1, were short-lived, whereas an FAD-linked variant that lacked exon 9 (ΔE9) and was not endoproteolytically cleaved exhibited a long half-life. These observations suggest that endoproteolytic cleavage and stability are not linked, leading us to propose a model in which wild-type full-length huPS1 molecules are first stabilized then subsequently endoproteolytically cleaved to generate the N- and C-terminal fragments. These fragments appear to represent the mature and functional forms of wild-type huPS1.

Presenilin-1 Regulates Intracellular Trafficking and Cell Surface Delivery of β-Amyloid Precursor Protein

Presenilins (PS1/PS2) play a critical role in proteolysis of β-amyloid precursor protein (βAPP) to generate β-amyloid, a peptide important in the pathogenesis of Alzheimers disease. Nevertheless, several regulatory functions of PS1 have also been reported. Here we demonstrate, in neuroblastoma cells, that PS1 regulates the biogenesis of βAPP-containing vesicles from thetrans-Golgi network and the endoplasmic reticulum. PS1 deficiency or the expression of loss-of-function variants leads to robust vesicle formation, concomitant with increased maturation and/or cell surface accumulation of βAPP. In contrast, release of vesicles containing βAPP is impaired in familial Alzheimers disease (FAD)-linked PS1 mutant cells, resulting in reduced βAPP delivery to the cell surface. Moreover, diminution of surface βAPP is profound at axonal terminals in neurons expressing a PS1 FAD variant. These results suggest that PS1 regulation of βAPP trafficking may represent an alternative mechanism by which FAD-linked PS1 variants modulate βAPP processing.

Multiple Effects of Aspartate Mutant Presenilin 1 on the Processing and Trafficking of Amyloid Precursor Protein

PS1 deficiency and expression of PS1 with substitutions of two conserved transmembrane aspartate residues (“PS1 aspartate variants”) leads to the reduction of Aβ peptide secretion and the accumulation of amyloid precursor protein (APP) C-terminal fragments. To define the nature of the “dominant negative” effect of the PS1 aspartate variants, we stably expressed PS1 harboring aspartate to alanine substitutions at codons 257 (D257A) or 385 (D385A), singly or in combination (D257A/D385A), in mouse neuroblastoma, N2a cells. Expression of the PS1 aspartate variants resulted in marked accumulation of intracellular and cell surface APP C-terminal fragments. While expression of the D385A PS1 variant reduced the levels of secreted Aβ peptides, we now show that neither the PS1 D257A nor D257A/D385A variants impair Aβ production. Surprisingly, the stability of both immature and mature forms of APP is dramatically elevated in cells expressing PS1 aspartate variants, commensurate with an increase in the cell surface levels of APP. These findings lead us to conclude that the stability and trafficking of APP can be profoundly modulated by coexpression of PS1 with mutations at aspartate 257 and aspartate 385.

Ectodomain phosphorylation of beta-amyloid precursor protein at two distinct cellular locations.

The β-amyloid precursor protein (βAPP) is a transmembrane protein that is exclusively phosphorylated on serine residues within its ectodomain. To identify the cellular site of βAPP phosphorylation, we took advantage of an antibody that specifically detects the free C terminus of β-secretase-cleaved βAPP containing the Swedish missense mutation (APPssw-β). This antibody previously established the cellular location of the β-secretase cleavage of Swedish βAPP as a post-Golgi secretory compartment (Haass, C., Lemere, C., Capell, A., Citron, M., Seubert, P., Schenk, D., Lannfelt, L., and Selkoe, D. J. (1995) Nature Med. 1, 1291-1296). We have now localized the selective ectodomain phosphorylation of βAPP to the same compartment. Moreover, the phosphorylation sites of βAPP were identified at Ser198 and Ser206 of βAPP695 by tryptic peptide mapping, mass spectrometry, and site-directed mutagenesis. Intracellular phosphorylation of βAPP was inhibited by Brefeldin A and by incubating cells at 20°C, thus excluding phosphorylation in the endoplasmic reticulum or trans-Golgi network. Ectodomain phosphorylation within a post-Golgi compartment occurred not only with mutant Swedish βAPP, but also with wild type βAPP. In addition to phosphorylation within a post-Golgi compartment, βAPP was also found to undergo phosphorylation at the cell surface by an ectoprotein kinase. Therefore, this study revealed two distinct cellular locations for βAPP phosphorylation.