Willem Annaert

TMEM165 deficiency causes a congenital disorder of glycosylation.

Protein glycosylation is a complex process that depends not only on the activities of several enzymes and transporters but also on a subtle balance between vesicular Golgi trafficking, compartmental pH, and ion homeostasis. Through a combination of autozygosity mapping and expression analysis in two siblings with an abnormal serum-transferrin isoelectric focusing test (type 2) and a peculiar skeletal phenotype with epiphyseal, metaphyseal, and diaphyseal dysplasia, we identified TMEM165 (also named TPARL) as a gene involved in congenital disorders of glycosylation (CDG). The affected individuals are homozygous for a deep intronic splice mutation in TMEM165. In our cohort of unsolved CDG-II cases, we found another individual with the same mutation and two unrelated individuals with missense mutations in TMEM165. TMEM165 encodes a putative transmembrane 324 amino acid protein whose cellular functions are unknown. Using a siRNA strategy, we showed that TMEM165 deficiency causes Golgi glycosylation defects in HEK cells.

Presenilin-1-mediated retention of APP derivatives in early biosynthetic compartments

Processing of the amyloid precursor protein (APP) leads to the production of amyloid‐β (Aβ), the major component of extracellular plaques in the brains of Alzheimers disease (AD) patients. Presenilin‐1 (PS‐1) plays a key role in the final step of Aβ formation, the γ‐secretase cleavage. Previously, we showed that PS‐1 is retained in pre‐Golgi compartments by incorporation into COPI‐coated membranes of the vesicular tubular clusters (VTCs) between endoplasmic reticulum (ER) and Golgi complex. Here, we show that PS‐1 also mediates the retention of the β‐cleavage‐derived APP‐C‐terminal fragment (CTFβ) and/or Aβ in pre‐Golgi membranes. Overexpression of PS‐1 increased the percentage of CTFβ and/or Aβ in VTCs as well as their distribution to COPI‐coated VTC membranes. By contrast, overexpression of the dominant‐negative aspartate mutant PS‐1D257A or PS‐knockout decreased incorporation of these APP derivatives into COPI‐coated membranes. Sorting of APP derivatives to COPI‐coated VTC membranes was not depending on the APP cytosolic tail. In post‐Golgi compartments, PS‐1 expression enhanced the association of full‐length APP/APPs with endosomal compartments at the expense of plasma membrane‐bound APP. We conclude that PS‐1, in addition to its role in γ‐secretase cleavage, is also required for the subcellular routing of APP and its derivatives. Malfunctioning of PS‐1 in this role may have important consequences for the progress of AD.

Amyloid precursor family proteins are expressed by thymic and lymph node stromal cells but are not required for lymphocyte development

Pharmacological inhibitors that block amyloid precursor protein (APP) cleavage and the formation of senile plaques are under development for the treatment of familial Alzheimers disease. Unfortunately, many inhibitors that block γ-secretase-mediated cleavage of APP also have immunosuppressive side effects. In addition to APP, numerous other proteins undergo γ-secretase-mediated cleavage. In order to develop safer inhibitors, it is necessary to determine which of the γ-secretase substrates contribute to the immunosuppressive effects. Because APP family members are widely expressed and are reported to influence calcium flux, transcription and apoptosis, they could be important for normal lymphocyte maturation. We find that APP and amyloid precursor-like protein 2 are expressed by stromal cells of thymus and lymph nodes, but not by lymphocytes. Although signals provided by thymic stromal cells are critical for normal T cell differentiation, lymphocyte development proceeds unperturbed in mice deficient for these APP family members.

P3-398: Cysteine scanning mutagenesis as a tool for structure-function analysis of presenilin 1

the active site domain located in its catalytic subunit presenilin (PS), where an additional substrate binding site has been proposed. Objective: To identify sequence determinants in the PS active site domain possibly involved in -secretase substrate identification. To further characterize the PS active site domain, in particular the role of the conserved GxGD protease active site motif. Methods: Mutants of the PS active site domain were generated. The constructs were assessed for -secretase activity towards APP and Notch substrates in embryonic fibroblast cells derived from PS1/2-/knockout mice cells or in HEK293 cells. In addition, the mutants were tested for their rescuing activity of a Notch-signaling deficient C. elegans sel-12 mutant. Results and Conclusions: When the active site domain of PS1 located in transmembrane domains 6 and 7 was exchanged with that of the C. elegans sperm protein SPE-4, the most distant PS homologue, the chimeric protein, PS1/SPE-46/7, supported APP but not Notch processing. In addition, PS1/SPE-46/7 was strongly impaired in C. elegans Notch signaling in vivo. Mapping experiments identified a single amino acid at position (amino acid 383 in PS1) of the GxGD active site motif in transmembrane domain 7 of PS to be responsible for the observed defect in Notch processing and signaling. Our data thus implicate a role of the PS active site domain in APP/Notch substrate selectivity of -secretase. We have also generated mutants of G382 of PS1 to further assess the functional role of the GxGD motif. The progress on these studies will be presented.

Missorting of the Dendritic Cell Adhesion Molecule Telencephalin in Presenilin-Deficient Neurons

Presenilin deficiency abrogates γ-secretase cleavage, thereby preventing the release of the amyloid peptide from the β-clipped APP fragment or the notch intracellular domain. The presenilins are therefore implicated in such diverse processes as the neurodegeneration in Alzheimer’s disease and the regulation of Notch signaling. The question remains whether other type I transmembrane proteins or pathways are subject to presenilin-mediated proteolysis. While the two aspartate residues in presenilin likely are essential for a functional γ-secretase complex, the highly conserved carboxyterminal tail also appears to be important for presenilin function. Therefore we screened a double hybrid library with a short presenilin 1 carboxyterminal fragment as a bait. We identified a type I transmembrane protein that belongs to the family of intercellular cell adhesion molecules, telencephalin. Telencephalin is exclusively expressed in neurons of the telencephalic region, where it is abundantly expressed at the somatodendritic plasmamembrane. Functionally, telencephalin is, like notch, involved in neurite outgrowth. Interestingly, telencephalin is also involved in long-term potentiation. We confirmed the presenilin 1/telencephalin interaction using two other independent approaches. Finally, functional evidence for the physiological relevance of this interaction comes from the observation that telencephalin becomes mislocalized in differentiated hippocampal neurons. Further research into the telencephalin and presenilin 1 interaction may shed some light on the way presenilin 1 influences the processing of other type I transmembrane proteins, such as APP and Notch.

The Putative Role of Presenilins in the Transmembrane Domain Cleavage of Amyloid Precursor Protein and Other Integral Membrane Proteins

Missense mutations in the presenilin (PS)-1 and -2 genes are major causes of familial Alzheimer’s disease (AD), acting apparently in a dominant fashion (Levy-Lahad et al. 1995; Rogaev et al. 1995; Sherrington et al. 1995). Although the exact pathogenic mechanism underlying the disease process remains to be further elucidated, it is fairly established that almost all PS missense mutations affect the processing of the amyloid precursor protein (APP). The result is an increased secretion of the longer form of the amyloid peptide (Borchelt et al. 1996; Duff et al. 1996; Lemere et al. 1996; Tomita et al. 1997; Xia et al. 1997). This peptide constitutes the major component of the amyloid plaques in patients. Interestingly several of the PS mutations appear also to enhance the sensitivity of cells, and in particular neurons, to apoptotic stimuli (Deng et al. 1996; Guo et al. 1997; Janicki and Monteiro 1997; Vito et al. 1997; Wolozin et al. 1996). In principle both mechanisms could contribute to the pathogenesis of AD (Zhang et al. 1998). Insight into the biological functions of the PS in the cell is probably not only important for understanding the pathogenesis of the familial form of AD, but also of the sporadic form of AD. Interestingly, recent findings suggest that PS is involved in the proteolysis of the transmembrane domains of APP, Notch, APLP-1, and possibly Ire-1, and could therefore function as a molecular switch linking proteolysis to intracullular signaling (Annaert and De Strooper 1999).