Restricted Location of PSEN2/γ-Secretase Determines Substrate Specificity and Generates an Intracellular Aß Pool.

γ-Secretases are a family of intramembrane-cleaving proteases involved in various signaling pathways and diseases, including Alzheimer's disease (AD). Cells co-express differing γ-secretase complexes, including two homologous presenilins (PSENs). We examined the significance of this heterogeneity and identified a unique motif in PSEN2 that directs this γ-secretase to late endosomes/lysosomes via a phosphorylation-dependent interaction with the AP-1 adaptor complex. Accordingly, PSEN2 selectively cleaves late endosomal/lysosomal localized substrates and generates the prominent pool of intracellular Aß that contains longer Aß; familial AD (FAD)-associated mutations in PSEN2 increased the levels of longer Aß further. Moreover, a subset of FAD mutants in PSEN1, normally more broadly distributed in the cell, phenocopies PSEN2 and shifts its localization to late endosomes/lysosomes. Thus, localization of γ-secretases determines substrate specificity, while FAD-causing mutations strongly enhance accumulation of aggregation-prone Aß42 in intracellular acidic compartments. The findings reveal potentially important roles for specific intracellular, localized reactions contributing to AD pathogenesis.

An Administrator's Perspective on the Organization of Physical Activity for Older Adults in Long-Term Care Facilities.

BACKGROUND: The positive influence of physical activity (PA) on health is well documented. Even at old age, PA remains useful but participation in PA decreases with age. In long-term care facilities (LTCFs), PA appears to be reduced to a bare minimum. Because administrators have a key role in developing the care policy of LTCFs, it is important that they support the organization of PA in LTCFs. OBJECTIVE: The main objective of this mixed-method study was to identify motivators and barriers for organizing PA in LTCFs according to administrators. A secondary goal was to examine the knowledge of the World Health Organization (WHO) guidelines regarding PA and to reveal potential motivators and barriers for the implementation of the guidelines. METHODS: First, 24 administrators completed semistructured interviews. Data were analyzed using the deductive approach of qualitative content analysis. The obtained motivators and barriers were categorized on 3 different levels (intrapersonal, interpersonal, and community) according to the socioecological model by 2 independent reviewers; conflicts were resolved with a third researcher. Next, 127 administrators of Flemish (Belgium) LTCFs completed an online questionnaire survey containing open-ended, unique, and multiple choice questions regarding the LTCFs, PA, and the WHO guidelines, as well as statements (scored on a 5-point Likert scale) regarding perceived motivators and barriers for organizing PA sessions in the LTCF. RESULTS: In the qualitative component, the administrators reported 31 motivators and 24 barriers for organizing PA in the LTCF. In the survey, maintaining or enhancing general health of the residents (98%) and improving the psychological well-being of the residents were marked as key motivators at the intrapersonal level. The administrators (97%) were convinced that PA is a useful way to spend time for LTCF residents. Encouraging social contact (94%) and countering loneliness (86%) are the motivators cited at the interpersonal level. At the community level, the infrastructure of the facility (91%) and adequate and sufficient material (88%) are the main motivators. The barriers that were presented to the participants were scored as less important. The majority of the administrators (83%) are not familiar with the WHO guidelines for PA; 70% of the participants believe that the guidelines are useful, but only 40% is convinced that it is realistic to implement the guidelines in an LTCF. CONCLUSIONS: This study described different motivators and barriers for administrators to organize PA in LTCFs. Contrary to other studies, lack of staff, lack of adequate equipment, and lack of financial resources were rejected as potential barriers for organizing PA. Despite the fact that administrators were not familiar with the WHO guidelines for PA, they believed that the guidelines are useful. The participants reported several barriers for implementation of the guidelines. Administrators of LTCFs are motivated to implement the guidelines if PA can be integrated in daily activities and education of LTCF staff regarding PA is provided.

Physiotherapists' perceived motivators and barriers for organizing physical activity for older long-term care facility residents.

BACKGROUND: Information regarding factors that hinder or stimulate older adults in long-term care facilities (LTCF) for being physically active is available in the literature, but much less is known regarding perceived motivators and barriers among physiotherapists (PTs) to organize physical activity (PA) in LTCF. OBJECTIVE: The main purpose of this study was to examine factors influencing PTs to organize PA in LTCF for older adults. A secondary goal was to examine the PTs' knowledge about and their barriers at the PA guidelines for older adults of the World Health Organization (WHO). METHODS: A mixed qualitative and quantitative study was carried out using semistructured interviews (n = 24) followed by an online survey (n = 254). As a frame the social-ecological model (McLeroy) was used, distinguishing factors at the intrapersonal, interpersonal, and community level. RESULTS: In the qualitative component the PTs reported 41 motivators and 35 barriers for organizing PA in LTCF. The survey revealed that although the majority of the respondents (71%) are convinced of the usefulness of PA in LTCF, 84% are not familiar with the WHO-guidelines. Seventy-five percent of the respondents believe that the WHO-guidelines are not feasible for LTCF-residents. The strongest motivators on the intrapersonal level were maintaining the independence of the residents (98%), reducing the risk of falling (98%), and improving the physical (93%) and psychological (90%) wellbeing of LTCF-residents. The social interaction among LTCF-residents (91%) during PA was the strongest motivator on the interpersonal level. Motivators on the community level are the belief that PA is the basis of their physiotherapeutic work (89%) and that offering varied activities avoids PA becoming monotonous (71%). Barriers on the intra- and interpersonal level were of less influence. On the community level, they felt hindered to organize PA because of lack of time (38%) and the overload of paperwork (33%). CONCLUSIONS: This study described different motivators and barriers for PTs to organize PA in LTCFs. The majority does not know the WHO guidelines regarding the amount of PA for adults aged 65 and over. Although they agree that the guidelines are useful, they believe the guidelines are not feasible. There is a need for a multifactorial strategy that acts on different determinants in order to stimulate PA in LTCF.

Motivators and barriers for physical activity in older adults with osteoporosis.

BACKGROUND AND PURPOSE: Although physical activity (PA) is an important tool to counter osteoporosis, too few older patients with osteoporosis (OPWO) engage in PA. Little is known about specific motivators for and barriers to PA in OPWO, hindering the development of targeted PA promotion campaigns for these persons. Therefore, the main objective of this study was to identify motivators for and barriers to PA specifically in OPWO. METHODS: This qualitative study identified specific motivators for and barriers to PA in OPWO through 2 different methods: focus groups with professionals and in-depth interviews with OPWO. RESULTS: The OPWO tended to give a broad interpretation of what they considered as PA (practicing sports, physical work, and performing household activities), whereas the professionals seemed to mainly focus on (therapeutic) exercise as PA. Fifteen different motivators and 18 barriers have been identified. Among others, health improvement, social contact, habit, feeling good, and receiving medical advice from a medical doctor were motivators. Pain, fear of falling, bad weather, lack of interest, and caring for an ill partner were barriers to PA. For some older respondents, osteoporosis acted as a trigger for PA, and for others it was a barrier. CONCLUSIONS: This study emphasizes the importance for health care professionals to give personalized PA advice regarding the nature and frequency of PA that is safe and beneficial for osteoporosis. It stands to reason that the information about PA needs to be clear and consistent. Furthermore, it is quintessential to mention that it can take some time to adapt to physical exercise and to experience the beneficial effects, because pain sensations during the first PA sessions can be perceived as barriers to OPWO. Misconceptions or barriers to PA should be countered by assessing motivators for and barriers to PA by the health care professional together with the older client so that barriers can be eliminated and motivators can be strengthened. Physical activity education should involve not only the OPWO but also their relatives, friends, and important peers. Different social aspects of PA and the encouragements from peers are stimulating for older adults to initiate and to continue PA. The results of our study can constitute a starting point for further research to identify the motivators for and barriers to PA with the highest impact on PA behavior in OPWO, thus enabling evidence-based PA promotion campaigns for this patient group.

Rer1p maintains ciliary length and signaling by regulating γ-secretase activity and Foxj1a levels.

Cilia project from the surface of most vertebrate cells and are important for several physiological and developmental processes. Ciliary defects are linked to a variety of human diseases, named ciliopathies, underscoring the importance of understanding signaling pathways involved in cilia formation and maintenance. In this paper, we identified Rer1p as the first endoplasmic reticulum/cis-Golgi-localized membrane protein involved in ciliogenesis. Rer1p, a protein quality control receptor, was highly expressed in zebrafish ciliated organs and regulated ciliary structure and function. Both in zebrafish and mammalian cells, loss of Rer1p resulted in the shortening of cilium and impairment of its motile or sensory function, which was reflected by hearing, vision, and left-right asymmetry defects as well as decreased Hedgehog signaling. We further demonstrate that Rer1p depletion reduced ciliary length and function by increasing γ-secretase complex assembly and activity and, consequently, enhancing Notch signaling as well as reducing Foxj1a expression.

Lysosomal calcium homeostasis defects, not proton pump defects, cause endo-lysosomal dysfunction in PSEN-deficient cells.

Presenilin (PSEN) deficiency is accompanied by accumulation of endosomes and autophagosomes, likely caused by impaired endo-lysosomal fusion. Recently, Lee et al. (2010. Cell. doi: http://dx.doi.org/10.1016/j.cell.2010.05.008) attributed this phenomenon to PSEN1 enabling the transport of mature V0a1 subunits of the vacuolar ATPase (V-ATPase) to lysosomes. In their view, PSEN1 mediates the N-glycosylation of V0a1 in the endoplasmic reticulum (ER); consequently, PSEN deficiency prevents V0a1 glycosylation, compromising the delivery of unglycosylated V0a1 to lysosomes, ultimately impairing V-ATPase function and lysosomal acidification. We show here that N-glycosylation is not a prerequisite for proper targeting and function of this V-ATPase subunit both in vitro and in vivo in Drosophila melanogaster. We conclude that endo-lysosomal dysfunction in PSEN(-/-) cells is not a consequence of failed N-glycosylation of V0a1, or compromised lysosomal acidification. Instead, lysosomal calcium storage/release is significantly altered in PSEN(-/-) cells and neurons, thus providing an alternative hypothesis that accounts for the impaired lysosomal fusion capacity and accumulation of endomembranes that accompanies PSEN deficiency.

ARF6-mediated endosomal transport of Telencephalin affects dendritic filopodia-to-spine maturation.

Dendritic filopodia are dynamic structures thought to be the precursors of spines during synapse development. Morphological maturation to spines is associated with the stabilization and strengthening of synapses, and can be altered in various neurological disorders. Telencephalin (TLN/intercellular adhesion molecule-5 (ICAM5)) localizes to dendritic filopodia, where it facilitates their formation/maintenance, thereby slowing spine morphogenesis. As spines are largely devoid of TLN, its exclusion from the filopodia surface appears to be required in this maturation process. Using HeLa cells and primary hippocampal neurons, we demonstrate that surface removal of TLN involves internalization events mediated by the small GTPase ADP-ribosylation factor 6 (ARF6), and its activator EFA6A. This endocytosis of TLN affects filopodia-to-spine transition, and requires Rac1-mediated dephosphorylation/release of actin-binding ERM proteins from TLN. At the somato-dendritic surface, TLN and EFA6A are confined to distinct, flotillin-positive membrane subdomains. The co-distribution of TLN with this lipid raft marker also persists during its endosomal targeting to CD63-positive late endosomes. This suggests a specific microenvironment facilitating ARF6-mediated mobilization of TLN that contributes to promotion of dendritic spine development.

Peptides based on the presenilin-APP binding domain inhibit APP processing and Aß production through interfering with the APP transmembrane domain.

Presenilins (PSENs) form the catalytic component of the γ-secretase complex, responsible for intramembrane proteolysis of amyloid precursor protein (APP) and Notch, among many other membrane proteins. Previously, we identified a PSEN1-binding domain in APP, encompassing half of the transmembrane domain following the amyloid ß (Aß) sequence. Based on this, we designed peptides mimicking this interaction domain with the aim to selectively block APP processing and Aß generation through interfering with enzyme-substrate binding. We identified a peptide sequence that, when fused to a virally derived translocation peptide, significantly lowered Aß production (IC(50): 317 nM) in cell-free and cell-based assays using APP-carboxy terminal fragment as a direct γ-secretase substrate. Being derived from the APP sequence, this inhibitory peptide did not affect NotchΔE γ-cleavage, illustrating specificity and potential therapeutic value. In cell-based assays, the peptide strongly suppressed APP shedding, demonstrating that it exerts the inhibitory effect already upstream of γ-secretase, most likely through steric hindrance.

Motivators and barriers for physical activity in the oldest old: a systematic review.

Worldwide, people engage insufficiently in physical activity, particularly subjects aged 80 years and over. For optimal life-style campaigns, knowledge of motivators and barriers for physical activity is mandatory. Given their specific needs, it is conceivable that these would be different for the oldest old compared to younger subjects. Pubmed, Web of Science and Psychinfo were systematically screened for articles reporting motivators and barriers for physical activity. Papers were excluded if data regarding elderly aged >79 years were absent. Forty-four relevant articles were included, involving a total of 28,583 subjects. Sixty one motivators and 59 barriers for physical activity in the elderly were identified, including those who are relevant for persons aged 80 years and over. Based on the results of our literature review, we recommend that when promoting physical activity in the oldest old, special attention is paid to the health benefits of physical activity, to the subject's fears, individual preferences and social support, and to constraints related to the physical environment. However, no studies were found exclusively describing people aged 80 years and over, and future research is necessary to differentiate the barriers or motivators that are specific for the oldest old from those of younger elderly.

Rer1p competes with APH-1 for binding to nicastrin and regulates gamma-secretase complex assembly in the early secretory pathway.

The gamma-secretase complex, consisting of presenilin, nicastrin, presenilin enhancer-2 (PEN-2), and anterior pharynx defective-1 (APH-1) cleaves type I integral membrane proteins like amyloid precursor protein and Notch in a process of regulated intramembrane proteolysis. The regulatory mechanisms governing the multistep assembly of this "proteasome of the membrane" are unknown. We characterize a new interaction partner of nicastrin, the retrieval receptor Rer1p. Rer1p binds preferentially immature nicastrin via polar residues within its transmembrane domain that are also critical for interaction with APH-1. Absence of APH-1 substantially increased binding of nicastrin to Rer1p, demonstrating the competitive nature of these interactions. Moreover, Rer1p expression levels control the formation of gamma-secretase subcomplexes and, concomitantly, total cellular gamma-secretase activity. We identify Rer1p as a novel limiting factor that negatively regulates gamma-secretase complex assembly by competing with APH-1 during active recycling between the endoplasmic reticulum (ER) and Golgi. We conclude that total cellular gamma-secretase activity is restrained by a secondary ER control system that provides a potential therapeutic value.

Presenilin-1 maintains a nine-transmembrane topology throughout the secretory pathway.

Presenilin-1 is a polytopic membrane protein that assembles with nicastrin, PEN-2, and APH-1 into an active gamma-secretase complex required for intramembrane proteolysis of type I transmembrane proteins. Although essential for a correct understanding of structure-function relationships, its exact topology remains an issue of strong controversy. We revisited presenilin-1 topology by inserting glycosylation consensus sequences in human PS1 and expressing the obtained mutants in a presenilin-1 and 2 knock-out background. Based on the glycosylation status of these variants we provide evidence that presenilin-1 traffics through the Golgi after a conformational change induced by complex assembly. Based on our glycosylation variants of presenilin-1 we hypothesize that complex assembly occurs during transport between the endoplasmic reticulum and the Golgi apparatus. Furthermore, our data indicate that presenilin-1 has a nine-transmembrane domain topology with the COOH terminus exposed to the lumen/extracellular surface. This topology is independently underscored by lysine mutagenesis, cell surface biotinylation, and cysteine derivation strategies and is compatible with the different physiological functions assigned to presenilin-1.

Coordinated and widespread expression of gamma-secretase in vivo: evidence for size and molecular heterogeneity.

Gamma-secretase is a high molecular weight protein complex composed of four subunits, namely, presenilin (PS; 1 or 2), nicastrin, anterior pharynx defective-1 (Aph-1; A or B), and presenilin enhancer-2 (Pen-2), and is responsible for the cleavage of a number of type-1 transmembrane proteins. A fundamental question is whether different gamma-secretase complexes exist in vivo. We demonstrate here by in situ hybridization and by Northern and Western blotting that the gamma-secretase components are widely distributed in all tissues investigated. The expression of the different subunits seems tightly coregulated. However, some variation in the expression of the Aph-1 proteins is observed, Aph-1A being more general and abundantly distributed than Aph-1B. The previously uncharacterized rodent-specific Aph-1C mRNA is highly expressed in the kidney and testis but not in brain or other tissues, indicating some tissue specificity for the Aph-1 component of the gamma-secretase complex. Blue-native electrophoresis revealed size heterogeneity of the mature gamma-secretase complex in various tissues. Using co-immunoprecipitations and blue-native electrophoresis at endogenous protein levels, we find evidence that several independent gamma-secretase complexes can coexist in the same cell type. In conclusion, our results suggest that gamma-secretase is a heterogeneous family of protein complexes widely expressed in the adult organism.

Presenilin 1 mediates the turnover of telencephalin in hippocampal neurons via an autophagic degradative pathway.

Presenilin 1 (PS1) interacts with telencephalin (TLN) and the amyloid precursor protein via their transmembrane domain (Annaert, W.G., C. Esselens, V. Baert, C. Boeve, G. Snellings, P. Cupers, K. Craessaerts, and B. De Strooper. 2001. Neuron. 32:579-589). Here, we demonstrate that TLN is not a substrate for gamma-secretase cleavage, but displays a prolonged half-life in PS1(-/-) hippocampal neurons. TLN accumulates in intracellular structures bearing characteristics of autophagic vacuoles including the presence of Apg12p and LC3. Importantly, the TLN accumulations are suppressed by adenoviral expression of wild-type, FAD-linked and D257A mutant PS1, indicating that this phenotype is independent from gamma-secretase activity. Cathepsin D deficiency also results in the localization of TLN to autophagic vacuoles. TLN mediates the uptake of microbeads concomitant with actin and PIP2 recruitment, indicating a phagocytic origin of TLN accumulations. Absence of endosomal/lysosomal proteins suggests that the TLN-positive vacuoles fail to fuse with endosomes/lysosomes, preventing their acidification and further degradation. Collectively, PS1 deficiency affects in a gamma-secretase-independent fashion the turnover of TLN through autophagic vacuoles, most likely by an impaired capability to fuse with lysosomes.