Assembly of γ-secretase occurs through stable dimers after exit from the endoplasmic reticulum.

γ-Secretase affects many physiological processes through targeting >100 substrates; malfunctioning links γ-secretase to cancer and Alzheimer's disease. The spatiotemporal regulation of its stoichiometric assembly remains unresolved. Fractionation, biochemical assays, and imaging support prior formation of stable dimers in the ER, which, after ER exit, assemble into full complexes. In vitro ER budding shows that none of the subunits is required for the exit of others. However, knockout of any subunit leads to the accumulation of incomplete subcomplexes in COPII vesicles. Mutating a DPE motif in presenilin 1 (PSEN1) abrogates ER exit of PSEN1 and PEN-2 but not nicastrin. We explain this by the preferential sorting of PSEN1 and nicastrin through Sec24A and Sec24C/D, respectively, arguing against full assembly before ER exit. Thus, dimeric subcomplexes aided by Sec24 paralog selectivity support a stepwise assembly of γ-secretase, controlling final levels in post-Golgi compartments.

Superparamagnetic Nanoparticles for Lysosome Isolation to Identify Spatial Alterations in Lysosomal Protein and Lipid Composition.

Lysosomes are dynamic organelles that serve as regulatory hubs in cellular homeostasis. Changes in lysosome morphology, composition, and turnover are typically linked to disease. These characteristics make enrichment protocols based on biophysical parameters challenging. However, organelle enrichment methods are essential to facilitate their biomolecular analysis. We describe the synthesis and use of superparamagnetic iron oxide nanoparticles (SPIONs) for high-yield purification of lysosomes compatible with "omics" analysis. NANOLYSE (Nanoparticles for Lysosome Isolation) provides a reliable strategy in fingerprinting the biomolecular composition of lysosomes. For complete details on the use and execution of this protocol, please refer to Tharkeshwar et al. (2017).

BACE1 levels correlate with phospho-tau levels in human cerebrospinal fluid.

Previous studies have investigated the activity and protein levels of BACE1, the ß-secretase, in the brain and cerebrospinal fluid (CSF) of Alzheimer's disease (AD) patients, however, results remain contradictory. We present here a highly specific and sensitive BACE1 ELISA, which allows measuring accurately BACE1 levels in human samples. We find that BACE1 levels in CSF of AD patients and other neurological disorder (OND) patients are slightly increased when compared to those of a non-neurological disorder control group (NND). BACE1 levels in CSF were well correlated with total-tau and hyperphosphorylated tau levels in the CSF, suggesting that the recorded alterations in BACE1 levels correlate with cell death and neurodegeneration.

Glycogen synthase kinase-3ß (GSK3ß) expression in a mouse model of Alzheimer's disease: a light and electron microscopy study.

Glycogen synthase kinase-3ß (GSK3ß) activity has been previously linked to Alzheimer's disease (AD) by its phosphorylation of tau and activation by amyloid. GSK3ß intracellular distribution is important in regulating its activity by restricting access to compartment-specific substrates. This study investigated regional and intracellular distribution of GSK3ß in a mouse model of AD, a bigenic mouse with combined amyloid and tau pathology (BiAT), and controls (FVB). At two different ages, the entire rostrocaudal extent of each brain was examined. Young (6-months-old) FVB and BiAT mice did not differ in GSK3ß expression and localization. In old (13-month-old) BiAT mice, neurons showed increased GSK3ß expression only in AD-relevant brain regions as compared with modest staining in region- and age-matched controls. Two regions with the most robust changes between FVB and BiAT mice, the amygdala and piriform cortex, were quantified at the light microscopic level. In both regions, the density of darkly labeled neurons was significantly greater in the old BiAT mice vs. the old FVB mice. Electron microscopy of the piriform cortex showed neuronal GSK3ß labeling in the rough endoplasmic reticulum, on ribosomes, and on microtubules in dendrites in both strains of mice. In old BiAT mice, GSK3ß labeling was qualitatively more robust compared to age-matched controls, and GSK3ß also appeared in neurofibrillary tangles. In conclusion, GSK3ß expression was increased in specific intracellular locations and was found in tangles in old BiAT mice, suggesting that GSK3ß overexpression in specific brain areas may be intrinsic to AD pathology.

Identification of glycogen synthase kinase-3 inhibitors with a selective sting for glycogen synthase kinase-3α.

The glycogen synthase kinase-3 (GSK-3) has been linked to the pathogenesis of colorectal cancer, diabetes, cardiovascular disease, acute myeloid leukemia (AML), and Alzheimer's disease (AD). The debate on the respective contributions of GSK-3α and GSK-3ß to AD pathology and AML is ongoing. Thus, the identification of potent GSK-3α-selective inhibitors, endowed with favorable pharmacokinetic properties, may elucidate the effect of GSK-3α inhibition in AD and AML models. The analysis of all available crystallized GSK-3 structures provided a simplified scheme of the relevant hot spots responsible for ligand binding and potency. This resulted in the identification of novel scorpion shaped GSK-3 inhibitors. It is noteworthy, compounds 14d and 15b showed the highest GSK-3α selectivity reported so far. In addition, compound 14d did not display significant inhibition of 48 out of 50 kinases in the test panel. The GSK-3 inhibitors were further profiled for efficacy and toxicity in the wild-type (wt) zebrafish embryo assay.

GSK-3α/ß kinases and amyloid production in vivo.

Arising from C. J. Phiel, C. A. Wilson, V. M.-Y. Lee & P. S. Klein 423, 435-439 (2003)A major unresolved issue in Alzheimer's disease is identifying the mechanisms that regulate proteolytic processing of amyloid precursor protein (APP)-glycogen synthase kinase-3 (GSK-3) isozymes are thought to be important in this regulation. Phiel et al. proposed that GSK-3α, but not GSK-3ß, controls production of amyloid. We analysed the proteolytic processing of mouse and human APP in mouse brain in vivo in five different genetic and viral models. Our data do not yield evidence for either GSK-3α-mediated or GSK-3ß-mediated control of APP processing in brain in vivo.

Early improved and late defective cognition is reflected by dendritic spines in Tau.P301L mice.

Cognitive demise correlates with progressive brain tauopathy in dementing patients. Improved cognition of young Tau.P301L mice contrasts with dysfunction later in life and remains unexplained (Boekhoorn et al., 2006). To unravel early mechanisms, we composed a correlative time line of clinical symptoms, cognitive defects, and biochemical and pathological traits, including comprehensive analysis of dendritic spines in specified regions of the cortex and hippocampus of young and adult Tau.P301L mice. Remarkably, young Tau.P301L mice have not more, but more mature spines than wild-type mice, revealing the anatomical substrate for their improved cognition and LTP. Spine maturation remained high in the hippocampus of adult Tau.P301L mice. However, spines regressed in length paralleling impaired cognition and increased Tau phosphorylation (Terwel et al., 2005). Conversely, spine maturation was unaffected in adult Tau.4R mice, while spine density was increased and length reduced similar to Tau.P301L mice. To explain how protein Tau promoted spinogenesis, we analyzed hippocampal synaptosomes and dendritic spines for mouse and human Tau. While synaptosomes were positive for both mouse and human Tau, weak variable reaction in spines was observed only after fixation according to Bouin. Mouse Tau was absent from spines in wild-type mice, dissociating the pathological actions of Tau in transgenic mice by relocalization into dendrites and spines from the physiological actions of protein Tau in axons only. We conclude that mutant protein Tau modulates cognition and morphology of spines similarly and in both directions, with pathology later in life coinciding with increased phosphorylation and relocalization of Tau from axons to soma and processes.

Dendritic degeneration, neurovascular defects, and inflammation precede neuronal loss in a mouse model for tau-mediated neurodegeneration.

Adeno-associated virus (AAV)-mediated expression of wild-type or mutant P301L protein tau produces massive degeneration of pyramidal neurons without protein tau aggregation. We probed this novel model for genetic and structural factors and early parameters of pyramidal neurodegeneration. In yellow fluorescent protein-expressing transgenic mice, intracerebral injection of AAV-tauP301L revealed early damage to apical dendrites of CA1 pyramidal neurons, whereas their somata remained normal. Ultrastructurally, more and enlarged autophagic vacuoles were contained in degenerating dendrites and manifested as dark, discontinuous, vacuolated processes surrounded by activated astrocytes. Dendritic spines were lost in AAV-tauP301L-injected yellow fluorescent protein-expressing transgenic mice, and ultrastructurally, spines appeared dark and degenerating. In CX3CR1(EGFP/EGFP)-deficient mice, microglia were recruited early to neurons expressing human tau. The inflammatory response was accompanied by extravasation of plasma immunoglobulins. α2-Macroglobulin, but neither albumin nor transferrin, became lodged in the brain parenchyma. Large proteins, but not Evans blue, entered the brain of mice injected with AAV-tauP301L. Ultrastructurally, brain capillaries were constricted and surrounded by swollen astrocytes with extensions that contacted degenerating dendrites and axons. Together, these data corroborate the hypothesis that neuroinflammation participates essentially in tau-mediated neurodegeneration, and the model recapitulates early dendritic defects reminiscent of "dendritic amputation" in Alzheimer's disease.

Amyloid precursor protein mutation E682K at the alternative ß-secretase cleavage ß'-site increases Aß generation.

BACE1 cleaves the amyloid precursor protein (APP) at the ß-cleavage site (Met(671) -Asp(672) ) to initiate the generation of amyloid peptide Aß. BACE1 is also known to cleave APP at a much less well-characterized ß'-cleavage site (Tyr(681) -Glu(682) ). We describe here the identification of a novel APP mutation E682K located at this ß'-site in an early onset Alzheimer's disease (AD) case. Functional analysis revealed that this E682K mutation blocked the ß'-site and shifted cleavage of APP to the ß-site, causing increased Aß production. This work demonstrates the functional importance of APP processing at the ß'-site and shows how disruption of the balance between ß- and ß'-site cleavage may enhance the amyloidogenic processing and consequentially risk for AD. Increasing exon- and exome-based sequencing efforts will identify many more putative pathogenic mutations without conclusive segregation-based evidence in a single family. Our study shows how functional analysis of such mutations allows to determine the potential pathogenic nature of these mutations. We propose to classify the E682K mutation as probable pathogenic awaiting further independent confirmation of its association with AD in other patients.

AAV-tau mediates pyramidal neurodegeneration by cell-cycle re-entry without neurofibrillary tangle formation in wild-type mice.

In Alzheimer's disease tauopathy is considered secondary to amyloid, and the duality obscures their relation and the definition of their respective contributions.Transgenic mouse models do not resolve this problem conclusively, i.e. the relative hierarchy of amyloid and tau pathology depends on the actual model and the genes expressed or inactivated. Here, we approached the problem in non-transgenic models by intracerebral injection of adeno-associated viral vectors to express protein tau or amyloid precursor protein in the hippocampus in vivo. AAV-APP mutant caused neuronal accumulation of amyloid peptides, and eventually amyloid plaques at 6 months post-injection, but with only marginal hippocampal cell-death. In contrast, AAV-Tau, either wild-type or mutant P301L, provoked dramatic degeneration of pyramidal neurons in CA1/2 and cortex within weeks. Tau-mediated neurodegeneration proceeded without formation of large fibrillar tau-aggregates or tangles, but with increased expression of cell-cycle markers.We present novel AAV-based models, which demonstrate that protein tau mediates pyramidal neurodegeneration in vivo. The data firmly support the unifying hypothesis that post-mitotic neurons are forced to re-enter the cell-cycle in primary and secondary tauopathies, including Alzheimer's disease.