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The presenilin-containing γ-secretase complex produces the amyloid β-peptide (Aβ) through intramembrane proteolysis, and>100 presenilin mutations are associated with familial early- onset Alzheimer disease (AD). The question of whether these mutations result in AD through a gain or a loss of function remains highly controversial. Mutations in presenilins increase ratios of 42- to 40-residue Aβ critical to pathogenesis, but other Aβs of 38-49 residues are also formed by γ-secretase. Evidence in cells suggests the protease first cleaves substrate within the transmembrane domain at the ϵ site to form 48- or 49-residue Aβ. Subsequent cleavage almost every three residues from the C terminus is thought to occur along two pathways toward shorter secreted forms of Aβ: Aβ49 → Aβ46 → Aβ43 → Aβ40 and Aβ48 → Aβ45 → Aβ42 → Aβ38. Here we show that the addition of synthetic long Aβ peptides (Aβ45-49) directly into purified preparations of γ-secretase leads to the formation of Aβ40 and Aβ42 whether the protease complex is detergent-solubilized or reconstituted into lipid vesicles, and the ratios of products Aβ42 to Aβ40 follow a pattern consistent with the dual-pathway hypothesis. Kinetic analysis of five different AD-causing mutations in presenilin-1 revealed that all result in drastic reduction of normal carboxypeptidase function. Altered trimming of long Aβ peptides to Aβ40 and Aβ42 by mutant proteases occurs at multiple levels, independent of the effects on initial endoproteolysis at the ϵ site, all conspiring to increase the critical Aβ42/Aβ40 ratio implicated in AD pathogenesis. Taken together, these results suggest that specific reduction of carboxypeptidase function of γ-secretase leads to the gain of toxic Aβ42/Aβ40. [ABSTRACT FROM AUTHOR]

Loss-of-function mutations in the X-linked endosomal Na+/H+ exchanger 6 (NHE6) cause Christianson syndrome in males. Christianson syndrome involves endosome dysfunction leading to early cerebellar degeneration, as well as later-onset cortical and subcortical neurodegeneration, potentially including tau deposition as reported in post-mortem studies. In addition, there is reported evidence of modulation of amyloid-β levels in experimental models wherein NHE6 expression was targeted. We have recently shown that loss of NHE6 causes defects in endosome maturation and trafficking underlying lysosome deficiency in primary mouse neurons in vitro. For in vivo studies, rat models may have an advantage over mouse models for the study of neurodegeneration, as rat brain can demonstrate robust deposition of endogenously-expressed amyloid-β and tau in certain pathological states. Mouse models generally do not show the accumulation of insoluble, endogenously-expressed (non-transgenic) tau or amyloid-β. Therefore, to study neurodegeneration in Christianson syndrome and the possibility of amyloid-β and tau pathology, we generated an NHE6-null rat model of Christianson syndrome using CRISPR-Cas9 genome-editing. Here, we present the sequence of pathogenic events in neurodegenerating NHE6-null male rat brains across the lifespan. NHE6-null rats demonstrated an early and rapid loss of Purkinje cells in the cerebellum, as well as a more protracted neurodegenerative course in the cerebrum. In both the cerebellum and cerebrum, lysosome deficiency is an early pathogenic event, preceding autophagic dysfunction. Microglial and astrocyte activation also occur early. In the hippocampus and cortex, lysosome defects precede loss of pyramidal cells. Importantly, we subsequently observed biochemical and in situ evidence of both amyloid-β and tau aggregation in the aged NHE6-null hippocampus and cortex (but not in the cerebellum). Tau deposition is widely distributed, including cortical and subcortical distributions. Interestingly, we observed tau deposition in both neurons and glia, as has been reported in Christianson syndrome post-mortem studies previously. In summary, this experimental model is among very few examples of a genetically modified animal that exhibits neurodegeneration with deposition of endogenously-expressed amyloid-β and tau. This NHE6-null rat will serve as a new robust model for Christianson syndrome. Furthermore, these studies provide evidence for linkages between endolysosome dysfunction and neurodegeneration involving protein aggregations, including amyloid-β and tau. Therefore these studies may provide insight into mechanisms of more common neurodegenerative disorders, including Alzheimer's disease and related dementias. [ABSTRACT FROM AUTHOR]

Presents a letter to the editor reporting the experience with a child who had deletion 13q syndrome.

The amyloid β-peptide (Aβ), strongly implicated in the pathogenesis of Alzheimer's disease (AD), is produced from the amyloid β-protein precursor (APP) through consecutive proteolysis by β- and y-secretases. The latter protease contains presenilin as the catalytic component of a membrane-embedded aspartyl protease complex. Missense mutations in presenilin are associated with early-onset familial AD, and these mutations generally both decrease Aβ production and increase the ratio of the aggregation-prone 42-residue form (Aβ42) to the 40-residue form (Aβ40). The connection between these two effects is not understood. Besides Aβ40 and Aβ42, ?-secretase produces a range of Aβ peptides, the result of initial cutting at the e site to form Aβ48 or Aβ49 and subsequent trimming every three or four residues. Thus, ?-secretase displays both overall proteolytic activity (e cutting) and processivity (trimming) toward its substrate APP. Here we tested whether a decrease in total activity correlates with decreased processivity using wild-type and AD-mutant presenilin-containing protease complexes. Changes in pH, temperature, and salt concentration that reduced the overall activity of the wild-type enzyme did not consistently result in increased proportions of longer Aβ peptides. Low salt concentrations and acidic pH were notable exceptions that subtly alter the proportion of individual Aβ peptides, suggesting that the charged state of certain residues may influence processivity. Five different AD mutant complexes, representing a broad range of effects on overall activity, Aβ42:Aβ40 ratios, and ages of disease onset, were also tested, revealing again that changes in total activity and processivity can be dissociated. Factors that control initial proteolysis of APP at the ϵ site apparently differ significantly from factors affecting subsequent trimming and the distribution of Aβ peptides. [ABSTRACT FROM AUTHOR]

Fourteen subtype B and C protease variants have been engineered in an effort to study whether the preexistent baseline polymorphisms, by themselves or in combination with drug resistance mutations, differentially alter the biochemical and structural features of the subtype C protease when compared with those of subtype B protease. The kinetic studies performed in this work showed that the preexistent polymorphisms in subtype C protease, by themselves, do not provide for a greater level of resistance. Inhibition analysis with eight clinically used protease inhibitors revealed that the natural polymorphisms found in subtype C protease, in combination with drug resistance mutations, can influence enzymatic catalytic efficiency and inhibitor resistance. Structural analyses of the subtype C protease bound to nelfinavir and indinavir showed that these inhibitors form similar interactions with the residues in the active site of subtype B and C proteases. It also revealed that the naturally occurring polymorphisms could alter the position of the outer loops of the subtype C protease, especially the 60's loop. [ABSTRACT FROM AUTHOR]

Amyloid plaques, a neuropathological hallmark of Alzheimer's disease, are largely composed of amyloid β (Aβ) peptide, derived from cleavage of amyloid precursor protein (APP) byβ- andβ-secretases. The endosome is increasingly recognized as an important crossroad for APP and these secretases, with major implications for APP processing and amyloidogenesis. Among various post-translational modifications affectingAPPaccumulation, ubiquitination of cytodomain lysines may represent a key signal controlling APP endosomal sorting. Here, we show that substitution of APP C-terminal lysines with arginine disrupts APP ubiquitination and that an increase in the number of substituted lysines tends to increase APP metabolism. An APP mutant lacking all C-terminal lysines underwent the most pronounced increase in processing, leading to accumulation of both secreted and intracellular Aβ40. Artificial APP ubiquitination with rapalog-mediated proximity inducers reduced Aβ40 generation. A lack of APP C-terminal lysines caused APP redistribution from endosomal intraluminal vesicles (ILVs) to the endosomal limiting membrane, with a subsequent decrease in APP C-terminal fragment (CTF) content in secreted exosomes, but had minimal effects on APP lysosomal degradation. Both the increases in secreted and intracellular Aβ40 were abolished by depletion of presenilin 2 (PSEN2), recently shown to be enriched on the endosomal limiting membrane compared with PSEN1. Our findings demonstrate that ubiquitin can act as a signal at five cytodomain-located lysines for endosomal sorting of APP. They further suggest that disruption of APP endosomal sorting reduces its sequestration in ILVs and results in PSEN2- mediated processing of a larger pool of APP-CTF on the endosomal membrane. [ABSTRACT FROM AUTHOR]

The crystal structure of the unbound form of HIV-1 subtype A protease (PR) has been determined to 1.7 Å resolution and refined as a homodimer in the hexagonal space group P61 to an Rcryst of 20.5%. The structure is similar in overall shape and fold to the previously determined subtype B, C and F PRs. The major differences lie in the conformation of the flap region. The flaps in the crystal structures of the unbound subtype B and C PRs, which were crystallized in tetragonal space groups, are either semi-open or wide open. In the present structure of subtype A PR the flaps are found in the closed position, a conformation that would be more anticipated in the structure of HIV protease complexed with an inhibitor. The amino-acid differences between the subtypes and their respective crystal space groups are discussed in terms of the differences in the flap conformations. [ABSTRACT FROM AUTHOR]

We have generated a controlled and manipulable resource that captures genetic risk for Alzheimer's disease: iPSC lines from 53 individuals coupled with RNA and proteomic profiling of both iPSC-derived neurons and brain tissue of the same individuals. Data collected for each person include genome sequencing, longitudinal cognitive scores, and quantitative neuropathology. The utility of this resource is exemplified here by analyses of neurons derived from these lines, revealing significant associations between specific Aβ and tau species and the levels of plaque and tangle deposition in the brain and, more importantly, with the trajectory of cognitive decline. Proteins and networks are identified that are associated with AD phenotypes in iPSC neurons, and relevant associations are validated in brain. The data presented establish this iPSC collection as a resource for investigating person-specific processes in the brain that can aid in identifying and validating molecular pathways underlying AD. [Display omitted] • Generation and characterization of iPSC lines from 53 deeply phenotyped aged humans • Degree of congruence in expression profiles between iNs and brain of the same person • Association of cognitive decline and measures of Aβ and tau in iPSC-derived neurons • Identification and validation of PP1 as a molecular link between LOAD PRS, Aβ, and tau In this study, Lagomarsino et al. describe the generation of an iPSC collection from 53 deeply phenotyped individuals. These lines are coupled to RNA-seq and proteomic profiling of both iPSC neurons and brain of the same individuals. Using this system, biological insights are uncovered regarding the molecular mechanisms underlying Alzheimer's disease. [ABSTRACT FROM AUTHOR]