Early lysosome defects precede neurodegeneration with amyloid-ß and tau aggregation in NHE6-null rat brain.

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-ß 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-ß and tau in certain pathological states. Mouse models generally do not show the accumulation of insoluble, endogenously-expressed (non-transgenic) tau or amyloid-ß. Therefore, to study neurodegeneration in Christianson syndrome and the possibility of amyloid-ß 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-ß 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-ß 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-ß and tau. Therefore these studies may provide insight into mechanisms of more common neurodegenerative disorders, including Alzheimer's disease and related dementias.

Disruption of amyloid precursor protein ubiquitination selectively increases amyloid ß (Aß) 40 levels via presenilin 2-mediated cleavage.

Amyloid plaques, a neuropathological hallmark of Alzheimer's disease, are largely composed of amyloid ß (Aß) peptide, derived from cleavage of amyloid precursor protein (APP) by ß- and γ-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 affecting APP accumulation, 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 Aß40. Artificial APP ubiquitination with rapalog-mediated proximity inducers reduced Aß40 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 Aß40 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.

Transmembrane Substrate Determinants for γ-Secretase Processing of APP CTFß.

The amyloid ß-peptide (Aß) of Alzheimer's disease (AD) is generated by proteolysis within the transmembrane domain (TMD) of a C-terminal fragment of the amyloid ß protein-precursor (APP CTFß) by the γ-secretase complex. This processing produces Aß ranging from 38 to 49 residues in length. Evidence suggests that this spectrum of Aß peptides is the result of successive γ-secretase cleavages, with endoproteolysis first occurring at the ε sites to generate Aß48 or Aß49, followed by C-terminal trimming mostly every three residues along two product lines to generate shorter, secreted forms of Aß: the primary Aß49-46-43-40 line and a minor Aß48-45-42-38 line. The major secreted Aß species are Aß40 and Aß42, and an increased proportion of the longer, aggregation-prone Aß42 compared to Aß40 is widely thought to be important in AD pathogenesis. We examined TMD substrate determinants of the specificity and efficiency of ε site endoproteolysis and carboxypeptidase trimming of CTFß by γ-secretase. We determined that the C-terminal negative charge of the intermediate Aß49 does not play a role in its trimming by γ-secretase. Peptidomimetic probes suggest that γ-secretase has S1', S2', and S3' pockets, through which trimming by tripeptides may be determined. However, deletion of residues around the ε sites demonstrates that a depth of three residues within the TMD is not a determinant of the location of endoproteolytic ε cleavage of CTFß. We also show that instability of the CTFß TMD helix near the ε site significantly increases endoproteolysis, and that helical instability near the carboxypeptidase cleavage sites facilitates C-terminal trimming by γ-secretase. In addition, we found that CTFß dimers are not endoproteolyzed by γ-secretase. These results support a model in which initial interaction of the array of residues along the undimerized single helical TMD of substrates dictates the site of initial ε cleavage and that helix unwinding is essential for both endoproteolysis and carboxypeptidase trimming.

Alzheimer presenilin-1 mutations dramatically reduce trimming of long amyloid ß-peptides (Aß) by γ-secretase to increase 42-to-40-residue Aß.

The presenilin-containing γ-secretase complex produces the amyloid ß-peptide (Aß) 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 Aß critical to pathogenesis, but other Aßs 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 Aß. Subsequent cleavage almost every three residues from the C terminus is thought to occur along two pathways toward shorter secreted forms of Aß: Aß49 → Aß46 → Aß43 → Aß40 and Aß48 → Aß45 → Aß42 → Aß38. Here we show that the addition of synthetic long Aß peptides (Aß45-49) directly into purified preparations of γ-secretase leads to the formation of Aß40 and Aß42 whether the protease complex is detergent-solubilized or reconstituted into lipid vesicles, and the ratios of products Aß42 to Aß40 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 Aß peptides to Aß40 and Aß42 by mutant proteases occurs at multiple levels, independent of the effects on initial endoproteolysis at the ϵ site, all conspiring to increase the critical Aß42/Aß40 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 Aß42/Aß40.

A neurodegeneration checkpoint mediated by REST protects against the onset of Alzheimer's disease.

Many aging individuals accumulate the pathology of Alzheimer's disease (AD) without evidence of cognitive decline. Here we describe an integrated neurodegeneration checkpoint response to early pathological changes that restricts further disease progression and preserves cognitive function. Checkpoint activation is mediated by the REST transcriptional repressor, which is induced in cognitively-intact aging humans and AD mouse models at the onset of amyloid ß-protein (Aß) deposition and tau accumulation. REST induction is mediated by the unfolded protein response together with ß-catenin signaling. A consequence of this response is the targeting of REST to genes involved in key pathogenic pathways, resulting in downregulation of gamma secretase, tau kinases, and pro-apoptotic proteins. Deletion of REST in the 3xTg and J20 AD mouse models accelerates Aß deposition and the accumulation of misfolded and phosphorylated tau, leading to neurodegeneration and cognitive decline. Conversely, viral-mediated overexpression of REST in the hippocampus suppresses Aß and tau pathology. Thus, REST mediates a neurodegeneration checkpoint response with multiple molecular targets that may protect against the onset of AD.

Loss of endosomal exchanger NHE6 leads to pathological changes in tau in human neurons.

Disruption of endolysosomal and autophagy-lysosomal systems is increasingly implicated in neurodegeneration. Sodium-proton exchanger 6 (NHE6) contributes to the maintenance of proper endosomal pH, and loss-of function mutations in the X-linked NHE6 lead to Christianson syndrome (CS) in males. Neurodegenerative features of CS are increasingly recognized, with postmortem and clinical data implicating a role for tau. We generated cortical neurons from NHE6 knockout (KO) and isogenic wild-type control human induced pluripotent stem cells. We report elevated phosphorylated and sarkosyl-insoluble tau in NHE6 KO neurons. We demonstrate that NHE6 KO leads to lysosomal and autophagy dysfunction involving reduced lysosomal number and protease activity, diminished autophagic flux, and p62 accumulation. Finally, we show that treatment with trehalose or rapamycin, two enhancers of autophagy-lysosomal function, each partially rescue this tau phenotype. We provide insight into the neurodegenerative processes underlying NHE6 loss of function and into the broader role of the endosome-lysosome-autophagy network in neurodegeneration.

Dissociation between the processivity and total activity of γ-secretase: implications for the mechanism of Alzheimer's disease-causing presenilin mutations.

The amyloid ß-peptide (Aß), strongly implicated in the pathogenesis of Alzheimer's disease (AD), is produced from the amyloid ß-protein precursor (APP) through consecutive proteolysis by ß- and γ-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 Aß production and increase the ratio of the aggregation-prone 42-residue form (Aß42) to the 40-residue form (Aß40). The connection between these two effects is not understood. Besides Aß40 and Aß42, γ-secretase produces a range of Aß peptides, the result of initial cutting at the ε site to form Aß48 or Aß49 and subsequent trimming every three or four residues. Thus, γ-secretase displays both overall proteolytic activity (ε 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 Aß peptides. Low salt concentrations and acidic pH were notable exceptions that subtly alter the proportion of individual Aß 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, Aß42:Aß40 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 Aß peptides.

Stem cell-derived neurons reflect features of protein networks, neuropathology, and cognitive outcome of their aged human donors.

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 Aß 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.

Structure of the unbound form of HIV-1 subtype A protease: comparison with unbound forms of proteases from other HIV subtypes.

The crystal structure of the unbound form of HIV-1 subtype A protease (PR) has been determined to 1.7 A resolution and refined as a homodimer in the hexagonal space group P6(1) to an R(cryst) 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.

Cell-type Dependent Alzheimer's Disease Phenotypes: Probing the Biology of Selective Neuronal Vulnerability.

Alzheimer's disease (AD) induces memory and cognitive impairment in the absence of motor and sensory deficits during its early and middle course. A major unresolved question is the basis for this selective neuronal vulnerability. Aß, which plays a central role in AD pathogenesis, is generated throughout the brain, yet some regions outside of the limbic and cerebral cortices are relatively spared from Aß plaque deposition and synapse loss. Here, we examine neurons derived from iPSCs of patients harboring an amyloid precursor protein mutation to quantify AD-relevant phenotypes following directed differentiation to rostral fates of the brain (vulnerable) and caudal fates (relatively spared) in AD. We find that both the generation of Aß and the responsiveness of TAU to Aß are affected by neuronal cell type, with rostral neurons being more sensitive than caudal neurons. Thus, cell-autonomous factors may in part dictate the pattern of selective regional vulnerability in human neurons in AD.

The contribution of naturally occurring polymorphisms in altering the biochemical and structural characteristics of HIV-1 subtype C protease.

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.

Water permeability properties of the human small intestine in vitro: effects of Escherichia coli heat-stable enterotoxin.

The net absorptive water flux (Jw), the transepithelial potential difference (PD) and the short-circuit current (Isc) were simultaneously measured in the human small intestine in vitro with the following results: 1) An absorptive Jw was observed when the jejunum or the ileum were mounted between two identical standard solutions in the presence of an hydrostatic pressure gradient (delta P) of 13 cm of water (mucosal side positive). 2) The absorptive Jw was a linear function of the applied delta P or the imposed osmotic transepithelial gradient (delta Osm) in both intestinal segments. The hydrostatic (Phydr) and osmotic (Posm) permeabilities to water for jejunum and ileum were: 0.349 +/- 0.049 cm/s vs. 0.156 +/- 0.022 cm/s and 0.0012 +/- 0.0001 cm/s vs. 0.0019 +/- 0.0003, respectively. 3) A fraction of this absorptive Jw was independent of the presence of any hydrostatic, osmotic or chemical gradient and represented the transport associated to movement of water (Jwt). 4) PD and Isc values were similar in the jejunum and in the ileum but the transepithelial resistance (Rt) was significantly greater in ileum than in jejunum. 5) 2 micrograms/ml of E. coli heat-stable enterotoxin (STa) caused a significant inhibition of the absorptive Jw without modification of Phydr, Posm or Isc. 6) After STa treatment, the absorptive Jwt reverted to a secretory one in the jejunum. In the ileum, STa action caused a 48% decrease in the absorptive Jwt values.

Tratamiento quirurgico de la hernia hiatal. / Surgical treatment of hiatal hernia

Se presentan 61 pacientes portadores de hernia hiatal sometidos a tratamiento quirurgico, con 88,5% de resultados excelentes. Se consideran las distintas tecnicas empleadas, reservandose la utilizacion de la gastropexia anterior en pacientes seleccionados