W. Taylor Kimberly

Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and gamma-secretase activity.

Accumulation of the amyloid-β protein (Aβ) in the cerebral cortex is an early and invariant event in the pathogenesis of Alzheimers disease. The final step in the generation of Aβ from the β-amyloid precursor protein is an apparently intramembranous proteolysis by the elusive γ-secretase(s). The most common cause of familial Alzheimers disease is mutation of the genes encoding presenilins 1 and 2, which alters γ-secretase activity to increase the production of the highly amyloidogenic Aβ42 isoform. Moreover, deletion of presenilin-1 in mice greatly reduces γ-secretase activity, indicating that presenilin-1 mediates most of this proteolytic event. Here we report that mutation of either of two conserved transmembrane (TM) aspartate residues in presenilin-1, Asp 257 (in TM6) and Asp 385 (in TM7), substantially reduces Aβ production and increases the amounts of the carboxy-terminal fragments of β-amyloid precursor protein that are the substrates of γ-secretase. We observed these effects in three different cell lines as well as in cell-free microsomes. Either of the Asp → Ala mutations also prevented the normal endoproteolysis of presenilin-1 in the TM6 → TM7 cytoplasmic loop. In a functional presenilin-1 variant (carrying a deletion in exon 9) that is associated with familial Alzheimers disease and which does not require this cleavage, the Asp 385 → Ala mutation still inhibited γ-secretase activity. Our results indicate that the two transmembrane aspartate residues are critical for both presenilin-1 endoproteolysis and γ-secretase activity, and suggest that presenilin 1 is either a unique diaspartyl cofactor for γ-secretase or is itself γ-secretase, an autoactivated intramembranous aspartyl protease.

γ-Secretase is a membrane protein complex comprised of presenilin, nicastrin, aph-1, and pen-2

γ-Secretase catalyzes the intramembrane proteolysis of Notch, β-amyloid precursor protein, and other substrates as part of a new signaling paradigm and as a key step in the pathogenesis of Alzheimers disease. This unusual protease has eluded identification, though evidence suggests that the presenilin heterodimer comprises the catalytic site and that a highly glycosylated form of nicastrin associates with it. The formation of presenilin heterodimers from the holoprotein is tightly gated by unknown limiting cellular factors. Here we show that Aph-1 and Pen-2, two recently identified membrane proteins genetically linked to γ-secretase, associate directly with presenilin and nicastrin in the active protease complex. Coexpression of all four proteins leads to marked increases in presenilin heterodimers, full glycosylation of nicastrin, and enhanced γ-secretase activity. These findings suggest that the four membrane proteins comprise the limiting components of γ-secretase and coassemble to form the active enzyme in mammalian cells.

Transition-state analogue inhibitors of γ-secretase bind directly to presenilin-1

The β-amyloid precursor protein (β-APP), which is involved in the pathogenesis of Alzheimer’s disease, and the Notch receptor, which is responsible for critical signalling events during development, both undergo unusual proteolysis within their transmembrane domains by unknown γ-secretases. Here we show that an affinity reagent designed to interact with the active site of γ-secretase binds directly and specifically to heterodimeric forms of presenilins, polytopic proteins that are mutated in hereditary Alzheimer’s and are known mediators of γ-secretase cleavage of both β-APP and Notch. These results provide evidence that heterodimeric presenilins contain the active site of γ-secretase, and validate presenilins as principal targets for the design of drugs to treat and prevent Alzheimer’s disease.

Activity-dependent isolation of the presenilin– γ-secretase complex reveals nicastrin and a γ substrate

Presenilin heterodimers apparently contain the active site of γ-secretase, a polytopic aspartyl protease involved in the transmembrane processing of both the Notch receptor and the amyloid-β precursor protein. Although critical to embryonic development and the pathogenesis of Alzheimers disease, this protease is difficult to characterize, primarily because it is a multicomponent complex of integral membrane proteins. Here the functional γ-secretase complex was isolated by using an immobilized active site-directed inhibitor of the protease. Presenilin heterodimers and nicastrin bound specifically to this inhibitor under conditions tightly correlating with protease activity, whereas several other presenilin-interacting proteins (β-catenin, calsenilin, and presenilin-associated protein) did not bind. Moreover, anti-nicastrin antibodies immunoprecipitated γ-secretase activity from detergent-solubilized microsomes. Unexpectedly, C83, the major endogenous amyloid-β precursor protein substrate of γ-secretase, was also quantitatively associated with the complex. These results provide direct biochemical evidence that nicastrin is a member of the active γ-secretase complex, indicate that β-catenin, calsenilin, and presenilin-associated protein are not required for γ activity, and suggest an unprecedented mechanism of substrate–protease interaction.

Transition-state analogue inhibitors of γ-secretase bind directlyto presenilin-1

The β-amyloid precursor protein (β-APP), which is involved in the pathogenesis of Alzheimer’s disease, and the Notch receptor, which is responsible for critical signalling events during development, both undergo unusual proteolysis within their transmembrane domains by unknown γ-secretases. Here we show that an affinity reagent designed to interact with the active site of γ-secretase binds directly and specifically to heterodimeric forms of presenilins, polytopic proteins that are mutated in hereditary Alzheimer’s and are known mediators of γ-secretase cleavage of both β-APP and Notch. These results provide evidence that heterodimeric presenilins contain the active site of γ-secretase, and validate presenilins as principal targets for the design of drugs to treat and prevent Alzheimer’s disease.

Assembly of the γ-Secretase Complex Involves Early Formation of an Intermediate Subcomplex of Aph-1 and Nicastrin

The γ-secretase complex is an unusual multimeric protease responsible for the intramembrane cleavage of a variety of type 1 transmembrane proteins, including the β-amyloid precursor protein and Notch. Genetic and biochemical data have revealed that this protease consists of the presenilin heterodimer, a highly glycosylated form of nicastrin, and the recently identified gene products, Aph-1 and Pen-2. Whereas current evidence supports the notion that presenilin comprises the active site of the protease and that the other three components are members of the active complex required for proteolytic activity, the individual roles of the three co-factors remain unclear. Here, we demonstrate that endogenous Aph-1 interacts with an immature species of nicastrin, forming a stable intermediate early in the assembly of the γ-secretase complex, prior to the addition of presenilin and Pen-2. Our data suggest 1) that Aph-1 is involved in the early stages of γ-secretase assembly through the stabilization and perhaps glycosylation of nicastrin and by scaffolding nicastrin to the immature γ-secretase complex, and 2) that presenilin, and later Pen-2, bind to this intermediate during the formation of the mature protease.

Recommendations for the Management of Cerebral and Cerebellar Infarction With Swelling A Statement for Healthcare Professionals From the American Heart Association/American Stroke Association

Background and Purpose— There are uncertainties surrounding the optimal management of patients with brain swelling after an ischemic stroke. Guidelines are needed on how to manage this major complication, how to provide the best comprehensive neurological and medical care, and how to best inform families facing complex decisions on surgical intervention in deteriorating patients. This scientific statement addresses the early approach to the patient with a swollen ischemic stroke in a cerebral or cerebellar hemisphere. Methods— The writing group used systematic literature reviews, references to published clinical and epidemiology studies, morbidity and mortality reports, clinical and public health guidelines, authoritative statements, personal files, and expert opinion to summarize existing evidence and to indicate gaps in current knowledge. The panel reviewed the most relevant articles on adults through computerized searches of the medical literature using MEDLINE, EMBASE, and Web of Science through March 2013. The evidence is organized within the context of the American Heart Association framework and is classified according to the joint American Heart Association/American College of Cardiology Foundation and supplementary American Heart Association Stroke Council methods of classifying the level of certainty and the class and level of evidence. The document underwent extensive American Heart Association internal peer review. Results— Clinical criteria are available for hemispheric (involving the entire middle cerebral artery territory or more) and cerebellar (involving the posterior inferior cerebellar artery or superior cerebellar artery) swelling caused by ischemic infarction. Clinical signs that signify deterioration in swollen supratentorial hemispheric ischemic stroke include new or further impairment of consciousness, cerebral ptosis, and changes in pupillary size. In swollen cerebellar infarction, a decrease in level of consciousness occurs as a result of brainstem compression and therefore may include early loss of corneal reflexes and the development of miosis. Standardized definitions should be established to facilitate multicenter and population-based studies of incidence, prevalence, risk factors, and outcomes. Identification of patients at high risk for brain swelling should include clinical and neuroimaging data. If a full resuscitative status is warranted in a patient with a large territorial stroke, admission to a unit with neurological monitoring capabilities is needed. These patients are best admitted to intensive care or stroke units attended by skilled and experienced physicians such as neurointensivists or vascular neurologists. Complex medical care includes airway management and mechanical ventilation, blood pressure control, fluid management, and glucose and temperature control. In swollen supratentorial hemispheric ischemic stroke, routine intracranial pressure monitoring or cerebrospinal fluid diversion is not indicated, but decompressive craniectomy with dural expansion should be considered in patients who continue to deteriorate neurologically. There is uncertainty about the efficacy of decompressive craniectomy in patients ≥60 years of age. In swollen cerebellar stroke, suboccipital craniectomy with dural expansion should be performed in patients who deteriorate neurologically. Ventriculostomy to relieve obstructive hydrocephalus after a cerebellar infarct should be accompanied by decompressive suboccipital craniectomy to avoid deterioration from upward cerebellar displacement. In swollen hemispheric supratentorial infarcts, outcome can be satisfactory, but one should anticipate that one third of patients will be severely disabled and fully dependent on care even after decompressive craniectomy. Surgery after a cerebellar infarct leads to acceptable functional outcome in most patients. Conclusions— Swollen cerebral and cerebellar infarcts are critical conditions that warrant immediate, specialized neurointensive care and often neurosurgical intervention. Decompressive craniectomy is a necessary option in many patients. Selected patients may benefit greatly from such an approach, and although disabled, they may be functionally independent.

γ-Secretase/presenilin inhibitors for Alzheimer’s disease phenocopy Notch mutations in Drosophila

Signaling from the Notch (N) receptor is essential for proper cell‐fate determinations and tissue patterning in all metazoans. N signaling requires a presenilin (PS)‐dependent transmembrane‐cleaving activity that is closely related or identical to the γ‐secretase proteolysis of the amyloid‐β precursor protein (APP) involved in Alzheimers disease pathogenesis. Here, we show that N‐[N‐(3,5‐difluorophenacetyl)‐L‐alanyl]‐(S)‐phenylglycine t‐butyl ester, a potent γ‐secretase inhibitor reported to reduce amyloid‐β levels in transgenic mice, prevents N processing, translocation, and signaling in cell culture. This compound also induces developmental defects in Drosophila remarkably similar to those caused by genetic reduction of N. The appearance of this phenocopy depends on the timing and dose of compound exposure, and effects on N‐dependent signaling molecules established its biochemical mechanism of action in vivo. Other γ‐secretase inhibitors caused similar effects. Thus, the three‐dimensional structure of the drug‐binding site(s) in Drosophila γ‐secretase is remarkably conserved vis‐à‐vis the same site(s) in the mammalian enzyme. These results show that genetics and developmental biology can help elucidate the in vivo site of action of pharmacological agents and suggest that organisms such as Drosophila may be used as simple models for in vivo prescreening of drug candidates.

Aspartate Mutations in Presenilin and γ‐Secretase Inhibitors Both Impair Notch1 Proteolysis and Nuclear Translocation with Relative Preservation of Notch1 Signaling

It has been hypothesized that a presenilin 1 (PS1)‐related enzymatic activity is responsible for proteolytic cleavage of the C‐terminal intracellular protein of Notch1, in addition to its role in β‐amyloid protein (Aβ) formation from the amyloid precursor protein (APP). We developed an assay to monitor ligand‐induced Notch1 proteolysis and nuclear translocation in individual cells : Treatment of full‐length Notch1‐enhanced green fluorescent protein‐transfected Chinese hamster ovary (CHO) cells with a soluble preclustered form of the physiologic ligand Delta leads to rapid accumulation of the C terminus of Notch1 in the nucleus and to transcriptional activation of a C‐promoter binding factor 1 (CBF1) reporter construct. Nuclear translocation was blocked by cotransfection with Notchs physiologic inhibitor Numb. Using this assay, we now confirm and extend the observation that PS1 is involved in Notch1 nuclear translocation and signaling in mammalian cells. We demonstrate that the D257A and the D385A PS1 mutations, which had been shown previously to block APP γ‐secretase activity, also prevent Notch1 cleavage and translocation to the nucleus but do not alter Notch1 trafficking to the cell surface. We also show that two APP γ‐secretase inhibitors block Notch1 nuclear translocation with an IC50 similar to that reported for APP γ‐secretase. Notch1 signaling, assessed by measuring the activity of CBF1, a downstream transcription factor, was impaired but not abolished by the PS1 aspartate mutations or γ‐secretase inhibitors. Our results support the hypotheses that (a) PS1‐dependent APP γ‐secretase‐like enzymatic activity is critical for both APP and Notch processing and (b) the Notch1 signaling pathway remains partially activated even when Notch1 proteolytic processing and nuclear translocation are markedly inhibited. The latter is an important finding from the perspective of therapeutic treatment of Alzheimers disease by targeting γ‐secretase processing of APP to reduce Aβ production.

Physiological Regulation of the β-Amyloid Precursor Protein Signaling Domain by c-Jun N-Terminal Kinase JNK3 during Neuronal Differentiation

β-Amyloid precursor protein (APP) is a conserved and ubiquitous transmembrane glycoprotein strongly implicated in the pathogenesis of Alzheimers disease but whose normal biological function is unknown. Analogy to the Notch protein suggests that APP is a cell-surface receptor that signals via sequential proteolytic cleavages that release its intracellular domain (AICD) to the nucleus. Because these cleavages are major targets for therapeutic inhibition, it is critical to elucidate their physiological function. AICD is stabilized by Fe65, interacts with the transcriptional factor Tip60, and translocates to the nucleus. Here, we show that endogenous AICD in primary neurons is detectable only during a short period of time during differentiation in culture. During this transient rise, a portion of AICD localizes to the nucleus. Subsequently, phosphorylation of the APP cytoplasmic domain at threonine 668 appears to disrupt the stabilizing interaction with Fe65 and thus downregulate AICD-mediated signaling. Furthermore, we find that the neuron-specific c-Jun N-terminal kinase JNK3, but not JNK1 or JNK2, mediates a substantial portion of this phosphorylation. We conclude that endogenous AICD undergoes tight temporal regulation during the differentiation of neurons and is negatively regulated by JNK3 via phosphorylation of APP at Thr668.