Extracellular Vesicle-Enriched miRNA-Biomarkers Show Improved Utility for Detecting Alzheimer's Disease Dementia and Medial Temporal Atrophy.

Background: Emerging diagnostic modalities suggest that miRNA profiles within extracellular vesicles (EVs) isolated from peripheral blood specimens may provide a non-invasive diagnostic alternative for dementia and neurodegenerative disorders. Given that EVs confer a protective environment against miRNA enzymatic degradation, the miRNAs enriched in the EV fraction of blood samples could serve as more stable and clinically relevant biomarkers compared to those obtained from serum. Objective: To compare miRNAs isolated from EVs versus serum in blood taken from Alzheimer's disease (AD) dementia patients and control cohorts. Methods: We compared 25 AD patients to 34 individuals who exhibited no cognitive impairments (NCI). Subjects were Singapore residents with Chinese heritage. miRNAs purified from serum versus blood-derived EVs were analyzed for associations with AD dementia and medial temporal atrophy detected by magnetic resonance imaging. Results: Compared to serum-miRNAs, we identified almost twice as many EV-miRNAs associated with AD dementia, and they also correlated more significantly with medial temporal atrophy, a neuroimaging marker of AD-brain pathology. We further developed combination panels of serum-miRNAs and EV-miRNAs with improved performance in identifying AD dementia. Dominant in both panels was miRNA-1290. Conclusions: This data indicates that miRNA profiling from EVs offers diagnostic superiority. This underscores the role of EVs as vectors harboring prognostic biomarkers for neurodegenerative disorders and suggests their potential in yielding novel biomarkers for AD diagnosis.

FUS-dependent microRNA deregulations identify TRIB2 as a druggable target for ALS motor neurons.

MicroRNAs (miRNAs) modulate mRNA expression, and their deregulation contributes to various diseases including amyotrophic lateral sclerosis (ALS). As fused in sarcoma (FUS) is a causal gene for ALS and regulates biogenesis of miRNAs, we systematically analyzed the miRNA repertoires in spinal cords and hippocampi from ALS-FUS mice to understand how FUS-dependent miRNA deregulation contributes to ALS. miRNA profiling identified differentially expressed miRNAs between different central nervous system (CNS) regions as well as disease states. Among the up-regulated miRNAs, miR-1197 targets the pro-survival pseudokinase Trib2. A reduced TRIB2 expression was observed in iPSC-derived motor neurons from ALS patients. Pharmacological stabilization of TRIB2 protein with a clinically approved cancer drug rescues the survival of iPSC-derived human motor neurons, including those from a sporadic ALS patient. Collectively, our data indicate that miRNA profiling can be used to probe the molecular mechanisms underlying selective vulnerability, and TRIB2 is a potential therapeutic target for ALS.

APP family member dimeric complexes are formed predominantly in synaptic compartments.

BACKGROUND: The amyloid precursor protein (APP), a key player in Alzheimer's disease (AD), is part of a larger gene family, including the APP like proteins APLP1 and APLP2. They share similar structures, form homo- and heterotypic dimers and exhibit overlapping functions. RESULTS: We investigated complex formation of the APP family members via two inducible dimerization systems, the FKBP-rapamycin based dimerization as well as cysteine induced dimerization, combined with co-immunoprecipitations and Blue Native (BN) gel analyses. Within the APP family, APLP1 shows the highest degree of dimerization and high molecular weight (HMW) complex formation. Interestingly, only about 20% of APP is dimerized in cultured cells whereas up to 50% of APP is dimerized in mouse brains, independent of age and splice forms. Furthermore, we could show that dimerized APP originates mostly from neurons and is enriched in synaptosomes. Finally, BN gel analysis of human cortex samples shows a significant decrease of APP dimers in AD patients compared to controls. CONCLUSIONS: Together, we suggest that loss of full-length APP dimers might correlate with loss of synapses in the process of AD.

Enhanced cleavage of APP by co-expressed Bace1 alters the distribution of APP and its fragments in neuronal and non-neuronal cells.

BACKGROUND: Alzheimer's disease amyloid-beta peptides (Aß) are generated via sequential cleavage of the amyloid precursor protein (APP) by ß-secretase (Bace1) and γ-secretase. Though the precise subcellular location(s) of Bace1-mediated APP cleavage remains unresolved, current models suggest APP internalization into Bace1-containing endosomes is a critical step. However, direct evidence for this model is lacking, and previous reports that probed the APP/Bace1 interaction (using co-expressed APP and Bace1 differentially labeled with fluorescent protein tags) did not determine if APP fluorescence originated from full-length APP (fl-APP) molecules that had internalized from the cell surface pool. METHODS: We adapted the bungarotoxin-ligand (BTX) system to label surface APP and track internalized fluorescent APP/BTX puncta in rodent primary neurons co-expressing fluorescently-tagged Bace1. Subsequently, we employed imaging and biochemical-based approaches to measure N- and C-terminal APP epitope levels in primary neurons, N2a neuroblastoma, and HeLa cell lines. RESULTS: We hypothesized that surface-labeled APP/BTX puncta would, upon internalization, colocalize with fluorescently-tagged Bace1. Unexpectedly, we observed a dramatic loss of internalized APP in co-transfected neurons and ~ 80-90% loss of surface-resident fl-APP, which we also observed in HeLa and N2a cells. Loss of surface fl-APP could be reversed by a Bace1 inhibitor, suggesting that enhanced Bace1-mediated APP cleavage was responsible for the altered processing and mis-sorting. Importantly, in a C-terminally-tagged APP construct, the majority of C-terminal fluorescence was preserved in HeLa cells despite the loss of N-terminal APP signal. This phenomenon was not only recapitulated in cultured neurons, but also showed a progressive disappearance of the APP N-terminal tag, reflecting continual cleavage of fl-APP by Bace1 away from the cell body. CONCLUSIONS: Our results strongly suggested that in APP/Bace1 co-expression approaches, there was significant early and aberrant Bace1-mediated APP cleavage that perturbed fl-APP trafficking from the secretory pathway onwards, resulting in a substantial loss of surface fl-APP, which in turn led to a marked reduction in APP internalization. In C-terminally-tagged APP constructs, a large fraction of the APP fluorescence signal therefore likely arose from fluorescently-tagged ß-C-terminal-fragment (ß-CTF) or downstream proteolytic derivatives instead of fl-APP. Thus, care is needed in interpreting results where APP is detected only with a C-terminal tag in the presence of Bace1 co-expression, and previous findings may need to be reinterpreted if it is unclear whether fl-APP is present in normal physiological levels.

Dysfunction in nonsense-mediated decay, protein homeostasis, mitochondrial function, and brain connectivity in ALS-FUS mice with cognitive deficits.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) represent two ends of the same disease spectrum of adult-onset neurodegenerative diseases that affect the motor and cognitive functions, respectively. Multiple common genetic loci such as fused in sarcoma (FUS) have been identified to play a role in ALS and FTD etiology. Current studies indicate that FUS mutations incur gain-of-toxic functions to drive ALS pathogenesis. However, how the disease-linked mutations of FUS affect cognition remains elusive. Using a mouse model expressing an ALS-linked human FUS mutation (R514G-FUS) that mimics endogenous expression patterns, we found that FUS proteins showed an age-dependent accumulation of FUS proteins despite the downregulation of mouse FUS mRNA by the R514G-FUS protein during aging. Furthermore, these mice developed cognitive deficits accompanied by a reduction in spine density and long-term potentiation (LTP) within the hippocampus. At the physiological expression level, mutant FUS is distributed in the nucleus and cytosol without apparent FUS aggregates or nuclear envelope defects. Unbiased transcriptomic analysis revealed a deregulation of genes that cluster in pathways involved in nonsense-mediated decay, protein homeostasis, and mitochondrial functions. Furthermore, the use of in vivo functional imaging demonstrated widespread reduction in cortical volumes but enhanced functional connectivity between hippocampus, basal ganglia and neocortex in R514G-FUS mice. Hence, our findings suggest that disease-linked mutation in FUS may lead to changes in proteostasis and mitochondrial dysfunction that in turn affect brain structure and connectivity resulting in cognitive deficits.

Presymptomatic Increase of an Extracellular RNA in Blood Plasma Associates with the Development of Alzheimer's Disease.

The extracellular RNAs (exRNAs) from human biofluid have recently been systematically characterized. However, the correlations of biofluid exRNA levels and human diseases remain largely untested. Here, considering the unmet need for presymptomatic biomarkers of sporadic Alzheimer's disease (AD), we leveraged the recently developed SILVER-seq (small-input liquid volume extracellular RNA sequencing) technology to generate exRNA profiles from a longitudinal collection of human plasma samples. These 164 plasma samples were collected from research subjects 70 years or older with up to 15 years of clinical follow-up prior to death and whose clinical diagnoses were confirmed by pathological analysis of their post mortem brains. The exRNAs of AD-activated genes and transposons in the brain exhibited a concordant trend of increase in AD plasma in comparison with age-matched control plasma. However, when we required statistical significance with multiple testing adjustments, phosphoglycerate dehydrogenase (PHGDH) was the only gene that exhibited consistent upregulation in AD brain transcriptomes from 3 independent cohorts and an increase in AD plasma as compared to controls. We validated PHGDH's serum exRNA and brain protein expression increases in AD by using 5 additional published cohorts. Finally, we compared the time-course exRNA trajectories between "converters" and controls. Plasma PHGDH exRNA exhibited presymptomatic increases in each of the 11 converters during their transitions from normal to cognitive impairment but remained stable over the entire follow-up period in 8 out of the 9 control elderly subjects. These data suggest the potential utilities of plasma exRNA levels for screening and longitudinal exRNA changes as a presymptomatic indication of sporadic AD.

PERK-mediated induction of microRNA-483 disrupts cellular ATP homeostasis during the unfolded protein response.

Endoplasmic reticulum (ER) stress activates the unfolded protein response (UPR), which reduces levels of misfolded proteins. However, if ER homeostasis is not restored and the UPR remains chronically activated, cells undergo apoptosis. The UPR regulator, PKR-like endoplasmic reticulum kinase (PERK), plays an important role in promoting cell death when persistently activated; however, the underlying mechanisms are poorly understood. Here, we profiled the microRNA (miRNA) transcriptome in human cells exposed to ER stress and identified miRNAs that are selectively induced by PERK signaling. We found that expression of a PERK-induced miRNA, miR-483, promotes apoptosis in human cells. miR-483 induction was mediated by a transcription factor downstream of PERK, activating transcription factor 4 (ATF4), but not by the CHOP transcription factor. We identified the creatine kinase brain-type (CKB) gene, encoding an enzyme that maintains cellular ATP reserves through phosphocreatine production, as being repressed during the UPR and targeted by miR-483. We found that ER stress, selective PERK activation, and CKB knockdown all decrease cellular ATP levels, leading to increased vulnerability to ER stress-induced cell death. Our findings identify miR-483 as a downstream target of the PERK branch of the UPR. We propose that disruption of cellular ATP homeostasis through miR-483-mediated CKB silencing promotes ER stress-induced apoptosis.

FUS-mediated dysregulation of Sema5a, an autism-related gene, in FUS mice with hippocampus-dependent cognitive deficits.

Pathological fused in sarcoma (FUS) inclusions are found in 10% of patients with frontotemporal dementia and those with amyotrophic lateral sclerosis (ALS) carrying FUS mutations. Current work indicates that FUS mutations may incur gain-of-toxic functions to drive ALS pathogenesis. However, how FUS dysfunction may affect cognition remains elusive. Using a mouse model expressing wild-type human FUS mimicking the endogenous expression pattern and level within the central nervous system, we found that they developed hippocampus-mediated cognitive deficits accompanied by an age-dependent reduction in spine density and long-term potentiation in their hippocampus. However, there were no apparent FUS aggregates, nuclear envelope defects and cytosolic FUS accumulation. These suggest that these proposed pathogenic mechanisms may not be the underlying causes for the observed cognitive deficits. Unbiased transcriptomic analysis identified expression changes in a small set of genes with preferential expression in the neurons and oligodendrocyte lineage cells. Of these, we focused on Sema5a, a gene involved in axon guidance, spine dynamics, Parkinson's disease and autism spectrum disorders. Critically, FUS binds directly to Sema5a mRNA and regulates Sema5a expression in a FUS-dose-dependent manner. Taken together, our data suggest that FUS-driven Sema5a deregulation may underlie the cognitive deficits in FUS transgenic mice.

Microinterventional endocapsular nucleus disassembly: novel technique and results of first-in-human randomised controlled study.

AIM: To assess the safety and efficacy of microinterventional endocapsular nuclear fragmentation in moderate to severe cataracts. METHODS: This was a prospective single-masked multisurgeon interventional randomised controlled trial (ClinicalTrials.gov NCT02843594) where 101 eyes of 101 subjects with grade 3‒4+ nuclear cataracts were randomised to torsional phacoemulsification alone (controls) or torsional phacoemulsification with adjunctive endocapsular nuclear fragmentation using a manual microinterventional nitinol filament loop device (miLOOP group). Outcome measures were phacoemulsification efficiency as measured by ultrasound energy (cumulative dispersed energy (CDE) units) and fluidics requirements (total irrigation fluid used) as well as incidence of intraoperative and postoperative complications. RESULTS: Only high-grade advanced cataracts were enrolled with more than 85% of eyes with baseline best corrected visual acuity (BCVA) of 20/200 or worse in either group. Mean CDE was 53% higher in controls (32.8±24.9 vs 21.4±13.1 with miLOOP assistance) (p=0.004). Endothelial cell loss after surgery was low and similar between groups (7‒8%, p=0.561) One-month BCVA averaged 20/27 Snellen in miLOOP eyes and 20/24 in controls. No direct complications were caused by the miLOOP. In two cases, capsular tears occurred during IOL implantation and in all remaining cases during phacoemulsification, with none occurring during the miLOOP nucleus disassembly part of the procedure. CONCLUSIONS: Microinterventional endocapsular fragmentation with the manual, disposable miLOOP device achieved consistent, ultrasound-free, full-thickness nucleus disassembly and significantly improved overall phaco efficiency in advanced cataracts. TRIAL REGISTRATION NUMBER: NCT02843594.

Tauopathy-associated PERK alleles are functional hypomorphs that increase neuronal vulnerability to ER stress.

Tauopathies are neurodegenerative diseases characterized by tau protein pathology in the nervous system. EIF2AK3 (eukaryotic translation initiation factor 2 alpha kinase 3), also known as PERK (protein kinase R-like endoplasmic reticulum kinase), was identified by genome-wide association study as a genetic risk factor in several tauopathies. PERK is a key regulator of the Unfolded Protein Response (UPR), an intracellular signal transduction mechanism that protects cells from endoplasmic reticulum (ER) stress. PERK variants had previously been identified in Wolcott-Rallison Syndrome, a rare autosomal recessive metabolic disorder, and these variants completely abrogated the function of PERK's kinase domain or prevented PERK expression. In contrast, the PERK tauopathy risk variants were distinct from the Wolcott-Rallison variants and introduced missense alterations throughout the PERK protein. The function of PERK tauopathy variants and their effects on neurodegeneration are unknown. Here, we discovered that tauopathy-associated PERK alleles showed reduced signaling activity and increased PERK protein turnover compared to protective PERK alleles. We found that iPSC-derived neurons carrying PERK risk alleles were highly vulnerable to ER stress-induced injury with increased tau pathology. We found that chemical inhibition of PERK in human iPSC-derived neurons also increased neuronal cell death in response to ER stress. Our results indicate that tauopathy-associated PERK alleles are functional hypomorphs during the UPR. We propose that reduced PERK function leads to neurodegeneration by increasing neuronal vulnerability to ER stress-associated damage. In this view, therapies to enhance PERK signaling would benefit at-risk carriers of hypomorphic alleles.

Microinterventional endocapsular nucleus disassembly for phacoemulsification-free full-thickness fragmentation.

We describe a new technique for nucleus disassembly using a microinterventional approach. This is the first report of a microinterventional phacoemulsification-free endocapsular lens fragmentation using a superelastic memory-shaped nickel and titanium (nitinol) microfilament (miLOOP). After standard capsulorhexis and hydrodissection, the microinterventional loop is inserted through the 2.3 mm clear corneal incision. An actuation mechanism is used to unfold the microloop, which glides horizontally on the anterior surface of the lens, expands below the anterior capsule, and then rotates vertically while traveling in the hydrodissection plane to encircle the entire nucleus. Then, the loop is mechanically retracted within the sleeve, achieving full-thickness centripetal segmentation of the nucleus. After rotation of the lens, the loop cuts can be repeated to create 4 or more segments without using phaco energy. The technique is independent of cataract grade and eliminates the need for phacoemulsification or femtosecond energy during the nucleus disassembly step of cataract surgery.

Short Aß peptides attenuate Aß42 toxicity in vivo.

Processing of amyloid-ß (Aß) precursor protein (APP) by γ-secretase produces multiple species of Aß: Aß40, short Aß peptides (Aß37-39), and longer Aß peptides (Aß42-43). γ-Secretase modulators, a class of Alzheimer's disease therapeutics, reduce production of the pathogenic Aß42 but increase the relative abundance of short Aß peptides. To evaluate the pathological relevance of these peptides, we expressed Aß36-40 and Aß42-43 in Drosophila melanogaster to evaluate inherent toxicity and potential modulatory effects on Aß42 toxicity. In contrast to Aß42, the short Aß peptides were not toxic and, when coexpressed with Aß42, were protective in a dose-dependent fashion. In parallel, we explored the effects of recombinant adeno-associated virus-mediated expression of Aß38 and Aß40 in mice. When expressed in nontransgenic mice at levels sufficient to drive Aß42 deposition, Aß38 and Aß40 did not deposit or cause behavioral alterations. These studies indicate that treatments that lower Aß42 by raising the levels of short Aß peptides could attenuate the toxic effects of Aß42.

γ-Secretase inhibitors and modulators.

γ-Secretase is a fascinating, multi-subunit, intramembrane cleaving protease that is now being considered as a therapeutic target for a number of diseases. Potent, orally bioavailable γ-secretase inhibitors (GSIs) have been developed and tested in humans with Alzheimer's disease (AD) and cancer. Preclinical studies also suggest the therapeutic potential for GSIs in other disease conditions. However, due to inherent mechanism based-toxicity of non-selective inhibition of γ-secretase, clinical development of GSIs will require empirical testing with careful evaluation of benefit versus risk. In addition to GSIs, compounds referred to as γ-secretase modulators (GSMs) remain in development as AD therapeutics. GSMs do not inhibit γ-secretase, but modulate γ-secretase processivity and thereby shift the profile of the secreted amyloid ß peptides (Aß) peptides produced. Although GSMs are thought to have an inherently safe mechanism of action, their effects on substrates other than the amyloid ß protein precursor (APP) have not been extensively investigated. Herein, we will review the current state of development of GSIs and GSMs and explore pertinent biological and pharmacological questions pertaining to the use of these agents for select indications. This article is part of a Special Issue entitled: Intramembrane Proteases.

Steroids as γ-secretase modulators.

Aggregation and accumulation of Aß42 play an initiating role in Alzheimer's disease (AD); thus, selective lowering of Aß42 by γ-secretase modulators (GSMs) remains a promising approach to AD therapy. Based on evidence suggesting that steroids may influence Aß production, we screened 170 steroids at 10 µM for effects on Aß42 secreted from human APP-overexpressing Chinese hamster ovary cells. Many acidic steroids lowered Aß42, whereas many nonacidic steroids actually raised Aß42. Studies on the more potent compounds showed that Aß42-lowering steroids were bonafide GSMs and Aß42-raising steroids were inverse GSMs. The most potent steroid GSM identified was 5ß-cholanic acid (EC50=5.7 µM; its endogenous analog lithocholic acid was virtually equipotent), and the most potent inverse GSM identified was 4-androsten-3-one-17ß-carboxylic acid ethyl ester (EC50=6.25 µM). In addition, we found that both estrogen and progesterone are weak inverse GSMs with further complex effects on APP processing. These data suggest that certain endogenous steroids may have the potential to act as GSMs and add to the evidence that cholesterol, cholesterol metabolites, and other steroids may play a role in modulating Aß production and thus risk for AD. They also indicate that acidic steroids might serve as potential therapeutic leads for drug optimization/development.

Lysine 624 of the amyloid precursor protein (APP) is a critical determinant of amyloid ß peptide length: support for a sequential model of γ-secretase intramembrane proteolysis and regulation by the amyloid ß precursor protein (APP) juxtamembrane region.

γ-Secretase is a multiprotein intramembrane cleaving aspartyl protease (I-CLiP) that catalyzes the final cleavage of the amyloid ß precursor protein (APP) to release the amyloid ß peptide (Aß). Aß is the primary component of senile plaques in Alzheimer's disease (AD), and its mechanism of production has been studied intensely. γ-Secretase executes multiple cleavages within the transmembrane domain of APP, with cleavages producing Aß and the APP intracellular domain (AICD), referred to as γ and ε, respectively. The heterogeneous nature of the γ cleavage that produces various Aß peptides is highly relevant to AD, as increased production of Aß 1-42 is genetically and biochemically linked to the development of AD. We have identified an amino acid in the juxtamembrane region of APP, lysine 624, on the basis of APP695 numbering (position 28 relative to Aß) that plays a critical role in determining the final length of Aß peptides released by γ-secretase. Mutation of this lysine to alanine (K28A) shifts the primary site of γ-secretase cleavage from 1-40 to 1-33 without significant changes to ε cleavage. These results further support a model where ε cleavage occurs first, followed by sequential proteolysis of the remaining transmembrane fragment, but extend these observations by demonstrating that charged residues at the luminal boundary of the APP transmembrane domain limit processivity of γ-secretase.

Mechanism of cytotoxicity mediated by the C31 fragment of the amyloid precursor protein.

The cytoplasmic tail of the amyloid precursor protein (APP) contains two putatively cytotoxic peptides, Jcasp and C31, derived by caspase cleavage of APP. Jcasp is a fragment starting from the epsilon-secretase site to position 664, while C31 is a fragment from position 665 to the C-terminus. Our studies now showed that compared to C31, Jcasp appeared to play a minor role in cytotoxicity. In particular, inhibition of Jcasp generation by treatment of gamma-secretase inhibitor did not lead to any attenuation of C31-induced toxicity. Secondly, because C31 toxicity is largely absent in cells lacking endogenous APP, we determined, using a split beta-galactosidase complementary assay to monitor protein-protein interactions, the presence of APP associated complexes. Our results demonstrated that both APP homomeric and C31/APP heteromeric complexes were correlated with cell death, indicating that C31 complexes with APP to recruit the interacting partners that initiate the signals related to cellular toxicity.

Induced dimerization of the amyloid precursor protein leads to decreased amyloid-beta protein production.

The amyloid precursor protein (APP) plays a central role in Alzheimer disease (AD) pathogenesis because sequential cleavages by beta- and gamma-secretase lead to the generation of the amyloid-beta (Abeta) peptide, a key constituent in the amyloid plaques present in brains of AD individuals. In several studies APP has recently been shown to form homodimers, and this event appears to influence Abeta generation. However, these studies have relied on APP mutations within the Abeta sequence itself that may affect APP processing by interfering with secretase cleavages independent of dimerization. Therefore, the impact of APP dimerization on Abeta production remains unclear. To address this question, we compared the approach of constitutive cysteine-induced APP dimerization with a regulatable dimerization system that does not require the introduction of mutations within the Abeta sequence. To this end we generated an APP chimeric molecule by fusing a domain of the FK506-binding protein (FKBP) to the C terminus of APP. The addition of the synthetic membrane-permeant drug AP20187 induces rapid dimerization of the APP-FKBP chimera. Using this system we were able to induce up to 70% APP dimers. Our results showed that controlled homodimerization of APP-FKBP leads to a 50% reduction in total Abeta levels in transfected N2a cells. Similar results were obtained with the direct precursor of beta-secretase cleavage, C99/SPA4CT-FKBP. Furthermore, there was no modulation of different Abeta peptide species after APP dimerization in this system. Taken together, our results suggest that APP dimerization can directly affect gamma-secretase processing and that dimerization is not required for Abeta production.

Formation of Pmel17 amyloid is regulated by juxtamembrane metalloproteinase cleavage, and the resulting C-terminal fragment is a substrate for gamma-secretase.

The formation of insoluble cross beta-sheet amyloid is pathologically associated with disorders such as Alzheimer, Parkinson, and Huntington diseases. One exception is the nonpathological amyloid derived from the protein Pmel17 within melanosomes to generate melanin pigment. Here we show that the formation of insoluble MalphaC intracellular fragments of Pmel17, which are the direct precursors to Pmel17 amyloid, depends on a novel juxtamembrane cleavage at amino acid position 583 between the furin-like proprotein convertase cleavage site and the transmembrane domain. The resulting Pmel17 C-terminal fragment is then processed by the gamma-secretase complex to release a short-lived intracellular domain fragment. Thus, by analogy to the Notch receptor, we designate this cleavage the S2 cleavage site, whereas gamma-secretase mediates proteolysis at the intramembrane S3 site. Substitutions or deletions at this S2 cleavage site, the use of the metalloproteinase inhibitor TAPI-2, as well as small interfering RNA-mediated knock-down of the metalloproteinases ADAM10 and 17 reduced the formation of insoluble Pmel17 fragments. These results demonstrate that the release of the Pmel17 ectodomain, which is critical for melanin amyloidogenesis, is initiated by S2 cleavage at a juxtamembrane position.

Biodistribution and toxicological safety of adenovirus type 5 and type 35 vectored vaccines against human immunodeficiency virus-1 (HIV-1), Ebola, or Marburg are similar despite differing adenovirus serotype vector, manufacturer's construct, or gene inserts.

The Vaccine Research Center has developed vaccine candidates for different diseases/infectious agents (including HIV-1, Ebola, and Marburg viruses) built on an adenovirus vector platform, based on adenovirus type 5 or 35. To support clinical development of each vaccine candidate, pre-clinical studies were performed in rabbits to determine where in the body they biodistribute and how rapidly they clear, and to screen for potential toxicities (intrinsic and immunotoxicities). The vaccines biodistribute only to spleen, liver (Ad5 only), and/or iliac lymph node (Ad35 only) and otherwise remain in the site of injection muscle and overlying subcutis. Though approximately 10(11) viral particles were inoculated, already by Day 9, all but 10(3) to 10(5) genome copies per mu g of DNA had cleared from the injection site muscle. By three months, the adenovector was cleared with, at most, a few animals retaining a small number of copies in the injection site, spleen (Ad5), or iliac lymph node (Ad35). This pattern of limited biodistribution and extensive clearance is consistent regardless of differences in adenovector type (Ad5 or 35), manufacturer's construct and production methods, or gene-insert. Repeated dose toxicology studies identified treatment-related toxicities confined primarily to the sites of injection, in certain clinical pathology parameters, and in body temperatures (Ad5 vectors) and food consumption immediately post-inoculation. Systemic reactogenicity and reactogenicity at the sites of injection demonstrated reversibility. These data demonstrate the safety and suitability for investigational human use of Ad5 or Ad35 adenovector-based vaccine candidates at doses of up to 2 x 10(11) given intramuscularly to prevent various infectious diseases.

Substrate-targeting gamma-secretase modulators.

Selective lowering of Abeta42 levels (the 42-residue isoform of the amyloid-beta peptide) with small-molecule gamma-secretase modulators (GSMs), such as some non-steroidal anti-inflammatory drugs, is a promising therapeutic approach for Alzheimer's disease. To identify the target of these agents we developed biotinylated photoactivatable GSMs. GSM photoprobes did not label the core proteins of the gamma-secretase complex, but instead labelled the beta-amyloid precursor protein (APP), APP carboxy-terminal fragments and amyloid-beta peptide in human neuroglioma H4 cells. Substrate labelling was competed by other GSMs, and labelling of an APP gamma-secretase substrate was more efficient than a Notch substrate. GSM interaction was localized to residues 28-36 of amyloid-beta, a region critical for aggregation. We also demonstrate that compounds known to interact with this region of amyloid-beta act as GSMs, and some GSMs alter the production of cell-derived amyloid-beta oligomers. Furthermore, mutation of the GSM binding site in the APP alters the sensitivity of the substrate to GSMs. These findings indicate that substrate targeting by GSMs mechanistically links two therapeutic actions: alteration in Abeta42 production and inhibition of amyloid-beta aggregation, which may synergistically reduce amyloid-beta deposition in Alzheimer's disease. These data also demonstrate the existence and feasibility of 'substrate targeting' by small-molecule effectors of proteolytic enzymes, which if generally applicable may significantly broaden the current notion of 'druggable' targets.