Proteases that degrade the amyloid ␤-protein (A␤) are important regulators of brain A␤ levels in health and in Alzheimer's disease, yet few practical methods exist to study their detailed kinetics. Here, we describe robust and quantitative A␤ degradation assays based on the novel substrate, fluorescein-A␤-(1-40)-Lys-biotin (FA␤B). Liquid chromatography/mass spectrometric analysis shows that FA␤B is hydrolyzed at closely similar sites as wild-type A␤ by neprilysin and insulindegrading enzyme, the two most widely studied A␤-degrading proteases. The derivatized peptide is an avid substrate and is suitable for use with biological samples and in high throughput compound screening. The assays we have developed are easily implemented and are particularly useful for the generation of quantitative kinetic data, as we demonstrate by determining the kinetic parameters of FA␤B degradation by several A␤-degrading proteases, including plasmin, which has not previously been characterized. The use of these assays should yield additional new insights into the biology of A␤-degrading proteases and facilitate the identification of activators and inhibitors of such enzymes.Progressive accumulation of the amyloid ␤-protein (A␤) 1 in brain regions important for memory and cognition is a defining pathogenic feature of Alzheimer's disease (AD). Nevertheless, the causes of elevated brain A␤ levels in the vast majority of AD patients remain unknown. With the exception of rare familial forms of the disease, there is little evidence that AD is attributable to the overproduction of A␤. Instead, failed clearance of the peptide, including defects in its proteolytic degradation, could underlie its accumulation with age, a possibility that is gaining increasing experimental support (1). Significant elevations in cerebral A␤ levels have now been observed in vivo in gene-targeted mice lacking each of several A␤-degrading proteases: neprilysin (NEP) (2), endothelin-converting enzyme-1 and -2 (3), and insulin-degrading enzyme (IDE) (4, 5). Moreover, several genetic studies have reported linkage and/or allelic association between late onset AD and polymorphisms near or within the IDE gene on chromosome 10q (6 -8).Progress in elucidating the mechanisms underlying the production of A␤ from its protein precursor, APP, by the ␤-and ␥-secretases has depended critically on the development of sensitive, reliable, and accessible assays for quantifying A␤ levels in biological samples. More recently, assays for directly measuring the activity of the secretases have been described (9 -11), enabling significant progress in the biochemical characterization of these proteases and the identification and characterization of small-molecule inhibitors. In contrast, relatively few techniques and assays for studying A␤ degradation have been reported, and the most commonly used general methods (e.g. measurement of radiolabeled peptides by trichloroacetic acid precipitation or HPLC) are cumbersome and ill suited for accurate quantification of kinetic constants or for hig...

Ubiquilin 1 (UBQLN1) is a ubiquitin-like protein, which has been shown to play a central role in regulating the proteasomal degradation of various proteins, including the presenilins. We recently reported that DNA variants in UBQLN1 increase the risk for Alzheimer disease, by influencing expression of this gene in brain. Here we present the first assessment of the effects of UBQLN1 on the metabolism of the amyloid precursor protein (APP). For this purpose, we employed RNA interference to down-regulate UBQLN1 in a variety of neuronal and non-neuronal cell lines. We demonstrate that down-regulation of UBQLN1 accelerates the maturation and intracellular trafficking of APP, while not interfering with ␣-, ␤-, or ␥-secretase levels or activity. UBQLN1 knockdown increased the ratio of APP mature/immature, increased levels of full-length APP on the cell surface, and enhanced the secretion of sAPP (␣-and ␤-forms). Moreover, UBQLN1 knockdown increased levels of secreted A␤40 and A␤42. Finally, employing a fluorescence resonance energy transfer-based assay, we show that UBQLN1 and APP come into close proximity in intact cells, independently of the presence of the presenilins. Collectively, our findings suggest that UBQLN1 may normally serve as a cytoplasmic "gatekeeper" that may control APP trafficking from intracellular compartments to the cell surface. These findings suggest that changes in UBQLN1 steady-state levels affect APP trafficking and processing, thereby influencing the generation of A␤. Alzheimer disease (AD)3 is the most common cause of progressive neurological disorder leading to dementia. It is neuropathologically characterized by extracellular deposits of amyloid beta (A␤) peptide and by the generation of intracellular neurofibrillary tangles. Mutations in the amyloid precursor protein (APP), presenilin-1 (PSEN1), and presenilin-2 (PSEN2) genes are responsible for roughly half of the rare autosomal dominant, early-onset forms of the disease, which usually occur before the age of 60 (1-4). Meanwhile, apolipoprotein E (APOE) is the only commonly accepted susceptibility factor for late-onset AD (5, 6). Most mutations in APP, PSEN1, and PSEN2 genes lead to the increased production of A␤42 (relative to A␤40). A␤ is released from APP via sequential proteolytic cleavage by the ␤-and ␥-secretases (7). In addition to APP, the presenilins, and APOE, it is evident that additional AD susceptibility genes exist; successfully identifying these novel risk genes is an extremely important task that will not only facilitate prediction and diagnosis of AD but can also elucidate novel therapeutic approaches to treating and preventing AD.We have recently shown that genetic variants in the ubiquilin 1 (UBQLN1) gene, located on chromosome 9q22, increase the risk for AD, possibly by altering the expression and alternative splicing of this gene in brain (8). As is often the case with gene variants exerting modest effects on disease risk, subsequent genetic studies have both supported (9, 10) and not supported (11, 12) the initial g...

In albino mammals, lack of pigment in the retinal pigment epithelium is associated with retinal defects, including poor visual acuity from a photoreceptor deficit in the central retina and poor depth perception from a decrease in ipsilaterally projecting retinal fibers. Possible contributors to these abnormalities are reported delays in neuronogenesis (Ilia and Jeffery, 1996) and retinal maturation (Webster and Rowe, 1991). To further determine possible perturbations in neuronogenesis and/or differentiation, we used cell-specific markers and refined birth dating methods to examine these events during retinal ganglion cell (RGC) genesis in albino and pigmented mice from embryonic day 11 (E11) to E18. Our data indicate that relative to pigmented mice, more ganglion cells are born in the early stages of neuronogenesis in the albino retina, although the initiation of RGC genesis in the albino is unchanged. The cellular organization of the albino retina is perturbed as early as E12. In addition, cell cycle kinetics and output along the nasotemporal axis differ in retinas of albino and pigmented mice, both absolutely, with the temporal aspect of the retina expanded in albino, and relative to the position of the optic nerve head. Finally, blocking melanin synthesis in pigmented eyecups in culture leads to an increase in RGC differentiation, consistent with a role for melanin formation in regulating RGC neuronogenesis. These results point to spatiotemporal defects in neuronal production in the albino retina, which could perturb expression of genes that specify cell fate, number, and/or projection phenotype.

The Alzheimer's disease (AD)-associated ubiquilin-1 regulates proteasomal degradation of proteins, including presenilin (PS). PS-dependent γ-secretase generates β-amyloid (Aβ) peptides, which excessively accumulate in AD brain. Here, we have characterized the effects of naturally occurring ubiquilin-1 transcript variants (TVs) on the levels and subcellular localization of PS1 and other γ-secretase complex components and subsequent γ-secretase function in human embryonic kidney 293, human neuroblastoma SH-SY5Y and mouse primary cortical cells. Full-length ubiquilin-1 TV1 and TV3 that lacks the proteasome-interaction domain increased fulllength PS1 levels as well as induced accumulation of high-molecular-weight PS1 and aggresome formation. Accumulated PS1 colocalized with TV1 or TV3 in the aggresomes. Electron microscopy indicated that aggresomes containing TV1 or TV3 were targeted to autophagosomes. TV1-and TV3-expressing cells did not accumulate other unrelated proteasome substrates, suggesting that the increase in PS1 levels was not because of a general impairment of the ubiquitinproteasome system. Furthermore, PS1 accumulation and aggresome formation coincided with alterations in Aβ levels, particularly in cells overexpressing TV3. These effects were not related to altered γ-secretase activity or PS1 binding to TV3. Collectively, our results indicate that specific ubiquilin-1 TVs can cause PS1 accumulation and aggresome formation, which may impact AD pathogenesis or susceptibility.

Insulin-degrading enzyme (IDE) is a zinc metalloprotease that degrades the amyloid ␤-peptide, the key component of Alzheimer disease (AD)-associated senile plaques. We have previously reported evidence for genetic linkage and association of AD on chromosome 10q23-24 in the region harboring the IDE gene. Here we have presented the first functional assessment of IDE in AD families showing the strongest evidence of the genetic linkage. We have examined the catalytic activity and expression of IDE in lymphoblast samples from 12 affected and unaffected members of three chromosome 10-linked AD pedigrees in the National Institute of Mental Health AD Genetics Initiative family sample. We have shown that the catalytic activity of cytosolic IDE to degrade insulin is reduced in affected versus unaffected subjects of these families. Further, we have shown the decrease in activity is not due to reduced IDE expression, suggesting the possible defects in IDE function in these AD families. In attempts to find potential mutations in the IDE gene in these families, we have found no coding region substitutions or alterations in splicing of the canonical exons and exon 15b of IDE. We have also found that total IDE mRNA levels are not significantly different in sporadic AD versus age-matched control brains. Collectively, our data suggest that the genetic linkage of AD in this set of chromosome 10-linked AD families may be the result of systemic defects in IDE activity in the absence of altered IDE expression, further supporting a role for IDE in AD pathogenesis.2 is the primary component of senile plaques, a pathological hallmark in the brains of patients with Alzheimer disease (AD). Elevated levels of cerebral A␤ have also been observed in AD patients (1, 2), implicating excessive accumulation of A␤ as a key pathogenic event in AD. Unlike early onset autosomal dominant AD, the vast majority of AD cases do not show any clear evidence of Mendelian transmission and predominantly present with late onset AD (LOAD) (onset age Ͼ65). However, there is evidence that genetic factors play a significant role in modifying the disease risk/age of onset in the majority of LOAD cases (3, 4). To date, only the ⑀4 allele of the apolipoprotein E gene (APOE) has been firmly established as a LOAD genetic risk factor and has been proposed to be involved in A␤ clearance (5). Cerebral A␤ accumulation has been proposed to greatly influence the age of onset of LOAD and is determined by the amount of A␤ generated versus the amount that is degraded and exported from the brain over one's lifetime (6, 7).Several proteases have been identified to degrade A␤, including neprilysin, plasmin, endothelin-converting enzyme-1 as well as insulin-degrading enzyme (IDE) (EC 3.4.24.56) (1). IDE, also called insulysin, is a zinc metalloprotease that cleaves small polypeptides, many of which share amyloid fibril-forming ability, including insulin, atrial naturetic peptide, amylin, calcitonin, and A␤ (8, 9). IDE is a major protease to degrade soluble, monomeric A␤ (10, 11) an...

Previous studies have implicated the unfolded protein response (UPR) in the pathogenesis of Alzheimer's disease (AD). We previously reported that DNA variants in the ubiquilin 1 (UBQLN1) gene increase the risk for AD. Since UBQLN1 has been shown to play a role in the UPR, we assessed the effects of overexpression and downregulation of UBQLN1 splice variants during tunicamycin-induced ER stress. In addition to previously described transcript variants, TV1 and TV2, we identified two novel transcript variants of UBQLN1 in brain: TV3 (lacking exons 2-4) and TV4 (lacking exon 4). Overexpression of TV1-3, but not TV4 significantly decreased the mRNA induction of UPR-inducible genes, C/EBP homologous protein (CHOP), BiP/GRP78, and protein disulfide isomerase (PDI) during the UPR. Stable overexpression of TV1-3, but not TV4, also significantly decreased the induction of CHOP protein and increased cell viability during the UPR. In contrast, downregulation of UBQLN1 did not affect CHOP mRNA induction, but instead increased PDI mRNA levels. These findings suggest that overexpression UBQLN1 transcript variants TV1-3, but not TV4, exert a protective effect during the UPR by attenuating CHOP induction and potentially increasing cell viability.

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Introduction: The number of forcibly displaced immigrants entering the United States continues to rapidly increase. Movement from Latin America across the southern border of the United States was the third-largest migration worldwide in 2017; the U.S. now serves as home to one-fifth of the world’s migrants (Budiman, 2020; Leyva-Flores et al., 2019). Reporting on the first two years of clients receiving forensic medical evaluations (FMEs) conducted by clinicians trained at University of California, San Francisco (UCSF), this descriptive study demonstrates the multiple layers and types of trauma in asylum seekers presenting to a student-run asylum clinic (SRAC) at an academic medical center.   Methods: A retrospective review of the first 102 asylum seekers presenting to a university-affiliated SRAC for forensic medical and psychological evaluations is summarized. Demographics, immigration history, medical and mental health histories, descriptions of extensive trauma and referral patterns are reported. Multivariate statistics were employed to investigate the relationship between past trauma and current mental health status.   Results: Clients reported extensive trauma histories, with an average of 4.4 different types of ill-treatment per person, including physical, psychological, and sexual violence. The current mental health burden was extensive with 86.9 percent of clients reporting symptoms of PTSD and/or depression. Clients were evaluated within a clinic structure that intentionally aligns with SAMHSA’s implementation domains of trauma-informed care using a continuous improvement model to reduce barriers to FMEs and promote longitudinal follow-up and referral access.   Discussion: This study demonstrates the profound trauma exposure reported by asylum seekers, as well as the adaptation of a SRAC to better respond to complex trauma through intentional structural and leadership decisions. The HRC experience provides a blueprint for other asylum clinics to implement systematic trauma-centered services.