Apo and Aß46-bound γ-secretase structures provide insights into amyloid-ß processing by the APH-1B isoform.

Deposition of amyloid-ß (Aß) peptides in the brain is a hallmark of Alzheimer's disease. Aßs are generated through sequential proteolysis of the amyloid precursor protein by the γ-secretase complexes (GSECs). Aß peptide length, modulated by the Presenilin (PSEN) and APH-1 subunits of GSEC, is critical for Alzheimer's pathogenesis. Despite high relevance, mechanistic understanding of the proteolysis of Aß, and its modulation by APH-1, remain incomplete. Here, we report cryo-EM structures of human GSEC (PSEN1/APH-1B) reconstituted into lipid nanodiscs in apo form and in complex with the intermediate Aß46 substrate without cross-linking. We find that three non-conserved and structurally divergent APH-1 regions establish contacts with PSEN1, and that substrate-binding induces concerted rearrangements in one of the identified PSEN1/APH-1 interfaces, providing structural basis for APH-1 allosteric-like effects. In addition, the GSEC-Aß46 structure reveals an interaction between Aß46 and loop 1PSEN1, and identifies three other H-bonding interactions that, according to functional validation, are required for substrate recognition and efficient sequential catalysis.

Electropositive Citric Acid-Polyethyleneimine Carbon Dots Carrying the PINK1 Gene Regulate ATP-Related Metabolic Dysfunction in APP/PS1-N2a Cells.

(1) Background: Alzheimer's disease (AD) is characterized by ß-amyloid (Aß) peptide accumulation and mitochondrial dysfunction during the early stage of disease. PINK1 regulates the balance between mitochondrial homeostasis and bioenergy supply and demand via the PINK1/Parkin pathway, Na+/Ca2+ exchange, and other pathways. (2) Methods: In this study, we synthesized positively charged carbon dots (CA-PEI CDs) using citric acid (CA) and polyethyleneimine (PEI) and used them as vectors to express PINK1 genes in the APP/PS1-N2a cell line to determine mitochondrial function, electron transport chain (ETC) activity, and ATP-related metabolomics. (3) Results: Our findings showed that the CA-PEI CDs exhibit the characteristics of photoluminescence, low toxicity, and concentrated DNA. They are ideal biological carriers for gene delivery. PINK1 overexpression significantly increased the mitochondrial membrane potential in APP/PS1-N2a cells and reduced reactive-oxygen-species generation and Aß1-40 and Aß1-42 levels. An increase in the activity of NADH ubiquinone oxidoreductase (complex I, CI) and cytochrome C oxidase (complex IV, CIV) induces the oxidative phosphorylation of mitochondria, increasing ATP generation. (4) Conclusions: These findings indicate that the PINK gene can alleviate AD by increasing bioenergetic metabolism, reducing Aß1-40 and Aß1-42, and increasing ATP production.

CNTN4 modulates neural elongation through interplay with APP.

The neuronal cell adhesion molecule contactin-4 (CNTN4) is genetically associated with autism spectrum disorder (ASD) and other psychiatric disorders. Cntn4-deficient mouse models have previously shown that CNTN4 plays important roles in axon guidance and synaptic plasticity in the hippocampus. However, the pathogenesis and functional role of CNTN4 in the cortex has not yet been investigated. Our study found a reduction in cortical thickness in the motor cortex of Cntn4 -/- mice, but cortical cell migration and differentiation were unaffected. Significant morphological changes were observed in neurons in the M1 region of the motor cortex, indicating that CNTN4 is also involved in the morphology and spine density of neurons in the motor cortex. Furthermore, mass spectrometry analysis identified an interaction partner for CNTN4, confirming an interaction between CNTN4 and amyloid-precursor protein (APP). Knockout human cells for CNTN4 and/or APP revealed a relationship between CNTN4 and APP. This study demonstrates that CNTN4 contributes to cortical development and that binding and interplay with APP controls neural elongation. This is an important finding for understanding the physiological function of APP, a key protein for Alzheimer's disease. The binding between CNTN4 and APP, which is involved in neurodevelopment, is essential for healthy nerve outgrowth.

Histone deacetylase-3 regulates the expression of the amyloid precursor protein and its inhibition promotes neuroregenerative pathways in Alzheimer's disease models.

HDAC3 inhibition has been shown to improve memory and reduce amyloid-ß (Aß) in Alzheimer's disease (AD) models, but the underlying mechanisms are unclear. We investigated the molecular effects of HDAC3 inhibition on AD pathology, using in vitro and ex vivo models of AD, based on our finding that HDAC3 expression is increased in AD brains. For this purpose, N2a mouse neuroblastoma cells as well as organotypic brain cultures (OBCSs) of 5XFAD and wild-type mice were incubated with various concentrations of the HDAC3 selective inhibitor RGFP966 (0.1-10 µM) for 24 h. Treatment with RGFP966 or HDAC3 knockdown in N2a cells was associated with an increase on amyloid precursor protein (APP) and mRNA expressions, without alterations in Aß42 secretion. In vitro chromatin immunoprecipitation analysis revealed enriched HDAC3 binding at APP promoter regions. The increase in APP expression was also detected in OBCSs from 5XFAD mice incubated with 1 µM RGFP966, without changes in Aß. In addition, HDAC3 inhibition resulted in a reduction of activated Iba-1-positive microglia and astrocytes in 5XFAD slices, which was not observed in OBCSs from wild-type mice. mRNA sequencing analysis revealed that HDAC3 inhibition modulated neuronal regenerative pathways related to neurogenesis, differentiation, axonogenesis, and dendritic spine density in OBCSs. Our findings highlight the complexity and diversity of the effects of HDAC3 inhibition on AD models and suggest that HDAC3 may have multiple roles in the regulation of APP expression and processing, as well as in the modulation of neuroinflammatory and neuroprotective genes.

Osteoclasts Link Dysregulated Peripheral Degradation Processes and Accelerated Progression in Alzheimer's Disease.

Background: The amyloid-ß (Aß) enhances the number and activity of blood monocyte-derived osteoclasts (OCs). Individuals with osteoporosis (OP) face an increased risk of developing dementia or Alzheimer's disease (AD). Despite this association, the contribution of bone-resorbing OCs to the progression of AD pathology remains unclear. Objective: Our objective was to investigate the potential impacts of OCs on the development of AD pathology. Methods: We conducted targeted analysis of publicly available whole blood transcriptomes from patients with AD to characterize the blood molecular signatures and pathways associated with hyperactive OCs. In addition, we used APP23 transgenic (APP23 TG) AD mouse model to assess the effects of OCs pharmacological blockade on AD pathology and behavior. Results: Patients with AD exhibited increased osteoclastogenesis signature in their blood cells, which appears to be positively correlated with dysfunction of peripheral clearance of Aß mediated by immune cells. Long-term anti-resorptive intervention with Alendronate inhibited OC activity in APP23 mice, leading to improvements in peripheral monocyte Aß-degrading enzyme expression, Aß-deposition, and memory decline. Conclusions: Our findings suggest that OCs have a disease-promoting role in the development and progression of AD, possibly linked to their modulation of peripheral immunity. These findings guide future research to further elucidate the connection between OP and AD pathogenesis, highlighting the potential benefits of preventing OP in alleviating cognitive burden.

Differential disruptions in population coding along the dorsal-ventral axis of CA1 in the APP/PS1 mouse model of Aß pathology.

Alzheimer's Disease (AD) is characterized by a range of behavioral alterations, including memory loss and psychiatric symptoms. While there is evidence that molecular pathologies, such as amyloid beta (Aß), contribute to AD, it remains unclear how this histopathology gives rise to such disparate behavioral deficits. One hypothesis is that Aß exerts differential effects on neuronal circuits across brain regions, depending on the neurophysiology and connectivity of different areas. To test this, we recorded from large neuronal populations in dorsal CA1 (dCA1) and ventral CA1 (vCA1), two hippocampal areas known to be structurally and functionally diverse, in the APP/PS1 mouse model of amyloidosis. Despite similar levels of Aß pathology, dCA1 and vCA1 showed distinct disruptions in neuronal population activity as animals navigated a virtual reality environment. In dCA1, pairwise correlations and entropy, a measure of the diversity of activity patterns, were decreased in APP/PS1 mice relative to age-matched C57BL/6 controls. However, in vCA1, APP/PS1 mice had increased pair-wise correlations and entropy as compared to age matched controls. Finally, using maximum entropy models, we connected the microscopic features of population activity (correlations) to the macroscopic features of the population code (entropy). We found that the models' performance increased in predicting dCA1 activity, but decreased in predicting vCA1 activity, in APP/PS1 mice relative to the controls. Taken together, we found that Aß exerts distinct effects across different hippocampal regions, suggesting that the various behavioral deficits of AD may reflect underlying heterogeneities in neuronal circuits and the different disruptions that Aß pathology causes in those circuits.

A novel APP splice variant-dependent marker system to precisely demarcate maturity in SH-SY5Y cell-derived neurons.

SH-SY5Y, a neuroblastoma cell line, can be converted into mature neuronal phenotypes, characterized by the expression of mature neuronal and neurotransmitter markers. However, the mature phenotypes described across multiple studies appear inconsistent. As this cell line expresses common neuronal markers after a simple induction, there is a high chance of misinterpreting its maturity. Therefore, sole reliance on common neuronal markers is presumably inadequate. The Alzheimer's disease (AD) central gene, amyloid precursor protein (APP), has shown contrasting transcript variant dynamics in various cell types. We differentiated SH-SY5Y cells into mature neuron-like cells using a concise protocol and observed the upregulation of total APP throughout differentiation. However, APP transcript variant-1 was upregulated only during the early to middle stages of differentiation and declined in later stages. We identified the maturity state where this post-transcriptional shift occurs, terming it "true maturity." At this stage, we observed a predominant expression of mature neuronal and cholinergic markers, along with a distinct APP variant pattern. Our findings emphasize the necessity of using a differentiation state-sensitive marker system to precisely characterize SH-SY5Y differentiation. Moreover, this study offers an APP-guided, alternative neuronal marker system to enhance the accuracy of the conventional markers.

Olfactory Dysfunction and Alzheimer's Disease: A Review.

 Alzheimer's disease is the most common cause of dementia, and it is one of the leading causes of death globally. Identification and validation of biomarkers that herald the onset and progression of Alzheimer's disease is of paramount importance for early reliable diagnosis and effective pharmacological therapy commencement. A substantial body of evidence has emerged demonstrating that olfactory dysfunction is a preclinical symptom of neurodegenerative diseases including Alzheimer's disease. While a correlation between olfactory dysfunction and Alzheimer's disease onset and progression in humans exists, the mechanism underlying this relationship remains unknown. The aim of this article is to review the current state of knowledge regarding the range of potential factors that may contribute to the development of Alzheimer's disease-related olfactory dysfunction. This review predominantly focuses on genetic mutations associated with Alzheimer's disease including amyloid-ß protein precursor, presenilin 1 and 2, and apolipoprotein E mutations, that may (in varying ways) drive the cellular events that lead to and sustain olfactory dysfunction.

Aplp1 interacts with Lag3 to facilitate transmission of pathologic α-synuclein.

Pathologic α-synuclein (α-syn) spreads from cell-to-cell, in part, through binding to the lymphocyte-activation gene 3 (Lag3). Here we report that amyloid ß precursor-like protein 1 (Aplp1) interacts with Lag3 that facilitates the binding, internalization, transmission, and toxicity of pathologic α-syn. Deletion of both Aplp1 and Lag3 eliminates the loss of dopaminergic neurons and the accompanying behavioral deficits induced by α-syn preformed fibrils (PFF). Anti-Lag3 prevents the internalization of α-syn PFF by disrupting the interaction of Aplp1 and Lag3, and blocks the neurodegeneration induced by α-syn PFF in vivo. The identification of Aplp1 and the interplay with Lag3 for α-syn PFF induced pathology deepens our insight about molecular mechanisms of cell-to-cell transmission of pathologic α-syn and provides additional targets for therapeutic strategies aimed at preventing neurodegeneration in Parkinson's disease and related α-synucleinopathies.

PLC-CN-NFAT1 signaling-mediated Aß and IL-1ß crosstalk synergistically promotes hippocampal neuronal damage.

Alzheimer's disease (AD) is a progressive neurodegenerative disease. Neuronal calcium overload plays an important role in Aß deposition and neuroinflammation, which are strongly associated with AD. However, the specific mechanisms by which calcium overload contributes to neuroinflammation and AD and the relationship between them have not been elucidated. Phospholipase C (PLC) is involved in regulation of calcium homeostasis, and CN-NFAT1 signaling is dependent on intracellular Ca2+ ([Ca2+]i) to regulate transcription of genes. Therefore, we hypothesized that the PLC-CN-NFAT1 signaling might mediate the interaction between Aß and inflammation to promote neuronal injury in AD. In this experiment, the results showed that the levels of Aß, IL-1ß and [Ca2+]i in the hippocampal primary neurons of APP/PS1 mice (APP neurons) were significantly increased. IL-1ß exposure also significantly increased Aß and [Ca2+]i in HT22 cells, suggesting a close association between Aß and IL-1ß in the development of AD. Furthermore, PLC activation induced significant calcium homeostasis imbalance, cell apoptosis, Aß and ROS production, and significantly increased expressions of CN and NFAT1, while PLC inhibitor significantly reversed these changes in APP neurons and IL-1ß-induced HT22 cells. Further results indicated that PLC activation significantly increased the expressions of NOX2, APP, BACE1, and NCSTN, which were inhibited by PLC inhibitor in APP neurons and IL-1ß-induced HT22 cells. All indications point to a synergistic interaction between Aß and IL-1ß by activating the PLC-CN-NFAT1 signal, ultimately causing a vicious cycle, resulting in neuronal damage in AD. The study may provide a new idea and target for treatment of AD.

Monocytes release cystatin F dimer to associate with Aß and aggravate amyloid pathology and cognitive deficits in Alzheimer's disease.

BACKGROUND: Understanding the molecular mechanisms of Alzheimer's disease (AD) has important clinical implications for guiding therapy. Impaired amyloid beta (Aß) clearance is critical in the pathogenesis of sporadic AD, and blood monocytes play an important role in Aß clearance in the periphery. However, the mechanism underlying the defective phagocytosis of Aß by monocytes in AD remains unclear. METHODS: Initially, we collected whole blood samples from sporadic AD patients and isolated the monocytes for RNA sequencing analysis. By establishing APP/PS1 transgenic model mice with monocyte-specific cystatin F overexpression, we assessed the influence of monocyte-derived cystatin F on AD development. We further used a nondenaturing gel to identify the structure of the secreted cystatin F in plasma. Flow cytometry, enzyme-linked immunosorbent assays and laser scanning confocal microscopy were used to analyse the internalization of Aß by monocytes. Pull down assays, bimolecular fluorescence complementation assays and total internal reflection fluorescence microscopy were used to determine the interactions and potential interactional amino acids between the cystatin F protein and Aß. Finally, the cystatin F protein was purified and injected via the tail vein into 5XFAD mice to assess AD pathology. RESULTS: Our results demonstrated that the expression of the cystatin F protein was specifically increased in the monocytes of AD patients. Monocyte-derived cystatin F increased Aß deposition and exacerbated cognitive deficits in APP/PS1 mice. Furthermore, secreted cystatin F in the plasma of AD patients has a dimeric structure that is closely related to clinical signs of AD. Moreover, we noted that the cystatin F dimer blocks the phagocytosis of Aß by monocytes. Mechanistically, the cystatin F dimer physically interacts with Aß to inhibit its recognition and internalization by monocytes through certain amino acid interactions between the cystatin F dimer and Aß. We found that high levels of the cystatin F dimer protein in blood contributed to amyloid pathology and cognitive deficits as a risk factor in 5XFAD mice. CONCLUSIONS: Our findings highlight that the cystatin F dimer plays a crucial role in regulating Aß metabolism via its peripheral clearance pathway, providing us with a potential biomarker for diagnosis and potential target for therapeutic intervention.

Upregulation of neuronal ER-phagy improves organismal fitness and alleviates APP toxicity.

ER-phagy, a selective autophagy targeting the endoplasmic reticulum (ER) for lysosomal degradation through cargo receptors, plays a critical role in ER quality control and is linked to various diseases. However, its physiological and pathological roles remain largely unclear due to a lack of animal model studies. This study establishes Drosophila as an in vivo ER-phagy model. Starvation triggers ER-phagy across multiple fly tissues. Disturbing ER-phagy by either globally upregulating or downregulating ER-phagy receptors, Atl or Rtnl1, harms the fly. Notably, moderate upregulation of ER-phagy in fly brains by overexpressing Atl or Rtnl1 significantly attenuates age-associated neurodegenerations. Furthermore, in a Drosophila model of Alzheimer's disease expressing human amyloid precursor protein (APP), impaired ER-phagy is observed. Enhancing ER-phagy in the APP-expressing fly brain facilitates APP degradation, significantly alleviating disease symptoms. Therefore, our findings suggest that modulating ER-phagy may offer a therapeutic strategy to treat aging and diseases associated with ER protein aggregation.

The amyloid precursor protein and its derived fragments concomitantly contribute to the alterations of mitochondrial transport machinery in Alzheimer's disease.

Mitochondria dysfunctions and mitophagy failure have been associated with several Alzheimer's disease (AD) related molecular actors including amyloid beta (Aß) and recently the amyloid precursor protein-C terminal fragments (APP-CTFs). The efficacy of the mitophagy process in neurons relies on regulated mitochondrial transport along axons involving a complex molecular machinery. The contribution of the amyloid precursor protein (APP) and its derived fragments to the mitochondrial transport machinery alterations in AD have not been investigated before. We report herein a change of the expression of mitochondrial transport proteins (SNPH and Miro1), motor adapters (TRANK1 and TRAK2), and components of the dynein and kinesin motors (i.e., IC1,2 and Kif5 (A, B, C) isoforms) by endogenous APP and by overexpression of APP carrying the familial Swedish mutation (APPswe). We show that APP-CTFs and Aß concomitantly regulate the expression of a set of transport proteins as demonstrated in APPswe cells treated with ß- and γ-secretase inhibitors and in cells Knock-down for presenilin 1 and 2. We further report the impact of APP-CTFs on the expression of transport proteins in AAV-injected C99 mice brains. Our data also indicate that both Aß oligomers (Aßo) and APP-CTFs impair the colocalization of mitochondria and transport proteins. This has been demonstrated in differentiated SH-SY5Y naive cells treated with Aßo and in differentiated SH-SY5Y and murine primary neurons expressing APPswe and treated with the γ-secretase inhibitor. Importantly, we uncover that the expression of a set of transport proteins is modulated in a disease-dependent manner in 3xTgAD mice and in human sporadic AD brains. This study highlights molecular mechanisms underlying mitochondrial transport defects in AD that likely contribute to mitophagy failure and disease progression.

Ferroptosis and its implications in treating cognitive impairment caused by aging: A study on the mechanism of repetitive transcranial magnetic stimulation.

OBJECTIVE: Ferroptosis has been recognized as being closely associated with cognitive impairment. Research has established that Alzheimer's disease (AD)-associated proteins, such as amyloid precursor protein (APP) and phosphorylated tau, are involved in brain iron metabolism. These proteins are found in high concentrations within senile plaques and neurofibrillary tangles. Repetitive transcranial magnetic stimulation (rTMS) offers a non-pharmacological approach to AD treatment. This study aims to explore the potential therapeutic effects of rTMS on cognitive impairment through the modulation of the ferroptosis pathway, thereby laying both a theoretical and experimental groundwork for the application of rTMS in treating Alzheimer's disease. METHODS: The study utilized senescence-accelerated mouse prone 8 (SAMP8) mice to model brain aging-related cognitive impairment, with senescence-accelerated-mouse resistant 1 (SAMR1) mice acting as controls. The SAMP8 mice were subjected to high-frequency rTMS at 25 Hz for durations of 14 and 28 days. Cognitive function was evaluated using behavioral tests. Resting-state functional magnetic resonance imaging (rs-fMRI) assessed alterations in cerebral activity by measuring the fractional amplitude of low-frequency fluctuations (fALFF) of the blood oxygen level-dependent signal. Neuronal recovery post-rTMS in the SAMP8 model was examined via HE and Nissl staining. Immunohistochemistry was employed to detect the expression of APP and Phospho-Tau (Thr231). Oxidative stress markers were quantified using biochemical assay kits. ELISA methods were utilized to measure hippocampal levels of Fe2+ and Aß1-42. Finally, the expression of proteins related to the ferroptosis pathway was determined through western blot analysis. RESULTS: The findings indicate that 25 Hz rTMS enhances cognitive function and augments cerebral activity in SAMP8 model mice. Treatment with rTMS in these mice resulted in diminished oxidative stress and safeguarded neurons against damage. Additionally, iron accumulation was mitigated, and the expression of ferroptosis pathway proteins Gpx4, system Xc-, and Nrf2 was elevated. CONCLUSIONS: The Tau/APP-Fe-GPX4/system Xc-/Nrf2 pathway is implicated in the remedial effects of rTMS on cognitive dysfunction, offering a theoretical and experimental basis for employing rTMS in AD treatment.

Dl-3-n-butylphthalide promotes microglial phagocytosis and inhibits microglial inflammation via regulating AGE-RAGE pathway in APP/PS1 mice.

Alzheimer's disease (AD) stands as the most prevalent neurodegenerative condition worldwide, and its correlation with microglial function is notably significant. Dl-3-n-butylphthalide (NBP), derived from the seeds of Apium graveolens L. (Chinese celery), has demonstrated the capacity to diminish Aß levels in the brain tissue of Alzheimer's transgenic mice. Despite this, its connection to neuroinflammation and microglial phagocytosis, along with the specific molecular mechanism involved, remains undefined. In this study, NBP treatment exhibited a substantial improvement in learning deficits observed in AD transgenic mice (APP/PS1 transgenic mice). Furthermore, NBP treatment significantly mitigated the total cerebral Aß plaque deposition. This effect was attributed to the heightened presence of activated microglia surrounding Aß plaques and an increase in microglial phagocytosis of Aß plaques. Transcriptome sequencing analysis unveiled the potential involvement of the AGE (advanced glycation end products) -RAGE (receptor for AGE) signaling pathway in NBP's impact on APP/PS1 mice. Subsequent investigation disclosed a reduction in the secretion of AGEs, RAGE, and proinflammatory factors within the hippocampus and cortex of NBP-treated APP/PS1 mice. In summary, NBP alleviates cognitive impairment by augmenting the number of activated microglia around Aß plaques and ameliorating AGE-RAGE-mediated neuroinflammation. These findings underscore the related mechanism of the crucial neuroprotective roles of microglial phagocytosis and anti-inflammation in NBP treatment for AD, offering a potential therapeutic target for the disease.

Loss of Cholinergic and Monoaminergic Afferents in APPswe/PS1ΔE9 Transgenic Mouse Model of Cerebral Amyloidosis Preferentially Occurs Near Amyloid Plaques.

Alzheimer's disease (AD) is characterized by a loss of neurons in the cortex and subcortical regions. Previously, we showed that the progressive degeneration of subcortical monoaminergic (MAergic) neurons seen in human AD is recapitulated in the APPswe/PS1ΔE9 (APP/PS) transgenic mouse model. Because degeneration of cholinergic (Ach) neurons is also a prominent feature of AD, we examined the integrity of the Ach system in the APP/PS model. The overall density of Ach fibers is reduced in APP/PS1 mice at 12 and 18 months of age but not at 4 months of age. Analysis of basal forebrain Ach neurons shows no loss of Ach neurons in the APP/PS model. Thus, since MAergic systems show overt cell loss at 18 months of age, the Ach system is less vulnerable to neurodegeneration in the APP/PS1 model. We also examined whether the proximity to Aß deposition affected the degeneration of Ach and 5-HT afferents. We found that the areas closer to the edges of compact Aß deposits exhibit a more severe loss of afferents than the areas that are more distal to Aß deposits. Collectively, the results indicate that the APP/PS model recapitulates the degeneration of multiple subcortical neurotransmitter systems, including the Ach system. In addition, the results indicate that Aß deposits cause global as well as local toxicity to subcortical afferents.

Cellular depletion of major cathepsin proteases reveals their concerted activities for lysosomal proteolysis.

Proteins delivered by endocytosis or autophagy to lysosomes are degraded by exo- and endoproteases. In humans 15 lysosomal cathepsins (CTS) act as important physiological regulators. The cysteine proteases CTSB and CTSL and the aspartic protease CTSD are the most abundant and functional important lysosomal proteinases. Whereas their general functions in proteolysis in the lysosome, their individual substrate, cleavage specificity, and their possible sequential action on substrate proteins have been previously studied, their functional redundancy is still poorly understood. To address a possible common role of highly expressed and functional important CTS proteases, we generated CTSB-, CTSD-, CTSL-, and CTSBDL-triple deficient (KO) human neuroblastoma-derived SH-SY5Y cells and CTSB-, CTSD-, CTSL-, CTSZ and CTSBDLZ-quadruple deficient (KO) HeLa cells. These cells with a combined cathepsin deficiency exhibited enlarged lysosomes and accumulated lipofuscin-like storage material. The lack of the three (SH-SY5Y) or four (HeLa) major CTSs caused an impaired autophagic flux and reduced degradation of endocytosed albumin. Proteome analyses of parental and CTS-depleted cells revealed an enrichment of cleaved peptides, lysosome/autophagy-associated proteins, and potentially endocytosed membrane proteins like the amyloid precursor protein (APP), which can be subject to endocytic degradation. Amino- and carboxyterminal APP fragments accumulated in the multiple CTS-deficient cells, suggesting that multiple CTS-mediated cleavage events regularly process APP. In summary, our analyses support the idea that different lysosomal cathepsins act in concert, have at least partially and functionally redundant substrates, regulate protein degradation in autophagy, and control cellular proteostasis, as exemplified by their involvement in the degradation of APP fragments.

STAU1 exhibits a dual function by promoting amyloidogenesis and tau phosphorylation in cultured cells.

Staufen-1 (STAU1) is a double-stranded RNA-binding protein (RBP) involved in a variety of pathological conditions. In this study, we investigated the potential role of STAU1 in Alzheimer's disease (AD), in which two hallmarks are well-established as cerebral ß-amyloid protein (Aß) deposition and Tau-centered neurofibrillary tangles. We found that STAU1 protein level was significantly increased in cells that stably express full-length APP and the brain of APP/PS1 mice, an animal model of AD. STAU1 knockdown, as opposed to overexpression, significantly decreased the protein levels of ß-amyloid converting enzyme 1 (BACE1) and Aß. We further found that STAU1 extended the half-life of the BACE1 mRNA through binding to the 3' untranslated region (3'UTR). Transcriptome analysis revealed that STAU1 enhanced the expression of growth arrest and DNA damage 45 ß (GADD45B) upstream of P38 MAPK signaling, which contributed to STAU1-induced regulation of Tau phosphorylation at Ser396 and Thr181. Together, STAU1 promoted amyloidogenesis by inhibiting BACE1 mRNA decay, and augmented Tau phosphorylation through activating GADD45B in relation to P38 MAPK. Targeting STAU1 that acts on both amyloidogenesis and tauopathy may serve as an optimistic approach for AD treatment.

Ergosterol promotes neurite outgrowth, inhibits amyloid-beta synthesis, and extends longevity: In vitro neuroblastoma and in vivo Caenorhabditis elegans evidence.

AIMS: Alzheimer's disease (AD), the most common neurodegenerative disorder associated with aging, is characterized by amyloid-ß (Aß) plaques in the hippocampus. Ergosterol, a mushroom sterol, exhibits neuroprotective activities; however, the underlying mechanisms of ergosterol in promoting neurite outgrowth and preventing Aß-associated aging have never been investigated. We aim to determine the beneficial activities of ergosterol in neuronal cells and Caenorhabditis elegans (C. elegans). MATERIALS AND METHODS: The neuritogenesis and molecular mechanisms of ergosterol were investigated in wild-type and Aß precursor protein (APP)-overexpressing Neuro2a cells. The anti-amyloidosis properties of ergosterol were determined by evaluating in vitro Aß production and the potential inhibition of Aß-producing enzymes. Additionally, AD-associated transgenic C. elegans was utilized to investigate the in vivo attenuating effects of ergosterol. KEY FINDINGS: Ergosterol promoted neurite outgrowth in Neuro2a cells through the upregulation of the transmembrane protein Teneurin-4 (Ten-4) mRNA and protein expressions, phosphorylation of the extracellular signal-regulated kinases (ERKs), activity of cAMP response element (CRE), and growth-associated protein-43 (GAP-43). Furthermore, ergosterol enhanced neurite outgrowth in transgenic Neuro2A cells overexpressing either the wild-type APP (Neuro2a-APPwt) or the Swedish mutant APP (Neuro2a-APPswe) through the Ten-4/ERK/CREB/GAP-43 signaling pathway. Interestingly, ergosterol inhibited Aß synthesis in Neuro2a-APPwt cells. In silico analysis indicated that ergosterol can interact with the catalytic sites of ß- and γ-secretases. In Aß-overexpressing C. elegans, ergosterol decreased Aß accumulation, increased chemotaxis behavior, and prolonged lifespan. SIGNIFICANCE: Ergosterol is a potential candidate compound that might benefit AD patients by promoting neurite outgrowth, inhibiting Aß synthesis, and enhancing longevity.

The Compartmentalization of Amyloid-ß in Idiopathic Normal Pressure Hydrocephalus Brain Biopsies.

Background: Amyloid-ß (Aß) is one of the hallmark lesions of Alzheimer's disease (AD). During the disease process, Aß undergoes biochemical changes, producing toxic Aß variants, proposed to be detected within the neurons. Idiopathic normal pressure hydrocephalus (iNPH) causes cognitive impairment, gait, and urinary symptoms in elderly, that can be reversed by a ventriculo-peritoneal shunt. Majority of iNPH subjects display different Aß variants in their brain biopsies, obtained during shunting. Objective: To study the cellular compartmentalization of different Aß variants in brain biopsies from iNPH subjects. Methods: We studied the cellular localization of different proteoforms of Aß using antibodies towards different amino acid sequences or post-translational modifications of Aß, including clones 4G8, 6F/3D, unmodified- (7H3D6), pyroglutamylated- (N3pE), phosphorylated-(1E4E11) Aß and Aß protein precursor (AßPP), in brain biopsies from 3 iNPH subjects, using immunohistochemistry and light microscopy (LM), light microscopy on semi-thin sections (LMst), and electron microscopy (EM). Results: In LM all Aß variants were detected. In LMst and EM, the Aß 4G8, 6F/3D, and the pyroglutamylated Aß were detected. The AßPP was visualized by all methods. The Aß labelling was located extracellularly with no specific signal within the intracellular compartment, whereas the AßPP was seen both intra- and extracellularly. Conclusions: The Aß markers displayed extracellular localization when visualized by three assessment techniques, reflecting the pathological extracellular accumulation of Aß in the human brain. No intracellular Aß pathology was seen. AßPP was visualized in intra- and extracellularly, which corresponds to the localization of the protein in the membranes of cells and organelles.