APP Processing Research Articles

BACE cleavage of APP does not drive the diabetic phenotype of PLB4 mice

BackgroundAlzheimer's Disease (AD) and Type 2 Diabetes Mellitus (T2DM), two prevalent diseases related to ageing, often share common pathologies including increased inflammation, endoplasmic reticulum (ER) stress, and impaired metabolic homeostasis predominantly affecting different organs. Therefore, it was unexpected to find in a previous study that neuronal hBACE1 knock-in (PLB4 mouse) leads to both an AD- and T2DM- like phenotype. The complexity of this co-morbidity phenotype required a deeper systems approach to explore the age-related changes in AD and T2DM-like pathologies of the PLB4 mouse. Therefore, we here analysed key neuronal and metabolic tissues comparing associated pathologies to those of normal ageing. MethodsGlucose tolerance, insulin sensitivity and protein turnover were assessed in 5-h fasted 3- and 8-month-old male PLB4 and wild-type mice. Western Blot and quantitative PCR were performed to determine regulation of homeostatic and metabolic pathways in insulin-stimulated brain, liver and muscle tissue. ResultsNeuronal hBACE1 expression caused early pathological cleavage of APP (increased monomeric Aβ (mAβ) levels at 3 months), in parallel with brain ER stress (increased phosphorylation of the translation regulation factor (p-eIF2α) and the chaperone binding immunoglobulin protein (BIP)). However, APP processing shifted over time (higher full-length APP and secreted APPβ levels, alongside lower mAβ and secreted APPα at 8 months), together with increased ER stress (phosphorylated/total inositol-requiring enzyme 1α (IRE1α)) in brain and liver. Metabolically, systemic glucose intolerance was evident from 3 months, yet metabolic signalling varied greatly between tissues and ages, and was confined to the periphery (increased muscle insulin receptors (IR), dipeptidyl-peptidase-4 (DPP4) levels, and decreased phosphorylated protein Kinase B (p-Akt), alongside increased liver DPP4 and fibroblast growth factor 21 (FGF21)), all of which normalised to wild-type levels at 8 months. ConclusionOur data suggest that the murine nervous system is affected early by APP misprocessing as a result of hBACE1 introduction, which coincided with ER stress, but not IR changes, and was alleviated with age. Peripheral metabolic alterations occurred early and revealed tissue-specific (liver vs. muscle) adaptations in metabolic markers but did not correlate with neuronal APP processing. Compensatory vs. contributory neuronal mechanisms associated with hBACE1 expression at different ages may explain why mice intrinsically do not develop AD pathologies and may offer new insights for future interventions.

Assessing the Co-Chaperone and Synaptic Protein DNAJC5 as a Therapeutic Target in Alzheimer’s Disease (P1-6.010)

<h3>Objective:</h3> Assess the therapeutic potential of <i>DNAJC5</i> in Alzheimer’s disease (AD). <h3>Background:</h3> <i>DNAJC5</i> encodes cysteine-string protein alpha (CSPα), whose mutations cause adult neuronal ceroid lipofuscinosis (ANCL). ANCL is characterized by endolysosomal dysfunction, synaptic loss, and protein aggregate accumulation, similar to AD. CSPα facilitates secretion of the neurodegenerative proteins tau, TDP-43, and α-synuclein and maintains synaptic integrity. Therefore, CSPα is a promising therapeutic target. <h3>Design/Methods:</h3> N2A695 cells stably expressing mutant <i>DNAJC5</i> or knocking down endogenous <i>Dnajc5</i> were analyzed through immunoblotting and ELISA for APP processing and Aβ production. Brains of ANCL patients and <i>Dnajc5</i>+/−; 5xFAD mice were assessed for amyloid pathology with immunohistochemistry. Transcriptomics from ANCL, AD, and control brains was analyzed for differentially expressed genes (DEGs). The Clue.IO database was used to identify potential targets for gene therapy or pharmacological intervention. <h3>Results:</h3> ANCL-patient brains exhibited low soluble or insoluble Aβ levels, but intra-neuronal Aβ accumulation. They exhibited reductions in synaptic protein levels (SNAP-25, Synaptobrevin, Syntaxin 1, and Synaptophysin). Transcriptomic analysis of ANCL-patient brains showed reduced <i>TSPAN14</i>, which facilitates ADAM10 maturation, elevated <i>APH1B</i>, a γ-secretase subunit, and elevated <i>GSAP</i>, the γ-secretase activating protein. There were 67 upregulated and 15 downregulated transcripts associated with processes essential for synaptic function, including the MAPK cascade (<i>FYN</i>, <i>SOS1</i>), dendritic spine organization (<i>FYN</i>), and endosomal vesicle fusion (<i>VPS11</i>, <i>VPS39</i>). Clue.IO identified heat-shock protein inhibitors and an opioid receptor antagonist (BNTX) as potential pharmacological agents to counteract the effects of mutant <i>DNAJC5</i>. AD patients and 5xFAD mice exhibited reduced <i>DNAJC5</i> expression. Preliminary results from <i>Dnajc5</i>+/−; 5xFAD mice showed increased plaques in the corpus collosum and hippocampus. N2A695 cells expressing <i>DNAJC5</i> mutants showed increased Aβ while <i>DNAJC5</i> knockdown decreased Aβ. <h3>Conclusions:</h3> Low levels of CSPα alter Ab secretion and dysregulate genes and synaptic proteins, suggesting that increasing CSPα levels could reduce Aβ and improve synaptic function. <b>Disclosure:</b> Dr. Rosene has nothing to disclose. Dr. Nykanen has nothing to disclose. Kevin O’Dell has nothing to disclose. Ms. Nunez has nothing to disclose. Dr. Harari has nothing to disclose. John Cirrito has nothing to disclose. Prof. Sands has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for M6P Therapeutics. Prof. Sands has received personal compensation in the range of $500-$4,999 for serving on a Speakers Bureau for Amicus. Prof. Sands has stock in JAYA Biosciences. The institution of Prof. Sands has received research support from JAYA Biosciences. Prof. Sands has received intellectual property interests from a discovery or technology relating to health care. Bruno Benitez has nothing to disclose.

E674Q (Shanghai APP mutant), a novel amyloid precursor protein mutation, in familial late-onset Alzheimer's disease

Identified as the pathogenic genes of Alzheimer's disease (AD), APP, PSEN1, and PSEN2 mainly lead to early-onset AD, whose course is more aggressive, and atypical symptoms are more common than sporadic AD. Here, a novel missense mutation, APP E674Q (also named “Shanghai APP”), was detected in a Chinese index patient with typical late-onset AD (LOAD) who developed memory decline in his mid-70s. The results from neuroimaging were consistent with AD, where widespread amyloid β deposition was demonstrated in 18F-florbetapir Positron Emission Tomography (PET). APP E674Q is close to the β-secretase cleavage site and the well-studied Swedish APP mutation (KM670/671NL), which was predicted to be pathogenic in silico. Molecular dynamics simulation indicated that the E674Q mutation resulted in a rearrangement of the interaction mode between APP and BACE1 and that the E674Q mutation was more prone to cleavage by BACE1. The in vitro results suggested that the E674Q mutation was pathogenic by facilitating the BACE1-mediated processing of APP and the production of Aβ. Furthermore, we applied an adeno-associated virus (AAV)-mediated transfer of the human E674Q mutant APP gene to the hippocampi of two-month-old C57Bl/6 J mice. AAV-E674Q-injected mice exhibited impaired learning behavior and increased pathological burden in the brain, implying that the E674Q mutation had a pathogenicity that bore a comparison with the classical Swedish mutation. Collectively, we report a strong amyloidogenic effect of the E674Q substitution in AD. To our knowledge, E674Q is the only pathogenic mutation within the amyloid processing sequence causing LOAD.

ADAM10 Gene Variants in AD Patients and Their Relationship to CSF Protein Levels

ADAM10 is the main α-secretase acting in the non-amyloidogenic processing of APP. We hypothesized that certain rare ADAM10 variants could increase the risk for AD by conferring the age-related downregulation of α-secretase. The ADAM10 gene was sequenced in 103 AD cases (82% familial) and 96 cognitively preserved nonagenarians. We examined rare variants (MAF < 0.01) and determined their potential association in the AD group with lower CSF protein levels, as analyzed by means of ELISA, and Western blot (species of 50 kDa, 55 kDa, and 80 kDa). Rare variants were found in 15.5% of AD cases (23% early-onset, 8% late-onset) and in 12.5% of nonagenarians, and some were group-specific. All were intronic variants except Q170H, found in three AD cases and one nonagenarian. The 3'UTR rs74016945 (MAF = 0.01) was found in 6% of the nonagenarians (OR 0.146, p = 0.057). Altogether, ADAM10 total levels or specific species were not significantly different when comparing AD with controls or carriers of rare variants versus non-carriers (except a Q170H carrier exhibiting low levels of all species), and did not differ according to the age at onset or APOE genotype. We conclude that ADAM10 exonic variants are uncommon in AD cases, and the presence of rare intronic variants (more frequent in early-onset cases) is not associated with decreased protein levels in CSF.

APPsα rescues CDK5 and GSK3β dysregulation and restores normal spine density in Tau transgenic mice.

The Tau protein can be phosphorylated by numerous kinases. In Alzheimer's disease (AD) hyperphosphorylated Tau species accumulate as neurofibrillary tangles that constitute a major hallmark of AD. AD is further characterized by extracellular Aβ plaques, derived from the β-amyloid precursor protein APP. Whereas Aβ is produced by amyloidogenic APP processing, APP processing along the competing non-amyloidogenic pathway results in the secretion of neurotrophic and synaptotrophic APPsα. Recently, we demonstrated that APPsα has therapeutic effects in transgenic AD model mice and rescues Aβ-dependent impairments. Here, we examined the potential of APPsα to regulate two major Tau kinases, GSK3β and CDK5 in THY-Tau22 mice, a widely used mouse model of tauopathy. Immunohistochemistry revealed a dramatic increase in pathologically phosphorylated (AT8 and AT180) or misfolded Tau species (MC1) in the hippocampus of THY-Tau22 mice between 3 and 12 months of age. Using a highly sensitive radioactive kinase assay with recombinant human Tau as a substrate and immunoblotting, we demonstrate an increase in GSK3β and CDK5 activity in the hippocampus of THY-Tau22 mice. Interestingly, AAV-mediated intracranial expression of APPsα in THY-Tau22 mice efficiently restored normal GSK3β and CDK5 activity. Western blot analysis revealed upregulation of the CDK5 regulatory proteins p35 and p25, indicating CDK5 hyperactivation in THY-Tau22 mice. Strikingly, AAV-APPsα rescued p25 upregulation to wild-type levels even at stages of advanced Tau pathology. Sarkosyl fractionation used to study the abundance of soluble and insoluble phospho-Tau species revealed increased soluble AT8-Tau and decreased insoluble AT100-Tau species upon AAV-APPsα injection. Moreover, AAV-APPsα reduced misfolded (MC1) Tau species, particularly in somatodendritic compartments of CA1 pyramidal neurons. Finally, we show that AAV-APPsα upregulated PSD95 expression and rescued deficits in spine density of THY-Tau22 mice. Together our findings suggest that APPsα holds therapeutic potential to mitigate Tau-induced pathology.

PICALM and Alzheimer's Disease: An Update and Perspectives.

Genome-wide association studies (GWAS) have identified the PICALM (Phosphatidylinositol binding clathrin-assembly protein) gene as the most significant genetic susceptibility locus after APOE and BIN1. PICALM is a clathrin-adaptor protein that plays a critical role in clathrin-mediated endocytosis and autophagy. Since the effects of genetic variants of PICALM as AD-susceptibility loci have been confirmed by independent genetic studies in several distinct cohorts, there has been a number of in vitro and in vivo studies attempting to elucidate the underlying mechanism by which PICALM modulates AD risk. While differential modulation of APP processing and Aβ transcytosis by PICALM has been reported, significant effects of PICALM modulation of tau pathology progression have also been evidenced in Alzheimer's disease models. In this review, we summarize the current knowledge about PICALM, its physiological functions, genetic variants, post-translational modifications and relevance to AD pathogenesis.

Mechanism of gamma‐secretase modulators

AbstractBackgroundAmyloid‐beta (Aβ) peptides are believed to be integral to Alzheimer’s disease (AD) pathogenesis through their role in the “amyloid hypothesis,” in which the accumulation of Aβ peptides initiates a cascade of pathological events leading to neurodegeneration and AD. γ‐Secretase is a transmembrane aspartyl protease which cleaves APP to generate Aβ peptides, making γ‐secretase an attractive drug target. However, inhibitors failed in clinical trials due to their unwanted side effects on other γ‐secretase substrates such as Notch. ɣ‐Secretase modulators (GSMs) selectively reduce levels of the pathogenic Aβ species without affecting Notch and overall APP processing. However, many questions on their mechanism still remain. Here we investigate the molecular mechanism of GSMs by (1) investigating the binding site of imidazole GSM E2012 using photoaffinity labeling and molecular docking and (2) investigating combination therapy with different GSMs on reducing Aβ production.MethodPart I: We incubated clickable GSM photoaffinity probes with cell membranes in the presence or absence of competitors, followed by UV irradiation to initiate photo‐crosslinking to nearby proteins and conjugation to biotin via click chemistry. Labeled proteins were isolated with affinity chromatography by streptavidin beads, eluted, and analyzed by western blot. Probes were docked onto γ‐secretase (PDB 7D8X) using tools from Schrodinger Suite 2021‐2. Part II: We treated N2a‐APP cells with GSM‐1 (acid GSM), E2012, or BPN‐15606 (imidazole GSMs) individually or in combination for 12h. Afterwards we collected cell culture media and measured secreted Aβ38, Aβ40, and Aβ42 levels using MesoScale Discovery.ResultWe demonstrate that GSM photoprobe E2012‐BPyne labels presenilin‐1 and nicastrin in the presence of transition state analog L‐685, 458, consistent with structural observations. From molecular docking studies we observe that GSM photoprobes differentially interacted with γ‐secretase and docking scores aligned with probe labeling efficiency. Furthermore, we demonstrate that acid and imidazole GSMs synergistically inhibit cellular Aβ production.ConclusionOur findings have revealed insight into the binding of GSMs and the effects of two GSM classes in combination. We are currently examining the effects of combined GSMs in primary neurons and on Notch production. These studies aim to better understand the mechanisms of GSMs to target γ‐secretase in AD.

Multigraph Cluster Analysis of AD Candidate Genes via Human Brain Mapping

AbstractBackgroundBrain imaging genetics examines associations between imaging quantitative traits (QTs) and genetic factors such as single nucleotide polymorphisms (SNPs) to provide important insights into the pathogenesis of Alzheimer’s diseases (AD). Given the high dimensionality, the individual level SNP‐QT signals typically have small effect sizes and are hard to be detected and replicated. To overcome this limitation, this work proposes a new approach to identify high‐level imaging genetic associations through applying multi‐graph clustering to SNP‐QT association maps.MethodsParticipants include 255 cognitive normal (CN) and 218 late mild cognitive impairment (LMCI) subjects from the ADNI data. Linear regression is performed to regress regional AV‐45 measures on each of the 54 AD SNPs to obtain a brain association map across 116 AAL‐ROIs. To quantify the similarity between each SNP pair, 5 scoring functions are used to calculate 5 different similarity measures between the respective brain maps of the two SNPs. Each scoring function yields a similarity graph of all 54 SNPs. Multigraph min‐max clustering is applied to 5 different graphs simultaneously to group similar SNPs together. Functional annotation is performed on each SNP cluster and the corresponding average brain association map to provide high level interpretations.ResultsFigure 1 shows the genetic effect on regional AV‐45 measures for each pair of SNP and ROI. The multi‐graph clustering result is shown in Figure 2, where 54 AD‐related SNPs are clustered into two groups. Figure 3a shows the results of Enrichr Elsevier pathway analysis. SNP group 1 is associated with pathways such as amyloid beta clearance, while SNP group 2 is associated with pathways including amyloid beta formation and APP processing in AD. Figure 3b shows the NeuroSynth annotation of the average brain effect maps associated with the two SNP clusters, respectively.ConclusionWe proposed an approach that transforms individual level imaging genetic associations (SNP vs ROI) to high‐level associations (i.e., SNP cluster vs brain association map), and applied it to an LMCI study. These high‐level findings have the potential to provide valuable insights into relevant genetic pathways and brain circuits, which can help form new hypotheses for more detailed imaging and genetics studies in independent cohorts.

Alternative Theory of AD Pathogenesis: Membrane Delimitation of the Histone Acetyltransferase Tip60/Kat5

AbstractBackgroundFor decades, mounting genetic and physiological data have pointed to the APP proteolysis as a seminal event in AD pathogenesis. This data has been viewed as supporting the amyloid hypothesis. However, growing data from multiple disciplines disputes that amyloid is a causal mechanism of neurodegeneration. Evidence suggests that FAD mutations are loss of function, inhibiting normal proteolytic processing of APP. APP extracellular and intracellular domains exhibit a broad array of binding interactions. Tip60 (a histone acetyltransferase, also known as Kat5) is an intracellular binding partner that supports the long‐standing speculation that APP functions as a signaling receptor. Recently, a conditional knockout (cKO) of Tip60 demonstrated neurodegenerative pathology and massive reactive gliosis, supporting a role for Tip60 in neurodegeneration. Reports that Tip60 is restricted from the nucleus in AD patient brains bolsters this idea.MethodTo assess the overlap of the cKO Tip60 model and human AD pathogenesis, we compared the dysregulated gene sets (DEGs) in the cKO Tip60 mouse and late‐onset AD postmortem brains. We break DEGs from mouse and human models into AD “biological domains” (BDs), and perform GSEA of GO enrichment within each BD. We then perform downstream analyses using network reconstruction applications and by examining the enriched BDs with pseudotime measures of disease progression in LOAD patients.ResultWe find significant overlap between DEGs in LOAD patients and the Tip60 cKO in multiple BDs, including, mitochondrial metabolism, immune response, structural stabilization, synaptic function, and endolysosomal trafficking. GSEA of LOAD and cKO Tip60 further implicates similar deficits in both models. The network analysis shows a convergence of gene dysregulation at core nodes, further implicating similar processes of dysregulation may impact particularly significant genes in human and mouse models. The pseudotime analysis shows that this dysfunction is an early signature of AD, suggesting Tip60 dysregulation may trigger early‐stage homeostatic destabilization.ConclusionWe propose that aberrant APP proteolysis traps Tip60 at the membrane, preventing the nuclear localization of Tip60, and altering both positive and negative gene regulation that is necessary for neuronal and glial homeostasis, and hypothesize that this is an explanation for the genetic linkage of APP proteolytic processing and AD.

Cell‐type specific alterations in intrinsic excitability and neurotransmission in short‐term virally induced model of Alzheimer’s Disease

AbstractBackgroundAlzheimer’s Disease (AD) is a neurodegenerative cascade resulting in neuronal dysfunction and memory loss, classically characterized by the deposition of amyloid plaques throughout the brain. Although deposition of these proteins correlates with cognitive loss in AD, these aggregates may not be causally related to initial neuronal and circuit pathology in AD. Interestingly, evidence in AD patients and mouse models has reported cortical circuit hyperexcitability prior to plaque pathology. The cell and molecular mechanisms underlying hyperexcitability in early‐stage AD remain unclear. In healthy brains, normal circuit operations are maintained through a fine‐tuned balance between excitatory (pyramidal) and inhibitory neuron activity. In several APP‐expressing mouse models, pyramidal and inhibitory neuron physiology are differentially affected and also evolve with disease severity. However, these parameters have not been explored in an adult‐onset APP model.MethodWe examined rapidly emerging physiological changes in cortical PV interneurons and neighboring pyramidal neurons in an adult‐onset AD model. We analyzed contributions of overexpressed wild‐type human amyloid precursor protein (WT hAPP) using an AAV viral approach. We fluorescently targeted cell types for whole‐cell patch‐clamp electrophysiology using a PV‐specific enhancer labeling technique or an excitatory neuron‐specific AAV. Lastly, we optogenetically stimulated PV interneurons to assess neurotransmission onto neighboring pyramidal cells.ResultWe report alterations in cortical PV interneuron and pyramidal neuron intrinsic excitability, as well as PV‐to‐pyramidal neurotransmission, after short‐term induction of hAPP virally. Interestingly, each of these two neuronal subtypes displayed unique changes suggesting different cell‐type‐specific molecular alterations.ConclusionThis breakdown of APP processing and its effects on PV interneuron and pyramidal neurons provides potential insights into early cell‐type‐specific mechanisms of the pathogenesis of Alzheimer’s Disease, thus providing specific interventions to ameliorate early symptoms and potentially slow the progression towards severe disease.

Alzheimer brains: increased soluble amyloid peptides, but decreased lipid rafts

AbstractBackgroundPET imaging studies of AD patients show progressive increases of fibrillar Aβ‐ amyloid. Because current PET ligands underestimate non‐fibrillar forms, we assayed soluble Aβ in AD and controls. To identify mechanisms responsible for soluble Aβ in AD brains, we examined lipid rafts (LR), where APP is enzymatically processed. Frontal cortex was compared with cerebellum, which has minimal AD pathology.MethodPostmortem samples (N=48) were age‐and sex matched for Braak scores. Soluble Aβ was extracted from homogenates of full thickness frontal cortex by isotonic buffer and mild detergent (RIPA; 1% NP40) and centrifuged (10,000g/1h). Supernates were assayed by dot blots with antibodies for Aβ40 and Aβ42 (Biolegend, San Diego, CA). Aβ was assayed as relative fluorescent units per gm brain. LR fractions were isolated from these same brains by kit (Invent Biotechnologies; LR‐039), validated by ultracentrifugal fractionation.ResultCompared with cognitively normal controls (Braak 0‐1), elevations of soluble Aβ40 and Aβ42 were similar for intermediate and later stage AD (Braak 2‐3 and 4‐6). Clinical grade AD had greater increase of soluble Aβ40 (+400%) than Aβ42 (+50%) (p&lt;0.05). LR raft yield per gram AD frontal cortex was 20% below controls (p&lt;0.05), while cerebellar LR did not differ by Braak score. The composition of LR had a similar pattern, with 35% AD decreases of total protein and cholesterol (p&lt;0.05); cerebellar LR again did not differ by Braak score. However, LR 4‐HNE, a marker of oxidative damage, was similarly increased in AD cortex and AD cerebellum.ConclusionThe extensive overlap of soluble Aβ levels in controls with AD contrasts with the PET findings on fibrillar Aβ. These findings further support fibrillar Aβ as a biomarker for AD treatments and show the need for more detailed postmortem analysis of diverse soluble and insoluble Aβ aggregates in relation to PET. The decreased LR in AD frontal cortex is consistent with its extensive neuronal loss, in contrast to the cerebellum. Further studies will examine LR enzymes of APP processing as a source of the increased soluble Aβ in AD, and subcellular clearance processes, which may be impaired in association with the oxidative damage indicated by increased 4‐HNE.

Late-long-term potentiation magnitude, but not Aβ levels and amyloid pathology, is associated with behavioral performance in a rat knock-in model of Alzheimer disease.

Cleavage of Amyloid precursor protein by β- and γ-secretases lead to Aβ formation. The widely accepted pathogenic model states that these mutations cause AD via an increase in Aβ formation and accumulation of Aβ in Amyloid plaques. APP mutations cause early onset familial forms of Alzheimer's disease (FAD) in humans. We generated App-Swedish (Apps ) knock-in rats, which carry a pathogenic APP mutation in the endogenous rat App gene. This mutation increases β-secretase processing of APP leading to both augmented Aβ production and facilitation of glutamate release in Apps/s rats, via a β-secretase and APP-dependent glutamate release mechanism. Here, we studied 11 to 14-month-old male and female Apps/s rats. To determine whether the Swedish App mutation leads to behavioral deficits, Apps/s knock-in rats were subjected to behavioral analysis using the IntelliCage platform, an automated behavioral testing system. This system allows behavioral assessment in socially housed animals reflecting a more natural, less stress-inducing environment and eliminates experimenter error and bias while increasing precision of measurements. Surprisingly, a spatial discrimination and flexibility task that can reveal deficits in higher order brain function showed that Apps/s females, but not Apps/s male rats, performed significantly worse than same sex controls. Moreover, female control rats performed significantly better than control and Apps/s male rats. The Swedish mutation causes a significant increase in Aβ production in 14-month-old animals of both sexes. Yet, male and female Apps/s rats showed no evidence of AD-related amyloid pathology. Finally, Apps/s rats did not show signs of significant neuroinflammation. Given that the APP Swedish mutation causes alterations in glutamate release, we analyzed Long-term potentiation (LTP), a long-lasting form of synaptic plasticity that is a cellular basis for learning and memory. Strikingly, LTP was significantly increased in Apps/s control females compared to both Apps/s sexes and control males. In conclusion, this study shows that behavioral performances are sex and App-genotype dependent. In addition, they are associated with LTP values and not Aβ or AD-related pathology. These data, and the failures of anti-Aβ therapies in humans, suggest that alternative pathways, such as those leading to LTP dysfunction, should be targeted for disease-modifying AD therapy.

Cathepsin D: Analysis of its potential role as an amyloid beta degrading protease

Proteolysis catalyzed by the major lysosomal aspartyl protease cathepsin-D (CTSD) appears to be of pivotal importance for proteostasis within the central nervous system and in neurodegeneration. Neuronal Ceroid Lipofuscinosis (NCL) type 10 is caused by a lack of CTSD leading to a defective autophagic flow and pathological accumulation of proteins. We previously demonstrated a therapeutic-relevant clearance of protein aggregates after dosing a NCL10 mouse model with recombinant human pro-cathepsin-D (proCTSD). Similar results could be achieved in cells and mice accumulating α-synuclein. Prompted by these positive effects and our in vitro findings showing that cathepsin-D can cleave the Alzheimer's Disease (AD)-causing amyloid beta peptides (Aβ), we envisaged that such a treatment with proCTSD could similarly be effective in clearance of potentially toxic Aβ species.We demonstrated that CTSD is able to cleave human Aβ1–42 by using liquid chromatography-mass spectrometry. Intracerebral dosing of proCTSD in a NCL10 (CTSD knockout) mouse model revealed uptake and processing of CTSD to its mature and active form. However, the re-addition of CTSD did not obviously affect intracellular APP processing or the generation of soluble APP and Aβ-species. ProCTSD treated HEK cells in comparison with untreated cells were found to contain comparable levels of soluble and membrane bound APP and Aβ-species. Also, the early intracranial application (P1 and P20) of proCTSD in the 5xFAD mouse model did not change Aβ pathology, plaque number and plaque composition and neuroinflammation, however we observed an increased level of Aβ1–42 in the CSF.Our data confirm proteolytic cleavage of human Aβ1–42 by CTSD but exclude a prominent role of CTSD in APP processing and Aβ degradation in our in vitro and in vivo models.

Regulation of the Soluble Amyloid Precursor Protein α (sAPPα) Levels by Acetylcholinesterase and Brain-Derived Neurotrophic Factor in Lung Cancer Cell Media.

In comparing two human lung cancer cells, we previously found lower levels of acetylcholinesterase (AChE) and intact amyloid-β40/42 (Aβ), and higher levels of mature brain-derived neurotrophic factor (mBDNF) in the media of H1299 cells as compared to A549 cell media. In this study, we hypothesized that the levels of soluble amyloid precursor protein α (sAPPα) are regulated by AChE and mBDNF in A549 and H1299 cell media. The levels of sAPPα were higher in the media of H1299 cells. Knockdown of AChE led to increased sAPPα and mBDNF levels and correlated with decreased levels of intact Aβ40/42 in A549 cell media. AChE and mBDNF had opposite effects on the levels of Aβ and sAPPα and were found to operate through a mechanism involving α-secretase activity. Treatment with AChE decreased sAPPα levels and simultaneously increased the levels of intact Aβ40/42 suggesting a role of the protein in shifting APP processing away from the non-amyloidogenic pathway and toward the amyloidogenic pathway, whereas treatment with mBDNF led to opposite effects on those levels. We also show that the levels of sAPPα are regulated by protein kinase C (PKC), extracellular signal-regulated kinase (ERK)1/2, phosphoinositide 3 Kinase (PI3K), but not by protein kinase A (PKA).

Cathepsin B-A Neuronal Death Mediator in Alzheimer's Disease Leading to Neurodegeneration.

The lysosomal cysteine protease enzyme, named Cathepsin B, mainly degrades the protein and manages its average turnover in our body. The Cathepsin B active form is mostly present inside the lysosomal part at a cellular level, providing the slightly acidic medium for its activation. Multiple findings on Cathepsin B reveal its involvement in neurons' degeneration and a possible role as a neuronal death mediator in several neurodegenerative diseases. In this review article, we highlight the participation of Cathepsin B in the etiology/progress of AD, along with various other factors. The enzyme is involved in producing neurotoxic Aβ amyloid in the AD brain by acting as the β-secretase enzyme in the regulated secretory pathways responsible for APP processing. Aβ amyloid accumulation and amyloid plaque formation lead to neuronal degeneration, one of the prominent pathological hallmarks of AD. Cathepsin B is also involved in the production of PGlu-Aβ, which is a truncated and highly neurotoxic form of Aβ. Some of the findings also revealed that Cathepsin B specific gene deletion decreases the level of PGlu-Aβ inside the brain of experimental mice. Therefore, neurotoxicity might be considered a new pathological indication of AD due to the involvement of Cathepsin B. It also damages neurons present in the CNS region by producing inflammatory responses and generating mitochondrial ROS. However, Cathepsin B inhibitors, i.e., CA-074, can prevent neuronal death in AD patients. The other natural inhibitors are also equally effective against neuronal damage with higher selectivity. Its synthetic inhibitors are specific for their target; however, they lose their selectivity in the presence of quite a few reducing agents. Therefore, a humanized monoclonal antibody is used as a selective Cathepsin B inhibitor to overcome the problem experienced. The use of Cathepsin B for the treatment of AD and other neurodegenerative diseases could be considered a rational therapeutic target.

DYRK1A Regulates the Bidirectional Axonal Transport of APP in Human-Derived Neurons.

Dyrk1a triplication in Down's syndrome and its overexpression in Alzheimer's disease suggest a role for increased DYRK1A activity in the abnormal metabolism of APP. Transport defects are early phenotypes in the progression of Alzheimer's disease, which lead to APP processing impairments. However, whether DYRK1A regulates the intracellular transport and delivery of APP in human neurons remains unknown. From a proteomic dataset of human cerebral organoids treated with harmine, a DYRK1A inhibitor, we found expression changes in protein clusters associated with the control of microtubule-based transport and in close interaction with the APP vesicle. Live imaging of APP axonal transport in human-derived neurons treated with harmine or overexpressing a dominant negative DYRK1A revealed a reduction in APP vesicle density and enhanced the stochastic behavior of retrograde vesicle transport. Moreover, harmine increased the fraction of slow segmental velocities and changed speed transitions supporting a DYRK1A-mediated effect in the exchange of active motor configuration. Contrarily, the overexpression of DYRK1A in human polarized neurons increased the axonal density of APP vesicles and enhanced the processivity of retrograde APP. In addition, increased DYRK1A activity induced faster retrograde segmental velocities together with significant changes in slow to fast anterograde and retrograde speed transitions, suggesting the facilitation of the active motor configuration. Our results highlight DYRK1A as a modulator of the axonal transport machinery driving APP intracellular distribution in neurons, and stress DYRK1A inhibition as a putative therapeutic intervention to restore APP axonal transport in Down's syndrome and Alzheimer's disease.SIGNIFICANCE STATEMENT Axonal transport defects are early events in the progression of neurodegenerative diseases, such as Alzheimer's disease. However, the molecular mechanisms underlying transport defects remain elusive. Dyrk1a kinase is triplicated in Down's syndrome and overexpressed in Alzheimer's disease, suggesting that DYRK1A dysfunction affects molecular pathways leading to early-onset neurodegeneration. Here, we show by live imaging of human-derived neurons that DYRK1A activity differentially regulates the intracellular trafficking of APP. Further, single-particle analysis revealed DYRK1A as a modulator of axonal transport and the configuration of active motors within the APP vesicle. Our work highlights DYRK1A as a regulator of APP axonal transport and metabolism, supporting DYRK1A inhibition as a therapeutic strategy to restore intracellular dynamics in Alzheimer's disease.

APPsα Rescues Tau-Induced Synaptic Pathology.

Alzheimer's disease (AD) is histopathologically characterized by Aβ plaques and the accumulation of hyperphosphorylated Tau species, the latter also constituting key hallmarks of primary tauopathies. Whereas Aβ is produced by amyloidogenic APP processing, APP processing along the competing nonamyloidogenic pathway results in the secretion of neurotrophic and synaptotrophic APPsα. Recently, we demonstrated that APPsα has therapeutic effects in transgenic AD model mice and rescues Aβ-dependent impairments. Here, we examined the potential of APPsα to mitigate Tau-induced synaptic deficits in P301S mice (both sexes), a widely used mouse model of tauopathy. Analysis of synaptic plasticity revealed an aberrantly increased LTP in P301S mice that could be normalized by acute application of nanomolar amounts of APPsα to hippocampal slices, indicating a homeostatic function of APPsα on a rapid time scale. Further, AAV-mediated in vivo expression of APPsα restored normal spine density of CA1 neurons even at stages of advanced Tau pathology not only in P301S mice, but also in independent THY-Tau22 mice. Strikingly, when searching for the mechanism underlying aberrantly increased LTP in P301S mice, we identified an early and progressive loss of major GABAergic interneuron subtypes in the hippocampus of P301S mice, which may lead to reduced GABAergic inhibition of principal cells. Interneuron loss was paralleled by deficits in nest building, an innate behavior highly sensitive to hippocampal impairments. Together, our findings indicate that APPsα has therapeutic potential for Tau-mediated synaptic dysfunction and suggest that loss of interneurons leads to disturbed neuronal circuits that compromise synaptic plasticity as well as behavior.SIGNIFICANCE STATEMENT Our findings indicate, for the first time, that APPsα has the potential to rescue Tau-induced spine loss and abnormal synaptic plasticity. Thus, APPsα might have therapeutic potential not only because of its synaptotrophic functions, but also its homeostatic capacity for neuronal network activity. Hence, APPsα is one of the few molecules which has proven therapeutic effects in mice, both for Aβ- and Tau-dependent synaptic impairments and might therefore have therapeutic potential for patients suffering from AD or primary tauopathies. Furthermore, we found in P301S mice a pronounced reduction of inhibitory interneurons as the earliest pathologic event preceding the accumulation of hyperphosphorylated Tau species. This loss of interneurons most likely disturbs neuronal circuits that are important for synaptic plasticity and behavior.

Secreted Amyloid Precursor Protein Alpha, a Neuroprotective Protein in the Brain Has Widespread Effects on the Transcriptome and Proteome of Human Inducible Pluripotent Stem Cell-Derived Glutamatergic Neurons Related to Memory Mechanisms.

Secreted amyloid precursor protein alpha (sAPPα) processed from a parent human brain protein, APP, can modulate learning and memory. It has potential for development as a therapy preventing, delaying, or even reversing Alzheimer’s disease. In this study a comprehensive analysis to understand how it affects the transcriptome and proteome of the human neuron was undertaken. Human inducible pluripotent stem cell (iPSC)-derived glutamatergic neurons in culture were exposed to 1 nM sAPPα over a time course and changes in the transcriptome and proteome were identified with RNA sequencing and Sequential Window Acquisition of All THeoretical Fragment Ion Spectra-Mass Spectrometry (SWATH-MS), respectively. A large subset (∼30%) of differentially expressed transcripts and proteins were functionally involved with the molecular biology of learning and memory, consistent with reported links of sAPPα to memory enhancement, as well as neurogenic, neurotrophic, and neuroprotective phenotypes in previous studies. Differentially regulated proteins included those encoded in previously identified Alzheimer’s risk genes, APP processing related proteins, proteins involved in synaptogenesis, neurotransmitters, receptors, synaptic vesicle proteins, cytoskeletal proteins, proteins involved in protein and organelle trafficking, and proteins important for cell signalling, transcriptional splicing, and functions of the proteasome and lysosome. We have identified a complex set of genes affected by sAPPα, which may aid further investigation into the mechanism of how this neuroprotective protein affects memory formation and how it might be used as an Alzheimer’s disease therapy.

APP processing: a biochemical competition influenced by exercise-induced signaling mediators?

Neurodegenerative diseases, such as Alzheimer's disease (AD), are becoming more common in aging our society. One specific neuropathological hallmark of this disease is excessive accumulation of amyloid-β (Aβ) peptides, which can aggregate to form the plaques commonly associated with this disease. These plaques are often observed well before clinical diagnosis of AD. At the cellular level, both the production and aggregation of Aβ peptides in the brain are detrimental to neuronal cell production, survival, and function, as well as often resulting in neuronal dysfunction and death. Exercise and physical activity have been shown to improve overall health, including brain health, and in the last several years there has been evidence to support that exercise may be able to regulate Aβ peptide production in the brain. Exercise promotes the release of a wide array of signaling mediators from various metabolically active tissues and organs in the body. These exercise-induced signaling mediators could be the driving force behind some of the beneficial effects observed in brain with exercise. This review will aim to discuss potential exercise-induced signaling mediators with the capacity to influence various proteins involved in the formation of Aβ peptide production in the brain.

Endosomal structure and APP biology are not altered in a preclinical mouse cellular model of Down syndrome.

Individuals who have Down syndrome (trisomy 21) are at greatly increased risk of developing Alzheimer’s disease, characterised by the accumulation in the brain of amyloid-β plaques. Amyloid-β is a product of the processing of the amyloid precursor protein, encoded by the APP gene on chromosome 21. In Down syndrome the first site of amyloid-β accumulation is within endosomes, and changes to endosome biology occur early in Alzheimer’s disease. Here, we determine if primary mouse embryonic fibroblasts isolated from a mouse model of Down syndrome can be used to study endosome and APP cell biology. We report that in this cellular model, endosome number, size and APP processing are not altered, likely because APP is not dosage sensitive in the model, despite three copies of App.