Rhei Undulati Rhizoma attenuates memory decline and reduces amyloid-ß induced neuritic dystrophy in 5xFAD mouse.

BACKGROUND: Alzheimer's disease (AD) is a common type of dementia characterized by amyloid-ß (Aß) accumulation, lysosomal dysfunction, and tau hyperphosphorylation, leading to neurite dystrophy and memory loss. This study aimed to investigate whether Rhei Undulati Rhizoma (RUR), which has been reported to have anti-neuroinflammatory effect, attenuates Aß-induced memory impairment, neuritic dystrophy, and tau hyperphosphorylation, and to reveal its mode of action. METHODS: Five-month-old 5xFAD mice received RUR (50 mg/kg) orally for 2 months. The Y-maze test was used to assess working memory. After behavioral testing, brain tissue was analyzed using thioflavin S staining, western blotting, and immunofluorescence staining to investigate the mode of action of RUR. To confirm whether RUR directly reduces Aß aggregation, a thioflavin T assay and dot blot were performed after incubating Aß with RUR. RESULTS: RUR administration attenuated the Aß-induced memory impairment in 5xFAD mice. Furthermore, decreased accumulation of Aß was observed in the hippocampus of the RUR-treated 5xFAD group compare to the vehicle-treated 5xFAD group. Moreover, RUR reduced the dystrophic neurites (DNs) that accumulate impaired endolysosomal organelles around Aß. In particular, RUR treatment downregulated the expression of ß-site amyloid precursor protein cleaving enzyme 1 and the hyperphosphorylation of tau within DNs. Additionally, RUR directly suppressed the aggregation of Aß, and eliminated Aß oligomers in vitro. CONCLUSIONS: This study showed that RUR could attenuate Aß-induced pathology and directly regulate the aggregation of Aß. These results suggest that RUR could be an efficient material for AD treatment through Aß regulation.

Myeloid ectopic viral integration site 2 accelerates the progression of Alzheimer's disease.

Amyloid plaques, a major pathological hallmark of Alzheimer's disease (AD), are caused by an imbalance between the amyloidogenic and non-amyloidogenic pathways of amyloid precursor protein (APP). BACE1 cleavage of APP is the rate-limiting step for amyloid-ß production and plaque formation in AD. Although the alteration of BACE1 expression in AD has been investigated, the underlying mechanisms remain unknown. In this study, we determined MEIS2 was notably elevated in AD models and AD patients. Alterations in the expression of MEIS2 can modulate the levels of BACE1. MEIS2 downregulation improved the learning and memory retention of AD mice and decreased the number of amyloid plaques. MEIS2 binds to the BACE1 promoter, positively regulates BACE1 expression, and accelerates APP amyloid degradation in vitro. Therefore, our findings suggest that MEIS2 might be a critical transcription factor in AD, since it regulates BACE1 expression and accelerates BACE1-mediated APP amyloidogenic cleavage. MEIS2 is a promising early intervention target for AD treatment.

Eta-secretase-like processing of the amyloid precursor protein (APP) by the rhomboid protease RHBDL4.

The amyloid precursor protein (APP) is a key protein in Alzheimer's disease synthesized in the endoplasmic reticulum (ER) and translocated to the plasma membrane where it undergoes proteolytic cleavages by several proteases. Conversely to other known proteases, we previously elucidated rhomboid protease RHBDL4 as a novel APP processing enzyme where several cleavages likely occur already in the ER. Interestingly, the pattern of RHBDL4-derived large APP C-terminal fragments resemble those generated by the η-secretase or MT5-MMP, which was described to generate so called Aη fragments. The similarity in large APP C-terminal fragments between both proteases raised the question whether RHBDL4 may contribute to η-secretase activity and Aη-like fragments. Here, we identified two cleavage sites of RHBDL4 in APP by mass spectrometry, which, intriguingly, lie in close proximity to the MT5-MMP cleavage sites. Indeed, we observed that RHBDL4 generates Aη-like fragments in vitro without contributions of α-, ß-, or γ-secretases. Such Aη-like fragments are likely generated in the ER since RHBDL4-derived APP-C-terminal fragments do not reach the cell surface. Inherited, familial APP mutations appear to not affect this processing pathway. In RHBDL4 knockout mice, we observed increased cerebral full length APP in comparison to wild type (WT) in support of RHBDL4 being a physiologically relevant protease for APP. Furthermore, we found secreted Aη fragments in dissociated mixed cortical cultures from WT mice, however significantly less Aη fragments in RHBDL4 knockout cultures. Our data underscores that RHBDL4 contributes to η-secretease-like processing of APP and that RHBDL4 is a physiologically relevant protease for APP.

Neuroprotective Strategies and Cell-Based Biomarkers for Manganese-Induced Toxicity in Human Neuroblastoma (SH-SY5Y) Cells.

Manganese (Mn) is an essential heavy metal in the human body, while excess Mn leads to neurotoxicity, as observed in this study, where 100 µM of Mn was administered to the human neuroblastoma (SH-SY5Y) cell model of dopaminergic neurons in neurodegenerative diseases. We quantitated pathway and gene changes in homeostatic cell-based adaptations to Mn exposure. Utilizing the Gene Expression Omnibus, we accessed the GSE70845 dataset as a microarray of SH-SY5Y cells published by Gandhi et al. (2018) and applied statistical significance cutoffs at p < 0.05. We report 74 pathway and 10 gene changes with statistical significance. ReactomeGSA analyses demonstrated upregulation of histones (5 out of 10 induced genes) and histone deacetylases as a neuroprotective response to remodel/mitigate Mn-induced DNA/chromatin damage. Neurodegenerative-associated pathway changes occurred. NF-κB signaled protective responses via Sirtuin-1 to reduce neuroinflammation. Critically, Mn activated three pathways implicating deficits in purine metabolism. Therefore, we validated that urate, a purine and antioxidant, mitigated Mn-losses of viability in SH-SY5Y cells. We discuss Mn as a hypoxia mimetic and trans-activator of HIF-1α, the central trans-activator of vascular hypoxic mitochondrial dysfunction. Mn induced a 3-fold increase in mRNA levels for antioxidant metallothionein-III, which was induced 100-fold by hypoxia mimetics deferoxamine and zinc.

Generation of Alzheimer's Disease Model Derived from Induced Pluripotent Stem Cells with APP Gene Mutation.

Alzheimer's disease (AD), the most common cause of dementia, is characterized by disruptions in memory, cognition, and personality, significantly impacting morbidity and mortality rates among older adults. However, the exact pathophysiological mechanism of AD remains unknown, and effective treatment options for AD are still lacking. Human induced pluripotent stem cells (iPSC) are emerging as promising platforms for disease research, offering the ability to model the genetic mutations associated with various conditions. Patient-derived iPSCs are useful for modeling neurodegenerative and neurodevelopmental disorders. In this study, we generated AD iPSCs from peripheral blood mononuclear cells obtained from a 65-year-old patient with AD carrying the E682K mutation in the gene encoding the amyloid precursor protein. Cerebral organoids derived from AD iPSCs recapitulated the AD phenotype, exhibiting significantly increased levels of tau protein. Our analysis revealed that an iPSC disease model of AD is a valuable assessment tool for pathophysiological research and drug screening.

Amino-terminally elongated Aß peptides are generated by the secreted metalloprotease ADAMTS4 and deposit in a subset of Alzheimer's disease brains.

AIMS: The aggregation and deposition of amyloid-ß (Aß) peptides in the brain is thought to be the initial driver in the pathogenesis of Alzheimer's disease (AD). Aside from full-length Aß peptides starting with an aspartate residue in position 1, both N-terminally truncated and elongated Aß peptides are produced by various proteases from the amyloid precursor protein (APP) and have been detected in brain tissues and body fluids. Recently, we demonstrated that the particularly abundant N-terminally truncated Aß4-x peptides are generated by ADAMTS4, a secreted metalloprotease that is exclusively expressed in the oligodendrocyte cell population. In this study, we investigated whether ADAMTS4 might also be involved in the generation of N-terminally elongated Aß peptides. METHODS: We used cell-free and cell-based assays in combination with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF) and electrochemiluminescence sandwich immunoassays to identify and quantify N-terminally elongated Aß peptide variants. Antibodies against these Aß variants were characterised by peptide microarrays and employed for the immunohistochemical analyses of human brain samples. RESULTS: In this study, we discovered additional ADAMTS4 cleavage sites in APP. These were located N-terminal to Asp-(1) in the Aß peptide sequence between residues Glu-(-7) and Ile-(-6) as well as Glu-(-4) and Val-(-3), resulting in the release of N-terminally elongated Aß-6-x and Aß-3-x peptides, of which the latter serve as a component in a promising Aß-based plasma biomarker. Aß-6/-3-40 peptides were detected in supernatants of various cell lines and in the cerebrospinal fluid (CSF), and ADAMTS4 enzyme activity promoted the release of Aß-6/-3-x peptides. Furthermore, by immunohistochemistry, a subset of AD cases displayed evidence of extracellular and vascular localization of N-terminally elongated Aß-6/-3-x peptides. DISCUSSION: The current findings implicate ADAMTS4 in both the pathological process of Aß peptide aggregation and in the early detection of amyloid pathology in AD.

Long term rescue of Alzheimer's deficits in vivo by one-time gene-editing of App C-terminus.

Gene-editing technologies promise to create a new class of therapeutics that can achieve permanent correction with a single intervention. Besides eliminating mutant alleles in familial disease, gene-editing can also be used to favorably manipulate upstream pathophysiologic events and alter disease-course in wider patient populations, but few such feasible therapeutic avenues have been reported. Here we use CRISPR-Cas9 to edit the last exon of amyloid precursor protein (App), relevant for Alzheimer's disease (AD). Our strategy effectively eliminates an endocytic (YENPTY) motif at APP C-terminus, while preserving the N-terminus and compensatory APP-homologues. This manipulation favorably alters events along the amyloid-pathway - inhibiting toxic APP-ß-cleavage fragments (including Aß) and upregulating neuroprotective APP-α-cleavage products. AAV-driven editing ameliorates neuropathologic, electrophysiologic, and behavioral deficits in an AD knockin mouse model. Effects persist for many months, and no abnormalities are seen in WT mice even after germline App-editing; underlining overall efficacy and safety. Pathologic alterations in the glial-transcriptome of App-KI mice, as seen by single nuclei RNA-sequencing (sNuc-Seq), are also normalized by App C-terminus editing. Our strategy takes advantage of innate transcriptional rules that render terminal exons insensitive to nonsense-decay, and the upstream manipulation is expected to be effective for all forms of AD. These studies offer a path for a one-time disease-modifying treatment for AD.

Optimizing NMR fragment-based drug screening for membrane protein targets.

NMR spectroscopy has played a pivotal role in fragment-based drug discovery by coupling detection of weak ligand-target binding with structural mapping of the binding site. Fragment-based screening by NMR has been successfully applied to many soluble protein targets, but only to a limited number of membrane proteins, despite the fact that many drug targets are membrane proteins. This is partly because of difficulties preparing membrane proteins for NMR-especially human membrane proteins-and because of the inherent complexity associated with solution NMR spectroscopy on membrane protein samples, which require the inclusion of membrane-mimetic agents such as micelles, nanodiscs, or bicelles. Here, we developed a generalizable protocol for fragment-based screening of membrane proteins using NMR. We employed two human membrane protein targets, both in fully protonated detergent micelles: the single-pass C-terminal domain of the amyloid precursor protein, C99, and the tetraspan peripheral myelin protein 22 (PMP22). For both we determined the optimal NMR acquisition parameters, protein concentration, protein-to-micelle ratio, and upper limit to the concentration of D6-DMSO in screening samples. Furthermore, we conducted preliminary screens of a plate-format molecular fragment mixture library using our optimized conditions and were able to identify hit compounds that selectively bound to the respective target proteins. It is hoped that the approaches presented here will be useful in complementing existing methods for discovering lead compounds that target membrane proteins.

APP fragment controls both ionotropic and non-ionotropic signaling of NMDA receptors.

NMDA receptors (NMDARs) are ionotropic receptors crucial for brain information processing. Yet, evidence also supports an ion-flux-independent signaling mode mediating synaptic long-term depression (LTD) and spine shrinkage. Here, we identify AETA (Aη), an amyloid-ß precursor protein (APP) cleavage product, as an NMDAR modulator with the unique dual regulatory capacity to impact both signaling modes. AETA inhibits ionotropic NMDAR activity by competing with the co-agonist and induces an intracellular conformational modification of GluN1 subunits. This favors non-ionotropic NMDAR signaling leading to enhanced LTD and favors spine shrinkage. Endogenously, AETA production is increased by in vivo chemogenetically induced neuronal activity. Genetic deletion of AETA production alters NMDAR transmission and prevents LTD, phenotypes rescued by acute exogenous AETA application. This genetic deletion also impairs contextual fear memory. Our findings demonstrate AETA-dependent NMDAR activation (ADNA), characterizing AETA as a unique type of endogenous NMDAR modulator that exerts bidirectional control over NMDAR signaling and associated information processing.

Sex differences in the extent of acute axonal pathologies after experimental concussion.

Although human females appear be at a higher risk of concussion and suffer worse outcomes than males, underlying mechanisms remain unclear. With increasing recognition that damage to white matter axons is a key pathologic substrate of concussion, we used a clinically relevant swine model of concussion to explore potential sex differences in the extent of axonal pathologies. At 24 h post-injury, female swine displayed a greater number of swollen axonal profiles and more widespread loss of axonal sodium channels than males. Axon degeneration for both sexes appeared to be related to individual axon architecture, reflected by a selective loss of small caliber axons after concussion. However, female brains had a higher percentage of small caliber axons, leading to more extensive axon loss after injury compared to males. Accordingly, sexual dimorphism in axonal size is associated with more extensive axonal pathology in females after concussion, which may contribute to worse outcomes.

Fine-Tuning Amyloid Precursor Protein Expression through Nonsense-Mediated mRNA Decay.

Studies on genetic robustness recently revealed transcriptional adaptation (TA) as a mechanism by which an organism can compensate for genetic mutations through activation of homologous genes. Here, we discovered that genetic mutations, introducing a premature termination codon (PTC) in the amyloid precursor protein-b (appb) gene, activated TA of two other app family members, appa and amyloid precursor-like protein-2 (aplp2), in zebrafish. The observed transcriptional response of appa and aplp2 required degradation of mutant mRNA and did not depend on Appb protein level. Furthermore, TA between amyloid precursor protein (APP) family members was observed in human neuronal progenitor cells; however, compensation was only present during early neuronal differentiation and could not be detected in a more differentiated neuronal stage or adult zebrafish brain. Using knockdown and chemical inhibition, we showed that nonsense-mediated mRNA decay (NMD) is involved in degradation of mutant mRNA and that Upf1 and Upf2, key proteins in the NMD pathway, regulate the endogenous transcript levels of appa, appb, aplp1, and aplp2 In conclusion, our results suggest that the expression level of App family members is regulated by the NMD pathway and that mutations destabilizing app/APP mRNA can induce genetic compensation by other family members through TA in both zebrafish and human neuronal progenitors.

ESCRT disruption provides evidence against transsynaptic signaling functions for extracellular vesicles.

Extracellular vesicles (EVs) are released by many cell types including neurons, carrying cargoes involved in signaling and disease. It is unclear whether EVs promote intercellular signaling or serve primarily to dispose of unwanted materials. We show that loss of multivesicular endosome-generating ESCRT (endosomal sorting complex required for transport) machinery disrupts release of EV cargoes from Drosophila motor neurons. Surprisingly, ESCRT depletion does not affect the signaling activities of the EV cargo Synaptotagmin-4 (Syt4) and disrupts only some signaling activities of the EV cargo Evenness Interrupted (Evi). Thus, these cargoes may not require intercellular transfer via EVs, and instead may be conventionally secreted or function cell autonomously in the neuron. We find that EVs are phagocytosed by glia and muscles, and that ESCRT disruption causes compensatory autophagy in presynaptic neurons, suggesting that EVs are one of several redundant mechanisms to remove cargoes from synapses. Our results suggest that synaptic EV release serves primarily as a proteostatic mechanism for certain cargoes.

Navigating the Maze of Alzheimer's disease by exploring BACE1: Discovery, current scenario, and future prospects.

Alzheimer's disease (AD) is a chronic neurological condition that has become a leading cause of cognitive decline in elder individuals. Hardly any effective medication has been developed to halt the progression of AD due to the disease's complexity. Several theories have been put forward to clarify the mechanisms underlying AD etiology. The identification of amyloid plaques as a hallmark of AD has sparked the development of numerous drugs targeting the players involved in the amyloidogenic pathway, such as the ß-site of amyloid precursor protein cleavage enzyme 1 (BACE1) blockers. Over the last ten years, preclinical and early experimental research has led several pharmaceutical companies to prioritize producing BACE1 inhibitors. Despite all these efforts, earlier discovered inhibitors were discontinued in consideration of another second-generation small molecules and recent BACE1 antagonists failed in the final stages of clinical trials because of the complications associated either with toxicity or effectiveness. In addition to discussing the difficulties associated with development of BACE1 inhibitors, this review aims to provide an overview of BACE1 and offer perspectives on the causes behind the failure of five recent BACE1 inhibitors, that would be beneficial for choosing effective treatment approaches in the future.

Small GTPases control macropinocytosis of amyloid precursor protein and cleavage to amyloid-ß.

The overproduction of the toxic peptide amyloid-beta (Aß) generated from the cleavage of amyloid precursor protein (APP) is proposed to be a critical event in the development of Alzheimer's disease. Evidence suggests that the cleavage of APP occurs after its internalization from the cell surface. Previously, we identified a novel pathway for APP internalization, which trafficks cell surface APP directly to lysosomes by macropinocytosis, leading to its processing into Aß. We also demonstrated that ADP-ribosylation factor 6 (Arf6) is required for the macropinocytosis of APP. Here, we characterized the roles of Arf6's downstream effectors Rac1, Cdc42 and RhoA. Both pharmacological inhibition and siRNA knockdown of these proteins reduced the amount of APP colocalized with LAMP1-labeled lysosomes without affecting APP transport to early endosomes. Decreases in the production of both Aß40 and Aß42 were also observed by ELISA in response to inhibitor treatment. These findings together demonstrate that Rac1, Cdc42 and RhoA are components of the mechanism regulating the macropinocytosis of APP and targeting these components can reduce the production of Aß.

Early Animal Origin of BACE1 APP/Aß Proteolytic Function.

Alzheimer's disease is characterized, in part, by the accumulation of ß-amyloid (Aß) in the brain. Aß is produced via the proteolysis of APP by BACE1 and γ-secretase. Since BACE1 is the rate-limiting enzyme in the production of Aß, and a target for therapeutics, it is of interest to know when its proteolytic function evolved and for what purpose. Here, we take a functional evolutionary approach to show that BACE1 likely evolved from a gene duplication event near the base of the animal clade and that BACE1 APP/Aß proteolytic function evolved during early animal diversification, hundreds of millions of years before the evolution of the APP/Aß substrate. Our examination of BACE1 APP/Aß proteolytic function includes cnidarians, ctenophores, and choanoflagellates. The most basal BACE1 ortholog is found in cnidarians, while ctenophores, placozoa, and choanoflagellates have genes equally orthologous to BACE1 and BACE2. BACE1 from a cnidarian (Hydra) can cleave APP to release Aß, pushing back the date of the origin of its function to near the origin of animals. We tested more divergent BACE1/2 genes from a ctenophore (Mnemiopsis) and a choanoflagellate (Monosiga), and neither has this activity. These findings indicate that the specific proteolytic function of BACE1 evolved during the very earliest diversification of animals, most likely after a gene-duplication event.

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.

Gastrodin ameliorates synaptic impairment, mitochondrial dysfunction and oxidative stress in N2a/APP cells.

Alzheimer's disease is characterized by abnormal ß-amyloid and tau accumulation, mitochondrial dysfunction, oxidative stress, and synaptic dysfunction. Here, we aimed to assess the mechanisms and signalling pathways in the neuroprotective effect of gastrodin, a phenolic glycoside, on murine neuroblastoma N2a cells expressing human Swedish mutant APP (N2a/APP). We found that gastrodin increased the levels of presynaptic-SNAP, synaptophysin, and postsynaptic-PSD95 and reduced phospho-tau Ser396, APP and Aß1-42 levels in N2a/APP cells. Gastrodin treatment reduced reactive oxygen species generation, lipid peroxidation, mitochondrial fragmentation and DNA oxidation; restored mitochondrial membrane potential and intracellular ATP production. Upregulated phospho-GSK-3ß and reduced phospho-ERK and phospho-JNK were involved in the protective effect of gastrodin. In conclusion, we demonstrated the neuroprotective effect of gastrodin in the N2a/APP cell line by ameliorating the impairment on synaptic and mitochondrial function, reducing tau phosphorylation, Aß1-42 levels as well as reactive oxygen species generation. These results provide new mechanistic insights into the potential effect of gastrodin in the treatment of Alzheimer's disease.

Spatiotemporal development of the neuronal accumulation of amyloid precursor protein and the amyloid plaque formation in the brain of 3xTg-AD mice.

The amyloid plaque is a hallmark of Alzheimer's disease. The accumulation of the amyloid precursor protein (APP) in the neuronal structure is assumed to lead to amyloid plaque formation through the excessive production of ß-amyloid protein. To study the relationship between the neuronal accumulation of APP and amyloid plaque formation, we histologically analyzed their development in the different brain regions in 3xTg-AD mice, which express Swedish mutated APP (APPSWE) in the neurons. Observation throughout the brain revealed APPSWE-positive somata in the broad regions. Quantitative model analysis showed that the somatic accumulation of APPSWE developed firstly in the hippocampus from a very early age (<1 month) and proceeded slower in the isocortex. In line with this, the hippocampus was the first region to form amyloid plaques at the age of 9-12 months, while amyloid plaques were rarely observed in the isocortex. Females had more APPSWE-positive somata and plaques than males. Furthermore, amyloid plaques were observed in the lateral septum and pontine grey, which did not contain APPSWE-positive somata but only the APPSWE-positive fibers. These results suggested that neuronal accumulation of APPSWE, both in somatodendritic and axonal domains, is closely related to the formation of amyloid plaques.

Short-term regulation of TSFM level does not alter amyloidogenesis and mitochondrial function in type-specific cells.

BACKGROUND: Mitochondrial Ts translation elongation factor (TSFM) is an enzyme that catalyzes exchange of guanine nucleotides. By forming a complex with mitochondrial Tu translation elongation factor (TUFM), TSFM participates in mitochondrial protein translation. We have previously reported that TUFM regulates translation of beta-site APP cleaving enzyme 1 (BACE1) via ROS (reactive oxygen species)-dependent mechanism, suggesting a potential role in amyloid precursor protein (APP) processing associated with Alzheimer's disease (AD), which led to the speculation that TSFM may regulate APP processing in a similar way to TUFM. METHODS AND RESULTS: Here, we report that in cultured cells, knockdown or overexpression TSFM did not change protein levels in BACE1 and APP. Besides, the levels of cytoplasmic ROS and mitochondrial superoxide, in addition to ATP level, cell viability and mitochondrial membrane potential were not significantly altered by TSFM knockdown in the short term. Further transcriptome analysis revealed that expression of majority of mitochondrial genes were not remarkably changed by TSFM silencing. The possibility of TSFM involved in cardiomyopathy and cancer development was uncovered using bioinformatics analysis. CONCLUSIONS: Collectively, short-term regulation of TSFM level in cultured cells does not cause a significant change in proteins involved in APP processing, levels in ROS and ATP associated with mitochondrial function. Whereas our study could contribute to comprehend certain clinical features of TSFM mutations, the roles of TSFM in cardiomyopathy and cancer development might deserve further investigation.

Dual-specificity tyrosine phosphorylation-regulated kinase 1A promotes the inclusion of amyloid precursor protein exon 7.

Extracellular amyloid plaques made of Amyloid-ß (Aß) derived from amyloid precursor protein (APP) is one of the major neuropathological hallmarks of Alzheimer's disease (AD). There are three major isoforms of APP, APP770, APP751, and APP695 generated by alternative splicing of exons 7 and 8. Exon 7 encodes the Kunitz protease inhibitor (KPI) domain. Its inclusion generates APP isoforms containing KPI, APPKPI+, which is elevated in AD and Down syndrome (DS) brains and associated with increased Aß deposition. Dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A) phosphorylates many splicing factors and regulates the alternative splicing of pre-mRNA. It is upregulated in DS and AD brain. However, it is not yet clear whether Dyrk1A could regulate APP alternative splicing. In the present study, we overexpressed or knocked down Dyrk1A in cultured cells and observed that Dyrk1A promoted the inclusion of both APP exons 7 and 8. Moreover, a significant increase in APP exon7 inclusion was also detected in the forebrain and hippocampus of human Dyrk1A transgenic mice - Tg/Dyrk1A. Screening for splicing factors regulated by Dyrk1A revealed that serine/arginine-rich protein 9G8 inhibited APP exon7 inclusion and interacted with APP pre-mRNA. In vitro, expression of exon 7 facilitated APP cleavage. In human Dyrk1A transgenic mice, we also found an increase in Aß production. These findings suggest that Dyrk1A inhibits the splicing factor 9G8 and promotes APP exon 7 inclusion, leading to more APPKPI+ expression and APP cleavage and potentially contributing to Aß production in vivo.