Quantitative association between gene expression and blood cell production of individual hematopoietic stem cells in mice.

Individual hematopoietic stem cells (HSCs) produce different amounts of blood cells upon transplantation. Taking advantage of the intercellular variation, we developed an experimental and bioinformatic approach to evaluating the quantitative association between gene expression and blood cell production across individual HSCs. We found that most genes associated with blood production exhibit the association only at some levels of blood production. By mapping gene expression with blood production, we identified four distinct patterns of their quantitative association. Some genes consistently correlate with blood production over a range of levels or across all levels, and these genes are found to regulate lymphoid but not myeloid production. Other genes exhibit one or more clear peaks of association. Genes with overlapping peaks are found to be coexpressed in other tissues and share similar molecular functions and regulatory motifs. By dissecting intercellular variations, our findings revealed four quantitative association patterns that reflect distinct dose-response molecular mechanisms modulating the blood cell production of HSCs.

Clinical applicability of external and internal body dimensions in predicting dystocia in late-gestation Holstein-Friesian heifers.

The objective of this study was to determine the clinical applicability of maternal intrapelvic area (PA) and selected morphometric parameters that can be measured before parturition in predicting dystocia in dairy heifers. The measurements were performed in 374 late-gestation Holstein-Friesian heifers. Inner pelvic height and width were measured using a pelvimeter, and PA was calculated. The heifers were monitored continuously around the time of calving, and calving difficulty was categorized as: unassisted calving (UC), slight assistance (SA), considerable difficulty (CD) and veterinary assistance (VA). Calving performance was analysed with the χ2 test, and the effect of body dimensions on the course of parturition was evaluated by one-way analysis of variance. Dystocia (CD + VA) was predicted with the use of the classification tree method. Dystocia accounted for 29.14% of all deliveries. The percentages of stillbirths and retained placenta increased (p < .01) with increasing calving difficulty. Average PA immediately before parturition was smaller (p < .01) in group VA (223.2 cm2 ) than in group UC (253.3 cm2 ). According to the classification tree, dystocia may occur (74.07% odds) in heifers with PA < 254.2 cm2 and a rump angle <5.68° before parturition. Measurements of heifer's cannon circumference and sire's body size improve the accuracy of dystocia prediction.

RNA splicing factor Rbm25 underlies heterogeneous preleukemic clonal expansion in mice.

Clonal expansion sets the stage for cancer genesis by allowing for the accumulation of molecular alterations. Although genetic mutations such as Tet2 that induce clonal expansion and malignancy have been identified, these mutations are also frequently found in healthy individuals. Here, we tracked preleukemic clonal expansion using genetic barcoding in an inducible Tet2 knockout mouse model and found that only a small fraction of hematopoietic stem cells (HSCs) expanded excessively upon Tet2 knockout. These overexpanded HSCs expressed significantly lower levels of genes associated with leukemia and RNA splicing than nonoverexpanded Tet2 knockout HSCs. Knocking down Rbm25, an identified RNA splicing factor, accelerated the expansion of Tet2-knockout hematopoietic cells in vitro and in vivo. Our data suggest that mutations of an epigenetic factor Tet2 induce variability in the expression of an RNA splicing factor Rbm25, which subsequently drives heterogeneous preleukemic clonal expansion. This heterogeneous clonal expansion could contribute to the variable disease risks across individuals.

Lineage tracking to reveal the fate of hematopoietic stem cells influenced by Flk2- multipotent progenitors after transplantation.

After transplantation, hematopoietic stem cells (HSCs) sustain blood cell regeneration throughout the patient's life. Recent studies suggest that several types of mature blood cells provide feedback signals to regulate HSC fate. However, the potential feedback effect of hematopoietic progenitor cells has not been characterized to date. The present investigation demonstrated that multipotent progenitors (MPPs) promoted T cell production of HSCs when both cell types were cotransplanted in mice. Using genetic barcodes to track individual HSCs in mice, we found that the increased T cell production by HSCs was associated with the combined effects of altered lineage bias and clonal expansion during HSC differentiation. We showed that MPP and HSC co-transplantation promoted the multilineage differentiation of HSCs in the short term while preserving lymphoid-specialized HSC differentiation in the long term. Our findings indicate that MPPs derived from HSCs regulate the fate of HSCs after bone marrow transplantation.

The Effect of Mealworm Frass on the Chemical and Microbiological Properties of Horticultural Peat in an Incubation Experiment.

Insect farming is growing in popularity, and in addition to insect meal, it generates waste products such as exuviae and frass, which can be recycled in agriculture. The aim of this incubation experiment was to evaluate the effect of Tenebrio molitor L. frass on selected chemical and biological properties of deacidified peat, which is widely used in horticulture. The optimal rate of frass fertilizer in peat for growing vegetables and ornamental plants was determined, with special emphasis on mineral nitrogen levels. Peat was fertilized with five nitrogen rates, 0, 50, 100, 200, and 400 mg dm-3, and supplied with frass or urea. The study demonstrated that frass can be used as organic fertilizer. An increase in the nitrogen rate significantly increased mineral nitrogen content and electrical conductivity and decreased Ca content in peat. Both frass and urea increased the ammonification rate at the beginning of incubation and the nitrification rate from the second week of the experiment. Higher frass rates (5 and 10 g dm-3) increased the content of plant-available nutrients (nitrogen, phosphorus, magnesium, potassium, and sodium) in peat as well as the abundance of microorganisms supporting organic matter mineralization. Unlike frass, urea increased the counts of nitrogen-fixing bacteria in peat.

Deciphering intratumoral heterogeneity using integrated clonal tracking and single-cell transcriptome analyses.

Cellular heterogeneity is a major cause of treatment resistance in cancer. Despite recent advances in single-cell genomic and transcriptomic sequencing, it remains difficult to relate measured molecular profiles to the cellular activities underlying cancer. Here, we present an integrated experimental system that connects single cell gene expression to heterogeneous cancer cell growth, metastasis, and treatment response. Our system integrates single cell transcriptome profiling with DNA barcode based clonal tracking in patient-derived xenograft models. We show that leukemia cells exhibiting unique gene expression respond to different chemotherapies in distinct but consistent manners across multiple mice. In addition, we uncover a form of leukemia expansion that is spatially confined to the bone marrow of single anatomical sites and driven by cells with distinct gene expression. Our integrated experimental system can interrogate the molecular and cellular basis of the intratumoral heterogeneity underlying disease progression and treatment resistance.

Clonal tracking using embedded viral barcoding and high-throughput sequencing.

Embedded viral barcoding in combination with high-throughput sequencing is a powerful technology with which to track single-cell clones. It can provide clonal-level insights into cellular proliferation, development, differentiation, migration, and treatment efficacy. Here, we present a detailed protocol for a viral barcoding procedure that includes the creation of barcode libraries, the viral delivery of barcodes, the recovery of barcodes, and the computational analysis of barcode sequencing data. The entire procedure can be completed within a few weeks. This barcoding method requires cells to be susceptible to viral transduction. It provides high sensitivity and throughput, and enables precise quantification of cellular progeny. It is cost efficient and does not require any advanced skills. It can also be easily adapted to many types of applications, including both in vitro and in vivo experiments.

Activation of the Unfolded Protein Response in Sporadic Inclusion-Body Myositis but Not in Hereditary GNE Inclusion-Body Myopathy.

Muscle fibers in patients with sporadic inclusion-body myositis (s-IBM),the most common age-associated myopathy, are characterized by autophagic vacuoles and accumulation of ubiquitinated and congophilic multiprotein aggregates that contain amyloid-ß and phosphorylated tau. Muscle fibers of autosomal-recessive hereditary inclusion-body myopathy caused by the GNE mutation (GNE-h-IBM) display similar pathologic features, except with less pronounced congophilia. Accumulation of unfolded/misfolded proteins inside the endoplasmic reticulum (ER) lumen leads to ER stress, which elicits the unfolded protein response (UPR) as a protective mechanism. Here we demonstrate for the first time that UPR is activated in s-IBM muscle biopsies, since there was 1) increased activating transcription factor 4 (ATF4) protein and increased mRNA of its target C/EBP homologous protein; 2) cleavage of the ATF6 and increased mRNA of its target glucose-regulated protein 78; and 3) an increase of the spliced form of X-box binding protein 1 and increased mRNA of ER degradation-enhancing α-mannosidase-like protein, target of heterodimer of cleaved ATF6 and spliced X-box binding protein 1. In contrast, we did not find similar evidence of the UPR induction in GNE-h-IBM patient muscle, suggesting that different intracellular mechanisms might lead to similar pathologic phenotypes. Interestingly, cultured GNE-h-IBM muscle fibers had a robust UPR response to experimental ER stress stimuli, suggesting that the GNE mutation per se is not responsible for the lack of UPR in GNE-h-IBM biopsied muscle.

Sporadic inclusion-body myositis: A degenerative muscle disease associated with aging, impaired muscle protein homeostasis and abnormal mitophagy.

Sporadic inclusion-body myositis (s-IBM) is the most common degenerative muscle disease in which aging appears to be a key risk factor. In this review we focus on several cellular molecular mechanisms responsible for multiprotein aggregation and accumulations within s-IBM muscle fibers, and their possible consequences. Those include mechanisms leading to: a) accumulation in the form of aggregates within the muscle fibers, of several proteins, including amyloid-ß42 and its oligomers, and phosphorylated tau in the form of paired helical filaments, and we consider their putative detrimental influence; and b) protein misfolding and aggregation, including evidence of abnormal myoproteostasis, such as increased protein transcription, inadequate protein disposal, and abnormal posttranslational modifications of proteins. Pathogenic importance of our recently demonstrated abnormal mitophagy is also discussed. The intriguing phenotypic similarities between s-IBM muscle fibers and the brains of Alzheimer and Parkinson's disease patients, the two most common neurodegenerative diseases associated with aging, are also discussed. This article is part of a Special Issue entitled: Neuromuscular Diseases: Pathology and Molecular Pathogenesis.

Sodium phenylbutyrate reverses lysosomal dysfunction and decreases amyloid-ß42 in an in vitro-model of inclusion-body myositis.

Sporadic inclusion-body myositis (s-IBM) is a severe, progressive muscle disease for which there is no enduring treatment. Pathologically characteristic are vacuolated muscle fibers having: accumulations of multi-protein aggregates, including amyloid-ß(Aß) 42 and its toxic oligomers; increased γ-secretase activity; and impaired autophagy. Cultured human muscle fibers with experimentally-impaired autophagy recapitulate some of the s-IBM muscle abnormalities, including vacuolization and decreased activity of lysosomal enzymes, accompanied by increased Aß42, Aß42 oligomers, and increased γ-secretase activity. Sodium phenylbutyrate (NaPB) is an orally bioavailable small molecule approved by the FDA for treatment of urea-cycle disorders. Here we describe that NaPB treatment reverses lysosomal dysfunction in an in vitro model of inclusion-body myositis, involving cultured human muscle fibers. NaPB treatment improved lysosomal activity, decreased Aß42 and its oligomers, decreased γ-secretase activity, and virtually prevented muscle-fiber vacuolization. Accordingly, NaPB might be considered a potential treatment of s-IBM patients.

Luminescent conjugated oligothiophenes for sensitive fluorescent assignment of protein inclusion bodies.

Small hydrophobic ligands identifying intracellular protein deposits are of great interest, as protein inclusion bodies are the pathological hallmark of several degenerative diseases. Here we report that fluorescent amyloid ligands, termed luminescent conjugated oligothiophenes (LCOs), rapidly and with high sensitivity detect protein inclusion bodies in skeletal muscle tissue from patients with sporadic inclusion body myositis (s-IBM). LCOs having a conjugated backbone of at least five thiophene units emitted strong fluorescence upon binding, and showed co-localization with proteins reported to accumulate in s-IBM protein inclusion bodies. Compared with conventional amyloid ligands, LCOs identified a larger fraction of immunopositive inclusion bodies. When the conjugated thiophene backbone was extended with terminal carboxyl groups, the LCO revealed striking spectral differences between distinct protein inclusion bodies. We conclude that 1) LCOs are sensitive, rapid and powerful tools for identifying protein inclusion bodies and 2) LCOs identify a wider range of protein inclusion bodies than conventional amyloid ligands.

Pathogenic considerations in sporadic inclusion-body myositis, a degenerative muscle disease associated with aging and abnormalities of myoproteostasis.

The pathogenesis of sporadic inclusion-body myositis (s-IBM) is complex; it involves multidimensional pathways and the most critical issues are still unresolved. The onset of muscle fiber damage is age related and the disease is slowly, but inexorably, progressive. Muscle fiber degeneration and mononuclear cell inflammation are major components of s-IBM pathology, but which is precedent and how they interrelate is not known. There is growing evidence that aging of the muscle fiber associated with intramyofiber accumulation of conformationally modified proteins plays a primary pathogenic role leading to muscle fiber destruction. Here, we review the presumably most important known molecular abnormalities that occur in s-IBM myofibers and that likely contribute to s-IBM pathogenesis. Abnormal accumulation within the fibers of multiprotein aggregates (several of which are congophilic and, therefore, generically called "amyloid") may result from increased transcription of several proteins, their abnormal posttranslational modifications and misfolding, and inadequate protein disposal, that is, abnormal "myoproteostasis," which is combined with and may be provoked or abetted by an aging intracellular milieu. The potential cytotoxicity of accumulated amyloid ß protein (Aß42) and its oligomers, phosphorylated tau in the form of paired helical filaments and α-synuclein, and the putative pathogenic role and cause of the mitochondrial abnormalities and oxidative stress are reviewed. On the basis of our experimental evidence, potential interventions in the complex, interwoven pathogenic cascade of s-IBM are suggested.

Activation of the γ-secretase complex and presence of γ-secretase-activating protein may contribute to Aß42 production in sporadic inclusion-body myositis muscle fibers.

The muscle-fiber phenotype of sporadic inclusion-body myositis (s-IBM), the most common muscle disease associated with aging, shares several pathological abnormalities with Alzheimer disease (AD) brain, including accumulation of amyloid-ß 42 (Aß42) and its cytotoxic oligomers. The exact mechanisms leading to Aß42 production within s-IBM muscle fibers are not known. Aß42 and Aß40 are generated after the amyloid-precursor protein (AßPP) is cleaved by ß-secretase and the γ-secretase complex. Aß42 is considered more cytotoxic than Aß40, and it has a higher propensity to oligomerize, form amyloid fibrils, and aggregate. Recently, we have demonstrated in cultured human muscle fibers that experimental inhibition of lysosomal enzyme activities leads to Aß42 oligomerization. In s-IBM muscle, we here demonstrate prominent abnormalities of the γ-secretase complex, as evidenced by: a) increase of γ-secretase components, namely active presenilin 1, presenilin enhancer 2, nicastrin, and presence of its mature, glycosylated form; b) increase of mRNAs of these γ-secretase components; c) increase of γ-secretase activity; d) presence of an active form of a newly-discovered γ-secretase activating protein (GSAP); and e) increase of GSAP mRNA. Furthermore, we demonstrate that experimental inhibition of lysosomal autophagic enzymes in cultured human muscle fibers a) activates γ-secretase, and b) leads to posttranslational modifications of AßPP and increase of Aß42. Since autophagy is impaired in biopsied s-IBM muscle, the same mechanism might be responsible for its having increased γ-secretase activity and Aß42 production. Accordingly, improving lysosomal function might be a therapeutic strategy for s-IBM patients.

Abnormalities of NBR1, a novel autophagy-associated protein, in muscle fibers of sporadic inclusion-body myositis.

Intra-muscle fiber accumulation of ubiquitinated protein aggregates containing several conformationally modified proteins, including amyloid-ß and phosphorylated tau, is characteristic of the pathologic phenotype of sporadic inclusion-body myositis (s-IBM), the most common progressive degenerative myopathy of older persons. Abnormalities of protein-degradation, involving both the 26S proteasome and autophagic-lysosomal pathways, were previously demonstrated in s-IBM muscle. NBR1 is a ubiquitin-binding scaffold protein importantly participating in autophagic degradation of ubiquitinated proteins. Whereas abnormalities of p62, a ubiquitin-binding protein, were previously described in s-IBM, abnormalities of NBR1 have not been reported in s-IBM. We have now identified in s-IBM muscle biopsies that NBR1, by: (a) immunohistochemistry, was strongly accumulated within s-IBM muscle-fiber aggregates, where it closely co-localized with p62, ubiquitin, and phosphorylated tau; (b) immunoblots, was increased threefold (p < 0.001); and (c) immunoprecipitation, was associated with p62 and LC3. By real-time PCR, NBR1 mRNA was increased twofold (p < 0.01). None of the various disease- and normal-control muscle biopsies had any NBR1 abnormality. In cultured human muscle fibers, NBR1 also physically associated with both p62 and LC3, and experimental inhibition of either the 26S proteasome or the lysosomal activity resulted in NBR1 increase. Our demonstration of NBR1 abnormalities in s-IBM provides further evidence that altered protein degradation pathways may be critically involved in the s-IBM pathogenesis. Accordingly, attempts to unblock defective protein degradation might be a therapeutic strategy for s-IBM patients.

Novel demonstration of conformationally modified tau in sporadic inclusion-body myositis muscle fibers.

s-IBM is the most common muscle disease of older persons. Its muscle fiber molecular phenotype has close similarities to Alzheimer disease (AD) brain, including intra-muscle-fiber accumulations of (a) Aß42 and its oligomers, and (b) large, squiggly or linear, clusters of paired-helical filaments (PHFs) that are immunoreactive with various antibodies directed against several epitopes of phosphorylated tau (p-tau), and thereby strongly resembling neurofibrillary tangles of AD brain. In AD brain, conformational changes of tau, including its modifications detectable with specific antibodies TG3 (recognizing phosphorylated-Thr231), and Alz50 and MC1 (both recognizing amino acids 5-15 and 312-322) are considered early and important modifications leading to tau's abnormal folding and assembly into PHFs. We have now identified conformationally modified tau in 14 s-IBM muscle biopsies by (a) light-and electron-microscopic immunohistochemistry, (b) immunoblots, and (c) dot-immunoblots, using TG3, Alz50 and MC1 antibodies. Our double-immunolabeling on the light- and electron-microscopic levels, which combined an antibody against p62 that recognizes s-IBM clusters of PHFs, revealed that TG3 immunodecorated, abundantly and exclusively, all p62 immunopositive clusters, while Alz50 labeling was less abundant, and MC1 was mainly diffusely immunoreactive. Interestingly, in the very atrophic degenerating fibers, TG3 co-localized with PHF-1 antibody that recognizes tau phosphorylated at Ser396/404, which is considered a later change in the formation of PHFs; however, most of TG3-positive inclusions in non-atrophic fibers were immunonegative with PHF-1. None of the 12 normal- and disease-control muscle biopsies contained conformational or PHF-1 immunoreactive tau. This first demonstration of conformational tau in s-IBM, because of its abundance in non-atrophic muscle fibers, suggests that it might play an early role in s-IBM PHFs formation and thus be pathogenically important.

Novel demonstration of amyloid-ß oligomers in sporadic inclusion-body myositis muscle fibers.

Accumulation of amyloid-ß (Aß) within muscle fibers has been considered an upstream step in the development of the s-IBM pathologic phenotype. Aß42, which is considered more cytotoxic than Aß40 and has a higher propensity to oligomerize, is preferentially increased in s-IBM muscle fibers. In Alzheimer disease (AD), low-molecular weight Aß oligomers and toxic oligomers, also referred to as "Aß-Derived Diffusible Ligands" (ADDLs), are considered strongly cytotoxic and proposed to play an important pathogenic role. ADDLs have been shown to be increased in AD brain. We now report for the first time that in s-IBM muscle biopsies Aß-dimer, -trimer, and -tetramer are identifiable by immunoblots. While all the s-IBM samples we studied had Aß-oligomers, their molecular weights and intensity varied between the patient samples. None of the control muscle biopsies had Aß oligomers. Dot-immunoblots using highly specific anti-ADDL monoclonal antibodies also showed highly increased ADDLs in all s-IBM biopsies studied, while controls were negative. By immunofluorescence, in some of the abnormal s-IBM muscle fibers ADDLs were accumulated in the form of plaque-like inclusions, and were often increased diffusely in very small fibers. Normal and disease-controls were negative. By gold-immuno-electron microscopy, ADDL-immunoreactivities were in close proximity to 6-10 nm amyloid-like fibrils, and also were immunodecorating amorphous and floccular material. In cultured human muscle fibers, we found that inhibition of autophagy led to the accumulation of Aß oligomers. This novel demonstration of Aß42 oligomers in s-IBM muscle biopsy provides additional evidence that intra-muscle fiber accumulation of Aß42 oligomers in s-IBM may contribute importantly to s-IBM pathogenic cascade.

Impaired autophagy in sporadic inclusion-body myositis and in endoplasmic reticulum stress-provoked cultured human muscle fibers.

The hallmark pathologies of sporadic inclusion-body myositis (s-IBM) muscle fibers are autophagic vacuoles and accumulation of ubiquitin-positive multiprotein aggregates that contain amyloid-beta or phosphorylated tau in a beta-pleated sheet amyloid configuration. Endoplasmic reticulum stress (ERS) and 26S proteasome inhibition, also associated with s-IBM, putatively aggrandize the accumulation of misfolded proteins. However, autophagosomal-lysosomal pathway formation and function, indicated by autophagosome maturation, have not been previously analyzed in this system. Here we studied the autophagosomal-lysosomal pathway using 14 s-IBM and 30 disease control and normal control muscle biopsy samples and our cultured human muscle fibers in a microenvironment modified to resemble aspects of s-IBM pathology. We report for the first time that in s-IBM, lysosomal enzyme activities of cathepsin D and B were decreased 60% (P < 0.01) and 40% (P < 0.05), respectively. We also detected two indicators of increased autophagosome maturation, the presence of LC3-II and decreased mammalian target of rapamycin-mediated phosphorylation of p70S6 kinase. Moreover, in cultured human muscle fibers, ERS induction significantly decreased activities of cathepsins D and B, increased levels of LC3-II, decreased phosphorylation of p70S6 kinase, and decreased expression of VMA21, a chaperone for assembly of lysosomal V-ATPase. We conclude that in s-IBM muscle, decreased lysosomal proteolytic activity might enhance accumulation of misfolded proteins, despite increased maturation of autophagosomes, and that ERS is a possible cause of s-IBM-impaired lysosomal function. Thus, unblocking protein degradation in s-IBM muscle fibers may be a desirable therapeutic strategy.

Increased BACE1 mRNA and noncoding BACE1-antisense transcript in sporadic inclusion-body myositis muscle fibers--possibly caused by endoplasmic reticulum stress.

Sporadic inclusion-body myositis (s-IBM) is the most common muscle disease of older persons. Its muscle-fiber phenotype shares several molecular similarities with Alzheimer-disease (AD) brain, including increased AbetaPP, accumulation of amyloid-beta (Abeta), and increased BACE1 protein. Abeta42 is prominently increased in AD brain and within s-IBM fibers, and its oligomers are putatively toxic to both tissues--accordingly, minimizing Abeta42 production can be a therapeutic objective in both tissues. The pathogenic development of s-IBM is unknown, including the mechanisms of BACE1 protein increase. BACE1 is an enzyme essential for production from AbetaPP of Abeta42 and Abeta40, which are proposed to be detrimental within s-IBM muscle fibers. Novel noncoding BACE1-antisense (BACE1-AS) was recently shown (a) to be increased in AD brain, and (b) to increase BACE1 mRNA and BACE1 protein. We studied BACE1-AS and BACE1 transcripts by real-time PCR (a) in 10 s-IBM and 10 age-matched normal muscle biopsies; and (b) in our established ER-Stress-Human-Muscle-Culture-IBM Model, in which we previously demonstrated increased BACE1 protein. Our study demonstrated for the first time that (a) in s-IBM biopsies BACE1-AS and BACE1 transcripts were significantly increased, suggesting that their increased expression can be responsible for the increase of BACE1 protein; and (b) experimental induction of ER stress significantly increased both BACE1-AS and BACE1 transcripts, suggesting that ER stress can participate in their induction in s-IBM muscle. Accordingly, decreasing BACE1 through a targeted downregulation of its regulatory BACE1-AS, or reducing ER stress, might be therapeutic strategies in s-IBM, assuming that it would not impair any normal cellular functions of BACE1.

In AbetaPP-overexpressing cultured human muscle fibers proteasome inhibition enhances phosphorylation of AbetaPP751 and GSK3beta activation: effects mitigated by lithium and apparently relevant to sporadic inclusion-body myositis.

Muscle fiber degeneration in sporadic inclusion-body myositis (s-IBM) is characterized by accumulation of multiprotein aggregates, including aggregated amyloid-beta (Abeta)-precursor protein 751 (AbetaPP751), Abeta, phosphorylated tau, and other 'Alzheimer-characteristic' proteins. Proteasome inhibition is an important component of the s-IBM pathogenesis. In brains of Alzheimer's disease (AD) patients and AD transgenic-mouse models, phosphorylation of neuronal AbetaPP695 (p-AbetaPP) on Thr668 (equivalent to T724 of AbetaPP751) is considered detrimental because it increases generation of cytotoxic Abeta and induces tau phosphorylation. Activated glycogen synthase kinase3beta (GSK3beta) is involved in phosphorylation of both AbetaPP and tau. Lithium, an inhibitor of GSK3beta, was reported to reduce levels of both the total AbetaPP and p-AbetaPP in AD animal models. In relation to s-IBM, we now show for the first time that (1) In AbetaPP-overexpressing cultured human muscle fibers (human muscle culture IBM model: (a) proteasome inhibition significantly increases GSK3beta activity and AbetaPP phosphorylation, (b) treatment with lithium decreases (i) phosphorylated-AbetaPP, (ii) total amount of AbetaPP, (iii) Abeta oligomers, and (iv) GSK3beta activity; and (c) lithium improves proteasome function. (2) In biopsied s-IBM muscle fibers, GSK3beta is significantly activated and AbetaPP is phosphorylated on Thr724. Accordingly, treatment with lithium, or other GSK3beta inhibitors, might benefit s-IBM patients.