Alzheimer's disease (AD) is a complex neurodegenerative disorder characterized by extracellular plaques containing amyloid β (Aβ)-protein and intracellular tangles containing hyperphosphorylated Tau ...protein. Here, we describe the generation of inducible pluripotent stem cell lines from patients harboring the London familial AD (fAD) amyloid precursor protein (APP) mutation (V717I). We examine AD-relevant phenotypes following directed differentiation to forebrain neuronal fates vulnerable in AD. We observe that over differentiation time to mature neuronal fates, APP expression and levels of Aβ increase dramatically. In both immature and mature neuronal fates, the APPV717I mutation affects both β- and γ-secretase cleavage of APP. Although the mutation lies near the γ-secretase cleavage site in the transmembrane domain of APP, we find that β-secretase cleavage of APP is elevated leading to generation of increased levels of both APPsβ and Aβ. Furthermore, we find that this mutation alters the initial cleavage site of γ-secretase, resulting in an increased generation of both Aβ42 and Aβ38. In addition to altered APP processing, an increase in levels of total and phosphorylated Tau is observed in neurons with the APPV717I mutation. We show that treatment with Aβ-specific antibodies early in culture reverses the phenotype of increased total Tau levels, implicating altered Aβ production in fAD neurons in this phenotype. These studies use human neurons to reveal previously unrecognized effects of the most common fAD APP mutation and provide a model system for testing therapeutic strategies in the cell types most relevant to disease processes.
Several protocols have been developed for human induced pluripotent stem cell neuronal differentiation. We compare several methods for forebrain cortical neuronal differentiation by assessing cell ...morphology, immunostaining and gene expression. We evaluate embryoid aggregate vs. monolayer with dual SMAD inhibition differentiation protocols, manual vs. AggreWell aggregate formation, plating substrates, neural progenitor cell (NPC) isolation methods, NPC maintenance and expansion, and astrocyte co-culture. The embryoid aggregate protocol, using a Matrigel substrate, consistently generates a high yield and purity of neurons. NPC isolation by manual selection, enzymatic rosette selection, or FACS all are efficient, but exhibit some differences in resulting cell populations. Expansion of NPCs as neural aggregates yields higher cell purity than expansion in a monolayer. Finally, co-culture of iPSC-derived neurons with astrocytes increases neuronal maturity by day 40. This study directly compares commonly employed methods for neuronal differentiation of iPSCs, and can be used as a resource for choosing between various differentiation protocols.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In injured tissues, regeneration is often associated with cell fate plasticity, in that cells deviate from their normal lineage paths. It is becoming increasingly clear that this plasticity often ...creates alternative strategies to restore damaged or lost cells. Alternatively, cell fate plasticity is also part and parcel of pathologic tissue transformations that accompany disease. In this Perspective, we summarize a few illustrative examples of physiologic and aberrant cellular plasticity. Then, we speculate on how one could enhance endogenous plasticity to promote regeneration and reverse pathologic plasticity, perhaps inspiring interest in a new class of therapies targeting cell fate modulation.
In this Perspective, Lin et al. highlight examples of plasticity during normal regeneration and in aberrant situations in a variety of tissues. The authors also discuss the merits of enhancing endogenous plasticity to promote regeneration and reversing pathologic plasticity as potential therapeutic strategies.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Background:
Fatigue can be a disabling multiple sclerosis (MS) symptom with no effective treatment options.
Objective:
Determine whether a low-fat diet improves fatigue in people with MS (PwMS).
...Methods:
We conducted a 16-week randomized controlled trial (RCT) and allocated PwMS to a low-fat diet (active, total daily fat calories not exceeding 20%) or wait-list (control) group. Subjects underwent 2 weeks of baseline diet data collection (24-hour diet recalls (24HDRs)), followed by randomization. The active group received 2 weeks of nutrition counseling and underwent a 12-week low-fat diet intervention. One set of three 24HDRs at baseline and week 16 were collected. We administered a food frequency questionnaire (FFQ) and Modified Fatigue Impact Scale (MFIS) every 4 weeks. The control group continued their pre-study diet and received diet training during the study completion.
Results:
We recruited 39 PwMS (20—active; 19—control). The active group decreased their daily caloric intake by 11% (95% confidence interval (CI): −18.5%, −3.0%) and the mean MFIS by 4.0 (95% CI: −12.0, 4.0) compared to the control (intent-to-treat). Sensitivity analysis strengthened the association with a mean MFIS difference of −13.9 (95% CI: −20.7, −7.2).
Conclusions:
We demonstrated a significant reduction in fatigue with a low-fat dietary intervention in PwMS.
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NUK, OILJ, SAZU, UKNU, UL, UM, UPUK
The development of three-dimensional culture methods has allowed for the study of developing cortical morphology in human cells. This provides a new tool to study the neurodevelopmental consequences ...of disease-associated mutations. Here, we study the effects of isogenic DISC1 mutation in cerebral organoids. DISC1 has been implicated in psychiatric disease based on genetic studies, including its interruption by a balanced translocation that increases the risk of major mental illness. Isogenic wild-type and DISC1-disrupted human-induced pluripotent stem cells were used to generate cerebral organoids, which were then examined for morphology and gene expression. We show that DISC1-mutant cerebral organoids display disorganized structural morphology and impaired proliferation, which is phenocopied by WNT agonism and rescued by WNT antagonism. Furthermore, there are many shared changes in gene expression with DISC1 disruption and WNT agonism, including in neural progenitor and cell fate markers, regulators of neuronal migration, and interneuron markers. These shared gene expression changes suggest mechanisms for the observed morphologic dysregulation with DISC1 disruption and points to new avenues for future studies. The shared changes in three-dimensional cerebral organoid morphology and gene expression with DISC1 interruption and WNT agonism further strengthens the link between DISC1 mutation, abnormalities in WNT signaling, and neuropsychiatric disease.
Genetic and clinical association studies have identified disrupted in schizophrenia 1 (DISC1) as a candidate risk gene for major mental illness. DISC1 is interrupted by a balanced chr(1;11) ...translocation in a Scottish family in which the translocation predisposes to psychiatric disorders. We investigate the consequences of DISC1 interruption in human neural cells using TALENs or CRISPR-Cas9 to target the DISC1 locus. We show that disruption of DISC1 near the site of the translocation results in decreased DISC1 protein levels because of nonsense-mediated decay of long splice variants. This results in an increased level of canonical Wnt signaling in neural progenitor cells and altered expression of fate markers such as Foxg1 and Tbr2. These gene expression changes are rescued by antagonizing Wnt signaling in a critical developmental window, supporting the hypothesis that DISC1-dependent suppression of basal Wnt signaling influences the distribution of cell types generated during cortical development.
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•We generate isogenic human iPSCs with an engineered disruption of the DISC1 gene•DISC1 disruption results in decreased expression of long DISC1 isoforms•DISC1 disruption affects the expression of NPC fate markers, including FoxG1 and Tbr2•DISC1 mutant NPCs show elevated baseline Wnt signaling and proliferation
Srikanth et al. report the generation of isogenic hiPSC lines with engineered mutations in two locations within the DISC1 gene. This disease-relevant disruption shows a loss of long isoforms, which, in turn, affects neural progenitor cell proliferation, baseline WNT signaling, and the expression of NPC fate markers such as FoxG1 and Tbr2.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Insulin/IGF-1 signaling plays a pivotal role in the regulation of cellular homeostasis through its control of glucose metabolism as well as due to its effects on cell proliferation. Aberrant ...regulation of insulin signaling has been repeatedly implicated in uncontrolled cell growth and malignant transformations. TBC1D3 is a hominoid specific gene previously identified as an oncogene in breast and prostate cancers. Our efforts to identify the molecular mechanisms of TBC1D3-induced oncogenesis revealed the role of TBC1D3 in insulin/IGF-1 signaling pathway. We document here that TBC1D3 intensifies insulin/IGF-1-induced signal transduction through intricate, yet elegant fine-tuning of signaling mechanisms. We show that TBC1D3 expression substantially delayed ubiquitination and degradation of insulin receptor substrate-1 (IRS-1). This effect is achieved through suppression of serine phosphorylation at S636/639, S307 and S312 of IRS-1, which are key phosphorylation sites required for IRS-1 degradation. Furthermore, we report that the effect of TBC1D3 on IRS-1:S636/639 phosphorylation is mediated through TBC1D3-induced activation of protein phosphatase 2A (PP2A), followed by suppression of T389 phosphorylation on p70 S6 kinase (S6K). TBC1D3 specifically interacts with PP2A regulatory subunit B56γ, indicating that TBC1D3 and PP2A B56γ operate jointly to promote S6K:T389 dephosphorylation. These findings suggest that TBC1D3 plays an unanticipated and potentially unique role in the fine-tuning of insulin/IGF-1 signaling, while providing novel insights into the regulation of tumorigenesis by a hominoid-specific protein.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Hominoid- and human-specific genes may have evolved to modulate signaling pathways of a higher order of complexity. TBC1D3 is a hominoid-specific oncogene encoded by a cluster of eight paralogs on ...chromosome 17. Initial work indicates that TBC1D3 is widely expressed in human tissues (Hodzic, D., Kong, C., Wainszelbaum, M. J., Charron, A. J., Su, X., and Stahl, P. D. (2006 Genomics 88, 731-736). In this study, we show that TBC1D3 expression has a powerful effect on cell proliferation that is further enhanced by epidermal growth factor (EGF) in both human and mouse cell lines. EGF activation of the Erk and protein kinase B/Akt pathways is enhanced, both in amplitude and duration, by TBC1D3 expression, whereas RNA interference silencing of TBC1D3 suppresses the activation. Light microscopy and Western blot experiments demonstrate that increased signaling in response to EGF is coupled with a significant delay in EGF receptor (EGFR) trafficking and degradation, which significantly extends the life span of EGFR. Moreover, TBC1D3 suppresses polyubiquitination of the EGFR and the recruitment of c-Cbl. Using the Ras binding domain of Raf1 to monitor GTP-Ras we show that TBC1D3 expression enhances Ras activation in quiescent cells, which is further increased by EGF treatment. We speculate that TBC1D3 may alter Ras GTP loading. We conclude that the expression of TBC1D3 generates a delay in EGFR degradation, a decrease in ubiquitination, and a failure to recruit adapter proteins that ultimately dysregulate EGFR signal transduction and enhance cell proliferation. Altered growth factor receptor trafficking and GTP-Ras turnover may be sites where recently evolved genes such as TBC1D3 selectively modulate signaling in hominoids and humans.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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