Zfp296 Is a Novel, Pluripotent-Specific Reprogramming Factor

Expression of the four transcription factors Oct4, Sox2, Klf4, and c-Myc (OSKM) is sufficient to reprogram somatic cells into induced pluripotent stem (iPSCs). However, this process is slow and inefficient compared with the fusion of somatic cells with embryonic stem cells (ESCs), indicating that ESCs express additional factors that can enhance the efficiency of reprogramming. We had previously developed a method to detect and isolate early neural induction intermediates during the differentiation of mouse ESCs. Using the gene expression profiles of these intermediates, we identified 23 ESC-specific transcripts and tested each for the ability to enhance iPSC formation. Of the tested factors, zinc finger protein 296 (Zfp296) led to the largest increase in mouse iPSC formation. We confirmed that Zfp296 was specifically expressed in pluripotent stem cells and germ cells. Zfp296 in combination with OSKM induced iPSC formation earlier and more efficiently than OSKM alone. Through mouse chimera and teratoma formation, we demonstrated that the resultant iPSCs were pluripotent. We showed that Zfp296 activates transcription of the Oct4 gene via the germ cell–specific conserved region 4 (CR4), and when overexpressed in mouse ESCs leads to upregulation of Nanog expression and downregulation of the expression of differentiation markers, including Sox17, Eomes, and T, which is consistent with the observation that Zfp296 enhances the efficiency of reprogramming. In contrast, knockdown of Zfp296 in ESCs leads to the expression of differentiation markers. Finally, we demonstrated that expression of Zfp296 in ESCs inhibits, but does not block, differentiation into neural cells

Neuronal Dysfunction in iPSC-Derived Medium Spiny Neurons from Chorea-Acanthocytosis Patients Is Reversed by Src Kinase Inhibition and F-Actin Stabilization

Chorea-acanthocytosis (ChAc) is a fatal neurological disorder characterized by red blood cell acanthocytes and striatal neurodegeneration. Recently, severe cell membrane disturbances based on depolymerized cortical actin and an elevated Lyn kinase activity in erythrocytes from ChAc patients were identified. How this contributes to the mechanism of neurodegeneration is still unknown. To gain insight into the pathophysiology, we established a ChAc patient-derived induced pluripotent stem cell model and an efficient differentiation protocol providing a large population of human striatal medium spiny neurons (MSNs), the main target of neurodegeneration in ChAc. Patient-derived MSNs displayed enhanced neurite outgrowth and ramification, whereas synaptic density was similar to controls. Electrophysiological analysis revealed a pathologically elevated synaptic activity in ChAc MSNs. Treatment with the F-actin stabilizer phallacidin or the Src kinase inhibitor PP2 resulted in the significant reduction of disinhibited synaptic currents to healthy control levels, suggesting a Src kinase- and actin-dependent mechanism. This was underlined by increased G/F-actin ratios and elevated Lyn kinase activity in patient-derived MSNs. These data indicate that F-actin stabilization and Src kinase inhibition represent potential therapeutic targets in ChAc that may restore neuronal function

Alteration of Mitochondrial Integrity as Upstream Event in the Pathophysiology of SOD1-ALS

Little is known about the early pathogenic events by which mutant superoxide dismutase 1 (SOD1) causes amyotrophic lateral sclerosis (ALS). This lack of mechanistic understanding is a major barrier to the development and evaluation of efficient therapies. Although protein aggregation is known to be involved, it is not understood how mutant SOD1 causes degeneration of motoneurons (MNs). Previous research has relied heavily on the overexpression of mutant SOD1, but the clinical relevance of SOD1 overexpression models remains questionable. We used a human induced pluripotent stem cell (iPSC) model of spinal MNs and three different endogenous ALS-associated SOD1 mutations (D90Ahom, R115Ghet or A4Vhet) to investigate early cellular disturbances in MNs. Although enhanced misfolding and aggregation of SOD1 was induced by proteasome inhibition, it was not affected by activation of the stress granule pathway. Interestingly, we identified loss of mitochondrial, but not lysosomal, integrity as the earliest common pathological phenotype, which preceded elevated levels of insoluble, aggregated SOD1. A super-elongated mitochondrial morphology with impaired inner mitochondrial membrane potential was a unifying feature in mutant SOD1 iPSC-derived MNs. Impaired mitochondrial integrity was most prominent in mutant D90Ahom MNs, whereas both soluble disordered and detergent-resistant misfolded SOD1 was more prominent in R115Ghet and A4Vhet mutant lines. Taking advantage of patient-specific models of SOD1-ALS in vitro, our data suggest that mitochondrial dysfunction is one of the first crucial steps in the pathogenic cascade that leads to SOD1-ALS and also highlights the need for individualized medical approaches for SOD1-ALS

Live cell imaging of ATP levels reveals metabolic compartmentalization within motoneurons and early metabolic changes in FUS ALS motoneurons

Motoneurons are one of the most energy-demanding cell types and a primary target in Amyotrophic lateral sclerosis (ALS), a debilitating and lethal neurodegenerative disorder without currently available effective treatments. Disruption of mitochondrial ultrastructure, transport, and metabolism is a commonly reported phenotype in ALS models and can critically affect survival and the proper function of motor neurons. However, how changes in metabolic rates contribute to ALS progression is not fully understood yet. Here, we utilize hiPCS-derived motoneuron cultures and live imaging quantitative techniques to evaluate metabolic rates in fused in sarcoma (FUS)-ALS model cells. We show that differentiation and maturation of motoneurons are accompanied by an overall upregulation of mitochondrial components and a significant increase in metabolic rates that correspond to their high energy-demanding state. Detailed compartment-specific live measurements using a fluorescent ATP sensor and FLIM imaging show significantly lower levels of ATP in the somas of cells carrying FUS-ALS mutations. These changes lead to the increased vulnerability of diseased motoneurons to further metabolic challenges with mitochondrial inhibitors and could be due to the disruption of mitochondrial inner membrane integrity and an increase in its proton leakage. Furthermore, our measurements demonstrate heterogeneity between axonal and somatic compartments, with lower relative levels of ATP in axons. Our observations strongly support the hypothesis that mutated FUS impacts the metabolic states of motoneurons and makes them more susceptible to further neurodegenerative mechanisms

<i>Zfp296</i> knockdown in ESCs.

<p>Lentiviral shRNA constructs were packaged and used to infect ESCs. Puromycin was added to select for infected cells beginning on day 4. Efficiency of <i>Zfp296</i> knockdown is shown on the indicated days after infection (A). Quantitative RT-PCR results are shown for the indicated genes on day 7 after infection (B).</p

<i>Zfp296</i> activates transcription from the <i>Oct4</i> promoter.

<p><i>Zfp296</i> and <i>Nanog</i> were tested by luciferase assays for transactivation of <i>Oct4</i> promoter fragments (A). The <i>P</i> value for <i>Zfp296</i>-induced transcription from CR4 compared with control was less than 0.1, as shown. CR1, CR2, CR3, and CR4 = evolutionary conserved regions 1, 2, 3, and 4, respectively, as defined by Nordhoff <i>et alia </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034645#pone.0034645-Nordhoff1" target="_blank">[20]</a>. ESCs were induced to express <i>Zfp296</i> for 18 hours followed by quantitative RT-PCR to assess the expression of transgenic <i>Zfp296</i> (B) and indicated marker genes (C) compared with uninduced cells (B). Expression of indicated marker genes on day 7 of neural differentiation in the presence of induced <i>Zfp296</i> expression relative to uninduced cells (D).</p

Distinct Neurodegenerative Changes in an Induced Pluripotent Stem Cell Model of Frontotemporal Dementia Linked to Mutant TAU Protein

Frontotemporal dementia (FTD) is a frequent form of early-onset dementia and can be caused by mutations in MAPT encoding the microtubule-associated protein TAU. Because of limited availability of neural cells from patients’ brains, the underlying mechanisms of neurodegeneration in FTD are poorly understood. Here, we derived induced pluripotent stem cells (iPSCs) from individuals with FTD-associated MAPT mutations and differentiated them into mature neurons. Patient iPSC-derived neurons demonstrated pronounced TAU pathology with increased fragmentation and phospho-TAU immunoreactivity, decreased neurite extension, and increased but reversible oxidative stress response to inhibition of mitochondrial respiration. Furthermore, FTD neurons showed an activation of the unfolded protein response, and a transcriptome analysis demonstrated distinct, disease-associated gene expression profiles. These findings indicate distinct neurodegenerative changes in FTD caused by mutant TAU and highlight the unique opportunity to use neurons differentiated from patient-specific iPSCs to identify potential targets for drug screening purposes and therapeutic intervention

Oct4 promoter methylation.

<p>Bisulfite sequencing results assessing the DNA methylation status of the <i>Oct4</i> promoter is shown for the indicated cell types. OSKM = <i>Oct4</i>, <i>Sox2</i>, <i>Klf4</i>, and <i>c-Myc</i>; ESCs = embryonic stem cells; NSCs = neural stem cells. Open and filled circles represent unmethylated and methylated CpGs, respectively.</p

Pluripotency of iPSCs induced with OSKM+<i>Zfp296</i>.

<p>An iPSC line induced with OSKM+<i>Zfp296</i> is shown in phase (A), DAPI (B), Oct4 immunostained (C), or Oct4-GFP (D). Teratomas formed with this iPSC line were composed of cartilage (mesoderm; E), neural rosettes (ectoderm; F), and epithelia (endoderm; G). A chimera formed with this iPSC line is shown (H), and offspring from this chimera carried the GFP transgene, which originated from the iPSCs (I).</p

Timing and efficiency of iPSC colony formation.

<p>NSCs were infected with either OSKM+<i>Zfp296</i> or with OSKM+empty vector. GFP-positive colonies from 30 fields from an automated microscope were counted in three independent replicates for each of the days shown (A). SSEA1-positive colonies from 60 fields from an automated microscope were counted in three independent replicates for each of the days shown (B). Mean and standard deviations are shown, and <i>P</i> values were calculated with t-tests.</p