Juan Carlos Polanco

Sox10 gain-of-function causes XX sex reversal in mice: implications for human 22q-linked disorders of sex development

Male development in mammals is normally initiated by the Y-linked gene Sry, which activates expression of Sox9, leading to a cascade of gene activity required for testis formation. Although defects in this genetic cascade lead to human disorders of sex development (DSD), only a dozen DSD genes have been identified, and causes of 46,XX DSD (XX maleness) other than SRY translocation are almost completely unknown. Here, we show that transgenic expression of Sox10, a close relative of Sox9, in gonads of XX mice resulted in development of testes and male physiology. The degree of sex reversal correlated with levels of Sox10 expression in different transgenic lines. Sox10 was expressed at low levels in primordial gonads of both sexes during normal mouse development, becoming male-specific during testis differentiation. SOX10 protein was able to activate transcriptional targets of SOX9, explaining at a mechanistic level its ability to direct male development. Because over-expression of SOX10 alone is able to mimic the XX DSD phenotypes associated with duplication of human chromosome 22q13, and given that human SOX10 maps to 22q13.1, our results functionally implicate SOX10 in the etiology of these DSDs.

Tau aggregation and its interplay with amyloid-β.

Neurofibrillary tangles and amyloid plaques constitute the hallmark brain lesions of Alzheimer’s disease (AD) patients. Tangles are composed of fibrillar aggregates of the microtubule-associated protein tau, and plaques comprise fibrillar forms of a proteolytic cleavage product, amyloid-β (Aβ). Although plaques and tangles are the end-stage lesions in AD, small oligomers of Aβ and tau are now receiving increased attention as they are shown to correlate best with neurotoxicity. One key question of debate, however, is which of these pathologies appears first and hence is upstream in the pathocascade. Studies suggest that there is an intense crosstalk between the two molecules and, based on work in animal models, there is increasing evidence that Aβ, at least in part, exerts its toxicity via tau, with the Src kinase Fyn playing a crucial role in this process. In other experimental paradigms, Aβ and tau have been found to exert both separate and synergistic modes of toxicity. The challenge, however, is to integrate these different scenarios into a coherent picture. Furthermore, the ability of therapeutic interventions targeting just one of these molecules, to successfully neutralize the toxicity of the other, needs to be ascertained to improve current therapeutic strategies, such as immunotherapy, for the treatment of AD. Although this article is not intended to provide a comprehensive review of the currently pursued therapeutic strategies, we will discuss what has been achieved by immunotherapy and, in particular, how the inherent limitations of this approach can possibly be overcome by novel strategies that involve single-chain antibodies.

Extracellular vesicles isolated from the brains of rTg4510 mice seed tau protein aggregation in a threshold-dependent manner

The microtubule-associated protein tau has a critical role in Alzheimer disease and related tauopathies. There is accumulating evidence that tau aggregates spread and replicate in a prion-like manner, with the uptake of pathological tau seeds causing misfolding and aggregation of monomeric tau in recipient cells. Here we focused on small extracellular vesicles enriched for exosomes that were isolated from the brains of tau transgenic rTg4510 and control mice. We found that these extracellular vesicles contained tau, although the levels were significantly higher in transgenic mice that have a pronounced tau pathology. Tau in the vesicles was differentially phosphorylated, although to a lower degree than in the brain cells from which they were derived. Several phospho-epitopes (AT8, AT100, and AT180) thought to be critical for tau pathology were undetected in extracellular vesicles. Despite this, when assayed with FRET tau biosensor cells, extracellular vesicles derived from transgenic mice were capable of seeding tau aggregation in a threshold-dependent manner. We also observed that the dye used to label extracellular vesicle membranes was still present during nucleation and formation of tau inclusions, suggesting either a role for membranes in the seeding or in the process of degradation. Together, we clearly demonstrate that extracellular vesicles can transmit tau pathology. This indicates a role for extracellular vesicles in the transmission and spreading of tau pathology. The characteristics of tau in extracellular vesicles and the seeding threshold we identified may explain why tau pathology develops very slowly in neurodegenerative diseases such as Alzheimer disease.

Functional analysis of the SRY—KRAB interaction in mouse sex determination

Background information. SRY (sex‐determining region Y), the master regulator of male development in mammals, has been studied extensively for more than 17 years, but how the SRY protein triggers the chain of events leading to testis development remains unclear. SRY probably requires a partner protein to elicit its molecular function. KRAB‐O, a novel protein containing a KRAB (Krüppel‐associated box) domain only, was suggested recently as a candidate SRY partner. In order to investigate the possible role of KRAB‐O in sex determination, we studied its expression and conducted functional assays of the SRY—KRAB interaction.

Identification of Unsafe Human Induced Pluripotent Stem Cell Lines Using a Robust Surrogate Assay for Pluripotency

Human induced pluripotent stem cells (hiPSC) have the potential to generate healthy cells and tissues for the study and medical treatment of a large number of diseases. The utility of putative hiPSC‐based therapies is constrained by a lack of robust quality‐control assays that address the stability of the cells or their capacity to form teratomas after differentiation. Here we report that virally derived hiPSC, but not human embryonic stem cells (hESC) or hiPSC derived using episomal nonintegrating vectors, exhibit a propensity to revert to a pluripotent phenotype following differentiation. This instability was revealed using our published method to identify pluripotent cells undergoing very early‐stage differentiation in standard hESC cultures, by fluorescence activated cell sorting (FACS) based on expression of the cell surface markers TG30 (CD9) and GCTM‐2. Differentiated cells cultured post‐FACS fractionation from virally derived hiPSC lines reacquired immunoreactivity to TG30 (CD9) and GCTM‐2, formed stem cell‐like colonies, and re‐expressed canonical pluripotency markers. Furthermore, differentiated cells from pluripotency‐reverting hiPSC lines generated teratomas in immunocompromised mice, raising concerns about their safety in downstream applications. In contrast, differentiated cell populations from hESC and episomally derived hiPSC did not show any of these abnormalities. Our assays may be used to identify “unsafe” hiPSC cell lines and this information should be considered when selecting hiPSC lines for clinical use and indicate that experiments using these “unsafe” hiPSC lines should be interpreted carefully. STEM Cells 2013;31:1498–1510

Extracellular Vesicles Containing P301L Mutant Tau Accelerate Pathological Tau Phosphorylation and Oligomer Formation but Do Not Seed Mature Neurofibrillary Tangles in ALZ17 Mice

In Alzheimers disease, the distribution of neurofibrillary tangles, a histological hallmark comprised of phosphorylated forms of the protein tau, follows a distinct pattern through anatomically connected brain regions. The well-documented correlation between the severity of tau pathology and disease progression implies a prion-like seeding and spreading mechanism for tau. Experimentally, this has been addressed in transgenic mice by the injection of protein lysates isolated from brains of transgenic mice or patients with tauopathies, including AD, that were shown to behave like seeds, accelerating tau pathology and tangle formation in predisposed mice. More specifically, in vivo data suggest that brain lysates from mice harboring the P301S mutation of tau can seed protein aggregation when injected into the hippocampi of human wild-type tau transgenic ALZ17 mice. Here, we compared the seeding potential of lysates and extracellular vesicles enriched for exosomes (EVs) from wild-type and human P301L tau transgenic rTg4510 mouse brains. We show that transgenic EVs cause increased tau phosphorylation and soluble oligomer formation in a manner comparable to that of freely available proteins in brain lysates, a prerequisite for the formation of mature protein aggregates.

Exosomes taken up by neurons hijack the endosomal pathway to spread to interconnected neurons

In Alzheimer disease and related disorders, the microtubule-associated protein tau aggregates and forms cytoplasmic lesions that impair neuronal physiology at many levels. In addition to affecting the host neuron, tau aggregates also spread to neighboring, recipient cells where the misfolded tau aggregates, in a manner similar to prions, actively corrupt the proper folding of soluble tau, and thereby impair cellular functions. One vehicle for the transmission of tau aggregates are secretory nanovesicles known as exosomes. Here, we established a simple model of a neuronal circuit using a microfluidics culture system in which hippocampal neurons A and B were seeded into chambers 1 and 2, respectively, extending axons via microgrooves in both directions and thereby interconnecting. This system served to establish two models to track exosome spreading. In the first model, we labeled the exosomal membrane by coupling tetraspanin CD9 with either a green or red fluorescent tag. This allowed us to reveal that interconnected neurons exchange exosomes only when their axons extend in close proximity. In the second model, we added exosomes isolated from the brains of tau transgenic rTg4510 mice (i.e. exogenous, neuron A-derived) to neurons in chamber 1 (neuron B) interconnected with neuron C in chamber 2. This allowed us to demonstrate that a substantial fraction of the exogenous exosomes were internalized by neuron B and passed then on to neuron C. This transportation from neuron B to C was achieved by a mechanism that is consistent with the hijacking of secretory endosomes by the exogenous exosomes, as revealed by confocal, super-resolution and electron microscopy. Together, these findings suggest that fusion events involving the endogenous endosomal secretory machinery increase the pathogenic potential and the radius of action of pathogenic cargoes carried by exogenous exosomes.

No full admission for tau to the exclusive prion club yet.

Aggregation of the microtubule‐associated protein tau is a key feature of Alzheimers disease and other so‐called tauopathies, yet what causes this protein to aggregate and what renders it toxic is only slowly being revealed. Because tau spreads in a stereotypical pattern through the diseased brain, it has been proposed that it possesses prion‐like properties, with aggregation‐prone tau facilitating the conversion of “naïve” tau into “toxic” forms. The current study by Wegmann et al (2015) addresses whether tau fulfils classical “prion criteria” by assessing its spreading and toxicity in the absence of endogenous tau. Using different transgenic and viral paradigms, the authors demonstrate that, although tau still propagates in this scenario, there is a decrease in its misfolding and neurotoxicity. They therefore conclude that tau is not a genuine prion, at least when the current definition of these infectious proteins is applied.

Safety Assessment of Reprogrammed Cells Prior to Clinical Applications: Potential Approaches to Eliminate Teratoma Formation

Human pluripotent stem cells (hPSC) include human embryonic stem cells (hESC) and hu‐ man induced pluripotent stem cells (hIPSC). Due to their inherent ability to self-renew in‐ definitely in vitro and to give rise to essentially all cell lineages, both cell types have enormous potential for applications in regenerative medicine, but differ in their origin. HESC are derived from early pre-implantation stage embryos and have the capacity, known as pluripotency, to generate any other cell type of the human body. HESC can be differentiat‐ ed in the laboratory, a procedure aimed at the generation of healthy somatic cells that even‐ tually could be used in a large variety of applications including therapeutic options. However, work with hESC raises ethical concerns regarding the use of human early pre-im‐ plantation embryos, as well as concerns regarding the future use of hESC-derived cells in non-autologous cell transplantation therapies due to immune rejection of hESC-derived tis‐ sues, given that hESC are non-self. These concerns appeared to be overcome when it was demonstrated that pluripotency could be induced in differentiated somatic (adult) cells of the body by introduction of a cocktail of pluripotency-associated transcription factors, usu‐ ally OCT4, SOX2, KLF4 and c-MYC [1]. This process is known as reprogramming, and gener‐ ates human induced pluripotent stem cells (hIPSC), which show an embryonic-like state similar to hESC (for review see [2]). Human iPSC are considered to have immense potential for regenerative medicine, do not require the use of donated human embryos for their gen‐ eration and may provide an alternative and suitable resource for autologous cell-based therapies, in which cells obtained from the patient could be used to generate self-hIPSC fol‐ lowed by differentiation to relevant lineages required for therapeutic intervention. Howev‐ er, disturbingly, mouse experiments have shown that autologous mouse iPSC can induce

ANALYSIS OF TAU PROTEIN IN EXOSOMES AND MICROVESICLES ISOLATED FROM A MOUSE MODEL OF ALZHEIMER'S DISEASE

a consistent result with in vivo findings of that the pharmacological activation of AMPK by AICAR inhibited the phosphorylation of Tau-Ser396, but increased the phosphorylation of Tau-Ser262.The reduced expression of SIRT1 and the increased expression of p-AMPK in the cortex of SAMP8 5 months group were also observed, as compared to same aged SAMR1 group. These results implicate that AMPK activation may be caused by reduced expression of SIRT1, and which may differentially regulate the phosphorylation of tau protein. Conclusions: A novel finding of significant suppression of SIRT1 and the concomitant increased activity of AMPK in the cortex of SAMP8 aged 2 months without increment of APP expressionis reported. In addition, we found that AMPK activation induce a differential phosphorylation of tau protein in vitro and in vivo models. In conclusion, further investigations are planned to test the hypothesis that the SIRT1 inhibition followed by AMPK activation may be one of the pathogenic mechanisms of tau-related neurodegeneration.