Maria Duran-Moreno

Ultrastructure of the subventricular zone in Macaca fascicularis and evidence of a mouse‐like migratory stream

Recent publications have shown that the lateral wall of the lateral ventricles in the Macaca fascicularis brain, in particular the subventricular zone (SVZ), contains neural stem cells throughout adulthood that migrate through a migratory pathway (RMS) to the olfactory bulb (OB). To date, a detailed and systematic cytoarchitectural and ultrastructural study of the monkey SVZ and RMS has not been done. We found that the organization of the SVZ was similar to that of humans, with the ependymal layer surrounding the lateral ventricles, a hypocellular GAP layer formed by astrocytic and ependymal expansions, and the astrocyte ribbon, composed of astrocytic bodies. We found no cells corresponding to the type C proliferating precursor of the rodent brain. Instead, proliferating cells, expressed as Ki‐67 immunoreactivity, were predominantly young neurons concentrated in the anterior regions, and occasional astrocytes of the ribbon. We observed displaced ependymal cells of still unknown significance. New neurons tended to organize in chain‐like structures, which were surrounded by astrocytes. This pattern was highly reminiscent of that observed in rodent RMS, but not in humans. These chains spread from the frontal SVZ along a GAP‐like layer, uniquely composed of astrocytic expansions, to the olfactory bulb (OB). The number of neuronal chains and the number of chain‐forming cells decreased gradually upon reaching the OB. The purpose of this work is to provide a reference for future studies in the field of adult neurogenesis that may lead to an understanding of the fate and functionality of newborn neurons in primates, and ultimately in humans. J. Comp. Neurol. 514:533–554, 2009.

Adult neural stem cells from the subventricular zone: a review of the neurosphere assay.

The possibility of obtaining large numbers of cells with potential to become functional neurons implies a great advance in regenerative medicine. A source of cells for therapy is the subventricular zone (SVZ) where adult neural stem cells (NSCs) retain the ability to proliferate, self‐renew, and differentiate into several mature cell types. The neurosphere assay, a method to isolate, maintain, and expand these cells has been extensively utilized by research groups to analyze the biological properties of aNSCs and to graft into injured brains from animal models. In this review we briefly describe the neurosphere assay and its limitations, the methods to optimize culture conditions, the identity and the morphology of aNSC‐derived neurospheres (including new ultrastructural data). The controversy regarding the identity and “stemness” of cells within the neurosphere is revised. The fine morphology of neurospheres, described thoroughly, allows for phenotypical characterization of cells in the neurospheres and may reveal slight changes that indirectly inform about cell integrity, cell damage, or oncogenic transformation. Along this review we largely highlight the critical points that researchers have to keep in mind before extrapolating results or translating experimental transplantation of neurosphere‐derived cells to the clinical setting. Anat Rec, 296:1435‐1452, 2013.

The LIM Homeodomain Factor Lhx2 Is Required for Hypothalamic Tanycyte Specification and Differentiation

Hypothalamic tanycytes, a radial glial-like ependymal cell population that expresses numerous genes selectively enriched in embryonic hypothalamic progenitors and adult neural stem cells, have recently been observed to serve as a source of adult-born neurons in the mammalian brain. The genetic mechanisms that regulate the specification and maintenance of tanycyte identity are unknown, but are critical for understanding how these cells can act as adult neural progenitor cells. We observe that LIM (Lin-11, Isl-1, Mec-3)-homeodomain gene Lhx2 is selectively expressed in hypothalamic progenitor cells and tanycytes. To test the function of Lhx2 in tanycyte development, we used an intersectional genetic strategy to conditionally delete Lhx2 in posteroventral hypothalamic neuroepithelium, both embryonically and postnatally. We observed that tanycyte development was severely disrupted when Lhx2 function was ablated during embryonic development. Lhx2-deficient tanycytes lost expression of tanycyte-specific genes, such as Rax, while also displaying ectopic expression of genes specific to cuboid ependymal cells, such as Rarres2. Ultrastructural analysis revealed that mutant tanycytes exhibited a hybrid identity, retaining radial morphology while becoming multiciliated. In contrast, postnatal loss of function of Lhx2 resulted only in loss of expression of tanycyte-specific genes. Using chromatin immunoprecipitation, we further showed that Lhx2 directly regulated expression of Rax, an essential homeodomain factor for tanycyte development. This study identifies Lhx2 as a key intrinsic regulator of tanycyte differentiation, sustaining Rax-dependent activation of tanycyte-specific genes while also inhibiting expression of ependymal cell-specific genes. These findings provide key insights into the transcriptional regulatory network specifying this still poorly characterized cell type.

Roles of p53 and p27 Kip1 in the regulation of neurogenesis in the murine adult subventricular zone

The tumor suppressor protein p53 (Trp53) and the cell cycle inhibitor p27 Kip1 (Cdknb1) have both been implicated in regulating proliferation of adult subventricular zone (aSVZ) cells. We previously reported that genetic ablation of Trp53 (Trp53 −/−) or Cdknb1 (p27 Kip1−/−) increased proliferation of cells in the aSVZ, but differentially affected the number of adult born neuroblasts. We therefore hypothesized that these molecules might play non‐redundant roles. To test this hypothesis we generated mice lacking both genes (Trp53 −/−;p27 Kip1−/−) and analysed the consequences on aSVZ cells and adult neuroblasts. Proliferation and self‐renewal of cultured aSVZ cells were increased in the double mutants compared with control, but the mice did not develop spontaneous brain tumors. In contrast, the number of adult‐born neuroblasts in the double mutants was similar to wild‐type animals and suggested a complementation of the p27 Kip1−/− phenotype due to loss of Trp53. Cellular differences detected in the aSVZ correlated with cellular changes in the olfactory bulb and behavioral data on novel odor recognition. The exploration time for new odors was reduced in p27 Kip1−/− mice, increased in Trp53 −/−mice and normalized in the double Trp53−/−;p27 Kip1−/− mutants. At the molecular level, Trp53 −/−aSVZ cells were characterized by higher levels of NeuroD and Math3 and by the ability to generate neurons more readily. In contrast, p27 Kip1−/− cells generated fewer neurons, due to enhanced proteasomal degradation of pro‐neural transcription factors. Together, these results suggest that p27 Kip1 and p53 function non‐redundantly to modulate proliferation and self‐renewal of aSVZ cells and antagonistically in regulating adult neurogenesis.

Longitudinally extensive transverse myelitis with AQP4 antibodies revealing ovarian teratoma

Paraneoplastic myelitis is a rare inflammatory disorder most frequently associated with solid tumors or lymphoproliferative disorders. Patients often harbor onconeuronal antibodies and their prognosis is usually poor. Here we report a 42-year old woman with longitudinally extensive transverse myelitis and aquaporin-4 (AQP4) antibodies that led to the diagnosis of ovarian teratoma. After tumor removal and immune therapy (including corticosteroids, plasma exchange, intravenous immunoglobulins and rituximab) the patient progressively improved achieving complete recovery. Histological study of the teratoma demonstrated neural tissue containing AQP4 expressing cells and intense inflammatory infiltrates, providing evidence for a possible paraneoplastic link between both disorders.

NIR excitation of upconversion nanohybrids containing a surface grafted Bodipy induces oxygen-mediated cancer cell death

We report the preparation of water-dispersible, ca. 30 nm-sized nanohybrids containing NaYF4:Er3+, Yb3+ up-conversion nanoparticles (UCNPs), capped with a polyethylene glycol (PEG) derivative and highly loaded with a singlet oxygen photosensitizer, specifically a diiodo-substituted Bodipy (IBDP). The photosensitizer, bearing a carboxylic group, was anchored to the UCNP surface and, at the same time, embedded in the PEG capping; the combined action of the UCNP surface and PEG facilitated the loading for an effective energy transfer and, additionally, avoided photosensitizer leaching from the nanohybrid (UCNP-IBDP@PEG). The effectiveness of the nanohybrids in generating singlet oxygen after near-infrared (NIR) excitation (975 nm) with a continuous wavelength (CW) laser was evidenced by using a probe molecule. In vitro assays demonstrated that the UCNP-IBDP@PEG nanohybrid was taken up by the SH-SY5Y human neuroblastoma-derived cells showing low cytotoxicity. Moreover, ca. 50% cancer cell death was observed after NIR irradiation (45 min, 239 mW).

Oxidative stress and mitochondrial dysfunction in Kindler syndrome

BackgroundKindler Syndrome (KS) is an autosomal recessive skin disorder characterized by skin blistering, photosensitivity, premature aging, and propensity to skin cancer. In spite of the knowledge underlying cause of this disease involving mutations of FERMT1 (fermitin family member 1), and efforts to characterize genotype-phenotype correlations, the clinical variability of this genodermatosis is still poorly understood. In addition, several pathognomonic features of KS, not related to skin fragility such as aging, inflammation and cancer predisposition have been strongly associated with oxidative stress. Alterations of the cellular redox status have not been previously studied in KS. Here we explored the role of oxidative stress in the pathogenesis of this rare cutaneous disease.MethodsPatient-derived keratinocytes and their respective controls were cultured and classified according to their different mutations by PCR and western blot, the oxidative stress biomarkers were analyzed by spectrophotometry and qPCR and additionally redox biosensors experiments were also performed. The mitochondrial structure and functionality were analyzed by confocal microscopy and electron microscopy.ResultsPatient-derived keratinocytes showed altered levels of several oxidative stress biomarkers including MDA (malondialdehyde), GSSG/GSH ratio (oxidized and reduced glutathione) and GCL (gamma-glutamyl cysteine ligase) subunits. Electron microscopy analysis of both, KS skin biopsies and keratinocytes showed marked morphological mitochondrial abnormalities. Consistently, confocal microscopy studies of mitochondrial fluorescent probes confirmed the mitochondrial derangement. Imbalance of oxidative stress biomarkers together with abnormalities in the mitochondrial network and function are consistent with a pro-oxidant state.ConclusionsThis is the first study to describe mitochondrial dysfunction and oxidative stress involvement in KS.

Alexander Disease Mutations Produce Cells with Coexpression of Glial Fibrillary Acidic Protein and NG2 in Neurosphere Cultures and Inhibit Differentiation into Mature Oligodendrocytes

Background Alexander disease (AxD) is a rare disease caused by mutations in the gene encoding glial fibrillary acidic protein (GFAP). The disease is characterized by presence of GFAP aggregates in the cytoplasm of astrocytes and loss of myelin. Objectives Determine the effect of AxD-related mutations on adult neurogenesis. Methods We transfected different types of mutant GFAP into neurospheres using the nucleofection technique. Results We find that mutations may cause coexpression of GFAP and NG2 in neurosphere cultures, which would inhibit the differentiation of precursors into oligodendrocytes and thus explain the myelin loss occurring in the disease. Transfection produces cells that differentiate into new cells marked simultaneously by GFAP and NG2 and whose percentage increased over days of differentiation. Increased expression of GFAP is due to a protein with an anomalous structure that forms aggregates throughout the cytoplasm of new cells. These cells display down-expression of vimentin and nestin. Up-expression of cathepsin D and caspase-3 in the first days of differentiation suggest that apoptosis as a lysosomal response may be at work. HSP27, a protein found in Rosenthal bodies, is expressed less at the beginning of the process although its presence increases in later stages. Conclusion Our findings seem to suggest that the mechanism of development of AxD may not be due to a function gain due to increase of GFAP, but to failure in the differentiation process may occur at the stage in which precursor cells transform into oligodendrocytes, and that possibility may provide the best explanation for the clinical and radiological images described in AxD.

La alteración de la mielina en la enfermedad de Alexander

INTRODUCTION Alexander disease (AxD) is a type of leukodystrophy. Its pathological basis, along with myelin loss, is the appearance of Rosenthal bodies, which are cytoplasmic inclusions in astrocytes. Mutations in the gene coding for GFAP have been identified as a genetic basis for AxD. However, the mechanism by which these variants produce the disease is not understood. DEVELOPMENT The most widespread hypothesis is that AxD develops when a gain of function mutation causes an increase in GFAP. However, this mechanism does not explain myelin loss, given that experimental models in which GFAP expression is normal or mutated do not exhibit myelin disorders. This review analyses other possibilities that may explain this alteration, such as epigenetic or inflammatory alterations, presence of NG2 (+) - GFAP (+) cells, or post-translational modifications in GFAP that are unrelated to increased expression. CONCLUSIONS The different hypotheses analysed here may explain the myelin alteration affecting these patients, and multiple mechanisms may coexist. These theories raise the possibility of designing therapies based on these mechanisms.

Immunoproteomic studies on paediatric opsoclonus-myoclonus associated with neuroblastoma

We aimed to identify new cell-membrane antigens implicated in opsoclonus-myoclonus with neuroblastoma. The sera of 3 out of 14 patients showed IgG electron-microscopy immunogold reactivity on SH-SY5Y neuroblastoma cells. Immunoprecipitation experiments using rat brain synaptosomes and SH-SY5Y cells led to the identification of: (1) thirty-one nuclear/cytoplasmic proteins (including antigens HuB, HuC); (2) seven neuronal membrane proteins, including the Shaw-potassium channel Kv3.3 (KCNC3), whose genetic disruption in mice causes ataxia and generalized muscle twitching. Although cell-based assays did not demonstrate direct antigenicity, our findings point to Shaw-related subfamily of the potassium voltage-gated channels complexed proteins as hypothetical antigenic targets.