Antonella Consiglio

Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes

The utility of induced pluripotent stem (iPS) cells for investigating the molecular logic of pluripotency and for eventual clinical application is limited by the low efficiency of current methods for reprogramming. Here we show that reprogramming of juvenile human primary keratinocytes by retroviral transduction with OCT4, SOX2, KLF4 and c-MYC is at least 100-fold more efficient and twofold faster compared with reprogramming of human fibroblasts. Keratinocyte-derived iPS (KiPS) cells appear indistinguishable from human embryonic stem cells in colony morphology, growth properties, expression of pluripotency-associated transcription factors and surface markers, global gene expression profiles and differentiation potential in vitro and in vivo. To underscore the efficiency and practicability of this technology, we generated KiPS cells from single adult human hairs. Our findings provide an experimental model for investigating the bases of cellular reprogramming and highlight potential advantages of using keratinocytes to generate patient-specific iPS cells.

Wnt signalling regulates adult hippocampal neurogenesis

The generation of new neurons from neural stem cells is restricted to two regions of the adult mammalian central nervous system: the subventricular zone of the lateral ventricle, and the subgranular zone of the hippocampal dentate gyrus. In both regions, signals provided by the microenvironment regulate the maintenance, proliferation and neuronal fate commitment of the local stem cell population. The identity of these signals is largely unknown. Here we show that adult hippocampal stem/progenitor cells (AHPs) express receptors and signalling components for Wnt proteins, which are key regulators of neural stem cell behaviour in embryonic development. We also show that the Wnt/β-catenin pathway is active and that Wnt3 is expressed in the hippocampal neurogenic niche. Overexpression of Wnt3 is sufficient to increase neurogenesis from AHPs in vitro and in vivo. By contrast, blockade of Wnt signalling reduces neurogenesis from AHPs in vitro and abolishes neurogenesis almost completely in vivo. Our data show that Wnt signalling is a principal regulator of adult hippocampal neurogenesis and provide evidence that Wnt proteins have a role in adult hippocampal function.

In vivo demonstration that alpha-synuclein oligomers are toxic.

The aggregation of proteins into oligomers and amyloid fibrils is characteristic of several neurodegenerative diseases, including Parkinson disease (PD). In PD, the process of aggregation of α-synuclein (α-syn) from monomers, via oligomeric intermediates, into amyloid fibrils is considered the disease-causative toxic mechanism. We developed α-syn mutants that promote oligomer or fibril formation and tested the toxicity of these mutants by using a rat lentivirus system to investigate loss of dopaminergic neurons in the substantia nigra. The most severe dopaminergic loss in the substantia nigra is observed in animals with the α-syn variants that form oligomers (i.e., E57K and E35K), whereas the α-syn variants that form fibrils very quickly are less toxic. We show that α-syn oligomers are toxic in vivo and that α-syn oligomers might interact with and potentially disrupt membranes.

Disease-corrected haematopoietic progenitors from Fanconi anaemia induced pluripotent stem cells

The generation of induced pluripotent stem (iPS) cells has enabled the derivation of patient-specific pluripotent cells and provided valuable experimental platforms to model human disease. Patient-specific iPS cells are also thought to hold great therapeutic potential, although direct evidence for this is still lacking. Here we show that, on correction of the genetic defect, somatic cells from Fanconi anaemia patients can be reprogrammed to pluripotency to generate patient-specific iPS cells. These cell lines appear indistinguishable from human embryonic stem cells and iPS cells from healthy individuals. Most importantly, we show that corrected Fanconi-anaemia-specific iPS cells can give rise to haematopoietic progenitors of the myeloid and erythroid lineages that are phenotypically normal, that is, disease-free. These data offer proof-of-concept that iPS cell technology can be used for the generation of disease-corrected, patient-specific cells with potential value for cell therapy applications.

In Vivo Fate Analysis Reveals the Multipotent and Self-Renewal Capacities of Sox2+ Neural Stem Cells in the Adult Hippocampus

To characterize the properties of adult neural stem cells (NSCs), we generated and analyzed Sox2-GFP transgenic mice. Sox2-GFP cells in the subgranular zone (SGZ) express markers specific for progenitors, but they represent two morphologically distinct populations that differ in proliferation levels. Lentivirus- and retrovirus-mediated fate-tracing studies showed that Sox2+ cells in the SGZ have potential to give rise to neurons and astrocytes, revealing their multipotency at the population as well as at a single-cell level. A subpopulation of Sox2+ cells gives rise to cells that retain Sox2, highlighting Sox2+ cells as a primary source for adult NSCs. In response to mitotic signals, increased proliferation of Sox2+ cells is coupled with the generation of Sox2+ NSCs as well as neuronal precursors. An asymmetric contribution of Sox2+ NSCs may play an important role in maintaining the constant size of the NSC pool and producing newly born neurons during adult neurogenesis.

Dentate gyrus-specific knockdown of adult neurogenesis impairs spatial and object recognition memory in adult rats

New granule cells are born throughout life in the dentate gyrus of the hippocampal formation. Given the fundamental role of the hippocampus in processes underlying certain forms of learning and memory, it has been speculated that newborn granule cells contribute to cognition. However, previous strategies aiming to causally link newborn neurons with hippocampal function used ablation strategies that were not exclusive to the hippocampus or that were associated with substantial side effects, such as inflammation. We here used a lentiviral approach to specifically block neurogenesis in the dentate gyrus of adult male rats by inhibiting WNT signaling, which is critically involved in the generation of newborn neurons, using a dominant-negative WNT (dnWNT). We found a level-dependent effect of adult neurogenesis on the long-term retention of spatial memory in the water maze task, as rats with substantially reduced levels of newborn neurons showed less preference for the target zone in probe trials >2 wk after acquisition compared with control rats. Furthermore, animals with strongly reduced levels of neurogenesis were impaired in a hippocampus-dependent object recognition task. Social transmission of food preference, a behavioral test that also depends on hippocampal function, was not affected by knockdown of neurogenesis. Here we identified a role for newborn neurons in distinct aspects of hippocampal function that will set the ground to further elucidate, using experimental and computational strategies, the mechanism by which newborn neurons contribute to behavior.

Synapse formation on neurons born in the adult hippocampus

Although new and functional neurons are produced in the adult brain, little is known about how they integrate into mature networks. Here we explored the mechanisms of synaptogenesis on neurons born in the adult mouse hippocampus using confocal microscopy, electron microscopy and live imaging. We report that new neurons, similar to mature granule neurons, were contacted by axosomatic, axodendritic and axospinous synapses. Consistent with their putative role in synaptogenesis, dendritic filopodia were more abundant during the early stages of maturation and, when analyzed in three dimensions, the tips of all filopodia were found within 200 nm of preexisting boutons that already synapsed on other neurons. Furthermore, dendritic spines primarily synapsed on multiple-synapse boutons, suggesting that initial contacts were preferentially made with preexisting boutons already involved in a synapse. The connectivity of new neurons continued to change until at least 2 months, long after the formation of the first dendritic protrusions.

Interplay of LRRK2 with chaperone-mediated autophagy

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinsons disease. We found LRRK2 to be degraded in lysosomes by chaperone-mediated autophagy (CMA), whereas the most common pathogenic mutant form of LRRK2, G2019S, was poorly degraded by this pathway. In contrast to the behavior of typical CMA substrates, lysosomal binding of both wild-type and several pathogenic mutant LRRK2 proteins was enhanced in the presence of other CMA substrates, which interfered with the organization of the CMA translocation complex, resulting in defective CMA. Cells responded to such LRRK2-mediated CMA compromise by increasing levels of the CMA lysosomal receptor, as seen in neuronal cultures and brains of LRRK2 transgenic mice, induced pluripotent stem cell–derived dopaminergic neurons and brains of Parkinsons disease patients with LRRK2 mutations. This newly described LRRK2 self-perpetuating inhibitory effect on CMA could underlie toxicity in Parkinsons disease by compromising the degradation of α-synuclein, another Parkinsons disease–related protein degraded by this pathway.

Signaling through BMPR-IA Regulates Quiescence and Long-Term Activity of Neural Stem Cells in the Adult Hippocampus

Neural stem cells (NSCs) in the adult hippocampus divide infrequently, and the molecules that modulate their quiescence are largely unknown. Here, we show that bone morphogenetic protein (BMP) signaling is active in hippocampal NSCs, downstream of BMPR-IA. BMPs reversibly diminish proliferation of cultured NSCs while maintaining their undifferentiated state. In vivo, acute blockade of BMP signaling in the hippocampus by intracerebral infusion of Noggin first recruits quiescent NSCs into the cycle and increases neurogenesis; subsequently, it leads to decreased stem cell division and depletion of precursors and newborn neurons. Consistently, selective ablation of Bmpr1a in hippocampal NSCs, or inactivation of BMP canonical signaling in conditional Smad4 knockout mice, transiently enhances proliferation but later leads to a reduced number of precursors, thereby limiting neuronal birth. BMPs are therefore required to balance NSC quiescence/proliferation and to prevent loss of the stem cell activity that supports continuous neurogenesis in the mature hippocampus.

In vivo gene therapy of metachromatic leukodystrophy by lentiviral vectors: correction of neuropathology and protection against learning impairments in affected mice

Metachromatic leukodystrophy (MLD) is a lipidosis caused by deficiency of arylsulfatase A (ARSA). Although the genetics of MLD are known, its pathophysiology is not understood. The disease leads to progressive demyelination and early death and no effective treatment is available. We used lentiviral vectors to deliver a functional ARSA gene (human ARSA) into the brain of adult mice with germ-line inactivation of the mouse gene encoding ARSA, As2. We report sustained expression of active enzyme throughout a large portion of the brain, with long-term protection from development of neuropathology and hippocampal-related learning impairments. We show that selective degeneration of hippocampal neurons is a central step in disease pathogenesis, and provide evidence that in vivo transfer of ARSA by lentiviral vectors reverts the disease phenotype in all investigated areas. Therefore, in vivo gene therapy offers a unique option for MLD and other storage diseases affecting the central nervous system.