Eva Porlan

Neural stem cells (NSCs) persist in the mammalian brain after development, but their potential for replacing neural populations lost in brain damage is dramatically restricted. Quiescence, or the ability to transition in and out of the cell cycle is a hallmark of adult NSCs. It deepens as the organism ages and is regarded as a strategy to protect the reservoirs of NSC for the long term. Deciphering the molecular code of dormancy is a major interest in cellular and molecular biology of ageing and in regenerative medicine since it has important implications for potentially manipulating NSCs to improve tissular homeostasis. In this work we have identified the mitotic kinase Polo like kinase 1 (Plk1) as a novel intrinsic regulator of the dynamic transition between NSC quiescence and activation in adult neurogenic niches. Using quantitative phosphoproteomics to pinpoint mechanistical mediators of the role of Plk1, we have identified the quiescence/activation molecular switch formed by E3 ubiquitin ligase Huwe1 and its target, the transcription factor Ascl1/Mash1, as novel effectors of Plk1 in NSCs. We discover that Plk1 restrains the accumulation of Ascl1/Mash1 by acting upstream of Huwe1, providing a direct molecular connection between completion of the cell cycle and the re-establishment of a low activation state following NSC division, which supports the concept that quiescence is an actively maintained state. Moreover we demonstrate that pharmacological inhibition of Plk1 is sufficient to enhance adult neurogenesis in vivo, identifying Plk1 as a viable target to acutely boost endogenous stem cell activation.

Huntington’s disease (HD) is a progressive, autosomal dominant neurodegenerative disorder characterized by the selective dysfunction and loss of neurons in the striatum and cerebral cortex. Experimental evidence suggests that GABAergic medium-sized spiny neurons (MSNs) in the striatum are particularly vulnerable to glutamate-induced toxicity (excitotoxicity) and its analogues. However, the molecular mechanisms underlying MSN-specific death in HD remain poorly understood. The serine/threonine protein kinase D1 (PKD1) confers neuroprotection in various neuropathological conditions, including ischemic stroke. While excitotoxicity inactivates PKD1 in cortical glutamatergic neurons without altering its levels, active PKD1 potentiates the survival of excitatory neurons in highly excitotoxic environments. Here, we investigated whether PKD1 activity dysregulation contributes to MSN death in HD and its association with neurodegeneration. We found an unexpected reduction in PKD1 protein levels in striatal neurons from HD patients. Similarly, the R6/1 mouse model of HD exhibited progressive PKD1 protein loss, commencing at early disease stages, accompanied by decreased Prkd1 transcript levels. PKD1 downregulation also occurred in the cerebral cortex of R6/1 mice, but only at late stages. Functionally, pharmacological PKD inhibition in primary striatal neurons exacerbated excitotoxic damage and apoptosis induced by glutamate N-methyl D-aspartate (NMDA) receptors, whereas expression of constitutively active PKD1 (PKD1-Ca) conferred neuroprotection. Furthermore, PKD1-Ca protected against polyQ-induced apoptosis in a cellular model of HD. In a translational approach, intrastriatal lentiviral delivery of PKD1-Ca in symptomatic R6/1 mice prevented the loss of DARPP-32, a molecular marker of MSNs. Collectively, our findings strongly suggest that PKD1 loss-of-function contributes to HD pathogenesis and the selective vulnerability of MSNs. These findings position PKD1 as a promising therapeutic target for mitigating MSN death in HD.

In the adult mammalian brain, neural stem cells (NSCs) located in highly restricted niches sustain the generation of new neurons that integrate into existing circuits. A reduction in adult neurogenesis is linked to ageing and neurodegeneration, whereas dysregulation of proliferation and survival of NSCs have been hypothesized to be at the origin of glioma. Thus, unravelling the molecular underpinnings of the regulated activation that NSCs must undergo to proliferate and generate new progeny is of considerable relevance. Current research has identified cues promoting or restraining NSCs activation. Yet, whether NSCs depend on external signals to survive or if intrinsic factors establish a threshold for sustaining their viability remains elusive, even if this knowledge could involve potential for devising novel therapeutic strategies. Kidins220 (Kinase D-interacting substrate of 220 kDa) is an essential effector of crucial pathways for neuronal survival and differentiation. It is dramatically altered in cancer and in neurological and neurodegenerative disorders, emerging as a regulatory molecule with important functions in human disease. Herein, we discover severe neurogenic deficits and hippocampal-based spatial memory defects accompanied by increased neuroblast death and high loss of newly formed neurons in Kidins220 deficient mice. Mechanistically, we demonstrate that Kidins220-dependent activation of AKT in response to EGF restraints GSK3 activity preventing NSCs apoptosis. We also show that NSCs with Kidins220 can survive with lower concentrations of EGF than the ones lacking this molecule. Hence, Kidins220 levels set a molecular threshold for survival in response to mitogens, allowing adult NSCs growth and expansion. Our study identifies Kidins220 as a key player for sensing the availability of growth factors to sustain adult neurogenesis, uncovering a molecular link that may help paving the way towards neurorepair.

Editorial: “Unravelling neural stem cell biology: players and strategies”

In the adult mammalian brain, neural stem cells (NSCs) located in highly restricted niches sustain the generation of new neurons that integrate into existing circuits. A reduction in adult neurogenesis is linked to ageing and neurodegeneration, whereas dysregulation of proliferation and survival of NSCs have been hypothesized to be at the origin of glioma. Thus, unravelling the molecular underpinnings of the regulated activation that NSCs must undergo to proliferate and generate new progeny is of considerable relevance. current research has identified cues promoting or restraining NSCs activation. Yet, whether NSCs depend on external signals to survive or if intrinsic factors establish a threshold for sustaining their viability remains elusive, even if this knowledge could involve potential for devising novel therapeutic strategies. Kidins220 (Kinase D-interacting substrate of 220 kDa) is an essential effector of crucial pathways for neuronal survival and differentiation. It is dramatically altered in cancer and in neurological and neurodegenerative disorders, emerging as a regulatory molecule with important functions in human disease. Herein, we discover severe neurogenic deficits and hippocampal-based spatial memory defects in Kidins220 deficient mice. Mechanistically, we demonstrate that Kidins220-dependent activation of AKT in response to EGF restraints GSK3 activity preventing NSCs apoptosis. Hence, Kidins220 levels set a molecular threshold for survival in response to mitogens, allowing adult NSCs to proliferate. Our study identifies Kidins220 as a key player for sensing the availability of growth factors to sustain adult neurogenesis, uncovering a molecular link that may help paving the way towards neurorepair.

Cell differentiation involves profound changes in global gene expression that often have to occur in coordination with cell cycle exit. Because cyclin-dependent kinase inhibitor p27 reportedly regulates proliferation of neural progenitor cells in the subependymal neurogenic niche of the adult mouse brain, but can also have effects on gene expression, we decided to molecularly analyze its role in adult neurogenesis and oligodendrogenesis. At the cell level, we show that p27 restricts residual cyclin-dependent kinase activity after mitogen withdrawal to antagonize cycling, but it is not essential for cell cycle exit. Contrasting gene expression with chromatin accessibility, we find that p27 is coincidentally necessary to globally repress many genes involved in the transit from multipotentiality to differentiation, including those coding for neural progenitor transcription factors SOX2, OLIG2, and ASCL1. Our data reveal both direct association of p27 with regulatory sequences in the three genes and an additional hierarchical relationship where p27 repression of the Sox2 gene leads to reduced levels of SOX2-downstream targets Olig2 and Ascl1. In vivo , p27 is also required for the regulation of the proper level of SOX2 necessary for neuroblasts and oligodendroglial progenitor cells to timely exit cell cycle in a lineage-dependent manner.

In the mammalian adult brain, neural stem cells persist in neurogenic niches. The subependymal zone is the most prolific neurogenic niche in adult rodents, where residing stem cells generate large numbers of immature neurons that migrate into the olfactory bulb, where they differentiate into different types of interneurons. Subependymal neural stem cells derive from embryonic radial glia and retain some of their features like apico-basal polarity, with apical processes piercing the ependymal layer, and a basal process contacting blood vessels, constituting an epithelial niche. Conservation of the cytoarchitecture of the niche is of crucial importance for the maintenance of stem cells and for their neurogenic potential. In this minireview we will focus on extracellular matrix and adhesion molecules in the adult subependymal zone, showing their involvement not only as structural elements sustaining the niche architecture and topology, but also in the maintenance of stemness and regulation of the quiescence-proliferation balance.

We used proximity‐dependent biotin identification (BioID) to find proteins that potentially interact with the major glial glutamate transporter, GLT‐1, and we studied how these interactions might affect its activity. GTPase Rac1 was one protein identified, and interfering with its GTP/GDP cycle in mixed primary rat brain cultures affected both the clustering of GLT‐1 at the astrocytic processes and the transport kinetics, increasing its uptake activity at low micromolar glutamate concentrations in a manner that was dependent on the effector kinase PAK1 and the actin cytoskeleton. Interestingly, the same manipulations had a different effect on another glial glutamate transporter, GLAST, inhibiting its activity. Importantly, glutamate acts through metabotropic receptors to stimulate the activity of Rac1 in astrocytes, supporting the existence of cross‐talk between extracellular glutamate and the astrocytic form of the GLT‐1 regulated by Rac1. CDC42EP4/BORG4 (a CDC42 effector) was also identified in the BioID screen, and it is a protein that regulates the assembly of septins and actin fibers, influencing the organization of the cytoskeleton. We found that GLT‐1 interacts with septins, which reduces its lateral mobility at the cell surface. Finally, the G‐protein subunit GNB4 dampens the activity of GLT‐1, as revealed by its response to the activator peptide mSIRK, both in heterologous systems and in primary brain cultures. This effect occurs rapidly and thus, it is unlikely to depend on cytoskeletal dynamics. These novel interactions shed new light on the events controlling GLT‐1 activity, thereby helping us to better understand how glutamate homeostasis is maintained in the brain.

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells...

Cell-cycle inhibitors of the Ink4 and Cip/Kip families are involved in cellular senescence and tumor suppression. These inhibitors are individually dispensable for the cell cycle and inactivation of specific family members results in increased proliferation and enhanced susceptibility to tumor development. We have now analyzed the consequences of eliminating a substantial part of the cell-cycle inhibitory activity in the cell by generating a mouse model, which combines the absence of both p21^Cip1 and p27^Kip1 proteins with the endogenous expression of a Cdk4 R24C mutant insensitive to Ink4 inhibitors. Pairwise combination of Cdk4 R24C, p21-null and p27-null alleles results in frequent hyperplasias and tumors, mainly in cells of endocrine origin such as pituitary cells and in mesenchymal tissues. Interestingly, complete abrogation of p21^Cip1 and p27^Kip1 in Cdk4 R24C mutant mice results in a different phenotype characterized by perinatal death accompanied by general hypoplasia in most tissues. This phenotype correlates with increased replicative stress in developing tissues such as the nervous system and subsequent apoptotic cell death. Partial inhibition of Cdk4/6 rescues replicative stress signaling as well as p53 induction in the absence of cell-cycle inhibitors. We conclude that one of the major physiological activities of cell-cycle inhibitors is to prevent replicative stress during development.