Transcriptome analysis reveals genes associated with stem cell activation by physical exercise in the dentate gyrus of aged p16Ink4a knockout mice.

Throughout adulthood neural stem cells divide in neurogenic niches-the dentate gyrus of the hippocampus and the subventricular zone-producing progenitor cells and new neurons. Stem cells self-renew, thus preserving their pool. Furthermore, the number of stem/progenitor cells in the neurogenic niches decreases with age. We have previously demonstrated that the cyclin-dependent kinase inhibitor p16Ink4a maintains, in aged mice, the pool of dentate gyrus stem cells by preventing their activation after a neurogenic stimulus such as exercise (running). We showed that, although p16Ink4a ablation by itself does not activate stem/progenitor cells, exercise strongly induced stem cell proliferation in p16Ink4a knockout dentate gyrus, but not in wild-type. As p16Ink4a regulates stem cell self-renewal during aging, we sought to profile the dentate gyrus transcriptome from p16Ink4a wild-type and knockout aged mice, either sedentary or running for 12 days. By pairwise comparisons of differentially expressed genes and by correlative analyses through the DESeq2 software, we identified genes regulated by p16Ink4a deletion, either without stimulus (running) added, or following running. The p16Ink4a knockout basic gene signature, i.e., in sedentary mice, involves upregulation of apoptotic, neuroinflammation- and synaptic activity-associated genes, suggesting a reactive cellular state. Conversely, another set of 106 genes we identified, whose differential expression specifically reflects the pattern of proliferative response of p16 knockout stem cells to running, are involved in processes that regulate stem cell activation, such as synaptic function, neurotransmitter metabolism, stem cell proliferation control, and reactive oxygen species level regulation. Moreover, we analyzed the regulation of these stem cell-specific genes after a second running stimulus. Surprisingly, the second running neither activated stem cell proliferation in the p16Ink4a knockout dentate gyrus nor changed the expression of these genes, confirming that they are correlated to the stem cell reactivity to stimulus, a process where they may play a role regulating stem cell activation.

Novel loss of function mutation in TUBA1A gene compromises tubulin stability and proteostasis causing spastic paraplegia and ataxia.

Microtubules are dynamic cytoskeletal structures involved in several cellular functions, such as intracellular trafficking, cell division and motility. More than other cell types, neurons rely on the proper functioning of microtubules to conduct their activities and achieve complex morphologies. Pathogenic variants in genes encoding for α and ß-tubulins, the structural subunits of microtubules, give rise to a wide class of neurological disorders collectively known as "tubulinopathies" and mainly involving a wide and overlapping range of brain malformations resulting from defective neuronal proliferation, migration, differentiation and axon guidance. Although tubulin mutations have been classically linked to neurodevelopmental defects, growing evidence demonstrates that perturbations of tubulin functions and activities may also drive neurodegeneration. In this study, we causally link the previously unreported missense mutation p.I384N in TUBA1A, one of the neuron-specific α-tubulin isotype I, to a neurodegenerative disorder characterized by progressive spastic paraplegia and ataxia. We demonstrate that, in contrast to the p.R402H substitution, which is one of the most recurrent TUBA1A pathogenic variants associated to lissencephaly, the present mutation impairs TUBA1A stability, reducing the abundance of TUBA1A available in the cell and preventing its incorporation into microtubules. We also show that the isoleucine at position 384 is an amino acid residue, which is critical for α-tubulin stability, since the introduction of the p.I384N substitution in three different tubulin paralogs reduces their protein level and assembly into microtubules, increasing their propensity to aggregation. Moreover, we demonstrate that the inhibition of the proteasome degradative systems increases the protein levels of TUBA1A mutant, promoting the formation of tubulin aggregates that, as their size increases, coalesce into inclusions that precipitate within the insoluble cellular fraction. Overall, our data describe a novel pathogenic effect of p.I384N mutation that differs from the previously described substitutions in TUBA1A, and expand both phenotypic and mutational spectrum related to this gene.

Mesoangioblasts at 20: From the embryonic aorta to the patient bed.

In 2002 we published an article describing a population of vessel-associated progenitors that we termed mesoangioblasts (MABs). During the past decade evidence had accumulated that during muscle development and regeneration things may be more complex than a simple sequence of binary choices (e.g., dorsal vs. ventral somite). LacZ expressing fibroblasts could fuse with unlabelled myoblasts but not among themselves or with other cell types. Bone marrow derived, circulating progenitors were able to participate in muscle regeneration, though in very small percentage. Searching for the embryonic origin of these progenitors, we identified them as originating at least in part from the embryonic aorta and, at later stages, from the microvasculature of skeletal muscle. While continuing to investigate origin and fate of MABs, the fact that they could be expanded in vitro (also from human muscle) and cross the vessel wall, suggested a protocol for the cell therapy of muscular dystrophies. We tested this protocol in mice and dogs before proceeding to the first clinical trial on Duchenne Muscular Dystrophy patients that showed safety but minimal efficacy. In the last years, we have worked to overcome the problem of low engraftment and tried to understand their role as auxiliary myogenic progenitors during development and regeneration.

Transcriptome Analysis in a Mouse Model of Premature Aging of Dentate Gyrus: Rescue of Alpha-Synuclein Deficit by Virus-Driven Expression or by Running Restores the Defective Neurogenesis.

The dentate gyrus of the hippocampus and the subventricular zone are neurogenic niches where neural stem and progenitor cells replicate throughout life to generate new neurons. The Btg1 gene maintains the stem cells of the neurogenic niches in quiescence. The deletion of Btg1 leads to an early transient increase of stem/progenitor cells division, followed, however, by a decrease during adulthood of their proliferative capability, accompanied by apoptosis. Since a physiological decrease of neurogenesis occurs during aging, the Btg1 knockout mouse may represent a model of neural aging. We have previously observed that the defective neurogenesis of the Btg1 knockout model is rescued by the powerful neurogenic stimulus of physical exercise (running). To identify genes responsible for stem and progenitor cells maintenance, we sought here to find genes underlying this premature neural aging, and whose deregulated expression could be rescued by running. Through RNA sequencing we analyzed the transcriptomic profiles of the dentate gyrus isolated from Btg1 wild-type or Btg1 knockout adult (2-month-old) mice submitted to physical exercise or sedentary. In Btg1 knockout mice, 545 genes were deregulated, relative to wild-type, while 2081 genes were deregulated by running. We identified 42 genes whose expression was not only down-regulated in the dentate gyrus of Btg1 knockout, but was also counter-regulated to control levels by running in Btg1 knockout mice, vs. sedentary. Among these 42 counter-regulated genes, alpha-synuclein (Snca), Fos, Arc and Npas4 showed significantly greater differential regulation. These genes control neural proliferation, apoptosis, plasticity and memory and are involved in aging. In particular, Snca expression decreases during aging. We tested, therefore, whether an Snca-expressing lentivirus, by rescuing the defective Snca levels in the dentate gyrus of Btg1 knockout mice, could also reverse the aging phenotype, in particular the defective neurogenesis. We found that the exogenous expression of Snca reversed the Btg1 knockout-dependent decrease of stem cell proliferation as well as the increase of progenitor cell apoptosis. This indicates that Snca has a functional role in the process of neural aging observed in this model, and also suggests that Snca acts as a positive regulator of stem cell maintenance.

Nerve Growth Factor Neutralization Promotes Oligodendrogenesis by Increasing miR-219a-5p Levels.

In the brain, the neurotrophin Nerve growth factor (NGF) regulates not only neuronal survival and differentiation, but also glial and microglial functions and neuroinflammation. NGF is known to regulate oligodendrogenesis, reducing myelination in the central nervous system (CNS). In this study, we found that NGF controls oligodendrogenesis by modulating the levels of miR-219a-5p, a well-known positive regulator of oligodendrocyte differentiation. We exploited an NGF-deprivation mouse model, the AD11 mice, in which the postnatal expression of an anti-NGF antibody leads to NGF neutralization and progressive neurodegeneration. Notably, we found that these mice also display increased myelination. A microRNA profiling of AD11 brain samples and qRT-PCR analyses revealed that NGF deprivation leads to an increase of miR-219a-5p levels in hippocampus and cortex and a corresponding down-regulation of its predicted targets. Neurospheres isolated from the hippocampus of AD11 mice give rise to more oligodendrocytes and this process is dependent on miR-219a-5p, as shown by decoy-mediated inhibition of this microRNA. Moreover, treatment of AD11 neurospheres with NGF inhibits miR-219a-5p up-regulation and, consequently, oligodendrocyte differentiation, while anti-NGF treatment of wild type (WT) oligodendrocyte progenitors increases miR-219a-5p expression and the number of mature cells. Overall, this study indicates that NGF inhibits oligodendrogenesis and myelination by down-regulating miR-219a-5p levels, suggesting a novel molecular circuitry that can be exploited for the discovery of new effectors for remyelination in human demyelinating diseases, such as Multiple Sclerosis.

UsnRNP trafficking is regulated by stress granules and compromised by mutant ALS proteins.

Activation of the integrated stress response (ISR), alterations in nucleo-cytoplasmic (N/C) transport and changes in alternative splicing regulation are all common traits of the pathogenesis of Amyotrophic Lateral Sclerosis (ALS). However, whether these processes act independently from each other, or are part of a coordinated mechanism of gene expression regulation that is affected in pathogenic conditions, is still rather undefined. To answer these questions, in this work we set out to characterise the functional connections existing between ISR activation and nucleo-cytosol trafficking and nuclear localization of spliceosomal U-rich small nuclear ribonucleoproteins (UsnRNPs), the core constituents of the spliceosome, and to study how ALS-linked mutant proteins affect this interplay. Activation of the ISR induces a profound reorganization of nuclear Gems and Cajal bodies, the membrane-less particles that assist UsnRNP maturation and storage. This effect requires the cytoplasmic assembly of SGs and is associated to the disturbance of the nuclear import of UsnRNPs by the snurportin-1/importin-ß1 system. Notably, these effects are reversed by both inhibiting the ISR or upregulating importin-ß1. This indicates that SGs are major determinants of Cajal bodies assembly and that the modulation of N/C trafficking of UsnRNPs might control alternative splicing in response to stress. Importantly, the dismantling of nuclear Gems and Cajal bodies by ALS-linked mutant FUS or C9orf72-derived dipeptide repeat proteins is halted by overexpression of importin-ß1, but not by inhibition of the ISR. This suggests that changes in the nuclear localization of the UsnRNP complexes induced by mutant ALS proteins are uncoupled from ISR activation, and that defects in the N/C trafficking of UsnRNPs might play a role in ALS pathogenesis.

Correction to: Impaired adult neurogenesis is an early event in Alzheimer's disease neurodegeneration, mediated by intracellular Aß oligomers.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Impaired adult neurogenesis is an early event in Alzheimer's disease neurodegeneration, mediated by intracellular Aß oligomers.

Alterations of adult neurogenesis have been reported in several Alzheimer's disease (AD) animal models and human brains, while defects in this process at presymptomatic/early stages of AD have not been explored yet. To address this, we investigated potential neurogenesis defects in Tg2576 transgenic mice at 1.5 months of age, a prodromal asymptomatic age in terms of Aß accumulation and neurodegeneration. We observe that Tg2576 resident and SVZ-derived adult neural stem cells (aNSCs) proliferate significantly less. Further, they fail to terminally differentiate into mature neurons due to pathological, tau-mediated, and microtubule hyperstabilization. Olfactory bulb neurogenesis is also strongly reduced, confirming the neurogenic defect in vivo. We find that this phenotype depends on the formation and accumulation of intracellular A-beta oligomers (AßOs) in aNSCs. Indeed, impaired neurogenesis of Tg2576 progenitors is remarkably rescued both in vitro and in vivo by the expression of a conformation-specific anti-AßOs intrabody (scFvA13-KDEL), which selectively interferes with the intracellular generation of AßOs in the endoplasmic reticulum (ER). Altogether, our results demonstrate that SVZ neurogenesis is impaired already at a presymptomatic stage of AD and is caused by endogenously generated intracellular AßOs in the ER of aNSCs. From a translational point of view, impaired SVZ neurogenesis may represent a novel biomarker for AD early diagnosis, in association to other biomarkers. Further, this study validates intracellular Aß oligomers as a promising therapeutic target and prospects anti-AßOs scFvA13-KDEL intrabody as an effective tool for AD treatment.

Running-Activated Neural Stem Cells Enhance Subventricular Neurogenesis and Improve Olfactory Behavior in p21 Knockout Mice.

In the subventricular zone (SVZ) of the adult brain, the neural stem cells (NSCs) ensure a continuous supply of new neurons to the olfactory bulb (OB), playing a key role in its plasticity and olfactory-related behavior. The activation and expansion of NSCs within the SVZ are finely regulated by environmental and intrinsic factors. Running represents one of the most powerful neurogenic stimuli, although is ineffective in enhancing SVZ neurogenesis. The cell cycle inhibitor p21 is an intrinsic inhibitor of NSCs' expansion through the maintenance of their quiescence and the restrain of neural progenitor proliferation. In this work, we decided to test whether running unveils the intrinsic neurogenic potential of p21-lacking NSCs. To test this hypothesis, we examined the effect of three different paradigms of voluntary running (5, 12, and 21 days) on SVZ neurogenesis of p21 knockout (KO) male mice at two different stages of development, 2 and 12 months of age. In vivo and in vitro data clearly demonstrate that physical activity is consistent with the activation and expansion of NSCs and with the enhancement of SVZ neurogenesis in p21 KO mice. We also found that 12 days of running contribute to the increase in the number of new neurons functionally active within the OB, which associates with an improvement in olfactory performance strictly dependent on adult SVZ neurogenesis, i.e., the odor detection threshold and short-term olfactory memory. These data suggest that in the adult SVZ of p21 KO mice, NSCs retain a high neurogenic potential, triggered by physical activity, with long-term consequences in olfactory-related behavior.

ProNGF Is a Cell-Type-Specific Mitogen for Adult Hippocampal and for Induced Neural Stem Cells.

The role of proNGF, the precursor of nerve growth factor (NGF), in the biology of adult neural stem cells (aNSCs) is still unclear. Here, we analyzed adult hippocampal neurogenesis in AD11 transgenic mice, in which the constitutive expression of anti-NGF antibody leads to an imbalance of proNGF over mature NGF. We found increased proliferation of progenitors but a reduced neurogenesis in the AD11 dentate gyrus (DG)-hippocampus (HP). Also in vitro, AD11 hippocampal neural stem cells (NSCs) proliferated more, but were unable to differentiate into morphologically mature neurons. By treating wild-type hippocampal progenitors with the uncleavable form of proNGF (proNGF-KR), we demonstrated that proNGF acts as mitogen on aNSCs at low concentration. The mitogenic effect of proNGF was specifically addressed to the radial glia-like (RGL) stem cells through the induction of cyclin D1 expression. These cells express high levels of p75NTR , as demonstrated by immunofluorescence analyses performed ex vivo on RGL cells isolated from freshly dissociated HP-DG or selected in vitro from NSCs by leukemia inhibitory factor. Clonogenic assay performed in the absence of mitogens showed that RGLs respond to proNGF-KR by reactivating their proliferation and thus leading to neurospheres formation. The mitogenic effect of proNGF was further exploited in the expansion of mouse-induced neural stem cells (iNSCs). Chronic exposure of iNSCs to proNGF-KR increased their proliferation. Altogether, we demonstrated that proNGF acts as mitogen on hippocampal and iNSCs. Stem Cells 2019;37:1223-1237.

p16Ink4a Prevents the Activation of Aged Quiescent Dentate Gyrus Stem Cells by Physical Exercise.

In the neurogenic niches-the dentate gyrus of the hippocampus and the subventricular zone (SVZ) adjacent to lateral ventricles-stem cells continue to divide during adulthood, generating progenitor cells and new neurons, and to self-renew, thus maintaining the stem cell pool. During aging, the numbers of stem/progenitor cells in the neurogenic niches are reduced. The preservation of the neurogenic pool is committed to a number of antiproliferative genes, with the role of maintaining the quiescence of neural cells. The cyclin-dependent kinase inhibitor p16Ink4a, whose expression increases with age, controls the expansion of SVZ aging stem cells, since in mice its deficiency prevents the decline of neurogenesis in SVZ. No change of neurogenesis is however observed in the p16Ink4a-null dentate gyrus. Here, we hypothesized that p16Ink4a plays a role as a regulator of the self-renewal of the stem cell pool also in the dentate gyrus, and to test this possibility we stimulated the dentate gyrus neural cells of p16Ink4a-null aging mice with physical exercise, a powerful neurogenic activator. We observed that running highly induced the generation of new stem cells in the p16Ink4a-null dentate gyrus, forcing them to exit from quiescence. Stem cells, notably, are not induced to proliferate by running in wild-type (WT) mice. Moreover, p16Ink4a-null progenitor cells were increased by running significantly above the number observed in WT mice. The new stem and progenitor cells generated new neurons, and continued to actively proliferate in p16Ink4a-null mice longer than in the WT after cessation of exercise. Thus, p16Ink4a prevents aging dentate gyrus stem cells from being activated by exercise. Therefore, p16Ink4a may play a role in the maintenance of dentate gyrus stem cells after stimulus, by keeping a reserve of their self-renewal capacity during aging.

Exploiting natural polysaccharides to enhance in vitro bio-constructs of primary neurons and progenitor cells.

Current strategies in Central Nervous System (CNS) repair focus on the engineering of artificial scaffolds for guiding and promoting neuronal tissue regrowth. Ideally, one should combine such synthetic structures with stem cell therapies, encapsulating progenitor cells and instructing their differentiation and growth. We used developments in the design, synthesis, and characterization of polysaccharide-based bioactive polymeric materials for testing the ideal composite supporting neuronal network growth, synapse formation and stem cell differentiation into neurons and motor neurons. Moreover, we investigated the feasibility of combining these approaches with engineered mesenchymal stem cells able to release neurotrophic factors. We show here that composite bio-constructs made of Chitlac, a Chitosan derivative, favor hippocampal neuronal growth, synapse formation and the differentiation of progenitors into the proper neuronal lineage, that can be improved by local and continuous delivery of neurotrophins. STATEMENT OF SIGNIFICANCE: In our work, we characterized polysaccharide-based bioactive platforms as biocompatible materials for nerve tissue engineering. We show that Chitlac-thick substrates are able to promote neuronal growth, differentiation, maturation and formation of active synapses. These observations support this new material as a promising candidate for the development of complex bio-constructs promoting central nervous system regeneration. Our novel findings sustain the exploitation of polysaccharide-based scaffolds able to favour neuronal network reconstruction. Our study shows that Chitlac-thick may be an ideal candidate for the design of biomaterial scaffolds enriched with stem cell therapies as an innovative approach for central nervous system repair.

Physical exercise rescues defective neural stem cells and neurogenesis in the adult subventricular zone of Btg1 knockout mice.

Adult neurogenesis occurs throughout life in the dentate gyrus (DG) and the subventricular zone (SVZ), where glia-like stem cells generate new neurons. Voluntary running is a powerful neurogenic stimulus triggering the proliferation of progenitor cells in the DG but, apparently, not in the SVZ. The antiproliferative gene Btg1 maintains the quiescence of DG and SVZ stem cells. Its ablation causes intense proliferation of DG and SVZ stem/progenitor cells in young mice, followed, during adulthood, by progressive decrease of the proliferative capacity. We have previously observed that running can rescue the deficit of DG Btg1-null neurogenesis. Here, we show that in adult Btg1-null SVZ stem and neuroblast cells, the reduction of proliferation is associated with a longer cell cycle and a more frequent entry into quiescence. Notably, running increases proliferation in Btg1-null SVZ stem cells highly above the levels of sedentary wild-type mice and restores normal values of cell cycle length and quiescence in stem and neuroblast cells, without affecting wild-type cells. Btg1-null SVZ neuroblasts show also increased migration throughout the rostral migratory stream and a deficiency of differentiated neurons in the olfactory bulb, possibly a consequence of premature exit from the cycle; running, however, normalizes migration and differentiation, increasing newborn neurons recruited to the olfactory circuitry. Furthermore, running increases the self-renewal of Btg1-null SVZ-derived neurospheres and, remarkably, in aged Btg1-null mice almost doubles the proliferating SVZ stem cells. Altogether, this reveals that SVZ stem cells are endowed with a hidden supply of self-renewal capacity, coupled to cell cycle acceleration and emerging after ablation of the quiescence-maintaining Btg1 gene and following exercise.

Increased cytoplasmic TDP-43 reduces global protein synthesis by interacting with RACK1 on polyribosomes.

TDP-43 is a well known RNA binding protein involved in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Dementia (FTLD). In physiological conditions, TDP-43 mainly localizes in the nucleus and shuttles, at least in neurons, to the cytoplasm to form TDP-43 RNA granules. In the nucleus, TDP-43 participates to the expression and splicing of RNAs, while in the cytoplasm its functions range from transport to translation of specific mRNAs. However, if loss or gain of these TDP-43 functions are affected in ALS/FTLD pathogenesis is not clear. Here, we report that TDP-43 localizes on ribosomes not only in primary neurons but also in SH-SY5Y human neuroblastoma cells. We find that binding of TDP-43 to the translational machinery is mediated by an interaction with a specific ribosomal protein, RACK1, and that an increase in cytoplasmic TDP-43 represses global protein synthesis, an effect which is rescued by overexpression of RACK1. Ribosomal loss of RACK1, which excludes TDP-43 from the translational machinery, remarkably reduces formation of TDP-43 cytoplasmic inclusions in neuroblastoma cells. Finally, we corroborate the interaction between TDP-43 and RACK1 on polyribosomes of neuroblastoma cells with mis-localization of RACK1 on TDP-43 positive cytoplasmic inclusions in motor neurons of ALS patients. In conclusions, results from this study suggest that TDP-43 represents a translational repressor not only for specific mRNAs but for overall translation and that its binding to polyribosomes through RACK1 may promote, under conditions inducing ALS pathogenesis, the formation of cytoplasmic inclusions.

Neutralization of nerve growth factor impairs proliferation and differentiation of adult neural progenitors in the subventricular zone.

Adult neurogenesis is a multistep process regulated by several extrinsic factors, including neurotrophins. Among them, little is known about the role of nerve growth factor (NGF) in the neurogenic niches of the mouse. Here we analyzed the biology of adult neural stem cells (NSCs) from the subventricular zone (SVZ) of AD11 anti-NGF transgenic mice, in which the expression of the recombinant antibody aD11 leads to a chronic postnatal neutralization of endogenous NGF. We showed that AD11-NSCs proliferate 10-fold less, with respect to their control counterparts, and display a significant impairment in their ability to differentiate into ß-tubulin positive neurons. We found a considerable reduction in the number of SVZ progenitors and neuroblasts also in vivo, which correlates with a lower number of newborn neurons in the olfactory bulbs of AD11 mice and a severe deficit in the ability of these mice to discriminate between different odors. We also demonstrated that, in AD11 mice, the morphology of both SVZ-resident and neurosphere-derived astrocytes is significantly altered. We were able to reproduce the AD11 phenotype in vitro, by acutely treating wild type NSCs with the anti-NGF antibody, further demonstrating that both the proliferation and the differentiation defects are due to the NGF deprivation. Consistently, the proliferative impairment of AD11 progenitors, as well as the atrophic morphology of AD11 astrocytes, can be partly rescued in vitro and in vivo by exogenous NGF addition. Altogether, our results demonstrate a causal link between NGF signaling and proper proliferation and differentiation of neural stem cells from the SVZ.

Tis21 is required for adult neurogenesis in the subventricular zone and for olfactory behavior regulating cyclins, BMP4, Hes1/5 and Ids.

Bone morphogenic proteins (BMPs) and the Notch pathway regulate quiescence and self-renewal of stem cells of the subventricular zone (SVZ), an adult neurogenic niche. Here we analyze the role at the intersection of these pathways of Tis21 (Btg2/PC3), a gene regulating proliferation and differentiation of adult SVZ stem and progenitor cells. In Tis21-null SVZ and cultured neurospheres, we observed a strong decrease in the expression of BMP4 and its effectors Smad1/8, while the Notch anti-neural mediators Hes1/5 and the basic helix-loop-helix (bHLH) inhibitors Id1-3 increased. Consistently, expression of the proneural bHLH gene NeuroD1 decreased. Moreover, cyclins D1/2, A2, and E were strongly up-regulated. Thus, in the SVZ Tis21 activates the BMP pathway and inhibits the Notch pathway and the cell cycle. Correspondingly, the Tis21-null SVZ stem cells greatly increased; nonetheless, the proliferating neuroblasts diminished, whereas the post-mitotic neuroblasts paradoxically accumulated in SVZ, failing to migrate along the rostral migratory stream to the olfactory bulb. The ability, however, of neuroblasts to migrate from SVZ explants was not affected, suggesting that Tis21-null neuroblasts do not migrate to the olfactory bulb because of a defect in terminal differentiation. Notably, BMP4 addition or Id3 silencing rescued the defective differentiation observed in Tis21-null neurospheres, indicating that they mediate the Tis21 pro-differentiative action. The reduced number of granule neurons in the Tis21-null olfactory bulb led to a defect in olfactory detection threshold, without effect on olfactory memory, also suggesting that within olfactory circuits new granule neurons play a primary role in odor sensitivity rather than in memory.

Btg1 is Required to Maintain the Pool of Stem and Progenitor Cells of the Dentate Gyrus and Subventricular Zone.

Btg1 belongs to a family of cell cycle inhibitory genes. We observed that Btg1 is highly expressed in adult neurogenic niches, i.e., the dentate gyrus and subventricular zone (SVZ). Thus, we generated Btg1 knockout mice to analyze the role of Btg1 in the process of generation of adult new neurons. Ablation of Btg1 causes a transient increase of the proliferating dentate gyrus stem and progenitor cells at post-natal day 7; however, at 2 months of age the number of these proliferating cells, as well as of mature neurons, greatly decreases compared to wild-type controls. Remarkably, adult dentate gyrus stem and progenitor cells of Btg1-null mice exit the cell cycle after completing the S phase, express p53 and p21 at high levels and undergo apoptosis within 5 days. In the SVZ of adult (two-month-old) Btg1-null mice we observed an equivalent decrease, associated to apoptosis, of stem cells, neuroblasts, and neurons; furthermore, neurospheres derived from SVZ stem cells showed an age-dependent decrease of the self-renewal and expansion capacity. We conclude that ablation of Btg1 reduces the pool of dividing adult stem and progenitor cells in the dentate gyrus and SVZ by decreasing their proliferative capacity and inducing apoptosis, probably reflecting impairment of the control of the cell cycle transition from G1 to S phase. As a result, the ability of Btg1-null mice to discriminate among overlapping contextual memories was affected. Btg1 appears, therefore, to be required for maintaining adult stem and progenitor cells quiescence and self-renewal.

Direct intracellular selection and biochemical characterization of a recombinant anti-proNGF single chain antibody fragment.

proNGF, the precursor of the neurotrophin NGF, is widely expressed in central and peripheral nervous system. Its physiological functions are still largely unknown, although it emerged from studies in the last decade that proNGF has additional and distinct functions with respect to NGF, besides acting chaperone-like for NGF folding during its biogenesis. The regulation of proNGF/NGF ratio represents a crucial process for homeostasis of brain and other tissues, and understanding the molecular aspects of these differences is important. We report the selection and characterization of a recombinant monoclonal anti-proNGF antibody in single chain Fv fragment (scFv) format. The selection exploited the Intracellular Antibody Capture Technology (IACT), starting from a naïve mouse SPLINT (Single Pot Library of INTracellular antibodies) library. This antibody (scFv FPro10) was expressed recombinantly in Escherichia coli, was proven to be highly soluble and stable, and thoroughly characterized from the biochemical-biophysical point of view. scFv FPro10 displays high affinity and specificity for proNGF, showing no cross-reactivity with other pro-neurotrophins. A structural model was obtained by SAXS. scFv FPro10 represents a new tool to be exploited for the selective immunoanalysis of proNGF, both in vitro and in vivo, and might help in understanding the molecular function of proNGF in neurodegeneration.

Bystander effect on brain tissue of mesoangioblasts producing neurotrophins.

Neurotrophic factors (NTFs) are involved in the regulation of neuronal survival and function and, thus, may be used to treat neurological diseases associated with neuronal death. A major hurdle for their clinical application is the delivery mode. We describe here a new strategy based on the use of progenitor cells called mesoangioblasts (MABs). MABs can be isolated from postnatal mesoderm tissues and, because of a high adhesin-dependent migratory capacity, can reach perivascular targets especially in damaged areas. We generated genetically modified MABs producing nerve growth factor (MABs-NGF) or brain-derived neurotrophic factor (MABs-BDNF) and assessed their bystander effects in vitro using PC12 cells, primary cultures, and organotypic cultures of adult hippocampal slices. MABs-NGF-conditioned medium induced differentiation of PC12 cells, while MABs-BDNF-conditioned medium increased viability of cultured neurons and slices. Slices cultured with MABs-BDNF medium also better retained their morphology and functional connections, and all these effects were abolished by the TrkB kinase blocker K252a or the BDNF scavenger TrkB-IgG. Interestingly, the amount of BDNF released by MABs-BDNF produced greater effects than an identical amount of recombinant BDNF, suggesting that other NTFs produced by MABs synergize with BDNF. Thus, MABs can be an effective vehicle for NTF delivery, promoting differentiation, survival, and functionality of neurons. In summary, MABs hold distinct advantages over other currently evaluated approaches for NTF delivery in the CNS, including synergy of MAB-produced NTF with the neurotrophins. Since MABs may be capable of homing into damaged brain areas, they represent a conceptually novel, promising therapeutic approach to treat neurodegenerative diseases.