No abstract

Adult neurogenesis in the dentate gyrus plays a critical role in hippocampus-dependent spatial learning. It remains unknown, however, how new neurons become functionally integrated into spatial circuits and contribute to hippocampus-mediated forms of learning and memory. To investigate these issues, we used a mouse model in which the differentiation of adult-generated dentate gyrus neurons can be anticipated by conditionally expressing the pro-differentiative gene PC3 (Tis21/BTG2) in nestin-positive progenitor cells. In contrast to previous studies that affected the number of newly generated neurons, this strategy selectively changes their timing of differentiation. New, adult-generated dentate gyrus progenitors, in which the PC3 transgene was expressed, showed accelerated differentiation and significantly reduced dendritic arborization and spine density. Functionally, this genetic manipulation specifically affected different hippocampus-dependent learning and memory tasks, including contextual fear conditioning, and selectively reduced synaptic plasticity in the dentate gyrus. Morphological and functional analyses of hippocampal neurons at different stages of differentiation, following transgene activation within defined time-windows, revealed that the new, adult-generated neurons up to 3–4 weeks of age are required not only to acquire new spatial information but also to use previously consolidated memories. Thus, the correct unwinding of these key memory functions, which can be an expression of the ability of adult-generated neurons to link subsequent events in memory circuits, is critically dependent on the correct timing of the initial stages of neuron maturation and connection to existing circuits.

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.

Neurogenesis in the dentate gyrus of the adult hippocampus has been implicated in neural plasticity and memory, but the molecular mechanisms controlling the proliferation and differentiation of newborn neurons and their integration into the synaptic circuitry are still largely unknown. To investigate this issue, we have analyzed the adult hippocampal neurogenesis in a PC3/Tis21-null mouse model. PC3/Tis21 is a transcriptional co-factor endowed with antiproliferative and prodifferentiative properties; indeed, its upregulation in neural progenitors has been shown to induce exit from cell cycle and differentiation. We demonstrate here that the deletion of PC3/Tis21 causes an increased proliferation of progenitor cells in the adult dentate gyrus and an arrest of their terminal differentiation. In fact, in the PC3/Tis21-null hippocampus postmitotic undifferentiated neurons accumulated, while the number of terminally differentiated neurons decreased of 40%. As a result, PC3/Tis21-null mice displayed a deficit of contextual memory. Notably, we observed that PC3/Tis21 can associate to the promoter of Id3, an inhibitor of proneural gene activity, and negatively regulates its expression, indicating that PC3/Tis21 acts upstream of Id3. Our results identify PC3/Tis21 as a gene required in the control of proliferation and terminal differentiation of newborn neurons during adult hippocampal neurogenesis and suggest its involvement in the formation of contextual memories.

Growing evidence indicates that cell cycle arrest and neurogenesis are highly coordinated and interactive processes, governed by cell cycle genes and neural transcription factors. The gene PC3 (Tis21/BTG2) is expressed in the neuroblast throughout the neural tube and inhibits cell cycle progression at the G 1 checkpoint by repressing cyclin D1 transcription. We generated inducible mouse models in which the expression of PC3 was upregulated in neuronal precursors of the neural tube and of the cerebellum. These mice exhibited a marked increase in the production of postmitotic neurons and impairment of cerebellar development. Cerebellar granule precursors of PC3 transgenic mice displayed inhibition of cyclin D1 expression and a strong increase in the expression of Math1, a transcription factor required for their differentiation. Furthermore, PC3, encoded by a recombinant adenovirus, also induced Math1 in postmitotic granule cells in vitro and stimulated the Math1 promoter activity. In contrast, PC3 expression was unaffected in the cerebellar primordium of Math1 null mice, suggesting that PC3 acts upstream to Math1. As a whole, our data suggest that cell cycle exit of cerebellar granule cell precursors and the onset of cerebellar neurogenesis are coordinated by PC3 through transcriptional control of cyclin D1 and Math1, respectively.

A failure in the control of proliferation of cerebellar granule neuron precursor cells (GCPs), located in the external granular layer (EGL) of the cerebellum, gives rise to medulloblastoma. To investigate the process of neoplastic transformation of GCPs, we generated a new medulloblastoma model by crossing Patched1 heterozygous mice, which develop medulloblastomas with low frequency, with mice lacking the Tis21 gene. Overexpression of Tis21 is known to inhibit proliferation and trigger differentiation of GCPs; its expression decreases in human medulloblastomas. Double-knock-out mice show a striking increase in the frequency of medulloblastomas and hyperplastic EGL lesions, formed by preneoplastic GCPs. Tis21 deletion does not affect the proliferation of GCPs but inhibits their differentiation and, chiefly, their intrinsic ability to migrate outside the EGL. This defect of migration may represent an important step in medulloblastoma formation, as GCPs, remaining longer in the EGL proliferative niche, may become more prone to transformation. By genome-wide analysis, we identified the chemokine Cxcl3 as a target of Tis21. Cxcl3 is downregulated in Tis21-null GCPs of EGL and lesions; addition of Cxcl3 to cerebellar slices rescues the defective migration of Tis21-null GCPs and, remarkably, reduces the area of hyperplastic lesions. As Tis21 activates Cxcl3 transcription, our results suggest that Tis21 induces migration of GCPs through Cxcl3, which may represent a novel target for medulloblastoma therapy.

As major components of neuronal membranes, omega-3 polyunsaturated acids (n-3 PUFA) exhibit a wide range of regulatory functions, modulating from synaptic plasticity to neuroinflammation, from oxidative stress to neuroprotection. Recent human and animal studies indicated the n-3 PUFA neuroprotective properties in aging, with a clear negative correlation between n-3 PUFA levels and hippocampal deficits. The present multidimensional study was aimed at associating cognition, hippocampal neurogenesis, volume, neurodegeneration and metabolic correlates to verify n-3 PUFA neuroprotective effects in aging. To this aim 19 month-old mice were given n-3 PUFA mixture, or olive oil or no dietary supplement for 8 weeks during which hippocampal-dependent mnesic functions were tested. At the end of behavioral testing morphological and metabolic correlates were analyzed. n-3 PUFA supplemented aged mice exhibited better object recognition memory, spatial and localizatory memory, and aversive response retention, without modifications in anxiety levels in comparison to controls. These improved hippocampal cognitive functions occurred in the context of an enhanced cellular plasticity and a reduced neurodegeneration. In fact, n-3 PUFA supplementation increased hippocampal neurogenesis and dendritic arborization of newborn neurons, volume, neuronal density and microglial cell number, while it decreased apoptosis, astrocytosis and lipofuscin accumulation in the hippocampus. The increased levels of some metabolic correlates (blood Acetyl-L-Carnitine and brain n-3 PUFA concentrations) found in n-3 PUFA supplemented mice also pointed toward an effective neuroprotection. On the basis of the present results n-3 PUFA supplementation appears to be a useful tool in health promotion and cognitive decline prevention during aging.

Environmental enrichment (EE) is a widely used paradigm for investigating the influence of complex stimulations on brain and behavior. Here we examined whether pre-reproductive exposure to EE of female rats may influence their maternal care and offspring cognitive performances. To this aim, from weaning to breeding age enriched females (EF) were reared in enriched environments. Females reared in standard conditions were used as controls. At 2.5 months of age all females were mated and reared in standard conditions with their offspring. Maternal care behaviors and nesting activity were assessed in lactating dams. Their male pups were also behaviorally evaluated at different post-natal days (pnd). Brain BDNF, reelin and adult hippocampal neurogenesis levels were measured as biochemical correlates of neuroplasticity. EF showed more complex maternal care than controls due to their higher levels of licking, crouching and nest building activities. Moreover, their offspring showed higher discriminative (maternal odor preference T-maze, pnd 10) and spatial (Morris Water Maze, pnd 45; Open Field with objects, pnd 55) performances, with no differences in social abilities (Sociability test, pnd 35), in comparison to controls. BDNF levels were increased in EF frontal cortex at pups' weaning and in their offspring hippocampus at pnd 21 and 55. No differences in offspring reelin and adult hippocampal neurogenesis levels were found. In conclusion, our study indicates that pre-reproductive maternal enrichment positively influences female rats' maternal care and cognitive development of their offspring, demonstrating thus a transgenerational transmission of EE benefits linked to enhanced BDNF-induced neuroplasticity.