Cognitive Decline and Modulation of Alzheimer's Disease-Related Genes After Inhibition of MicroRNA-101 in Mouse Hippocampal Neurons.

MicroRNAs have emerged as regulators of brain development and function. Reduction of miR-101 expression has been reported in rodent hippocampus during ageing, in the brain of Alzheimer's disease (AD) patients and in AD animal models. In this study, we investigated the behavioral and molecular consequences of inhibition of endogenous miR-101 in 4-5-month-old C57BL/6J mice, infused with lentiviral particles expressing a miR-101 sponge (pLSyn-miR-101 sponge) in the CA1 field of the hippocampus. The sponge-infected mouse model showed cognitive impairment. The pLSyn-miR-101 sponge-infected mice were unable to discriminate either a novel object location or a novel object as assessed by object place recognition (OPR) and novel object recognition (NOR) tasks, respectively. Moreover, the sponge-infected mice evaluated for contextual memory in inhibitory avoidance task showed shorter retention latency compared to control pLSyn mice. These cognitive impairment features were associated with increased hippocampal expression of relevant miR-101 target genes, amyloid precursor protein (APP), RanBP9 and Rab5 and overproduction of amyloid beta (Aß) 42 levels, the more toxic species of Aß peptide. Notably, phosphorylation-dependent AMP-activated protein kinase (AMPK) hyperactivation is associated with AD pathology and age-dependent memory decline, and we found AMPK hyperphosphorylation in the hippocampus of pLSyn-miR-101 sponge mice. This study demonstrates that mimicking age-associated loss of miR-101 in hippocampal neurons induces cognitive decline and modulation of AD-related genes in mice.

ProNGF\NGF imbalance triggers learning and memory deficits, neurodegeneration and spontaneous epileptic-like discharges in transgenic mice.

ProNGF, the precursor of mature nerve growth factor (NGF), is the most abundant form of NGF in the brain. ProNGF and mature NGF differ significantly in their receptor interaction properties and in their bioactivity. ProNGF increases markedly in the cortex of Alzheimer's disease (AD) brains and proNGF\NGF imbalance has been postulated to play a role in neurodegeneration. However, a direct proof for a causal link between increased proNGF and AD neurodegeneration is lacking. In order to evaluate the consequences of increased levels of proNGF in the postnatal brain, transgenic mice expressing a furin cleavage-resistant form of proNGF, under the control of the neuron-specific mouse Thy1.2 promoter, were derived and characterized. Different transgenic lines displayed a phenotypic gradient of neurodegenerative severity features. We focused the analysis on the two lines TgproNGF#3 and TgproNGF#72, which shared learning and memory impairments in behavioral tests, cholinergic deficit and increased Aß-peptide immunoreactivity. In addition, TgproNGF#3 mice developed Aß oligomer immunoreactivity, as well as late diffuse astrocytosis. Both TgproNGF lines also display electrophysiological alterations related to spontaneous epileptic-like events. The results provide direct evidence that alterations in the proNGF/NGF balance in the adult brain can be an upstream driver of neurodegeneration, contributing to a circular loop linking alterations of proNGF/NGF equilibrium to excitatory/inhibitory synaptic imbalance and amyloid precursor protein (APP) dysmetabolism.

Crossregulation between Neurogenin2 and pathways specifying neuronal identity in the spinal cord.

We have examined how genetic pathways that specify neuronal identity and regulate neurogenesis interface in the vertebrate neural tube. Here, we demonstrate that expression of the proneural gene Neurogenin2 (Ngn2) in the ventral spinal cord results from the modular activity of three enhancers active in distinct progenitor domains, suggesting that Ngn2 expression is controlled by dorsoventral patterning signals. Consistent with this hypothesis, Ngn2 enhancer activity is dependent on the function of Pax6, a homeodomain factor involved in specifying the identity of ventral spinal cord progenitors. Moreover, we show that Ngn2 is required for the correct expression of Pax6 and several homeodomain proteins expressed in defined neuronal populations. Thus, neuronal differentiation involves crossregulatory interactions between a bHLH-driven program of neurogenesis and genetic pathways specifying progenitor and neuronal identity in the spinal cord.

Pax6 is required for the multipotent state of retinal progenitor cells.

The molecular mechanisms mediating the retinogenic potential of multipotent retinal progenitor cells (RPCs) are poorly defined. Prior to initiating retinogenesis, RPCs express a limited set of transcription factors implicated in the evolutionary ancient genetic network that initiates eye development. We elucidated the function of one of these factors, Pax6, in the RPCs of the intact developing eye by conditional gene targeting. Upon Pax6 inactivation, the potential of RPCs becomes entirely restricted to only one of the cell fates normally available to RPCs, resulting in the exclusive generation of amacrine interneurons. Our findings demonstrate furthermore that Pax6 directly controls the transcriptional activation of retinogenic bHLH factors that bias subsets of RPCs toward the different retinal cell fates, thereby mediating the full retinogenic potential of RPCs.

Development of a murine orthotopic model of leukemia: evaluation of TP53 gene therapy efficacy.

The onco-suppressor gene TP53 has potential use in the gene therapy of many human cancers including leukemias. The latter indication derived from numerous experimental reports of p53-mediated suppressing effects on human and murine leukemia cells in vitro. However, few in vivo experiments have been performed, and those that have used a subcutaneous injection of p53-transduced leukemia cells. Thus, we developed an orthotopic leukemia model in adult, syngenic mice to evaluate the feasibility of TP53-mediated therapeutic approaches. We found that among other cells, v-src-transformed 32D myeloid progenitors induce leukemia when injected intravenously in syngenic mice. The resulting malignancy resembles the clinical manifestations of human acute myeloid leukemia because it is characterized by a massive invasion of bone marrow compartments, splenomegaly, generalized lymphadenopathy, and a macroscopic or microscopic infiltration of the kidneys, liver, and lungs. When these 32Dv-src cells were infected with a TP53-recombinant retrovirus before intravenous injection, we found a decreased mortality and, in those animals that develop leukemia, a drastic reduction of the generalized organ infiltration, suggesting that exogenous TP53 expression might be used for ex vivo bone marrow purging from leukemia cells.

Wt-p53 action in human leukaemia cell lines corresponding to different stages of differentiation.

Recent studies support the potential application of the wt-p53 gene in cancer therapy. Expression of exogenous wt-p53 suppresses a variety of leukaemia phenotypes by acting on cell survival, proliferation and/or differentiation. As for tumour gene therapy, the final fate of the neoplastic cells is one of the most relevant points. We examined the effects of exogenous wt-p53 gene expression in several leukaemia cell lines to identify p53-responsive leukaemia. The temperature-sensitive p53Val135 mutant or the human wt-p53 cDNA was transduced in leukaemia cell lines representative of different acute leukaemia FAB subtypes, including M1 (KG1), M2 (HL-60), M3 (NB4), M5 (U937) and M6 (HEL 92.1.7), as well as blast crisis of chronic myelogenous leukaemia (BC-CML: K562, BV173) showing diverse differentiation features. By morphological, molecular and biochemical analyses, we have shown that exogenous wt-p53 gene expression induces apoptosis only in cells corresponding to M1, M2 and M3 of the FAB classification and in BC-CML showing morphological and cytochemical features of undifferentiated blast cells. In contrast, it promotes differentiation in the others. Interestingly, cell responsiveness was independent of the vector used and the status of the endogenous p53 gene.

Oncogenes belonging to the CSF-1 transduction pathway direct p53 tumor suppressor effects to monocytic differentiation in 32D cells.

Expression of exogenous wt-p53 in different tumor cell lines can induce growth arrest, apoptosis, or differentiation. Several experimental works have highlighted the relevance of cellular context in the determination of p53-mediated final outcomes. We recently observed that these diverse wt-p53 effects can also be induced by overexpressing wt-p53 in a single cell type-the 32D myeloid progenitors-transformed with different activated oncogenes. Here we show that 32D cells transformed with two different oncogenes, v-src or c-fms [S301,F969], both belonging to the CSF-1 transduction pathway, respond to exogenous wt-p53 expression with the same final outcome-monocytic differentiation. This result is particularly significant since 32D cells do not spontaneously express the CSF-1 receptor, whereas they undergo granulocytic differentiation upon G-CSF stimulation. These data strongly support the idea that wt-p53 suppressing effects result from interactions between p53 activity and the signaling pathways activated in different transformed cells.

Expression of exogenous wt-p53 does not affect normal hematopoiesis: implications for bone marrow purging.

Some gene therapy approaches for cancer treatment attempt to transduce onco-suppressor genes into tumor cells. A central problem of this strategy is the targeting of tumor cells to avoid damage to normal ones. It has been noticed that transduction of wt-p53 into a large number of cancer cells induces tumor suppression. In contrast, some observations suggest that introduction of exogenous wt-p53 into nontransformed cells does not impair proliferation. If normal bone marrow (BM) cells are not affected by wt-p53 transduction, BM purging from p53-responding leukemic cells might be achieved in vitro by delivering the wild-type onco-suppressor to all marrow cells. We undertook a series of experiments to assess whether transduction of wt-p53 into normal hematopoietic cells is harmful. Two different wt-p53-recombinant retroviruses were used to infect primary, murine BM cells. Expression of exogenous wt-p53 in these cells did not affect in vitro colony formation, and did not induce any observable effects on morphology and differentiation. In contrast, the same viruses suppressed the tumor phenotype of v-src-transformed 32D cells. These results might open the way to gene therapy approaches to leukemias with the p53 gene without the need to target specifically and uniquely the tumor cells, sparing the normal ones.

The beta 4 integrin subunit is expressed in mouse fibroblasts and modulated by transforming growth factor-beta 1.

Integrin beta 4 subunit is present in association with alpha 6 chain on both normal and transformed epithelial cells. Recently alpha 6 beta 4 heterodimer was found on the endothelium of medium-sized blood vessels and on immature thymocytes. In this report we show, by Northern blotting, indirect immunofluorescence, immunoprecipitation, and Western blotting, that beta 4 subunit is expressed also on cells of mesenchymal origin such as fibroblasts, myoblasts, and myotubes. Increased expression of alpha 6 beta 4 has been related to the aggressive metastatic phenotype of human and murine carcinomas. The transforming growth factor beta 1 (TGF-beta 1) has been found to modulate the expression of several integrins and intracellular matrix proteins, as well as to stimulate cell invasion and metastatic potential. To evaluate whether alpha 6 beta 4 expression is modulated by TGF-beta 1, we transfected 3T3 fibroblasts with an expression vector carrying the human TGF-beta 1 cDNA driven by the SV40 early promoter. We observed by indirect immunofluorescence a modification in the subcellular distribution of beta 4 subunit, which acquires a perinuclear localization. This finding suggests this integrin subunit correlates with the cytoskeletal reorganization induced by TGF-beta 1.

Interference with p53 protein inhibits hematopoietic and muscle differentiation.

The involvement of p53 protein in cell differentiation has been recently suggested by some observations made with tumor cells and the correlation found between differentiation and increased levels of p53. However, the effect of p53 on differentiation is in apparent contrast with the normal development of p53-null mice. To test directly whether p53 has a function in cell differentiation, we interfered with the endogenous wt-p53 protein of nontransformed cells of two different murine histotypes: 32D myeloid progenitors, and C2C12 myoblasts. A drastic inhibition of terminal differentiation into granulocytes or myotubes, respectively, was observed upon expression of dominant-negative p53 proteins. This inhibition did not alter the cell cycle withdrawal typical of terminal differentiation, nor p21(WAF1/CIP1) upregulation, indicating that interference with endogenous p53 directly affects cell differentiation, independently of the p53 activity on the cell cycle. We also found that the endogenous wt-p53 protein of C2C12 cells becomes transcriptionally active during myogenesis, and this activity is inhibited by p53 dominant-negative expression. Moreover, we found that p53 DNA-binding and transcriptional activities are both required to induce differentiation in p53-negative K562 cells. Taken together, these data strongly indicate that p53 is a regulator of cell differentiation and it exerts this role, at least in part, through its transcriptional activity.

Wild-type p53 induces diverse effects in 32D cells expressing different oncogenes.

Expression of exogenous wild-type (wt) p53 in different leukemia cell lines can induce growth arrest, apoptotic cell death, or cell differentiation. The hematopoietic cell lines that have been used so far to study wt p53 functions have in common the characteristic of not expressing endogenous p53. However, the mechanisms involved in the transformation of these cells are different, and the cells are at different stages of tumor progression. It can be postulated that each type of neoplastic cell offers a particular environment in which p53 might generate different effects. To test this hypothesis, we introduced individual oncogenes into untransformed, interleukin-3 (IL-3)-dependent myeloid precursor 32D cells to have a single transforming agent at a time. The effects induced by wt p53 overexpression were subsequently evaluated in each oncogene-expressing 32D derivative. We found that in not fully transformed, v-ras-expressing 32D cells, as already shown for the parental 32D cells, overexpression of the wt p53 gene caused no phenotypic changes and no reduction of the proliferative rate as long as the cells were maintained in their normal culture conditions (presence of IL-3 and serum). An accelerated rate of apoptosis was observed after IL-3 withdrawal. In contrast, in transformed, IL-3-independent 32D cells, wt p53 overexpression induced different effects. The v-abl-transformed cells manifested a reduction in growth rate, while the v-src-transformed cells underwent monocytic differentiation. These results show that the phenotype effects of wt p53 action(s) can vary as a function of the cellular environment.

Wild-type p53 modulates apoptosis of normal, IL-3 deprived, hematopoietic cells.

Apoptotic cell death is an active process which regulates the maintenance of the hematopoietic homeostasis. It has been reported that wild-type p53 (wt-p53) protein induces apoptosis in leukemia cells. To assess whether p53 is involved in the apoptotic process of normal hematopoietic cells, we introduced the temperature-sensitive p53Val135 mutant into the murine myeloid precursor cell line 32Dcl3. These are diploid, non-tumorigenic cells whose survival and proliferation are dependent upon growth factor supply (IL-3 and serum). Overexpression of wt-p53 protein does not affect morphology and proliferation of 32D cells as long as they are maintained in the presence of IL-3. However, after IL-3 withdrawal, wt-p53 overexpression significantly accelerates apoptosis. This phenomenon is IL-3 specific since no differences in death rates induced by serum starvation are found between parental cells and p53-transfectants. When the latter experiments are carried out at 37 degrees C with p53 protein in mutant conformation, an extended survival of 32D cells is observed after IL-3 deprivation, but not after serum withdrawal. Taken together, these results show that wt-p53 actively mediates the apoptosis due to the absence of specific growth factors, such as IL-3, suggesting that p53 might be involved in the response of myeloid precursors to environmental cytokines for the maintenance of the hematopoietic homeostasis.

Wild-type p53 gene expression induces granulocytic differentiation of HL-60 cells.

Overexpression of wild-type p53 gene in malignant cell lines has been shown to inhibit cell proliferation in a number of cases. However, endogenous p53 protein seems to play little role in normal cell-cycle control as suggested by the normal development of p53 null mice, and by the low p53 protein levels expressed in most cell types. Recently, increased expression of endogenous p53 protein has been observed during the cellular response to DNA damage, as well as during differentiation of human hematopoietic cells. To study the role of the p53 gene in hematopoietic differentiation, we introduced the wild-type p53 gene or the temperature-sensitive p53(Val135) mutant into p53-deficient HL-60 promyelocytic leukemia cells. Morphological analysis, flow-cytometric determination of granulocytic or monocytic surface markers, and ability to reduce nitroblue tetrazolium (NBT) demonstrated that expression of exogenous wild-type p53 gene in HL-60 cells induces differentiation through the granulocytic pathway. Proliferation and cell-cycle analysis performed early after expression of wild-type p53 showed that induction of differentiation is not coupled with growth arrest, which suggests that p53 is involved in differentiation independently of its activity on the cell cycle.