Miguel A. López-Toledano

Inhibition of natural antisense transcripts in vivo results in gene-specific transcriptional upregulation

The ability to specifically upregulate genes in vivo holds great therapeutic promise. Here we show that inhibition or degradation of natural antisense transcripts (NATs) by single-stranded oligonucleotides or siRNAs can transiently and reversibly upregulate locus-specific gene expression. Brain-derived neurotrophic factor (BDNF) is normally repressed by a conserved noncoding antisense RNA transcript, BDNF-AS. Inhibition of this transcript upregulates BDNF mRNA by two- to sevenfold, alters chromatin marks at the BDNF locus, leads to increased protein levels and induces neuronal outgrowth and differentiation both in vitro and in vivo. We also show that inhibition of NATs leads to increases in glial-derived neurotrophic factor (GDNF) and ephrin receptor B2 (EPHB2) mRNA. Our data suggest that pharmacological approaches targeting NATs can confer locus-specific gene upregulation effects.Here we demonstrate that natural antisense transcripts (NATs), which are abundant in mammalian genomes, can function as repressors of specific genomic loci and that their removal or inhibition by AntagoNAT oligonucleotides leads to transient and reversible upregulation of sense gene expression. As one example, we show that Brain-Derived Neurotrophic Factor (BDNF) is under the control of a conserved noncoding antisense RNA transcript, BDNF-AS, both in vitro and in vivo. BDNF-AS tonically represses BDNF sense RNA transcription by altering chromatin structure at the BDNF locus, which in turn reduces endogenous BDNF protein and function. By providing additional and analogous examples of endogenous mRNA upregulation, we suggest that antisense RNA mediated transcriptional suppression is a common phenomenon. In sum, we demonstrate a novel pharmacological strategy by which endogenous gene expression can be upregulated in a locus-specific manner.

MicroRNA-219 modulates NMDA receptor-mediated neurobehavioral dysfunction

N-methyl-d-aspartate (NMDA) glutamate receptors are regulators of fast neurotransmission and synaptic plasticity in the brain. Disruption of NMDA-mediated glutamate signaling has been linked to behavioral deficits displayed in psychiatric disorders such as schizophrenia. Recently, noncoding RNA molecules such as microRNAs (miRNAs) have emerged as critical regulators of neuronal functions. Here we show that pharmacological (dizocilpine) or genetic (NR1 hypomorphism) disruption of NMDA receptor signaling reduces levels of a brain-specific miRNA, miR-219, in the prefrontal cortex (PFC) of mice. Consistent with a role for miR-219 in NMDA receptor signaling, we identify calcium/calmodulin-dependent protein kinase II γ subunit (CaMKIIγ), a component of the NMDA receptor signaling cascade, as a target of miR-219. In vivo inhibition of miR-219 by specific antimiR in the murine brain significantly modulated behavioral responses associated with disrupted NMDA receptor transmission. Furthermore, pretreatment with the antipsychotic drugs haloperidol and clozapine prevented dizocilpine-induced effects on miR-219. Taken together, these data support an integral role for miR-219 in the expression of behavioral aberrations associated with NMDA receptor hypofunction.

Neurogenic Effect of β-Amyloid Peptide in the Development of Neural Stem Cells

The adult mammalian brain contains neural stem cells (NSCs) with self-renewal and multilineage potential in the hippocampus and subventricular zone. However, neurogenesis from these areas does not compensate for neuronal loss in age-related neurodegenerative disorders such as Alzheimers disease (AD). To test whether an impairment of neurogenesis could contribute to the pathogenesis of AD, we examined the effects of amyloid-β peptide (Aβ) on the survival and neuronal differentiation of cultured NSCs from striatum and hippocampus. We show that Aβ peptide does not impair the neurogenic rate in NSC progeny, but that it increases the total number of neurons in vitro in a dose-dependent manner. The neurogenic effect of Aβ peptide is not dependent on soluble factors released from the NSC progeny. Neurogenesis is induced by Aβ42 and not Aβ40 or Aβ 25-35, and the activity appears to be a property of Aβ oligomers and not fibrils. These results suggest that Aβ may have positive as well as deleterious actions, and that a knowledge of the mechanisms involved in the former could be valuable in exploiting the regenerative and plastic potential of the brain in preventing and treating Alzheimers disease.

Increased Neurogenesis in Young Transgenic Mice Overexpressing Human APP Sw,Ind

APP overexpressing mice have been widely used in the study of Alzheimers disease (AD), focusing mainly at older ages, with higher accumulation of amyloid-beta peptide (Abeta). A decrease in hippocampal adult neurogenesis has been described in these models and proposed to be a consequence of Abeta accumulation. Only one study demonstrates increased neurogenesis in the hippocampus of APP-overexpressing J20 mice, and suggests it is a compensatory effect due to a subtle Abeta-induced damage. We have previously reported that a specific aggregation of Abeta has neurogenic potential on neural stem cells (NSC) in vitro. In order to clarify the contradicting data reported in vivo, we investigated NSC proliferation and neuronal differentiation in the hippocampi of J20 mice at a broader range of ages. Using immunohistochemistry, we show increased proliferation and neuronal differentiation in the hippocampi of 3 month-old J20 mice that reverted when animals became older. The increase in neurogenesis correlated with detectable levels of oligomeric Abeta, measured by ELISA and western blot. We suggest that oligomeric Abeta directly induces neurogenesis in vivo as has been demonstrated in vitro. Understanding the mechanisms underlying these changes could lead to treatments to control the neuronal differentiation of endogenous precursors through the progress of AD.

Mutations in the neurofilament light gene linked to Charcot‐Marie‐Tooth disease cause defects in transport

Neurofilament light gene mutations have been linked to a subset of patients with Charcot‐Marie‐Tooth disease, the most common inherited motor and sensory neuropathy. We have previously shown that Charcot‐Marie‐Tooth‐linked mutant neurofilament light assembles abnormally in non‐neuronal cells. In this study, we have characterized the effects of expression of mutant neurofilament light proteins on axonal transport in a neuronal cell culture model. We demonstrated that the Charcot‐Marie‐Tooth‐linked neurofilament light mutations: (i) affect the axonal transport of mutant neurofilaments; (ii) have a dominant‐negative effect on the transport of wild‐type neurofilaments; (iii) affect the transport of mitochondria and the anterograde axonal transport marker human amyloid precursor protein; (iv) result in alterations of retrograde axonal transport and (v) cause fragmentation of the Golgi apparatus. Increased neuritic degeneration was observed in neuronal cells overexpressing neurofilament light mutants. Our results suggest that these generalized axonal transport defects could be responsible for the neuropathy in Charcot‐Marie‐Tooth disease.

Cellular Characterization of Epidermal Growth Factor-expanded Free-floating Neurospheres

Neural stem cells proliferate in liquid culture as cell clusters (neurospheres). This study was undertaken to characterize the epidermal growth factor (EGF)-expanded free-floating neurospheres derived from rat fetal striatum. We examined the ultrastructural and antigenic characteristics of these spheres. They consisted of two cell types, electron-dense and electron-lucent cells. Lucent cells were immunopositive to actin, vimentin, and nestin, whereas dense cells were immunopositive to actin, weakly positive to vimentin, and nestin-negative. Neurospheres contained healthy, apoptotic, and necrotic cells. Healthy cells were attached to each other by adherens junctions. They showed many pseudopodia and occasionally a single cilium. Sphere cells showed phagocytic capability because healthy cells phagocytosed the cell debris derived from dead cells in a particular process that involves the engulfment of dying cells by cell processes from healthy cells. Sphere cells showed a cytoplasmic and a nuclear pool of fibroblast growth factor (FGF) receptors. They expressed E- and N-cadherin, α- and β-catenin, EGF receptor, and a specific subset of FGF receptors. Because sphere cells expressed this factor in the absence of exogenous FGF-2, we propose that they are able to synthesize FGF-2.

Knockdown of BACE1-AS Nonprotein-Coding Transcript Modulates Beta-Amyloid-Related Hippocampal Neurogenesis

Background. Alzheimers disease (AD) is a devastating neurological disorder and the main cause of dementia in the elderly population worldwide. Adult neurogenesis appears to be upregulated very early in AD pathogenesis in response to some specific aggregates of beta-amyloid (Aβ) peptides, exhausting the neuronal stem cell pools in the brain. Previously, we characterized a conserved nonprotein-coding antisense transcript for β-secretase-1 (BACE1), a critical enzyme in AD pathophysiology. We showed that the BACE1-antisense transcript (BACE1-AS) is markedly upregulated in brain samples from AD patients and promotes the stability of the (sense) BACE1 transcript. In the current paper, we examine the relationship between BACE1, BACE1-AS, adult neurogenesis markers, and amyloid plaque formation in amyloid precursor protein (APP) transgenic mice (Tg-19959) of various ages. Results. Consistent with previous publications in other APP overexpressing mouse models, we found adult neurogenesis markers to be noticeably upregulated in Tg-19959 mice very early in the development of the disease. Knockdown of either one of BACE1 or BACE1-AS transcripts by continuous infusion of locked nucleic acid- (LNA-) modified siRNAs into the third ventricle over the period of two weeks caused concordant downregulation of both transcripts in Tg-19959 mice. Downregulation of BACE1 mRNA was followed by reduction of BACE1 protein and insoluble Aβ. Modulation of BACE1 and BACE1-AS transcripts also altered oligomeric Aβ aggregation pattern, which was in turn associated with an increase in neurogenesis markers at the RNA and protein level. Conclusion. We found alterations in the RNA and protein concentrations of several adult neurogenesis markers, as well as non-protein-coding BACE1-AS transcripts, in parallel with the course of β-amyloid synthesis and aggregation in the brain of Tg15999 mice. In addition, by knocking down BACE1 or BACE1-AS (thereby reducing Aβ production and plaque deposition), we were able to modulate expression of these neurogenesis markers. Our findings suggest a distortion of adult neurogenesis that is associated with Aβ production very early in amyloid pathogenesis. We believe that these alterations, at the molecular level, could prove useful as novel therapeutic targets and/or as early biomarkers of AD.

Developmental expression of fibroblast growth factor (FGF) receptors in neural stem cell progeny. Modulation of neuronal and glial lineages by basic FGF treatment

Abstract Neural stem cells (NSCs) are self-renewable, multipotential cells capable of differentiating into the three major neural cell types, but the mechanisms which regulate their development are not fully understood. Both basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) promote the proliferation of NSCs. However, studies on the role of FGFs in the differentiation of EGF-expanded NSCs are still incomplete. We have studied the expression of distinct FGF receptors (FGFRs) in the progeny of EGFexpanded NSCs isolated from E15 rat striatum. In situ hybridization analysis and immunocytochemistry showed a developmentally related expression pattern and a cell lineage-specific distribution of these receptors. FGFR1 and FGFR2 were identified in many early precursors and in the oligodendrocyte lineage. The latter receptor was also present in a subpopulation of astrocytes. FGFR3 was detected in a restricted population of early precursors, in oligodendroglial progenitors, and in neurons and protoplasmic astrocytes of late-term cultures. Basic FGF treatment of the progeny of NSCs increased the proliferative rate of precursors and the number of oligodendrocytes generated, whereas the number of differentiating neurons was significantly reduced. Together these data provide evidence that FGFs modulate the development of EGF-expanded NSCs, and that this is at least partly determined by a cell lineage-specific expression of multiple FGFRs. [Neurol Res 2001; 23: 612-621]

RTP801/REDD1 Regulates the Timing of Cortical Neurogenesis and Neuron Migration

The generation, differentiation, and migration of newborn neurons are critical features of normal brain development that are subject to both extracellular and intracellular regulation. However, the means of such control are only partially understood. Here, we show that expression of RTP801/REDD1, an inhibitor of mTOR (mammalian target of rapamycin) activation, is regulated during neuronal differentiation and that RTP801 functions to influence the timing of both neurogenesis and neuron migration. RTP801 levels are high in embryonic cortical neuroprogenitors, diminished in newborn neurons, and low in mature neurons. Knockdown of RTP801 in vitro and in vivo accelerates cell cycle exit by neuroprogenitors and their differentiation into neurons. It also disrupts migration of rat newborn neurons to the cortical plate and results in the ectopic localization of mature neurons. On the other hand, RTP801 overexpression delays neuronal differentiation. These findings suggest that endogenous RTP801 plays an essential role in temporal control of cortical development and in cortical patterning.

Homocysteine inhibits proliferation of neuronal precursors in the mouse adult brain by impairing the basic fibroblast growth factor signaling cascade and reducing extracellular regulated kinase 1/2-dependent cyclin E expression

Hyperhomocysteinemia (HHcy)—abnormally elevated plasma levels of homocysteine (Hcy)—has been associated with the development of neurodegenerative dementia and mild cognitive impairment. This association suggests that HHcy might facilitate memory loss in the elderly. As memory loss can occur through a deteriorated neurogenic capacity, we have studied the effects of Hcy on neural progenitor cells (NPCs) both in vitro and in vivo. We show that Hcy exerts an antiproliferative effect on basic fibroblast growth factor (bFGF) ‐stimulated NPCs isolated from the postnatal subventricular zone (SVZ), accompanied by inactivation of the extracellular signal‐regulated kinase (Erk1/2) and inhibition of Erk1/ 2‐dependent expression of cyclin E. Using a mice model we show that, under normal folate conditions, HHcy exerts an inhibitory effect on adult brain neurogenesis. This inhibition occurs in the caudal areas of the dentate gyrus (DG) of the hippocampus, a neurogenic area mainly involved in learning and memory performance, and in the SVZ, recently implicated in olfactory learning performance. In both areas reduced number of proliferative neuroblasts were found. Since neuroblasts are primarily bFGF‐responsive progenitors already committed to a neuronal phenotype, our results strongly suggest that excess Hcyinhibits neurogenesis in the DG and SVZ byinhibiting the bFGF‐dependent activation of Erk1/2 in these cells.— Rabaneda, L. G., Carrasco, M., Lopez‐Toledano, M. A., Murillo‐Carretero, M., Ruiz, F. A., Estrada, C., Castro, C. Homocysteine inhibits proliferation of neuronal precursors in the mouse adult brain by impairing the basic fibroblast growth factor signaling cascade and reducing extracellular regulated kinase 1/2‐dependent cyclin E expression. FASEB J. 22, 3823–3835 (2008)