Anne Chiaramello

The neurogenic basic helix–loop–helix transcription factor NeuroD6 confers tolerance to oxidative stress by triggering an antioxidant response and sustaining the mitochondrial biomass

Preserving mitochondrial mass, bioenergetic functions and ROS (reactive oxygen species) homoeostasis is key to neuronal differentiation and survival, as mitochondria produce most of the energy in the form of ATP to execute and maintain these cellular processes. In view of our previous studies showing that NeuroD6 promotes neuronal differentiation and survival on trophic factor withdrawal, combined with its ability to stimulate the mitochondrial biomass and to trigger comprehensive antiapoptotic and molecular chaperone responses, we investigated whether NeuroD6 could concomitantly modulate the mitochondrial biomass and ROS homoeostasis on oxidative stress mediated by serum deprivation. In the present study, we report a novel role of NeuroD6 as a regulator of ROS homoeostasis, resulting in enhanced tolerance to oxidative stress. Using a combination of flow cytometry, confocal fluorescence microscopy and mitochondrial fractionation, we found that NeuroD6 sustains mitochondrial mass, intracellular ATP levels and expression of specific subunits of respiratory complexes upon oxidative stress triggered by withdrawal of trophic factors. NeuroD6 also maintains the expression of nuclear-encoded transcription factors, known to regulate mitochondrial biogenesis, such as PGC-1α (peroxisome-proliferator-activated receptor γ co-activator-1α), Tfam (transcription factor A, mitochondrial) and NRF-1 (nuclear respiratory factor-1). Finally, NeuroD6 triggers a comprehensive antioxidant response to endow PC12-ND6 cells with intracellular ROS scavenging capacity. The NeuroD6 effect is not limited to the classic induction of the ROS-scavenging enzymes, such as SOD2 (superoxide dismutase 2), GPx1 (glutathione peroxidase 1) and PRDX5 (peroxiredoxin 5), but also to the recently identified powerful ROS suppressors PGC-1α, PINK1 (phosphatase and tensin homologue-induced kinase 1) and SIRT1. Thus our collective results support the concept that the NeuroD6–PGC-1α–SIRT1 neuroprotective axis may be critical in co-ordinating the mitochondrial biomass with the antioxidant reserve to confer tolerance to oxidative stress.

Constitutive Overexpression of the Basic Helix-Loop-Helix Nex1/MATH-2 Transcription Factor Promotes Neuronal Differentiation of PC12 Cells and Neurite Regeneration

Elucidation of the intricate transcriptional pathways leading to neural differentiation and the establishment of neuronal identity is critical to the understanding and design of therapeutic approaches. Among the important players, the basic helix‐loop‐helix (bHLH) transcription factors have been found to be pivotal regulators of neurogenesis. In this study, we investigate the role of the bHLH differentiation factor Nex1/MATH‐2 in conjunction with the nerve growth factor (NGF) signaling pathway using the rat phenochromocytoma PC12 cell line. We report that the expression of Nex1 protein is induced after 5 hr of NGF treatment and reaches maximal levels at 24 hr, when very few PC12 cells have begun extending neurites and ceased cell division. Furthermore, our study demonstrates that Nex1 has the ability to trigger neuronal differentiation of PC12 cells in the absence of neurotrophic factor. We show that Nex1 plays an important role in neurite outgrowth and has the capacity to regenerate neurite outgrowth in the absence of NGF. These results are corroborated by the fact that Nex1 targets a repertoire of distinct types of genes associated with neuronal differentiation, such as GAP‐43, βIII‐tubulin, and NeuroD. In addition, our findings show that Nex1 up‐regulates the expression of the mitotic inhibitor p21WAF1, thus linking neuronal differentiation to cell cycle withdrawal. Finally, our studies show that overexpression of a Nex1 mutant has the ability to block the execution of NGF‐induced differentiation program, suggesting that Nex1 may be an important effector of the NGF signaling pathway.

Cloning and characterization of the 5'-flanking region of the rat neuron-specific Class III β-tubulin gene

Abstract The promoter regions of several neuron-specific structural proteins (e.g. neurofilaments, peripherin, Tα1-tubulin) have revealed potential regulatory elements that could contribute to the choice of a neuronal phenotype during development. We initiated study of the 5′-flanking region of the rat Class III neuron-specific β-tubulin gene ( βIII-tubulin ) because this gene is expressed at the time of terminal mitosis only in neurons and thus its promoter should be an excellent tool for studying neuron-specific gene expression during the transition from proliferative progenitor cell to early neuronal differentiation. We identified the minimal promoter region needed to drive expression of the βIII-tubulin gene. This minimal region contains multiple putative binding sites for the transcription factors SP1 and AP2, as well as a central nervous system enhancer regulatory element and an E-box. A primer extension analysis identifies a single transcription start site. We highlight several putative regulatory elements that may modulate the expression of the βIII-tubulin gene in a stage- and tissue-specific manner. In addition, we show that the first 490 bp of the promoter are sufficient to regulate βIII-tubulin gene expression during neuronal differentiation of PCC7 cells.

Mitochondrial Biogenesis: A Therapeutic Target for Neurodevelopmental Disorders and Neurodegenerative Diseases

In the developing and mature brain, mitochondria act as central hubs for distinct but interwined pathways, necessary for neural development, survival, activity, connectivity and plasticity. In neurons, mitochondria assume diverse functions, such as energy production in the form of ATP, calcium buffering and generation of reactive oxygen species. Mitochondrial dysfunction contributes to a range of neurodevelopmental and neurodegenerative diseases, making mitochondria a potential target for pharmacological-based therapies. Pathogenesis associated with these diseases is accompanied by an increase in mitochondrial mass, a quantitative increase to overcome a qualitative deficiency due to mutated mitochondrial proteins that are either nuclear- or mitochondrial-encoded. This compensatory biological response is maladaptive, as it fails to sufficiently augment the bioenergetically functional mitochondrial mass and correct for the ATP deficit. Since regulation of neuronal mitochondrial biogenesis has been scantily investigated, our current understanding on the network of transcriptional regulators, co-activators and signaling regulators mainly derives from other cellular systems. The purpose of this review is to present the current state of our knowledge and understanding of the transcriptional and signaling cascades controlling neuronal mitochondrial biogenesis and the various therapeutic approaches to enhance the functional mitochondrial mass in the context of neurodevelopmental disorders and adult-onset neurodegenerative diseases.

The basic helix-loop-helix differentiation factor Nex1/MATH-2 functions as a key activator of the GAP-43 gene.

Nex1/MATH‐2 is a neurogenic basic Helix‐Loop‐Helix (bHLH) transcription factor that belongs to the NeuroD subfamily. Its expression parallels that of the GAP‐43 gene and peaks during brain development, when neurite outgrowth and synaptogenesis are highly active. We previously observed a direct correlation between the levels of expression of Nex1 and GAP‐43 proteins, which resulted in extensive neurite outgrowth and neuronal differentiation of PC12 cells in the absence of nerve growth factor. Since the GAP‐43 gene is a target for bHLH regulation, we investigated whether Nex1 could regulate the activity of the GAP‐43 promoter. We found that among the members of the NeuroD subfamily, Nex1 promoted maximal activity of the GAP‐43 promoter. The Nex1‐mediated activity is restricted to the conserved E1–E2 cluster located near the major transcription start sites. By electrophoretic mobility shift assay and site‐directed mutagenesis, we showed that Nex1 binds as homodimers and that the E1 E‐box is a high affinity binding site. We further found that Nex1 released the ME1 E‐protein‐mediated repression in a concentration dependent manner. Thus, the E1–E2 cluster has a dual function: it can mediate activation or repression depending on the interacting bHLH proteins. Finally, a series of N‐terminal and C‐terminal deletions revealed that Nex1 transcriptional activity is linked to two distinct transactivation domains, TAD1 and TAD2, with TAD1 being unique to Nex1. Together, our results suggest that Nex1 may engage in selective interactions with components of the core transcriptional machinery whose assembly is dictated by the architecture of the GAP‐43 promoter and cellular environment.

5′UTR of the Neurogenic bHLH Nex1/MATH-2/NeuroD6 Gene Is Regulated by Two Distinct Promoters Through CRE and C/EBP Binding Sites

Expression of the bHLH transcription factor Nex1/MATH‐2/NeuroD6, a member of the NeuroD subfamily, parallels overt neuronal differentiation and synaptogenesis during brain development. Our previous studies have shown that Nex1 is a critical effector of the NGF pathway and promotes neuronal differentiation and survival of PC12 cells in the absence of growth factors. In this study, we investigated the transcriptional regulation of the Nex1 gene during NGF‐induced neuronal differentiation. We found that Nex1 expression is under the control of two conserved promoters, Nex1‐P1 and Nex1‐P2, located in two distinct non‐coding exons. Both promoters are TATA‐less with multiple transcription start sites, and are activated on NGF or cAMP exposure. Luciferase‐reporter assays showed that the Nex1‐P2 promoter activity is stronger than the Nex1‐P1 promoter activity, which supports the previously reported differential expression levels of Nex1 transcripts throughout brain development. Using a combination of DNaseI footprinting, EMSA assays, and site‐directed mutagenesis, we identified the essential regulatory elements within the first 2 kb of the Nex1 5′UTR. The Nex1‐P1 promoter is mainly regulated by a conserved CRE element, whereas the Nex1‐P2 promoter is under the control of a conserved C/EBP binding site. Overexpression of wild‐type C/EBPβ resulted in increased Nex1‐P2 promoter activity in NGF‐differentiated PC12 cells. The fact that Nex1 is a target gene of C/EBPβ provides new insight into the C/EBP transcriptional cascade known to promote neurogenesis, while repressing gliogenesis.

Differential expression patterns of the basic helix-loop-helix transcription factors during aging of the murine brain.

In this study, we investigated the expression pattern of the basic Helix-Loop-Helix transcription factors during brain aging. We provide the first evidence that NeuroD and ME2 are differentially expressed during brain aging. Modulation of their expression is specific to distinct areas of the aging brain. NeuroD expression is sustained at high levels in aging cerebellum, whereas it severely declines in aging hippocampus. In contrast, the bHLH E-protein ME2 remains expressed in both aged cerebellum and hippocampus, although at lower levels. These observations support the idea that a shift in the transcriptional dynamics controlling gene expression is associated with the progressive functional decline observed during brain aging.

Expression of the bHLH gene NSCL-1 suggests a role in regulating cerebellar granule cell growth and differentiation.

We report that the neuronal‐specific basic helix‐loop‐helix (bHLH) gene NSCL‐1 is expressed at multiple and distinct stages of cerebellar granule cell differentiation. During embryonic development, NSCI‐1 expression is initially evenly distributed in the cerebellar primordium and then becomes restricted to the ventricular zone. At the early steps of granule cell development, NSCL‐1 is not expressed in rhombic lip cells, but instead in migrating granule cell precursors. Its expression culminates during postnatal proliferation of the external germinal layer, and remains only transiently in the newly formed internal granular layer, and at a much lower level. Thus, NSCL‐1 expression is linked to the onset of granule cell differentiation, but is not involved in the maintenance of the differentiated state. These findings suggest that NSCL‐1 does not behave as a specification factor, but rather as a factor promoting expansion of progenitor external germinal layer (EGL) cells. Gel mobility shift assays show that NSCL‐1 only binds DNA as a heterodimeric complex with the ME1a E‐protein. We also provide the first evidence that NSCL‐1 functions as a transcriptional activator when heterodimerized with the ME1a E‐protein. Taken together, these results suggest that NSCL‐1 participates in the regulatory network controlling gene expression during cerebellar granule cell differentiation. J. Neurosci. Res. 57:770–781, 1999.

NeuroD6 genomic signature bridging neuronal differentiation to survival via the molecular chaperone network.

During neurogenesis, expression of the basic helix‐loop‐helix NeuroD6/Nex1/MATH‐2 transcription factor parallels neuronal differentiation and is maintained in differentiated neurons in the adult brain. To dissect NeuroD6 differentiation properties further, we previously generated a NeuroD6‐overexpressing stable PC12 cell line, PC12‐ND6, which displays a neuronal phenotype characterized by spontaneous neuritogenesis, accelerated NGF‐induced differentiation, and increased regenerative capacity. Furthermore, we reported that NeuroD6 promotes long‐term neuronal survival upon serum deprivation. In this study, we identified the NeuroD6‐mediated transcriptional regulatory pathways linking neuronal differentiation to survival, by conducting a genome‐wide microarray analysis using PC12‐ND6 cells and serum deprivation as a stress paradigm. Through a series of filtering steps and a gene‐ontology analysis, we found that NeuroD6 promotes distinct but overlapping gene networks, consistent with the differentiation, regeneration, and survival properties of PC12‐ND6 cells. By using a gene‐set‐enrichment analysis, we provide the first evidence of a compelling link between NeuroD6 and a set of heat shock proteins in the absence of stress, which may be instrumental in conferring stress tolerance on PC12‐ND6 cells. Immunocytochemistry results showed that HSP27 and HSP70 interact with cytoskeletal elements, consistent with their roles in neuritogenesis and preserving cellular integrity. HSP70 also colocalizes with mitochondria located in the soma, growing neurites, and growth cones of PC12‐ND6 cells prior to and upon stress stimulus, consistent with its neuroprotective functions. Collectively, our findings support the notion that NeuroD6 links neuronal differentiation to survival via the network of molecular chaperones and endows the cells with increased stress tolerance.

The basic helix-loop-helix transcription factor Nex-1/Math-2 promotes neuronal survival of PC12 cells by modulating the dynamic expression of anti-apoptotic and cell cycle regulators

The basic helix‐loop‐helix transcription factor Nex1/Math‐2 belongs to the NeuroD subfamily, which plays a critical role during neuronal differentiation and maintenance of the differentiated state. Previously, we demonstrated that Nex1 is a key regulatory component of the nerve growth factor (NGF) pathway. Further supporting this hypothesis, this study shows that Nex1 has survival‐inducing properties similar to NGF, as Nex1‐overexpressing PC12 cells survive in the absence of trophic factors. We dissected the molecular mechanism by which Nex1 confers neuroprotection upon serum removal and found that constitutive expression of Nex1 maintained the expression of specific G1 phase cyclin‐dependent kinase inhibitors and concomitantly induced a dynamic expression profile of key anti‐apoptotic regulators. This study provides the first evidence of the underlying mechanism by which a member of the NeuroD‐subfamily promotes an active anti‐apoptotic program essential to the survival of neurons. Our results suggest that the survival program may be viewed as an integral component of the intrinsic programming of the differentiated state.