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.

Two promoters for the Escherichia coli operon that contains the four genes dnaA, dnaN, recF, and gyrB were found to be growth rate regulated and under stringent control. Transcript abundance relative to total RNA increased with the growth rate. Changes in transcription from the dnaApl and dnaAp2 promoters that were induced by amino acid starvation and chloramphenicol and were reUl dependent were correlated with the stringent response. The abundance of these transcripts per total RNA also decreased in spoT mutants as the severity of the mutation increased (guanosine 5'-diphosphate 3'-diphosphate [ppGpp] basal levels increased).Because expression of these promoters appears to be inhibited by ppGpp, it is proposed that one mechanism for coupling DNA replication to the growth rate of bacteria is through ppGpp synthesis at the ribosome.The overall rate of DNA replication in Escherichia coli is determined by the initiation frequency at the origin of replication, oriC (12) (27), and the last gene, gyrB, encodes the ,B subunit of DNA gyrase, an enzyme required for maintenance of chromosomal superhelical density (2).Promoter identification studies and deletion analysis of the dnaA operon suggest that the genes in this operon are expressed in a coordinate and independent manner. Of the eight promoters in this operon (see Fig. 1 rRNA synthesis and because rRNA genes are under both growth rate and stringent controls (14,15, 43,44), we examined whether DnaA protein expression might also be under stringent control. The stringent response is induced in bacteria by amino acid starvation and depends on the presence of the relA gene product, (p)ppGpp synthetase I (10). This enzyme is an ATP:GTP pyrophosphoryltransferase that synthesizes the phosphorylated nucleotides guanosine 5'-triphosphate 3'-diphosphate (pppGpp) and guanosine 5'-diphosphate 3'-diphosphate (ppGpp). The purified enzyme is active only when bound to ribosomes which contain bound mRNA and codon-specified uncharged tRNA bound at the acceptor (A) site (18,33). The model that has emerged is that charged/uncharged tRNA ratios, which change in response to shifts in nutrient availability and amino acid starvation, are sensed by cells because uncharged tRNA stimulates the activity of (p)ppGpp synthetase I. This results in changes in pppGpp and ppGpp concentrations that affect expression of many promoters (10), including the promoter for mioC (38,39), which enhances the activity of oriC on plasmids (25). The intracellular concentration of ppGpp is inversely correlated with the activity of the rRNA operon promoters and with the growth rate (6, 45), as well as being directly correlated with the expression of the his and lac promoters (49). To explain these correlations, an RNA polymerase partitioning model has been proposed that suggests that the enzyme exists either bound to ppGpp or unbound and that the two forms of RNA polymerase differ in their promoter affinities (6, 51, 52).Methods that can change the concentration of ppGpp up to 500-fold and that were used in this st...

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, betaIII-tubulin, and NeuroD. In addition, our findings show that Nex1 up-regulates the expression of the mitotic inhibitor p21(WAF1), 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.

Mitochondria play a central role during neurogenesis by providing energy in the form of ATP for cytoskeletal remodelling, outgrowth of neuronal processes, growth cone activity and synaptic activity. However, the fundamental question of how differentiating neurons control mitochondrial biogenesis remains vastly unexplored. Since our previous studies have shown that the neurogenic bHLH (basic helix–loop–helix) transcription factor NeuroD6 is sufficient to induce differentiation of the neuronal progenitor-like PC12 cells and that it triggers expression of mitochondrial-related genes, we investigated whether NeuroD6 could modulate the mitochondrial biomass using our PC12-ND6 cellular paradigm. Using a combination of flow cytometry, confocal microscopy and mitochondrial fractionation, we demonstrate that NeuroD6 stimulates maximal mitochondrial mass at the lamellipodia stage, thus preceding axonal growth. NeuroD6 triggers remodelling of the actin and microtubule networks in conjunction with increased expression of the motor protein KIF5B, thus promoting mitochondrial movement in developing neurites with accumulation in growth cones. Maintenance of the NeuroD6-induced mitochondrial mass requires an intact cytoskeletal network, as its disruption severely reduces mitochondrial mass. The present study provides the first evidence that NeuroD6 plays an integrative role in co-ordinating increase in mitochondrial mass with cytoskeletal remodelling, suggestive of a role of this transcription factor as a co-regulator of neuronal differentiation and energy metabolism.

The GAP-43 promoter region contains seven E-boxes (E1 to E7) that are organized in two clusters, a distal cluster (E3 to E7) and a proximal cluster (E1 and E2). Deletion analysis and site-directed mutagenesis of the GAP-43 promoter region showed that only the most proximal E1 E-box significantly modulates GAP-43 promoter activity. This E-box is conserved between the rat and human GAP-43 promoter sequences in terms of flanking sequence, core sequence (CAGTTG), and position. We found that endogenous E-box-binding proteins present in neuronal N18 cells recognize the E1 E-box and activate the GAP-43 promoter. The transcriptional activity of the GAP-43 promoter was repressed not only by the negative regulator Id2 protein, but also by two class A basic helix-loop-helix proteins, E12 and ME1a. In vitro analyses showed that both ME1a and E12 bind to the E1 E-box as homodimers. By Northern analyses, we established an inverse correlation between the level of E12 and ME1a mRNAs and GAP-43 mRNA in various neuronal cell lines as well as in ME1a-overexpressing PC12 cells. Therefore, we have identified a cis-acting element, the E1 E-box, located in the GAP-43 promoter region that modulates either positively or negatively the expression of the GAP-43 gene depending on which E-box-binding proteins occupy this site. Together, these data indicate that basic helix-loop-helix transcription factors regulate the expression of the GAP-43 gene and that the class A ME1a and E12 proteins act as down-regulators of GAP-43 expression.

In this study, we focused on the potential function of the murine gene Tcf12 (also known as ME1 or HEB) encoding the bHLH E-protein ME1 during brain development. An exencephaly phenotype of low penetrance has consistently been observed in both Tcf12 null mice and Tcf12(dm) homozygous mice. Thus, to address the possible underlying mechanism of the Tcf12 gene during the early steps of brain development, we performed a detailed analysis of its spatio-temporal expression pattern at distinct steps of gastrulation and neurogenesis. We found that Tcf12 transcripts are detected in the embryonic ectoderm prior to neural induction during gastrulation. During neurulation, Tcf12 transcripts are evident at high levels in the proliferating neuroepithelium of the neural folds and the cephalic mesenchyme. Thus, Tcf12 gene expression coincides with the massive proliferation occurring in the forming neuroepithelium and cephalic mesenchyme during neural tube formation, which is consistent with the exencephaly phenotype of Tcf12 null mice. In the developing cortex and spinal cord, Tcf12 expression is restricted to the proliferative ventricular zones, indicating that Tcf12 expression is down regulated when these neuronal cells undergo their final differentiation. Interestingly, we found that the postnatal Tcf12 expression parallels the ongoing adult neurogenesis in the mitotically active subventricular zone. Thus, the timing and location of Tcf12 expression combined with this severe neurulation defect support our hypothesis that the Tcf12 gene may be involved in the control of proliferating neural stem cells and progenitor cells and that it may be critical to sustain their undifferentiated state during embryonic and adult neurogenesis.

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.

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 further dissect NeuroD6 differentiation properties, 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. 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 to confer stress tolerance to 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.