Redox-Mediated Rewiring of Signalling Pathways: The Role of a Cellular Clock in Brain Health and Disease.

Metazoan signalling pathways can be rewired to dampen or amplify the rate of events, such as those that occur in development and aging. Given that a linear network topology restricts the capacity to rewire signalling pathways, such scalability of the pace of biological events suggests the existence of programmable non-linear elements in the underlying signalling pathways. Here, we review the network topology of key signalling pathways with a focus on redox-sensitive proteins, including PTEN and Ras GTPase, that reshape the connectivity profile of signalling pathways in response to an altered redox state. While this network-level impact of redox is achieved by the modulation of individual redox-sensitive proteins, it is the population by these proteins of critical nodes in a network topology of signal transduction pathways that amplifies the impact of redox-mediated reprogramming. We propose that redox-mediated rewiring is essential to regulate the rate of transmission of biological signals, giving rise to a programmable cellular clock that orchestrates the pace of biological phenomena such as development and aging. We further review the evidence that an aberrant redox-mediated modulation of output of the cellular clock contributes to the emergence of pathological conditions affecting the human brain.

Cellular self-organization: An overdrive in Cambrian diversity?

During the early Cambrian period metazoan life forms diverged at an accelerated rate to occupy multiple ecological niches on earth. A variety of explanations have been proposed to address this major evolutionary phenomenon termed the "Cambrian explosion." While most hypotheses address environmental, developmental, and ecological factors that facilitated evolutionary innovations, the biological basis for accelerated emergence of species diversity in the Cambrian period remains largely conjectural. Herein, we posit that morphogenesis by self-organization enables the uncoupling of genomic mutational landscape from phenotypic diversification. Evidence is provided for a two-tiered interpretation of genomic changes in metazoan animals wherein mutations not only impact upon function of individual cells, but also alter the self-organization outcome during developmental morphogenesis. We provide evidence that the morphological impacts of mutations on self-organization could remain repressed if associated with an unmet negative energetic cost. We posit that accelerated morphological diversification in transition to the Cambrian period has occurred by emergence of dormant (i.e., reserved) morphological novelties whose molecular underpinnings were seeded in the Precambrian period.

Notch ankyrin domain: evolutionary rise of a thermodynamic sensor.

Notch signalling pathway plays a key role in metazoan biology by contributing to resolution of binary decisions in the life cycle of cells during development. Outcomes such as proliferation/differentiation dichotomy are resolved by transcriptional remodelling that follows a switch from Notchon to Notchoff state, characterised by dissociation of Notch intracellular domain (NICD) from DNA-bound RBPJ. Here we provide evidence that transitioning to the Notchoff state is regulated by heat flux, a phenomenon that aligns resolution of fate dichotomies to mitochondrial activity. A combination of phylogenetic analysis and computational biochemistry was utilised to disclose structural adaptations of Notch1 ankyrin domain that enabled function as a sensor of heat flux. We then employed DNA-based micro-thermography to measure heat flux during brain development, followed by analysis in vitro of the temperature-dependent behaviour of Notch1 in mouse neural progenitor cells. The structural capacity of NICD to operate as a thermodynamic sensor in metazoans stems from characteristic enrichment of charged acidic amino acids in ß-hairpins of the ankyrin domain that amplify destabilising inter-residue electrostatic interactions and render the domain thermolabile. The instability emerges upon mitochondrial activity which raises the perinuclear and nuclear temperatures to 50 °C and 39 °C, respectively, leading to destabilization of Notch1 transcriptional complex and transitioning to the Notchoff state. Notch1 functions a metazoan thermodynamic sensor that is switched on by intercellular contacts, inputs heat flux as a proxy for mitochondrial activity in the Notchon state via the ankyrin domain and is eventually switched off in a temperature-dependent manner. Video abstract.

Neural pericytes: A genomic archival state programmed by CHromatin topology.

Accumulating evidence suggests that mural pericytes, apart from stabilizing the associated microvessels, play additional roles in regeneration of local cellular elements. Herein, the mechanistic basis for such diverse and at times contradictory roles adopted by pericytes in the brain is reviewed. Core concepts of an emerging model are discussed wherein mural pericytes reside in a metastable "archival state" that conceals a neural progenitor identity. Upon angiogenic remodeling, a selected subpopulation of pericytes reclaim the progenitor state during transdifferentiation and contribute to neural regeneration. The genomic basis for neural transdifferentiation of pericytes is reviewed with reference to the extant literature.

Programmed genomic instability regulates neural transdifferentiation of human brain microvascular pericytes.

BACKGROUND: Transdifferentiation describes transformation in vivo of specialized cells from one lineage into another. While there is extensive literature on forced induction of lineage reprogramming in vitro, endogenous mechanisms that govern transdifferentiation remain largely unknown. The observation that human microvascular pericytes transdifferentiate into neurons provided an opportunity to explore the endogenous molecular basis for lineage reprogramming. RESULTS: We show that abrupt destabilization of the higher-order chromatin topology that chaperones lineage memory of pericytes is driven by transient global transcriptional arrest. This leads within minutes to localized decompression of the repressed competing higher-order chromatin topology and expression of pro-neural genes. Transition to neural lineage is completed by probabilistic induction of R-loops in key myogenic loci upon re-initiation of RNA polymerase activity, leading to depletion of the myogenic transcriptome and emergence of the neurogenic transcriptome. CONCLUSIONS: These findings suggest that the global transcriptional landscape not only shapes the functional cellular identity of pericytes, but also stabilizes lineage memory by silencing the competing neural program within a repressed chromatin state.

The fate of notch-1 transcript is linked to cell cycle dynamics by activity of a natural antisense transcript.

A core imprint of metazoan life is that perturbations of cell cycle are offset by compensatory changes in successive cellular generations. This trait enhances robustness of multicellular growth and requires transmission of signaling cues within a cell lineage. Notably, the identity and mode of activity of transgenerational signals remain largely unknown. Here we report the discovery of a natural antisense transcript encoded in exon 25 of notch-1 locus (nAS25) by which mother cells control the fate of notch-1 transcript in daughter cells to buffer against perturbations of cell cycle. The antisense transcript is transcribed at G1 phase of cell cycle from a bi-directional E2F1-dependent promoter in the mother cell where the titer of nAS25 is calibrated to the length of G1. Transmission of the antisense transcript from mother to daughter cells stabilizes notch-1 sense transcript in G0 phase of daughter cells by masking it from RNA editing and resultant nonsense-mediated degradation. In consequence, nAS25-mediated amplification of notch-1 signaling reprograms G1 phase in daughter cells to compensate for the altered dynamics of the mother cell. The function of nAS25/notch-1 in integrating G1 phase history of the mother cell into that of daughter cells is compatible with the predicted activity of a molecular oscillator, slower than cyclins, that coordinates cell cycle within cell lineage.

Cannibalized erythroblasts accelerate developmental neurogenesis by regulating mitochondrial dynamics.

Metabolic support was long considered to be the only developmental function of hematopoiesis, a view that is gradually changing. Here, we disclose a mechanism triggered during neurulation that programs brain development by donation of sacrificial yolk sac erythroblasts to neuroepithelial cells. At embryonic day (E) 8.5, neuroepithelial cells transiently integrate with the endothelium of yolk sac blood vessels and cannibalize intravascular erythroblasts as transient heme-rich endosymbionts. This cannibalistic behavior instructs precocious neuronal differentiation of neuroepithelial cells in the proximity of blood vessels. By experiments in vitro, we show that access to erythroblastic heme accelerates the pace of neurogenesis by induction of a truncated neurogenic differentiation program from a poised state. Mechanistically, the poised state is invoked by activation of the mitochondrial electron transport chain that leads to amplified production of reactive oxygen species in addition to omnipresent guanosine triphosphate (GTP) with consequential upregulation of pro-differentiation ß-catenin.

Genomic competition for noise reduction shaped evolutionary landscape of mir-4673.

The genomic platform that informs evolution of microRNA cascades remains unknown. Here we capitalised on the recent evolutionary trajectory of hominin-specific miRNA-4673, encoded in intron 4 of notch-1, to uncover the identity of one such precursor genomic element and the selective forces acting upon it. The miRNA targets genes that regulate Wnt/ß-catenin signalling cascade. Primary sequence of the microRNA and its target region in Wnt modulating genes evolved from homologous signatures mapped to homotypic cis-clusters recognised by TCF3/4 and TFAP2A/B/C families. Integration of homologous TFAP2A/B/C cis-clusters (short range inhibitor of ß-catenin) into the transcriptional landscape of Wnt cascade genes can reduce noise in gene expression. Probabilistic adoption of miRNA secondary structure by one such cis-signature in notch-1 reflected selection for superhelical curvature symmetry of precursor DNA to localise a nucleosome that overlapped the latter cis-cluster. By replicating the cis-cluster signature, non-random interactions of the miRNA with key Wnt modulator genes expanded the transcriptional noise buffering capacity via a coherent feed-forward loop mechanism. In consequence, an autonomous transcriptional noise dampener (the cis-cluster/nucleosome) evolved into a post-transcriptional one (the miRNA). The findings suggest a latent potential for remodelling of transcriptional landscape by miRNAs that capitalise on non-random distribution of genomic cis-signatures.

Coupled cycling and regulation of metazoan morphogenesis.

Metazoan animals are characterized by restricted phenotypic heterogeneity (i.e. morphological disparity) of organisms within various species, a feature that contrasts sharply with intra-species morphological diversity observed in the plant kingdom. Robust emergence of morphogenic blueprint in metazoan animals reflects restricted autonomy of individual cells in adoption of fate outcomes such as differentiation. Fates of individual cells are linked to and influenced by fates of neighboring cells at the population level. Such coupling is a common property of all self-organising systems and propels emergence of order from simple interactions between individual cells without supervision by external directing forces. As a consequence of coupling, expected functional relationship between the constituent cells of an organ system is robustly established concurrent with multiple rounds of cell division during morphogenesis. Notably, the molecular regulation of multicellular coupling during morphogenic self-organisation remains largely unexplored. Here, we review the existing literature on multicellular self-organisation with particular emphasis on recent discovery that ß-catenin is the key coupling factor that programs emergence of multi-cellular self-organisation by regulating synchronised cycling of individual cells.

ß-Catenin: A Metazoan Filter for Biological Noise?

Molecular noise refers to fluctuations of biological signals that facilitate phenotypic heterogeneity in a population. While endogenous mechanisms exist to limit genetic noise in biological systems, such restrictions are sometimes removed to propel phenotypic variability as an adaptive strategy. Herein, we review evidence for the potential role of ß-catenin in restricting gene expression noise by transcriptional and post-transcriptional mechanisms. We discuss mechanisms that restrict intrinsic noise subsequent to nuclear mobilization of ß-catenin. Nuclear ß-catenin promotes initiation of transcription but buffers against the resultant noise by restraining transcription elongation. Acceleration of cell cycle, mediated via Wnt/ß-catenin downstream signals, further diminishes intrinsic noise by curtailing the efficiency of protein synthesis. Extrinsic noise, on the other hand, is restricted by ß-catenin-mediated regulation of major cellular stress pathways.

Notch pathway: a bistable inducer of biological noise?

Notch signalling pathway is central to development of metazoans. The pathway codes a binary fate switch. Upon activation, downstream signals contribute to resolution of fate dichotomies such as proliferation/differentiation or sub-lineage differentiation outcome. There is, however, an interesting paradox in the Notch signalling pathway. Despite remarkable predictability of fate outcomes instructed by the Notch pathway, the associated transcriptome is versatile and plastic. This inconsistency suggests the presence of an interface that compiles input from the plastic transcriptome of the Notch pathway but communicates only a binary output in biological decisions. Herein, we address the interface that determines fate outcomes. We provide an alternative hypothesis for the Notch pathway as a biological master switch that operates by induction of genetic noise and bistability in order to facilitate resolution of dichotomous fate outcomes in development.

Coupled cycling programs multicellular self-organization of neural progenitors.

Self-organization is central to the morphogenesis of multicellular organisms. However, the molecular platform that coordinates the robust emergence of complex morphological patterns from local interactions between cells remains unresolved. Here we demonstrate that neural self- organization is driven by coupled cycling of progenitor cells. In a coupled cycling mode, intercellular contacts relay extrinsic cues to override the intrinsic cycling rhythm of an individual cell and synchronize the population. The stringency of coupling and hence the synchronicity of the population is programmed by recruitment of a key coupler, ß-catenin, into junctional complexes. As such, multicellular self-organization is driven by the same basic mathematical principle that governs synchronized behavior of macro-scale biological systems as diverse as the synchronized chirping of crickets, flashing of fireflies and schooling of fish; that is synchronization by coupling. It is proposed that coupled cycling foreshadows a fundamental adaptive change that facilitated evolution and diversification of multicellular life forms.

Evolution of Resistance in Cancer: A Cell Cycle Perspective.

Resistance of neoplastic cells to therapy is considered a key challenge in the treatment of cancer. Emergence of resistance is commonly attributed to the gradual mutational evolution of neoplastic cells. However, accumulating evidence suggests that exogenous stressors could significantly accelerate the emergence of resistant clones during the course of treatment. Herein, we review molecular mechanisms that regulate the evolution of resistance in a tumor with particular emphasis on the role of cell cycle.

Neural microvascular pericytes contribute to human adult neurogenesis.

Consistent adult neurogenic activity in humans is observed in specific niches within the central nervous system. However, the notion of an adult neurogenic niche is challenged by accumulating evidence for ectopic neurogenic activity in other cerebral locations. Herein we interface precision of ultrastructural resolution and anatomical simplicity of accessible human dental pulp neurogenic zone to address this conflict. We disclose a basal level of adult neurogenic activity characterized by glial invasion of terminal microvasculature followed by release of individual platelet-derived growth factor receptor-ß mural pericytes and subsequent reprogramming into NeuN+ local interneurons. Concomitant angiogenesis, a signature of adult neurogenic niches, accelerates the rate of neurogenesis by amplifying release and proliferation of the mural pericyte population by ≈10-fold. Subsequent in vitro and in vivo experiments confirmed gliogenic and neurogenic capacities of human neural pericytes. Findings foreshadow the bimodal nature of the glio-vascular assembly where pericytes, under instruction from glial cells, can stabilize the quiescent microvasculature or enrich local neuronal microcircuits upon differentiation.

miR4673 improves fitness profile of neoplastic cells by induction of autophagy.

Therapeutic resistance of neoplasms is mainly attributed to gradual evolution of mutational profile1. Here, we demonstrate a microRNA-mediated mechanism that effectively improves fitness of SKBR3 mammary carcinoma cells by cytoplasmic reprogramming. The reprogramming is triggered by endogenous miR4673 transcribed from notch-1 locus. The miRNA downregulates cdk-18, a cyclin-dependent kinase that regulates M-G1 transition in cycling cells2,3. Suppression of cdk-18 triggers mitophagy and autophagy. Due to high autophagic flux, oestrogen receptor-1+/progesterone receptor+/p53+ (Esr1+/Pr+/p53+) SKBR3 cells are coerced into an Esr1-/Prlow/p53-profile. Increased mitophagy in combination with proteasomal degradation of p53 transiently arrests the cycling cells at G0 and enhances radio-resistance of the SKBR3 population. These findings highlight the impact on cancer therapy of non-encoded neoplastic resistance, arising as a consequence of miRNA-mediated autophagic reprogramming that uncouples phenotype and genotype.

A method for investigating the cellular response to cyclic tension or compression in three-dimensional culture.

We have an interest in the cellular response to mechanical stimuli, and here describe an in-vitro method to examine the response of cells cultured in a three-dimensional matrix to mechanical compressive and tensile stress. Synthetic aliphatic polyester scaffolds coated with 45S5 bioactive glass were seeded with human dental follicular cells (HDFC), and attached to well inserts and magnetic endplates in six well palates. Scaffolds were subjected to either cyclic 10% tensile deformation, or 8% compression, at 1 Hz and 2 Hz respectively for 6, 24 or 48 h, by uniaxial motion of magnetically-coupled endplates. It was possible to isolate high quality mRNA from cells in these scaffolds, as demonstrated by high RNA integrity numbers scores, and ability to perform meaningful cRNA microarray analysis, in which 669 and 727 genes were consistently upregulated, and 662 and 518 genes down regulated at all times studied under tensile and compressive loading conditions respectively. MetaCore analysis revealed the most regulated gene ontogenies under both loading conditions to be for: cytoskeletal remodelling; cell adhesion-chemokines and adhesion; cytoskeleton remodelling-TGF WNT and cytoskeletal remodelling pathways. We believe the method here described will be of value for analysis of the cellular response to cyclic loading.

Characterization of inter-crystallite peptides in human enamel rods reveals contribution by the Y allele of amelogenin.

Proteins of the inter-rod sheath and peptides within the narrow inter-crystallite space of the rod structure are considered largely responsible for visco-elastic and visco-plastic properties of enamel. The present study was designed to investigate putative peptides of the inter-crystallite space. Entities of 1-6 kDa extracted from enamel rods of erupted permanent teeth were analysed by mass spectrometry (MS) and shown to comprise N-terminal amelogenin (AMEL) peptides either containing or not containing exon 4 product. Other dominant entities consisted of an N-terminal peptide from ameloblastin (AMBN) and a series of the most hydrophobic peptides from serum albumin (ALBN). Amelogenin peptides encoded by the Y-chromosome allele were strongly detected in Enamel from male teeth. Location of N-terminal AMEL peptides as well as AMBN and ALBN, between apatite crystallites, was disclosed by immunogold scanning electron microscopy (SEM). Density plots confirmed the relative abundance of these products including exon 4+ AMEL peptides that have greater capacity for binding to hydroxyapatite. Hydrophilic X and Y peptides encoded in exon 4 differ only in substitution of non-polar isoleucine in Y for polar threonine in X with reduced disruption of the hydrophobic N-terminal structure in the Y form. Despite similarity of X and Y alleles of AMEL the non-coding region upstream from exon 4 shows significant variation with implications for segregation of processing of transcripts from exon 4. Detection of fragments from multiple additional proteins including keratins (KER), fetuin A (FETUA), proteinases and proteinase inhibitors, likely reflect biochemical events during enamel formation.

Uncovering system-specific stress signatures in primate teeth with multimodal imaging.

Early life stress can disrupt development and negatively impact long-term health trajectories. Reconstructing histories of early life exposure to external stressors is hampered by the absence of retrospective time-specific biomarkers. Defects in tooth enamel have been used to reconstruct stress but the methods used are subjective and do not identify the specific biological systems impacted by external stressors. Here we show that external physical and social stressors impart biochemical signatures in primate teeth that can be retrieved to objectively reconstruct the timing of early life developmental disruptions. Using teeth from captive macaques, we uncovered elemental imprints specific to disruptions of skeletal growth, including major disruptions in body weight trajectory and moderate to severe illnesses. Discrete increases in heat shock protein-70 expression in dentine coincided with elemental signatures, confirming that elemental signals were associated with activation of stress-related pathways. To overcome limitations of conventional light-microscopic analysis, we used high resolution Raman microspectral imaging to identify structural and compositional alterations in enamel and dentine that coincided with elemental signatures and with detailed medical and behavioural data. Integrating these objective biochemical markers with temporal mapping of teeth enables the retrospective study of early life developmental disruptions and their ensuing health sequelae.

Platelet-Derived Growth Factor Receptor Alpha as a Marker of Mesenchymal Stem Cells in Development and Stem Cell Biology.

Three decades on, the mesenchymal stem cells (MSCs) have been intensively researched on the bench top and used clinically. However, ambiguity still exists in regard to their anatomical locations, identities, functions, and extent of their differentiative abilities. One of the major impediments in the quest of the MSC research has been lack of appropriate in vivo markers. In recent years, this obstacle has been resolved to some degree as PDGFRα emerges as an important mesenchymal stem cell marker. Accumulating lines of evidence are showing that the PDGFRα (+) cells reside in the perivascular locations of many adult interstitium and fulfil the classic concepts of MSCs in vitro and in vivo. PDGFRα has long been recognised for its roles in the mesoderm formation and connective tissue development during the embryogenesis. Current review describes the lines of evidence regarding the role of PDGFRα in morphogenesis and differentiation and its implications for MSC biology.

Directed glia-assisted angiogenesis in a mature neurosensory structure: pericytes mediate an adaptive response in human dental pulp that maintains blood-barrier function.

The specialized tightly controlled microcirculation of craniofacial neurosensory organs is an essential evolutionary adaptation and yet a dilemma where angiogenic remodeling occurs. Despite extreme plasticity of neurosensory structures, the capacity to reconcile barrier phenotype of the microcirculation with an angiogenic cascade is not known. Here we provide primary evidence for such a response in an elemental neurosensory structure, human dental pulp, following chronic carious insult. In response to hypoxic challenge neurosensory odontoblasts express hypoxia-inducible factor-1α and notch-1. Associated radial rearrangement of astrocyte-like telacytes that communicate through a cell-poor zone with the microvasculature is observed. Activated pericytes characterized by expression of α-smooth muscle actin are located adjacent to the telacyte attachment to the vasculature. In this location, endothelial expression of sonic hedgehog parallels expression of notch-1 by pericytes. The angiogenic response is initiated by pericyte contraction and altered endothelial polarity and proliferation leading to intussusception of endothelial cells and extensive remodeling of basement membrane with upregulation of laminin-8 and laminin-5. These responses guide intravascular loop formation that maintains both intact basement membrane and tight junctions. This initial phase is followed by formation of anastomoses that enhance the hemodynamic capacity of the intravascular loops. The formation of anastomoses is mediated by extension of cytonemes from pericytes guided by MHC-II(+)/CD-163(+) microglia aligned with the telacytes. The cytonemes seek out pericytes on adjacent intravascular loops to initiate migration of endothelial cells. These findings support a new paradigm for understanding angiogenic capacity of neurosensory structures and aberrations of this response manifest as neurovasculopathies.