Biochemistry and Biology of GDF11 and Myostatin: Similarities, Differences, and Questions for Future Investigation.

Growth differentiation factor 11 (GDF11) and myostatin (or GDF8) are closely related members of the transforming growth factor ß superfamily and are often perceived to serve similar or overlapping roles. Yet, despite commonalities in protein sequence, receptor utilization and signaling, accumulating evidence suggests that these 2 ligands can have distinct functions in many situations. GDF11 is essential for mammalian development and has been suggested to regulate aging of multiple tissues, whereas myostatin is a well-described negative regulator of postnatal skeletal and cardiac muscle mass and modulates metabolic processes. In this review, we discuss the biochemical regulation of GDF11 and myostatin and their functions in the heart, skeletal muscle, and brain. We also highlight recent clinical findings with respect to a potential role for GDF11 and/or myostatin in humans with heart disease. Finally, we address key outstanding questions related to GDF11 and myostatin dynamics and signaling during development, growth, and aging.

Neuronal control of locomotor handedness in Drosophila.

Genetically identical individuals display variability in their physiology, morphology, and behaviors, even when reared in essentially identical environments, but there is little mechanistic understanding of the basis of such variation. Here, we investigated whether Drosophila melanogaster displays individual-to-individual variation in locomotor behaviors. We developed a new high-throughout platform capable of measuring the exploratory behavior of hundreds of individual flies simultaneously. With this approach, we find that, during exploratory walking, individual flies exhibit significant bias in their left vs. right locomotor choices, with some flies being strongly left biased or right biased. This idiosyncrasy was present in all genotypes examined, including wild-derived populations and inbred isogenic laboratory strains. The biases of individual flies persist for their lifetime and are nonheritable: i.e., mating two left-biased individuals does not yield left-biased progeny. This locomotor handedness is uncorrelated with other asymmetries, such as the handedness of gut twisting, leg-length asymmetry, and wing-folding preference. Using transgenics and mutants, we find that the magnitude of locomotor handedness is under the control of columnar neurons within the central complex, a brain region implicated in motor planning and execution. When these neurons are silenced, exploratory laterality increases, with more extreme leftiness and rightiness. This observation intriguingly implies that the brain may be able to dynamically regulate behavioral individuality.

Behavioral idiosyncrasy reveals genetic control of phenotypic variability.

Quantitative genetics has primarily focused on describing genetic effects on trait means and largely ignored the effect of alternative alleles on trait variability, potentially missing an important axis of genetic variation contributing to phenotypic differences among individuals. To study the genetic effects on individual-to-individual phenotypic variability (or intragenotypic variability), we used Drosophila inbred lines and measured the spontaneous locomotor behavior of flies walking individually in Y-shaped mazes, focusing on variability in locomotor handedness, an assay optimized to measure variability. We discovered that some lines had consistently high levels of intragenotypic variability among individuals, whereas lines with low variability behaved as although they tossed a coin at each left/right turn decision. We demonstrate that the degree of variability is itself heritable. Using a genome-wide association study (GWAS) for the degree of intragenotypic variability as the phenotype across lines, we identified several genes expressed in the brain that affect variability in handedness without affecting the mean. One of these genes, Ten-a, implicates a neuropil in the central complex of the fly brain as influencing the magnitude of behavioral variability, a brain region involved in sensory integration and locomotor coordination. We validated these results using genetic deficiencies, null alleles, and inducible RNAi transgenes. Our study reveals the constellation of phenotypes that can arise from a single genotype and shows that different genetic backgrounds differ dramatically in their propensity for phenotypic variabililty. Because traditional mean-focused GWASs ignore the contribution of variability to overall phenotypic variation, current methods may miss important links between genotype and phenotype.

Recovery of locomotion after injury in Drosophila melanogaster depends on proprioception.

Locomotion is necessary for survival in most animal species. However, injuries to the appendages mediating locomotion are common. We assess the recovery of walking in Drosophila melanogaster following leg amputation. Whereas flies pre-amputation explore open arenas in a symmetric fashion on average, foreleg amputation induces a strong turning bias away from the side of the amputation. However, we find that unbiased walking behavior returns over time in wild-type flies, while recovery is significantly impaired in proprioceptive mutants. To identify the biomechanical basis of this locomotor impairment and recovery, we then examine individual leg motion (gait) at a fine scale. A minimal mathematical model that links neurodynamics to body mechanics during walking shows that redistributing leg forces between the right and left side enables the observed recovery. Altogether, our study suggests that proprioceptive input from the intact limbs plays a crucial role in the behavioral plasticity associated with locomotor recovery after injury.

Proteolytic processing of protocadherin proteins requires endocytosis.

The α-, ß-, and γ-protocadherins (Pcdhα, Pcdhß, and Pcdhγ) comprise a large family of single-pass transmembrane proteins predominantly expressed in the nervous system. These proteins contain six cadherin-like extracellular domains, and proteolysis of Pcdhα and Pcdhγ by the γ-secretase complex releases their intracellular domains into the cytoplasm where they may function locally and/or enter the nucleus and affect gene expression. Thus, cleavage of Pcdhs may function to link intercellular contacts and intracellular signaling. Here we report that shedding of the Pcdhα extracellular domain and subsequent processing by γ-secretase require endocytosis and that Pcdhs interact with the regulator of vesicular sorting ESCRT-0 in undifferentiated cells. We also find that the accumulation of Pcdh cleavage products is regulated during development. Differentiation leads to an increase in the interactions between Pcdh proteins and a decrease in the accumulation of cleavage products. We conclude that Pcdh processing requires endocytosis and that the level of cleavage products is regulated during neuronal differentiation.

Phosphorylation of protocadherin proteins by the receptor tyrosine kinase Ret.

The clustered protocadherins (Pcdhs) are a large family of cadherin-like transmembrane proteins expressed in the nervous system. Stochastic expression of Pcdh genes and alternative splicing of their pre-mRNAs have the potential to generate enormous protein diversity at the cell surface of neurons. At present, the regulation and function of Pcdh proteins are largely unknown. Here, we show that Pcdhs form a heteromeric signaling complex(es), consisting of multiple Pcdh isoforms, receptor tyrosine kinases, phosphatases, and cell adhesion molecules. In particular, we find that the receptor tyrosine kinase rearranged during transformation (Ret) binds to Pcdhs in differentiated neuroblastoma cells and is required for stabilization and differentiation-induced phosphorylation of Pcdh proteins. In addition, the Ret ligand glial cell line-derived neurotrophic factor induces phosphorylation of Pcdhgamma in motor neurons and phosphorylation of Pcdhalpha and Pcdhgamma in sympathetic neurons. Conversely, Pcdh proteins are also required for the stabilization of activated Ret in neuroblastoma cells and sympathetic ganglia. Thus, Pcdhs and Ret are functional components of a phosphorylation-dependent signaling complex.

Heterochronic parabiosis reprograms the mouse brain transcriptome by shifting aging signatures in multiple cell types.

Aging is a complex process involving transcriptomic changes associated with deterioration across multiple tissues and organs, including the brain. Recent studies using heterochronic parabiosis have shown that various aspects of aging-associated decline are modifiable or even reversible. To better understand how this occurs, we performed single-cell transcriptomic profiling of young and old mouse brains after parabiosis. For each cell type, we cataloged alterations in gene expression, molecular pathways, transcriptional networks, ligand-receptor interactions and senescence status. Our analyses identified gene signatures, demonstrating that heterochronic parabiosis regulates several hallmarks of aging in a cell-type-specific manner. Brain endothelial cells were found to be especially malleable to this intervention, exhibiting dynamic transcriptional changes that affect vascular structure and function. These findings suggest new strategies for slowing deterioration and driving regeneration in the aging brain through approaches that do not rely on disease-specific mechanisms or actions of individual circulating factors.

GDF11 expressed in the adult brain negatively regulates hippocampal neurogenesis.

Growth differentiation factor 11 (GDF11) is a transforming factor-ß superfamily member that functions as a negative regulator of neurogenesis during embryonic development. However, when recombinant GDF11 (rGDF11) is administered systemically in aged mice, it promotes neurogenesis, the opposite of its role during development. The goal of the present study was to reconcile this apparent discrepancy by performing the first detailed investigation into the expression of endogenous GDF11 in the adult brain and its effects on neurogenesis. Using quantitative histological analysis, we observed that Gdf11 is most highly expressed in adult neurogenic niches and non-neurogenic regions within the hippocampus, choroid plexus, thalamus, habenula, and cerebellum. To investigate the role of endogenous GDF11 during adult hippocampal neurogenesis, we generated a tamoxifen inducible mouse that allowed us to reduce GDF11 levels. Depletion of Gdf11 during adulthood increased proliferation of neural progenitors and decreased the number of newborn neurons in the hippocampus, suggesting that endogenous GDF11 remains a negative regulator of hippocampal neurogenesis in adult mice. These findings further support the idea that circulating systemic GDF11 and endogenously expressed GDF11 in the adult brain have different target cells or mechanisms of action. Our data describe a role for GDF11-dependent signaling in adult neurogenesis that has implications for how GDF11 may be used to treat CNS disease.

Pro-myogenic small molecules revealed by a chemical screen on primary muscle stem cells.

Satellite cells are the canonical muscle stem cells that regenerate damaged skeletal muscle. Loss of function of these cells has been linked to reduced muscle repair capacity and compromised muscle health in acute muscle injury and congenital neuromuscular diseases. To identify new pathways that can prevent loss of skeletal muscle function or enhance regenerative potential, we established an imaging-based screen capable of identifying small molecules that promote the expansion of freshly isolated satellite cells. We found several classes of receptor tyrosine kinase (RTK) inhibitors that increased freshly isolated satellite cell numbers in vitro. Further exploration of one of these compounds, the RTK inhibitor CEP-701 (also known as lestaurtinib), revealed potent activity on mouse satellite cells both in vitro and in vivo. This expansion potential was not seen upon exposure of proliferating committed myoblasts or non-myogenic fibroblasts to CEP-701. When delivered subcutaneously to acutely injured animals, CEP-701 increased both the total number of satellite cells and the rate of muscle repair, as revealed by an increased cross-sectional area of regenerating fibers. Moreover, freshly isolated satellite cells expanded ex vivo in the presence of CEP-701 displayed enhanced muscle engraftment potential upon in vivo transplantation. We provide compelling evidence that certain RTKs, and in particular RET, regulate satellite cell expansion during muscle regeneration. This study demonstrates the power of small molecule screens of even rare adult stem cell populations for identifying stem cell-targeting compounds with therapeutic potential.

Growth differentiation factor 11 (GDF11) has pronounced effects on skin biology.

Growth differentiation factor 11 (GDF11) belongs to the TGF-ß superfamily of proteins and is closely related to myostatin. Recent findings show that GDF11 has rejuvenating properties with pronounced effects on the cardiovascular system, brain, skeletal muscle, and skeleton in mice. Several human studies were also conducted, some implicating decreasing levels of circulating GDF11 with age. To date, however, there have not been any reports on its role in human skin. This study examined the impact of GDF11 on human skin, specifically related to skin aging. The effect of recombinant GDF11 on the function of various skin cells was examined in human epidermal keratinocytes, dermal fibroblasts, melanocytes, dermal microvascular endothelial cells and 3D skin equivalents, as well as in ex vivo human skin explants. GDF11 had significant effects on the production of dermal matrix components in multiple skin models in vitro and ex vivo. In addition, it had a pronounced effect on expression of multiple skin related genes in full thickness 3D skin equivalents. This work, for the first time, demonstrates an important role for GDF11 in skin biology and a potential impact on skin health and aging.

Single-cell transcriptomic profiling of the aging mouse brain.

The mammalian brain is complex, with multiple cell types performing a variety of diverse functions, but exactly how each cell type is affected in aging remains largely unknown. Here we performed a single-cell transcriptomic analysis of young and old mouse brains. We provide comprehensive datasets of aging-related genes, pathways and ligand-receptor interactions in nearly all brain cell types. Our analysis identified gene signatures that vary in a coordinated manner across cell types and gene sets that are regulated in a cell-type specific manner, even at times in opposite directions. These data reveal that aging, rather than inducing a universal program, drives a distinct transcriptional course in each cell population, and they highlight key molecular processes, including ribosome biogenesis, underlying brain aging. Overall, these large-scale datasets (accessible online at https://portals.broadinstitute.org/single_cell/study/aging-mouse-brain ) provide a resource for the neuroscience community that will facilitate additional discoveries directed towards understanding and modifying the aging process.

Growth Differentiation Factor 11 treatment leads to neuronal and vascular improvements in the hippocampus of aged mice.

Aging is the biggest risk factor for several neurodegenerative diseases. Parabiosis experiments have established that old mouse brains are improved by exposure to young mouse blood. Previously, our lab showed that delivery of Growth Differentiation Factor 11 (GDF11) to the bloodstream increases the number of neural stem cells and positively affects vasculature in the subventricular zone of old mice. Our new study demonstrates that GDF11 enhances hippocampal neurogenesis, improves vasculature and increases markers of neuronal activity and plasticity in the hippocampus and cortex of old mice. Our experiments also demonstrate that systemically delivered GDF11, rather than crossing the blood brain barrier, exerts at least some of its effects by acting on brain endothelial cells. Thus, by targeting the cerebral vasculature, GDF11 has a very different mechanism from that of previously studied circulating factors acting to improve central nervous system (CNS) function without entering the CNS.

Differential requirements of novel A1PiZ degradation deficient (ADD) genes in ER-associated protein degradation.

In the eukaryotic cell, a protein quality control process termed endoplasmic reticulum-associated degradation (ERAD) rids the ER of aberrant proteins and unassembled components of protein complexes that fail to reach a transport-competent state. To identify novel genes required for ERAD, we devised a rapid immunoassay to screen yeast lacking uncharacterized open reading frames that were known targets of the unfolded protein response (UPR), a cellular response that is induced when aberrant proteins accumulate in the ER. Six genes required for the efficient degradation of the Z variant of the alpha1-proteinase inhibitor (A1PiZ), a known substrate for ERAD, were identified, and analysis of other ERAD substrates in the six A1PiZ-degradation-deficient (add) mutants suggested diverse requirements for the Add proteins in ERAD. Finally, we report on bioinformatic analyses of the new Add proteins, which will lead to testable models to elucidate their activities.