William A. Tyler

Activation of the Mammalian Target of Rapamycin (mTOR) Is Essential for Oligodendrocyte Differentiation

Although both extrinsic and intrinsic factors have been identified that orchestrate the differentiation and maturation of oligodendrocytes, less is known about the intracellular signaling pathways that control the overall commitment to differentiate. Here, we provide evidence that activation of the mammalian target of rapamycin (mTOR) is essential for oligodendrocyte differentiation. Specifically, mTOR regulates oligodendrocyte differentiation at the late progenitor to immature oligodendrocyte transition as assessed by the expression of stage specific antigens and myelin proteins including MBP and PLP. Furthermore, phosphorylation of mTOR on Ser 2448 correlates with myelination in the subcortical white matter of the developing brain. We demonstrate that mTOR exerts its effects on oligodendrocyte differentiation through two distinct signaling complexes, mTORC1 and mTORC2, defined by the presence of the adaptor proteins raptor and rictor, respectively. Disrupting mTOR complex formation via siRNA mediated knockdown of raptor or rictor significantly reduced myelin protein expression in vitro. However, mTORC2 alone controlled myelin gene expression at the mRNA level, whereas mTORC1 influenced MBP expression via an alternative mechanism. In addition, investigation of mTORC1 and mTORC2 targets revealed differential phosphorylation during oligodendrocyte differentiation. In OPC-DRG cocultures, inhibiting mTOR potently abrogated oligodendrocyte differentiation and reduced numbers of myelin segments. These data support the hypothesis that mTOR regulates commitment to oligodendrocyte differentiation before myelination.

IGF-II promotes stemness of neural restricted precursors.

Insulin‐like growth factor (IGF)‐I and IGF‐II regulate brain development and growth through the IGF type 1 receptor (IGF‐1R). Less appreciated is that IGF‐II, but not IGF‐I, activates a splice variant of the insulin receptor (IR) known as IR‐A. We hypothesized that IGF‐II exerts distinct effects from IGF‐I on neural stem/progenitor cells (NSPs) via its interaction with IR‐A. Immunofluorescence revealed high IGF‐II in the medial region of the subventricular zone (SVZ) comprising the neural stem cell niche, with IGF‐II mRNA predominant in the adjacent choroid plexus. The IGF‐1R and the IR isoforms were differentially expressed with IR‐A predominant in the medial SVZ, whereas the IGF‐1R was more abundant laterally. Similarly, IR‐A was more highly expressed by NSPs, whereas the IGF‐1R was more highly expressed by lineage restricted cells. In vitro, IGF‐II was more potent in promoting NSP expansion than either IGF‐I or standard growth medium. Limiting dilution and differentiation assays revealed that IGF‐II was superior to IGF‐I in promoting stemness. In vivo, NSPs propagated in IGF‐II migrated to and took up residence in periventricular niches while IGF‐I‐treated NSPs predominantly colonized white matter. Knockdown of IR or IGF‐1R using shRNAs supported the conclusion that the IGF‐1R promotes progenitor proliferation, whereas the IR is important for self‐renewal. Q‐PCR revealed that IGF‐II increased Oct4, Sox1, and FABP7 mRNA levels in NSPs. Our data support the conclusion that IGF‐II promotes the self‐renewal of neural stem/progenitors via the IR. By contrast, IGF‐1R functions as a mitogenic receptor to increase precursor abundance. STEM CELLS2012;30:1265–1276

Multiplex Genetic Fate Mapping Reveals a Novel Route of Neocortical Neurogenesis, Which Is Altered in the Ts65Dn Mouse Model of Down Syndrome

While several major classes of neocortical neural precursor cells have been identified, the lineal relationships and molecular profiles of these cells are still largely unknown. Furthermore, the individual contribution of each cell class to neocortical growth during normal development and in neurodevelopmental disorders has not been determined. Using a novel fate-mapping approach, we demonstrate that precursors in the embryonic ventricular (VZ) and subventricular zones (SVZ), which give rise to excitatory neurons, are divided into distinct subtypes based on lineage profile, morphology, and transcription factor expression in vivo. Using this technique, we show that short neural precursors are a unique class of VZ intermediate progenitors derived from radial glial cells and are distinct from the multipolar Tbr2(+) intermediate progenitors, which divide in the SVZ. To test whether these multiple groups of intermediate progenitors are redundant or whether they are necessary for proper neocortical growth, we measured precursor cell diversity in the Ts65Dn mouse model of Down syndrome (DS), which exhibits reduced neurogenesis and postnatal microcephaly. We report that SNP generation is markedly reduced in the Ts65Dn VZ during mid-neurogenesis, indicating that faulty specification of this progenitor pool is a central component of the neocortical abnormality in DS. Together, these findings demonstrate that neocortical neurons are produced via multiple indirect routes during embryonic development and that these parallel streams of neurogenesis collectively contribute to the proper growth and development of the neocortex.

Down Syndrome Developmental Brain Transcriptome Reveals Defective Oligodendrocyte Differentiation and Myelination

Trisomy 21, or Down syndrome (DS), is the most common genetic cause of developmental delay and intellectual disability. To gain insight into the underlying molecular and cellular pathogenesis, we conducted a multi-region transcriptome analysis of DS and euploid control brains spanning from mid-fetal development to adulthood. We found genome-wide alterations in the expression of a large number of genes, many of which exhibited temporal and spatial specificity and were associated with distinct biological processes. In particular, we uncovered co-dysregulation of genes associated with oligodendrocyte differentiation and myelination that were validated via cross-species comparison to Ts65Dn trisomy mice. Furthermore, we show that hypomyelination present in Ts65Dn mice is in part due to cell-autonomous effects of trisomy on oligodendrocyte differentiation and results in slower neocortical action potential transmission. Together, these results identify defects in white matter development and function in DS, and they provide a transcriptional framework for further investigating DS neuropathogenesis.

Proteomic Identification of Novel Targets Regulated by the Mammalian Target of Rapamycin Pathway during Oligodendrocyte Differentiation

Previous work from our laboratory demonstrated that the mammalian target of rapamycin (mTOR) is active during and required for oligodendrocyte progenitor cell (OPC) differentiation. Here, we applied an iTRAQ mass spectrometry‐based proteomic approach to identify novel targets of the mTOR pathway during OPC differentiation. Among the 978 proteins identified in this study, 328 (34%) exhibited a greater than 20% change (P < 0.05) in control versus rapamycin‐treated cultures following 4 days of differentiation in vitro. Interestingly, 197 (20%) proteins were elevated in rapamycin‐treated cultures, while 131 (13%) proteins were downregulated by rapamycin. In support of our previous data, inhibiting mTOR caused a dramatic reduction in the expression of myelin proteins. mTOR also was required for the induction of proteins involved in cholesterol and fatty acid synthesis, as well as the expression of many cytoskeletal proteins, cell signaling components, and nuclear/transcriptional regulators. Of particular interest was the identification of several critical mediators of oligodendrocyte differentiation. Specifically, mTOR activity controls the developmentally programmed upregulation of the prodifferentiation factors Fyn and Quaking, whereas the expression of the differentiation repressor Gpr17 was elevated by mTOR inhibition. These data reveal a distinct signature of mTOR‐regulated protein expression during OPC differentiation.

Neural Precursor Lineages Specify Distinct Neocortical Pyramidal Neuron Types

Several neural precursor populations contemporaneously generate neurons in the developing neocortex. Specifically, radial glial stem cells of the dorsal telencephalon divide asymmetrically to produce excitatory neurons, but also indirectly to produce neurons via three types of intermediate progenitor cells. Why so many precursor types are needed to produce neurons has not been established; whether different intermediate progenitor cells merely expand the output of radial glia or instead generate distinct types of neurons is unknown. Here we use a novel genetic fate mapping technique to simultaneously track multiple precursor streams in the developing mouse brain and show that layer 2 and 3 pyramidal neurons exhibit distinctive electrophysiological and structural properties depending upon their precursor cell type of origin. These data indicate that individual precursor subclasses synchronously produce functionally different neurons, even within the same lamina, and identify a primary mechanism leading to cortical neuronal diversity.

Longitudinal measures of cognition in the Ts65Dn mouse: Refining windows and defining modalities for therapeutic intervention in Down syndrome.

Mouse models have provided insights into adult changes in learning and memory in Down syndrome, but an in-depth assessment of how these abnormalities develop over time has never been conducted. To address this shortcoming, we conducted a longitudinal behavioral study from birth until late adulthood in the Ts65Dn mouse model to measure the emergence and continuity of learning and memory deficits in individuals with a broad array of tests. Our results demonstrate for the first time that the pace at which neonatal and perinatal milestones are acquired is correlated with later cognitive performance as an adult. In addition, we find that life-long behavioral indexing stratifies mice within each genotype. Our expanded assessment reveals that diminished cognitive flexibility, as measured by reversal learning, is the most robust learning and memory impairment in both young and old Ts65Dn mice. Moreover, we find that reversal learning degrades with age and is therefore a useful biomarker for studying age-related decline in cognitive ability. Altogether, our results indicate that preclinical studies aiming to restore cognitive function in Ts65Dn should target both neonatal milestones and reversal learning in adulthood. Here we provide the quantitative framework for this type of approach.

CLASP2 Links Reelin to the Cytoskeleton during Neocortical Development

The Reelin signaling pathway plays a crucial role in regulating neocortical development. However, little is known about how Reelin controls the cytoskeleton during neuronal migration. Here, we identify CLASP2 as a key cytoskeletal effector in the Reelin signaling pathway. We demonstrate that CLASP2 has distinct roles during neocortical development regulating neuron production and controlling neuron migration, polarity, and morphogenesis. We found downregulation of CLASP2 in migrating neurons leads to mislocalized cells in deeper cortical layers, abnormal positioning of the centrosome-Golgi complex, and aberrant length/orientation of the leading process. We discovered that Reelin regulates several phosphorylation sites within the positively charged serine/arginine-rich region that constitute consensus GSK3β phosphorylation motifs of CLASP2. Furthermore, phosphorylation of CLASP2 regulates its interaction with the Reelin adaptor Dab1 and this association is required for CLASP2 effects on neurite extension and motility. Together, our data reveal that CLASP2 is an essential Reelin effector orchestrating cytoskeleton dynamics during brain development.

A new contribution to brain convolution: progenitor cell logistics during cortex development

New work identifies a neurogenic progenitor population in the developing human and ferret cortex that may have contributed substantially to the evolution of cortical expansion and folding in higher mammals.

Distinct Neocortical Progenitor Lineages Fine-tune Neuronal Diversity in a Layer-specific Manner

&NA; How the variety of neurons that organize into neocortical layers and functional areas arises is a central question in the study of cortical development. While both intrinsic and extrinsic cues are known to influence this process, whether distinct neuronal progenitor groups contribute to neuron diversity and allocation is poorly understood. Using in vivo genetic fate‐mapping combined with whole‐cell patch clamp recording, we show that the firing pattern and apical dendritic morphology of excitatory neurons in layer 4 of the barrel cortex are specified in part by their neural precursor lineage. Further, we show that separate precursors contribute to unique features of barrel cortex topography including the intralaminar position and thalamic innervation of the neurons they generate. Importantly, many of these lineage‐specified characteristics are different from those previously measured for pyramidal neurons in layers 2‐3 of the frontal cortex. Collectively, our data elucidate a dynamic temporal program in neuronal precursors that fine‐tunes the properties of their progeny according to the lamina of destination.