The Neuroplasticity Project

This project involves investigations of signaling mechanisms that regulate stem cell self-renewal,  neurogenesis, neurite outgrowth, synaptic plasticity and neuronal survival (Figure 3).  This project has evolved from discoveries made in my laboratory during the past 3 decades which revealed roles for neurodegenerative disease-related proteins (e.g., APP, presenilin-1, DJ-1, TNF, and TLRs) in neural plasticity, and vice-versa (e.g., glutamate, BDNF and Notch).

2a.  Characterization of human embryonic stem cells (hESC).  We developed a reproducible protocol to generate highly homogenous neural progenitors or a mixed population of neural progenitors and neurons from hESC (36). This defined adherent culture system allowed us to examine the effect of extracellular matrix molecules on neural differentiation of hESCs.   We found that five different growth substrates (Poly-D-Lysine (PDL), PDL/fibronectin, PDL/laminin, type I collagen and Matrigel) instructed neural progenitors followed by neuronal differentiation to differing degrees.  Neuronal generation and neurite outgrowth were significantly greater on laminin and laminin-rich Matrigel substrates than on other 3 substrates. Laminin stimulated hESC-derived neural progenitor expansion and neurite outgrowth in a concentration-dependent manner. The laminin-induced neural progenitor expansion was partially blocked by antibodies against integrin a6 or b1 subunits. These findings advance understanding of cell-matrix interactions in neural derivation of hESCs.

In a second study we made a side-by-side comparison of three NIH registered hESC lines (I3 (TE03); I6 (TE06); and BG01V) to determine:  1) their ability to maintain an undifferentiated state and to self-renew under standard conditions; 2) their ability to spontaneously differentiate into three primary embryonic germ lineages in differentiating embryoid bodies; and 3) their responses to directed neural differentiation (65a). Lines I3 and I6 possess normal XX and a normal XY karyotype while BG01V is a variant cell line with an abnormal karyotype derived from the karyotypically normal cell line BG01. Using immunocytochemistry, flow cytometry, qRT-PCR and MPSS, we found that all three cell lines actively proliferated and expressed similar "stemness" markers including transcription factors POU5F1/Oct3/4 and NANOG, glycolipids SSEA4 and TRA-1-81, and alkaline phosphatase activity.   However, a profound variation in colony morphology, growth rate, BrdU incorporation, and relative abundance of gene expression in undifferentiated and differentiated states of the cell lines was observed. Under the same neural differentiation-promoting conditions, the ability of each cell line to differentiate into neural progenitors varied.   Our comparative analysis provides further evidence for similarities and differences among hESC lines in self-renewal, and spontaneous and directed differentiation. 
 
2b. Integrins play roles in regulating neural progenitor cell fate and brain injury responses
We discovered a novel signaling mechanism that regulates neurogenesis in the developing mouse telencephalon.  To understand the role of the extracellular matrix (ECM) in regulating the behavior of neural stem cells (NSC), we first established the localization of laminins and their corresponding receptors in the embryonic murine ventricular zone (VZ) within which the NSCs undergo symmetrical and asymmetrical divisions required for cortical development (27). In addition to the presence of laminins containing both the a2 and a4 chains, we found distinct patterns of ECM receptor expression in the VZ and in the overlying cortex. NSC derived from the VZ express high levels of the integrin laminin receptor a6b1. At developmental stages at which NSC undergo asymmetrical divisions, integrin b1 was unevenly distributed in some mitotic pairs at the ventricular wall.  Next, we used antibody-blocking and genetic experiments to reveal a novel requirement for laminin/integrin interactions in apical process adhesion and NSC regulation (33). Transient abrogation of integrin binding and signaling using blocking antibodies to specifically target the ventricular region in utero resulted in abnormal NSC migration and alterations in the orientation of NSC divisions. These defects were also observed in laminin a2-deficient mice. More detailed analyses revealed that the transient embryonic disruption of laminin/integrin signaling at the VZ surface resulted in apical process detachment from the ventricular surface, dystrophic radial glia fibers, and substantial layering defects in the postnatal neocortex.  These data reveal novel roles for the laminin/integrin interaction in anchoring embryonic NSC to the ventricular surface and maintaining the physical integrity of the neocortical niche, with even transient perturbations resulting in long-lasting cortical defects.

With our interest in understanding if and how developmental signaling mechanisms are involved in brain aging and disease we next employed in vivo and in vitro models to elucidate the role of b1 integrin in vascular remodeling and stroke outcomes (28a). At 24 h after cerebral ischemia and reperfusion (I/R), the ischemic cortex exhibited modest b1 integrin immunoreactivity and a robust increase was observed at 72 h. Double-label immunohistochemical analysis for b1 integrin with neuronal (NeuN), microglial (Iba-1), astrocyte (GFAP), progenitor cell (Ng2) and blood vessel (collagen 4) markers showed that b1 integrin expression only localized to blood vessels. Cell culture studies showed that b1 integrin is required for endothelial cell migration, proliferation and blood vessel formation. In vivo studies in the cerebral I/R model using a b1 integrin blocking antibody confirmed that b1 integrin signaling is involved in vascular formation and recovery following ischemic stroke. Finally, we found that b1 integrin is critically involved in functional deficits and survival after a stroke. These results suggest that b1 integrin plays important roles in neurovascular remodeling and functional outcomes following stroke, and that targeting the b1 integrin signaling may provide a novel strategy for modulating angiogenesis in the CNS.

2c. Toll-like receptors are expressed in neurons and modify neuronal responses to ischemic brain injury. The innate immune system senses the invasion of pathogenic microorganisms and tissue injury through Toll-like receptors (TLR), a mechanism thought to be limited to immune cells. We discovered that neurons express several TLRs, and that the levels of TLR2 and -4 are increased in neurons in response to IFN-g stimulation and energy deprivation (64). Neurons from both TLR2 and TLR4 mutant mice were protected against energy deprivation-induced cell death. TLR2 and TLR4 expression were increased in cerebral cortical neurons in response to ischemic stroke, and the amount of brain damage and neurological deficits caused by a stroke were significantly less in mice deficient in TLR2 or TLR4 compared with wild type control mice.  TLR2- and TLR4-mediated ischemic neuronal death involved jun N-terminal kinase and the transcription factor AP-1.  These findings establish a pro-apoptotic signaling pathway for TLR2 and TLR4 in neurons that may contribute to their demise in ischemic stroke.

2d. Toll-like receptors 2, 3 and 4 play roles in developmental and adult neuroplasticity.
To address the possibility that TLRs play a functional role in development of the nervous system, we analyzed the expression of TLRs during different stages of mouse brain development and assessed the role of TLRs in cell proliferation.  Because TLR3 protein is present at particularly high levels in brain cells in early embryonic stages of development, and in cultured neural stem  cells (NSC) we initially focused on TLR3 (28). NSC from TLR3-deficient embryos formed greater numbers of neurospheres compared with neurospheres from wild-type embryos. Numbers of proliferating cells, as assessed by phospho histone H3 and proliferating cell nuclear antigen labeling, were also increased in the developing cortex of TLR3-deficient mice compared with wild-type mice in vivo. Treatment of cultured embryonic cortical neurospheres with a TLR3 ligand (polyIC) significantly reduced proliferating (BrdU-labeled) cells and neurosphere formation in wild type but not TLR3(-/-)-derived NSC. Our findings reveal a novel role for TLR3 in the negative regulation of NSC proliferation in the developing brain.

In a second study we focused on TLR2 and characterized its expression throughout mouse cortical development, and its possible role in regulating NSC fate (49). TLR2 mRNA and protein were expressed in the cortex in embryonic and early postnatal stages of development, and in cultured cortical NSC. While NSC from TLR2-deficient and wild type embryos had the same proliferative capacity, TLR2 activation by the synthetic bacterial lipopeptides Pam(3)CSK(4) and FSL1, or low molecular weight hyaluronan (an endogenous ligand for TLR2) inhibited neurosphere formation in vitro. Intracerebral in utero administration of TLR2 ligands resulted in ventricular dysgenesis characterized by increased ventricle size, reduced proliferative area around the ventricles, increased cell density, an increase in phospho-histone 3 cells, and a decrease in BrdU+ cells in the sub-ventricular zone. Our findings indicate that loss of TLR2 does not result in defects in cerebral development, and further suggest a role for TLR2 in adverse effects of infection, ischemia, and inflammation adversely affect brain development.

In light of our discoveries that some TLRs influence neural plasticity during embryonic development, and that TLRs 2, 3 and 4 are expressed in neurons, we asked whether one or more TLRs modify hippocampal plasticity in adult mice. We investigated learning and memory in TLR3-deficient (TLR3-/-) mice using both hippocampus-dependent and –independent behavioral tests (50).  Adult TLR3-/- mice exhibited enhanced hippocampus-dependent working memory in the Morris water maze, novel object recognition and contextual fear conditioning tasks.  In contrast, TLR3-/- mice demonstrated reduced amygdala-related behavior and anxiety in the cued fear conditioning, open field and elevated plus maze tasks.  TLR3-/- mice exhibited increased hippocampal CA1 and dentate gyrus volumes, increased hippocampal neurogenesis and elevated levels of the AMPA receptor subunit GluR1 in the CA1 region of the hippocampus. In addition, levels of activated forms of the kinase ERK and the transcription factor CREB were elevated in the hippocampus of TLR3-deficient mice, suggesting that constitutive TLR3 signaling negatively regulates pathways known to play important roles in hippocampal plasticity. Our findings reveal novel roles for TLR3 as a suppressor of hippocampal cellular plasticity and memory retention.
 
2e. The clathrin assembly proteins AP180 and CALM differentially regulate axogenesis and dendrite outgrowth. The clathrin assembly proteins AP180 and CALM (clathrin assembly lymphoid myeloid protein) are known to be involved in clathrin-mediated endocytosis, but their roles in mammalian neurons and, in particular, in developmental processes before synaptogenesis are unknown.  We found that AP180 and CALM play critical roles in establishing the polarity and controlling the growth of axons and dendrites in embryonic hippocampal neurons (6). Knockdown of AP180 primarily impairs axonal development, whereas reducing CALM levels results in dendritic dystrophy. Conversely, neurons that overexpress AP180 or CALM generate multiple axons. Ultrastructural analysis showed that CALM affiliates with a wider range of intracellular trafficking organelles than does AP180. Functional analysis shows that endocytosis is reduced in both AP180-deficient and CALM-deficient neurons. Additionally, CALM-deficient neurons show disrupted secretory transport. Our data demonstrate previously unknown functions for AP180 and CALM in intracellular trafficking that are essential in the growth of neurons.

2f. TRF2 interacts with the transcriptional silencer REST to control stem cell self-renewal and neuronal differentiation: implications for neurogenesis and cancer stem cell biology
Telomere repeat-binding factor 2 (TRF2) plays an important role in preventing damage to telomeric DNA.  We recently discovered an extra-telomeric function of TRF2 in the regulation of neuronal genes mediated by the interaction of TRF2 with repressor element 1-silencing transcription factor (REST), a master repressor of gene networks devoted to neuronal phenotypes (77). TRF2-REST complexes were readily detected by co-immunoprecipitation assays and are localized to aggregated PML-nuclear bodies in undifferentiated pluripotent human NTera2 stem cells. Inhibition of TRF2, either by a dominant-negative mutant or by RNA interference, reduces TRF2-REST complexes resulting in ubiquitin-mediated proteasomal degradation of REST. Consequentially, REST-targeted neural genes (e.g., BDNF, L1CAM, beta3-tubulin, synaptophysin, and others) are de-repressed, resulting in acquisition of neuronal phenotypes. Selective damage to telomeres without affecting TRF2 levels causes neither REST degradation nor cell differentiation. Thus, in addition to protecting telomeres, TRF2 possesses a novel role in stabilization of REST thereby controlling neural tumor and stem cell fate.

2g.  Two advances on the synaptic plasticity front.  Mutations in DJ-1 cause inherited Parkinson's disease (PD) in several families. The normal function of DJ-1 is unknown, but mice lacking DJ-1 exhibit a deficit in dopaminergic signaling in the striatum. Since the hippocampus contains relatively high levels of DJ-1, and PD patients are often cognitively impaired, we evaluated the effects of DJ-1 deficiency on the plasticity of hippocampal CA1 synapses (69). LTP was slightly impaired and LTD was abolished in DJ-1-/- mice, whereas DJ-1+/- mice exhibited no alterations in synaptic plasticity. The dopamine receptor D2/3 agonist quinpirole rescued LTD in DJ-1-/- mice, suggesting a role for impaired dopaminergic signaling in the hippocampal LTD deficit.

In the hippocampus, glucocorticoids bind to two types of receptors: the mineralocorticoid receptor (MR), which binds corticosterone with high affinity and is tonically occupied; and the glucocorticoid receptor (GR), which is occupied during stress and at certain phases in the circadian cycle.  Levels of glucocorticoids in diabetic humans and animal models of diabetes. To explore the contributions of hippocampal corticosteroid receptors to the diabetes-induced suppression of neuroplasticity, we manipulated these receptors in hippocampal slices from streptozocin-diabetic rats, a model of Type 1 diabetes mellitus (60). STZ-diabetes reduced LTP at medial perforant path synapses in the dentate gyrus, and induced a bias in favor of LTD following intermediate stimulation frequencies. Bath application of the MR agonist aldosterone restored LTP in slices from diabetic animals. These results suggest additional mechanisms for diabetes-induced functional alterations and support a restorative role for dentate gyrus MR.

Schematic diagram showing several biochemical cascades that either promote cell death (apoptosis) or protect cells against death. Adverse conditions occur in the environment of neurons in the brain during aging and in neurodegenerative disorders. Such conditions include reduced levels of trophic factors (TFW) and increased oxidative stress. Increased levels of intracellular calcium and oxyradicals induce production of pro-apoptotic gene products such as prostate apoptosis response (Par-4) and pro-apoptotic Bcl-2 family members. These proteins may interact with mitochondria resulting in mitochondrial membrane depolarization, uptake of calcium, and cytochrome c release. Mitochondrial dysfunction leads to caspase activation and cleavage of a variety of protein substrates that effect the cell death program. Counteracting these cell death-promoting cascades are anti-apoptotic signals such as transduction pathways activated by neurotrophic factors, as well as conditioning responses such as can be stimulated by dietary restriction, for example. These anti-apoptotic signals can interrupt the cell death cascade at several different levels. It should be noted that most of the biochemical machinery for either promoting or preventing cell death is present in synapses and neurites, as well as the cell body. Activation of these pathways in synaptic terminals is believed to be critical to neuronal survival or death in aging brain. This diagram summarizes our current state of understanding of the mechanisms whereby the anti-aging enzyme telomerase may promote cell survival and might guard against age-related degenerative disorders including Alzheimer's disease and stroke. Telomerase consists of a catalytic subunit, TERT, an RNA component, and several telomere or telomerase-associated proteins, including proteins including TRF-1, TRF-2 and TEP-1. The well-established function of telomerase is to add a 6-base DNA repeat onto the ends of chromosomes. This activity of telomerase may protect the chromosome ends against damage and thereby suppress apoptotic signals emanating from damaged DNA. In addition, the TERT protein has been shown to suppress apoptosis of neurons at an early step in the cell death process prior to mitochondrial dysfunction and caspase activation. Modulation of the activity of the tumor suppressor p53 might play a role in this anti-apoptotic function of telomerase.