Tammy McGuire

LIF and BMP signaling generate separate and discrete types of GFAP-expressing cells

Bone morphogenetic protein (BMP) and leukemia inhibitory factor (LIF) signaling both promote the differentiation of neural stem/progenitor cells into glial fibrillary acidic protein (GFAP) immunoreactive cells. This study compares the cellular and molecular characteristics, and the potentiality, of GFAP+ cells generated by these different signaling pathways. Treatment of cultured embryonic subventricular zone (SVZ) progenitor cells with LIF generates GFAP+ cells that have a bipolar/tripolar morphology, remain in cell cycle, contain progenitor cell markers and demonstrate self-renewal with enhanced neurogenesis - characteristics that are typical of adult SVZ and subgranular zone (SGZ) stem cells/astrocytes. By contrast, BMP-induced GFAP+ cells are stellate, exit the cell cycle, and lack progenitor traits and self-renewal - characteristics that are typical of astrocytes in the non-neurogenic adult cortex. In vivo, transgenic overexpression of BMP4 increases the number of GFAP+ astrocytes but depletes the GFAP+ progenitor cell pool, whereas transgenic inhibition of BMP signaling increases the size of the GFAP+ progenitor cell pool but reduces the overall numbers of astrocytes. We conclude that LIF and BMP signaling generate different astrocytic cell types, and propose that these cells are, respectively, adult progenitor cells and mature astrocytes.

Noggin Expands Neural Stem Cells in the Adult Hippocampus

New neurons are added to the adult hippocampus throughout life and contribute to cognitive functions, including learning and memory. It remains unclear whether ongoing neurogenesis arises from self-renewing neural stem cells (NSCs) or from multipotential progenitor cells that cannot self-renew in the hippocampus. This is primarily based on observations that neural precursors derived from the subventricular zone (SVZ) can be passaged long term, whereas hippocampal subgranular zone (SGZ) precursors are rapidly depleted by passaging. We demonstrate here that high levels of bone morphogenetic protein (BMP) signaling occur in hippocampal but not SVZ precursors in vitro, and blocking BMP signaling with Noggin is sufficient to foster hippocampal cell self-renewal, proliferation, and multipotentiality using single-cell clonal analysis. Moreover, NSC maintenance requires continual Noggin exposure, which implicates BMPs as crucial regulators of NSC aging. In vivo, Noggin is expressed in the adult dentate gyrus and limits BMP signaling in proliferative cells of the SGZ. Transgenic Noggin overexpression in the SGZ increases multiple precursor cell populations but proportionally increases the glial fibrillary acidic protein-positive cell population at the expense of other precursors, suggesting that Noggin acts on NSCs in vivo. To confirm this, we used a dual thymidine analog paradigm to repeatedly label slowly dividing cells over a long duration. We find that small populations of label-retaining cells exist in the SGZ and that Noggin overexpression increases their numbers. Thus, we propose that the adult hippocampus contains a population of NSCs, which can be expanded both in vitro and in vivo by blocking BMP signaling.

microRNA-21 Regulates Astrocytic Response Following Spinal Cord Injury

Astrogliosis following spinal cord injury (SCI) involves an early hypertrophic response that serves to repair damaged blood–brain barrier and a subsequent hyperplastic response that results in a dense scar that impedes axon regeneration. The mechanisms regulating these two phases of astrogliosis are beginning to be elucidated. In this study, we found that microRNA-21 (miR-21) increases in a time-dependent manner following SCI in mouse. Astrocytes adjacent to the lesion area express high levels of miR-21 whereas astrocytes in uninjured spinal cord express low levels of miR-21. To study the role of miR-21 in astrocytes after SCI, transgenic mice were generated that conditionally overexpress either the primary miR-21 transcript in astrocytes or a miRNA sponge designed to inhibit miR-21 function. Overexpression of miR-21 in astrocytes attenuated the hypertrophic response to SCI. Conversely, expression of the miR-21 sponge augmented the hypertrophic phenotype, even in chronic stages of SCI recovery when astrocytes have normally become smaller in size with fine processes. Inhibition of miR-21 function in astrocytes also resulted in increased axon density within the lesion site. These findings demonstrate a novel role for miR-21 in regulating astrocytic hypertrophy and glial scar progression after SCI, and suggest miR-21 as a potential therapeutic target for manipulating gliosis and enhancing functional outcome.

BMPR1a and BMPR1b Signaling Exert Opposing Effects on Gliosis after Spinal Cord Injury

Astrogliosis following spinal cord injury (SCI) involves an early hypertrophic response that is beneficial and a subsequent formation of a dense scar. We investigated the role of bone morphogenetic protein (BMP) signaling in gliosis after SCI and find that BMPR1a and BMPR1b signaling exerts opposing effects on hypertrophy. Conditional ablation of BMPR1a from glial fibrillary acidic protein (GFAP)-expressing cells leads to defective astrocytic hypertrophy, increased infiltration by inflammatory cells, and reduced axon density. BMPR1b-null mice conversely develop “hyperactive” reactive astrocytes and consequently have smaller lesion volumes. The effects of ablation of either receptor are reversed in the double knock-out animals. These findings indicate that BMPR1a and BMPR1b exert directly opposing effects on the initial reactive astrocytic hypertrophy. Also, BMPR1b knock-out mice have an attenuated glial scar in the chronic stages following injury, suggesting that it has a greater role in glial scar progression. To elucidate the differing roles of the two receptors in astrocytes, we examined the effects of ablation of either receptor in serum-derived astrocytes in vitro. We find that the two receptors exert opposing effects on the posttranscriptional regulation of astrocytic microRNA-21. Further, overexpression of microRNA-21 in wild-type serum-derived astrocytes causes a dramatic reduction in cell size accompanied by reduction in GFAP levels. Hence, regulation of microRNA-21 by BMP signaling provides a novel mechanism for regulation of astrocytic size. Targeting specific BMPR subunits for therapeutic purposes may thus provide an approach for manipulating gliosis and enhancing functional outcomes after SCI.

Dysregulation of Local Stem/Progenitor Cells as a Common Cellular Mechanism for Heterotopic Ossification

Heterotopic ossification (HO), the abnormal formation of true marrow‐containing bone within extraskeletal soft tissues, is a serious bony disorder that may be either acquired or hereditary. We utilized an animal model of the genetic disorder fibrodysplasia ossificans progressiva to examine the cellular mechanisms underlying HO. We found that HO in these animals was triggered by soft tissue injuries and that the effects were mediated by macrophages. Spreading of HO beyond the initial injury site was mediated by an abnormal adaptive immune system. These observations suggest that dysregulation of local stem/progenitor cells could be a common cellular mechanism for typical HO irrespective of the signal initiating the bone formation. STEM CELLS 2009;27:150–156

Substance P signaling mediates BMP‐dependent heterotopic ossification

Heterotopic ossification (HO) is a disabling condition associated with neurologic injury, inflammation, and overactive bone morphogenetic protein (BMP) signaling. The inductive factors involved in lesion formation are unknown. We found that the expression of the neuro‐inflammatory factor Substance P (SP) is dramatically increased in early lesional tissue in patients who have either fibrodysplasia ossificans progressiva (FOP) or acquired HO, and in three independent mouse models of HO. In Nse‐BMP4, a mouse model of HO, robust HO forms in response to tissue injury; however, null mutations of the preprotachykinin (PPT) gene encoding SP prevent HO. Importantly, ablation of SP+ sensory neurons, treatment with an antagonist of SP receptor NK1r, deletion of NK1r gene, or genetic down‐regulation of NK1r‐expressing mast cells also profoundly inhibit injury‐induced HO. These observations establish a potent neuro‐inflammatory induction and amplification circuit for BMP‐dependent HO lesion formation, and identify novel molecular targets for prevention of HO. J. Cell. Biochem. 112: 2759–2772, 2011.

BMPR1a Signaling Determines Numbers of Oligodendrocytes and Calbindin-Expressing Interneurons in the Cortex

Progenitor cells that express the transcription factor olig1 generate several neural cell types including oligodendrocytes and GABAergic interneurons in the dorsal cortex. The fate of these progenitor cells is regulated by a number of signals including bone morphogenetic proteins (BMPs) secreted in the dorsal forebrain. BMPs signal by binding to heteromeric serine–threonine kinase receptors formed by type I (BMPR1a, BMPR1b, Alk2) and type II (BMPRII) subunits. To determine the specific role of the BMPR1a subunit in lineage commitment by olig1-expressing cells, we used a cre/loxP genetic approach to ablate BMPR1a in these cells while leaving signaling from other subunits intact. There was a reduction in numbers of immature oligodendrocytes in the BMPR1a-null mutant brains at birth. However, by postnatal day 20, the BMPR1a-null mice had a significant increase in the number of mature and immature oligodendrocytes compared with wild-type littermates. There was also an increase in the proportion of calbindin-positive interneurons in the dorsomedial cortex of BMPR1a-null mice at birth without any change in the number of parvalbumin- or calretinin-positive cells. These effects were attributable, at least in part, to a decrease in the length of the cell cycle in subventricular zone progenitor cells. Thus, our findings indicate that BMPR1a mediates the suppressive effects of BMP signaling on oligodendrocyte lineage commitment and on the specification of calbindin-positive interneurons in the dorsomedial cortex.

Differential effects of BMP signaling on parvalbumin and somatostatin interneuron differentiation

Several different populations of interneurons in the murine cortex, including somatostatin (SST)- or parvalbumin (PV)-expressing cells, are born in the ventral ganglionic eminences during mid-gestation and then migrate tangentially to the cortex. SST is expressed by some interneuron progenitors in the cerebral cortex and in migrating populations in the ventrolateral cortex at birth. However, PV (also known as PVALB) is not expressed by interneurons until the second postnatal week after reaching the cortex, suggesting that molecular cues in the cerebral cortex might be involved in the differentiation process. BMP4 is expressed at high levels in the somatosensory cortex at the time when the PV+ interneurons differentiate. Treatment of cortical cultures containing interneuron precursors is sufficient to generate PV+ interneurons prematurely and inhibit SST differentiation. Furthermore, overexpression of BMP4 in vivo increases the number of interneurons expressing PV, with a reduction in the number of SST+ interneurons. PV+ interneurons in the cortex express BMP type I receptors and a subpopulation displays activated BMP signaling, assessed by downstream molecules including phosphorylated SMAD1/5/8. Conditional mutation of BMP type I receptors in interneuron precursors significantly reduces the number of cortical PV+ interneurons in the adult brain. Thus, BMP4 signaling through type I receptors regulates the differentiation of two major medial ganglionic eminence-derived interneuron populations and defines their relative numbers in the cortex.

BMP Signaling Regulates the Tempo of Adult Hippocampal Progenitor Maturation at Multiple Stages of the Lineage

Novel environmental stimuli, such as running and learning, increase proliferation of adult hippocampal neural stem cells (NSCs) and enlarge the population of new neurons. However, it remains unclear how increased numbers of new neurons can be generated in a time frame far shorter than the time required for proliferating stem cells to generate these neurons. Here, we show that bone morphogenetic protein (BMP) signaling in the subgranular zone regulates the tempo of neural progenitor cell (NPC) maturation by directing their transition between states of quiescence and activation at multiple stages along the lineage. Virally mediated overexpression of BMP4 caused NPC cell cycle exit and slowed the normal maturation of NPCs, resulting in a long‐term reduction in neurogenesis. Conversely, overexpression of the BMP inhibitor noggin promoted NPC cell cycle entry and accelerated NPC maturation. Similarly, BMP receptor type 2 (BMPRII) ablation in Ascl1+ intermediate NPCs accelerated their maturation into neurons. Importantly, ablation of BMPRII in GFAP+ stem cells accelerated maturation without depleting the NSC pool, indicating that an increased rate of neurogenesis does not necessarily diminish the stem cell population. Thus, inhibition of BMP signaling is a mechanism for rapidly expanding the pool of new neurons in the adult hippocampus by tipping the balance between quiescence/activation of NPCs and accelerating the rate at which they mature into neurons. Stem Cells 2014;32:2201–2214

β1-Integrin and integrin linked kinase regulate astrocytic differentiation of neural stem cells.

Astrogliosis with glial scar formation after damage to the nervous system is a major impediment to axonal regeneration and functional recovery. The present study examined the role of β1-integrin signaling in regulating astrocytic differentiation of neural stem cells. In the adult spinal cord β1-integrin is expressed predominantly in the ependymal region where ependymal stem cells (ESCs) reside. β1-integrin signaling suppressed astrocytic differentiation of both cultured ESCs and subventricular zone (SVZ) progenitor cells. Conditional knockout of β1-integrin enhanced astrogliogenesis both by cultured ESCs and by SVZ progenitor cells. Previous studies have shown that injection into the injured spinal cord of a self-assembling peptide amphiphile that displays an IKVAV epitope (IKVAV-PA) limits glial scar formation and enhances functional recovery. Here we find that injection of IKVAV-PA induced high levels of β1-integrin in ESCs in vivo, and that conditional knockout of β1-integrin abolished the astroglial suppressive effects of IKVAV-PA in vitro. Injection into an injured spinal cord of PAs expressing two other epitopes known to interact with β1-integrin, a Tenascin C epitope and the fibronectin epitope RGD, improved functional recovery comparable to the effects of IKVAV-PA. Finally we found that the effects of β1-integrin signaling on astrogliosis are mediated by integrin linked kinase (ILK). These observations demonstrate an important role for β1-integrin/ILK signaling in regulating astrogliosis from ESCs and suggest ILK as a potential target for limiting glial scar formation after nervous system injury.