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RESEARCH OUTLINE

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  Communication between neurons and non-neural cells is critical to integrated functional responses in the brain. Astrocytes can modulate the environment around neurons and/or endothelial cells, regulate neuronal survival during development, and regulate brain homeostasis, which facilitates axonal signals in synaptic transmission and information processing throughout the adult brain. We found that some specific cells can express Bone Morphogenetic Proteins (BMPs) in mature brain, and their receptors are express in astrocytes. It is described that BMPs play an important role in different stages of brain development. BMPs exert their biological effects by binding to type I and type II receptor subunits. BMP type I receptor (Bmpr1a and Bmpr1b) and type II receptor (Bmpr2) are expressed in multiple regions of the CNS. To explore the physiological functions of astrocytic BMP signaling, we used gene-targeting technology to generate mice deficient in BMP receptors in a tissue specific manner. Our goal of the research described and proposed here is to identify astrocytic BMPs receptor signaling pathways to develop the astrocytic functions. These attempts will reveal that BMPRIA signaling, together with BMPRIB signaling in astrocytes, may critical for cerebral angiogenesis, synaptic activity, global ischemia, and CNS injury. Emphasis will be placed on identifying and understanding neural pathology that results from disruption of these mechanism and processes, which may lead to developmental neural dysfunction or cognitive deficit.

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PROJECTS

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1. Astrocytic BMP signaling and angiogenesis
   Angiogenesis is a multistep process that involves proliferation and migration of endothelial cells (ECs) and generation of extracellular matrix. Angiogenesis is regulated by multiple positive- and negative-regulatory factors. Astrocytes, whose endfeet encircle neural capillaries, produce angiogenesis-modulating factors, and together with ECs, play a key role in forming and maintaining the blood-brain-barrier (BBB). It has been suggested that closure of the BBB is correlated to maturation of astrocytes. Recently we found that BMPRIA in astrocytes played a critical role in interactions between ECs and astrocytes to form a functional BBB.

BMPRIA signaling in astrocytes is essential for proper cerebral angiogenesis    Astrocytes adhere to most of the capillaries in the brain and promote formation of the blood-brain-barrier (BBB). It is recently shown that Bone morphogenetic protein (BMP) signaling through BMPRIA is important for cell fate determination in neural stem cells towards glial lineage, and for astrocytic maturation.    Furthermore, activation of BMPs signaling is thought to play a role in regulating proliferation, differentiation, and survival on vascular cells, but its involvement in regulating endothelial-astrocyte interactions is not yet understood. The study examines the role of BMP signaling through BMP type IA receptor (BMPRIA) in early neural development using a conditional mouse knockout model, in which Bmpr1a is selectively disrupted in telencephalic neural stem cells. The conditional mutant mice show a significant increase in the number of cerebral blood vessels and the level of vascular endothelial growth factor (VEGF) is significantly upregulated in the mutant astrocytes. The mutant mice also show leakage of immunoglobulin around cerebral microvessels in neonatal mice, suggesting a defect in formation of the blood-brain-barrier. Astrocytic endfeet fail to encircle cortical blood vessels in the mutant mice. These results suggest that BMPRIA signaling in astrocytes regulates expression of VEGF for proper cerebrovascular angiogenesis and has a role on formation of the BBB. Molecular and Cellular Neuroscience 2008.

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2. Global ischemia and astrocytic functions
   The mechanism for how astrocyte influences appropriate changes in neuronal network remains unclear during the reduced cerebral blood flow. We address the ability of vasoconstrictions influences on the astrocytic and neural state of the mouse brain. The M5 muscarinic acetylcholine receptor (M5R) has been shown to play a crucial role in mediating acetylcholine-dependent dilation of cerebral blood vessels. We reported that male M5R knockout mice (M5R-/- mice) suffer from a constitutive constriction of cerebral arteries, reduced cerebral blood flow, dendritic atrophy, and short-term memory loss. We proposed that male M5R-/- mouse is a novel animal model of cerebrovascular insufficiency.
   We show that Male M5R-/- mice showed gliosis in cortex and hippocampus. However, female M5R-/- mice did not show these phenotypic changes. However, ovariectomized female M5R-/- mice displayed phenotypic changes similar to male M5R-/- mice, strongly suggesting that estrogen plays a key role in the observed gender differences. We found that 17 -estradiol (E2) induced nitric oxide release and MAPK activation in endothelial cell line. Treatment with E2 also improved the performance of male M5R-/- mice in a cognitive test. This phenotype was reversed by E2 treatment, similar to the observed deficits in dendrite morphology and astrocyte swelling in the cerebral cortex and hippocampus. Astrocytic swelling are known to associate with an increase of extra-cellular space volume, which lead to the impairment of diffusion of neuroactive substances, extra-synaptic transmission between neuron-glia communications. Our observations strongly support the concept that the beneficial effects of estrogen on neural function observed in the present study are due to cerebral vasodilation as well as a direct neuroprotective effect.
   Recent work has suggested that the E2 may exert protective effects in neurodegenerative disorders such as acute ischemia. While E2 has consistently been shown to be protective in vivo, direct protection of neurons remains controversial, suggesting that while direct protection of neurons may occur in some instances, an alternative or parallel pathway for protection may exist which could involve another cell type in the brain. We propose a possible role for interaction neural cells in the mediation of neuroprotection by E2.

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	The volume of Cerebral blood vessel and Learning Ability    The M5 muscarinic acetylcholine receptor (M5R) has been shown to play a crucial role in mediating acetylcholine-dependent dilation of cerebral blood vessels.    In the present study, we demonstrated that M5R-/- mice showed pronounced constitutive constriction of cerebral arteries using magnetic resonance angiography in vivo, including reductions in resting cerebral blood flow (CBF).    Moreover, cortical and hippocampal pyramidal neurons from M5R-/- mice showed a reduced number of spines and dendritic atrophy.    In the absence of M5Rs, CA3 pyramidal cells displayed a significantly attenuated frequency of the spontaneous postsynaptic current, and long-term potentiation was significantly impaired at the mossy fiber-CA3 synapse.    Finally, hippocampus-dependent spatial and non-spatial memory was also impaired in M5R-/- mice.    Our findings strongly suggest that impaired M5R signaling may play a role in the pathophysiology of cerebrovascular deficits. Neurobiology of Disease, 24(2), 334-344, 2006
	Representative angiograms of the basilar artery from M5R+/+ and M5R-/- mice    High-resolution magnetic resonance angiography (MRA) was used as a means to investigate the arterial cerebrovascular hemodynamics noninvasively in adult mice.   The adjacent vessels emerging from the basilar artery. M5R-/- mice showed reduced diameters of the basilar artery. Arrows indicate the blood vessel regions.
	Acetylcholine has a well-known function for vasodialation through the muscarinic acetylcholine receptor. However, it was unclear which receptor was responsible for such a function.    M5 muscarinic receptor knockout mice showed the perfect loss of vasodilating ability by achetylcholine in the basilar artery, which revealed that the acetylcholine vasodilation was caused through the M5 muscarinic receptor. Yamada M. et al., Proc. Natl. Acad. Sci. USA, 98, 14096-14101, 2001.
	Our results indicated that the acetylcholine vasodialation through muscarinic acetylcholine receptor worked as an important role for relaxing blood vessels.    There were two results in different arteries of the knockout mice; "the vasodialating ability" by acetylcholine was lost in the brain artery, such as the basilar artery, but was preserved in peripheral one, such as the coronal artery.    After more precise analyses, M5 muscarinic receptor knockout mice showed chronic decrease of the amount of cerebral blood flow suffered by the impaired vasodilation.

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3. Astrocytic BMP receptor signaling and brain injury
   BMPs are believed to play roles in formation of glial scarring because they are highly expressed at the sites of brain injury and show potent ability to stimulate reactive astrocytic responses. However, mechanisms of how BMP signaling regulates astrocytic responses are not well understood. We study that mice deficient for Bmpr1a displayed decrease in astrocytic proliferation at damaged area of brain cortex, whereas mice deficient for Bmpr1b displayed enhanced gliosis. Inhibition of BMPRIA mediated BMP signaling would be an attractive therapeutic target.

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4. Development of stem cell differentiation system
   We report that mild electrical stimulation strongly influences embryonic stem cells to assume a neuronal fate. Although the resulting neuronal cells showed no sign of specific terminal differentiation in culture, they showed potential to differentiate into various types of neurons in vivo, and contributed to injured spinal cord in adult mice. Induction of calcium ionic influx has significant importance in this differentiation system. This phenomenon opens up possibilities for understanding novel mechanisms underlying cellular differentiation and early development, and perhaps more importantly, suggests possibilities for treatments in medical contexts.

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5. Development of gene delivery system
   We have developed virus-like nanoparticles in which gene and liposomal complexes or drugs are packaged in an artificial lipid bilayer membrane envelope. The surfaces are further modified with functional elements such as membrane permeability peptides like octa-arginine. These multifunctional envelope-type nano devices (MEND) have less toxicity than adenoviruses. Application of genes inserted into these particles on the skin of mice produced gene expression in the body hair. These MEND particles are useful as in vivo carriers of genes and proteins. Patents have been filed for use as a hair growth agent and to prevent glial scar formation (Domestic patent 2005-64687 [name of patent: hair growth composition]; Domestic patent 2005-181699 [name of patent: nerve regeneration promotion agent). This technology is useful for drug delivery to a variety of tissues in addition to skin. For example, by modifying the lipid bilayer membrane surface of MEND particles with functional elements such as antibodies, they can be used as a tool to control cell selection.

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