Amyloid‐beta 1‐40 is associated with alterations in NG2+ pericyte population ex vivo and in vitro

The population of brain pericytes, a cell type important for vessel stability and blood brain barrier function, has recently been shown altered in patients with Alzheimer's disease (AD). The underlying reason for this alteration is not fully understood, but progressive accumulation of the AD characteristic peptide amyloid‐beta (Aβ) has been suggested as a potential culprit. In the current study, we show reduced number of hippocampal NG2+ pericytes and an association between NG2+ pericyte numbers and Aβ1‐40 levels in AD patients. We further demonstrate, using in vitro studies, an aggregation‐dependent impact of Aβ1‐40 on human NG2+ pericytes. Fibril‐EP Aβ1‐40 exposure reduced pericyte viability and proliferation and increased caspase 3/7 activity. Monomer Aβ1‐40 had quite the opposite effect: increased pericyte viability and proliferation and reduced caspase 3/7 activity. Oligomer‐EP Aβ1‐40 had no impact on either of the cellular events. Our findings add to the growing number of studies suggesting a significant impact on pericytes in the brains of AD patients and suggest different aggregation forms of Aβ1‐40 as potential key regulators of the brain pericyte population size.     

Pericyte deficiencies lead to aberrant tumor vascularizaton in the brain of the NG2 null mouse

Tightly regulated crosstalk between endothelial cells and pericytes is required for formation and maintenance of functional blood vessels. When the NG2 proteoglycan is absent from pericyte surfaces, vascularization of syngeneic tumors growing in the C57Bl/6 mouse brain is aberrant in several respects, resulting in retardation of tumor progression. In the NG2 null mouse brain, pericyte investment of the tumor vascular endothelium is reduced, causing deficiencies in both pericyte and endothelial cell maturation, as well as reduced basal lamina assembly. While part of this deficit may be due to the previously-identified role of NG2 in β1 integrin-dependent periyte/endothelial cell crosstalk, the ablation of NG2 also appears responsible for loss of collagen VI anchorage, in turn leading to reduced collagen IV deposition. Poor functionality of tumor vessels in NG2 null brain is reflected by reduced vessel patency and increased vessel leakiness, resulting in large increases in tumor hypoxia. These findings demonstrate the importance of NG2-dependent pericyte/endothelial cell interaction in the development and maturation of tumor blood vessels, identifying NG2 as a potential target for anti-angiogenic cancer therapy.     

Targeting the NG2/CSPG4 proteoglycan retards tumour growth and angiogenesis in preclinical models of GBM and melanoma

Aberrant expression of the progenitor marker Neuron-glia 2 (NG2/CSPG4) or melanoma proteoglycan on cancer cells and angiogenic vasculature is associated with an aggressive disease course in several malignancies including glioblastoma multiforme (GBM) and melanoma. Thus, we investigated the mechanism of NG2 mediated malignant progression and its potential as a therapeutic target in clinically relevant GBM and melanoma animal models. Xenografting NG2 overexpressing GBM cell lines resulted in increased growth rate, angiogenesis and vascular permeability compared to control, NG2 negative tumours. The effect of abrogating NG2 function was investigated after intracerebral delivery of lentivirally encoded shRNAs targeting NG2 in patient GBM xenografts as well as in established subcutaneous A375 melanoma tumours. NG2 knockdown reduced melanoma proliferation and increased apoptosis and necrosis. Targeting NG2 in two heterogeneous GBM xenografts significantly reduced tumour growth and oedema levels, angiogenesis and normalised vascular function. Vascular normalisation resulted in increased tumour invasion and decreased apoptosis and necrosis. We conclude that NG2 promotes tumour progression by multiple mechanisms and represents an amenable target for cancer molecular therapy.     

Nerve/glial antigen (NG) 2 is a crucial regulator of intercellular adhesion molecule (ICAM)-1 expression

The proteoglycan nerve/glial antigen (NG) 2 is expressed on multiple cell types and mediates cell proliferation and migration. However, little is known about its function in gene regulation. In this study, we demonstrate that in pericytes and glioblastoma cells intercellular adhesion molecule (ICAM)-1, an essential protein for leukocyte adhesion and transmigration, underlies a NG2-dependent expression. As shown by flow cytometry, Western blot analysis and quantitative real-time polymerase chain reaction (qRT-PCR), silencing of NG2 in human placenta-derived pericytes increased the expression of ICAM-1. Pathway analyses revealed that this is mediated by extracellular-regulated-kinases (ERK) 1/2 signaling. Moreover, leukocyte adhesion to NG2 siRNA-treated pericytes was significantly enhanced when compared to scrambled (scr) siRNA-treated control cells. In vivo, we detected increased ICAM-1 protein levels in the retina of mice lacking NG2 expression. To exclude that this novel mechanism is pericyte-specific, we additionally analyzed the expression of ICAM-1 in dependency of NG2 in two glioblastoma cell lines. We found that A1207 and M059K cells exhibit an inverse expression pattern of NG2 and ICAM-1. Finally, downregulation of NG2 in A1207 cells significantly increased ICAM-1 expression. Taken together, these findings indicate that NG2 may represent a promising target for the modulation of ICAM-1-mediated immune responses.     

Changes in pericytic expression of NG2 and PDGFRB and vascular permeability in the sensory circumventricular organs of adult mouse by osmotic stimulation

The blood–brain barrier (BBB) is a barrier that prevents free access of blood‐derived substances to the brain through the tight junctions and maintains a specialized brain environment. Circumventricular organs (CVOs) lack the typical BBB. The fenestrated vasculature of the sensory CVOs, including the organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO) and area postrema (AP), allows parenchyma cells to sense a variety of blood‐derived information, including osmotic ones. In the present study, we utilized immunohistochemistry to examine changes in the expression of NG2 and platelet‐derived growth factor receptor beta (PDGFRB) in the OVLT, SFO and AP of adult mice during chronic osmotic stimulation. The expression of NG2 and PDGFRB was remarkably prominent in pericytes, although these angiogenesis‐associated proteins are highly expressed at pericytes of developing immature vasculature. The chronic salt loading prominently increased the expression of NG2 in the OVLT and SFO and that of PDGFRB in the OVLT, SFO and AP. The vascular permeability of low‐molecular‐mass tracer fluorescein isothiocyanate was increased significantly by chronic salt loading in the OVLT and SFO but not AP. In conclusion, the present study demonstrates changes in pericyte expression of NG2 and PDGFRB and vascular permeability in the sensory CVOs by chronic osmotic stimulation, indicating active participation of the vascular system in osmotic homeostasis. Copyright © 2013 John Wiley & Sons, Ltd.     

Peripheral nerve pericytes modify the blood–nerve barrier function and tight junctional molecules through the secretion of various soluble factors

The objectives of this study were to establish pure blood–nerve barrier (BNB) and blood–brain barrier (BBB)‐derived pericyte cell lines of human origin and to investigate their unique properties as barrier‐forming cells. Brain and peripheral nerve pericyte cell lines were established via transfection with retrovirus vectors incorporating human temperature‐sensitive SV40 T antigen (tsA58) and telomerase. These cell lines expressed several pericyte markers such as α‐smooth muscle actin, NG2, platelet‐derived growth factor receptor β, whereas they did not express endothelial cell markers such as vWF and PECAM. In addition, the inulin clearance was significantly lowered in peripheral nerve microvascular endothelial cells (PnMECs) through the up‐regulation of claudin‐5 by soluble factors released from brain or peripheral nerve pericytes. In particular, bFGF secreted from peripheral nerve pericytes strengthened the barrier function of the BNB by increasing the expression of claudin‐5. Peripheral nerve pericytes may regulate the barrier function of the BNB, because the BNB does not contain cells equivalent to astrocytes which regulate the BBB function. Furthermore, these cell lines expressed several neurotrophic factors such as NGF, BDNF, and GDNF. The secretion of these growth factors from peripheral nerve pericytes might facilitate axonal regeneration in peripheral neuropathy. Investigation of the characteristics of peripheral nerve pericytes may provide novel strategies for modifying BNB functions and promoting peripheral nerve regeneration. J. Cell. Physiol. 226: 255–266, 2010. © 2010 Wiley‐Liss, Inc.     

Rat choroidal pericytes as a target of the autonomic nervous system

Pericytes are contractile cells that surround blood vessels. When contracting, they change the diameter of the vessel and therefore influence blood flow homeostasis; however, mechanisms controlling pericyte action are less well understood. Since blood flow regulation per se is controlled by the autonomic nervous system, the latter might also be involved in pericyte action. Hence, rat choroidal pericytes were analyzed for such a connection by using appropriate markers. Rat choroidal wholemounts and sections were prepared for immunohistochemistry of the pericyte marker chondroitin-sulfate-proteoglycan (NG2) and the pan-neuronal marker PGP9.5 or of tyrosine hydroxylase (TH), vasoactive intestinal polypeptide (VIP) and choline acetyl transferase (ChAT). Additionally, PGP9.5 and TH were analyzed in the choroid of DCX-dsRed2 transgenic rats, displaying red-fluorescent perivascular cells and serving as a putative model for studying pericyte function in vivo. Confocal laser-scanning microscopy revealed NG2-immunoreactive cells and processes surrounding the blood vessels. These NG2-positive cells were not co-localized with PGP9.5 but received close appositions of PGP9.5-, TH-, VIP- and ChAT-immunoreactive boutons and fibers. In the DCX-dsRed2 transgenic rat, PGP9.5 and TH were also densely apposed on the dsRed-positive cells adjacent to blood vessels. These cells were likewise immunoreactive for NG2, suggesting their pericyte identity. In addition to the innervation of vascular smooth muscle cells, the close relationship of PGP9.5 and further sympathetic (TH) and parasympathetic (VIP, ChAT) nerve fibers on NG2-positive pericytes indicated an additional target of the autonomic nervous system for choroidal blood flow regulation. Similar findings in the DCX-dsRed transgenic rat indicate the potential use of this animal model for in vivo experiments revealing the role of pericytes in blood flow regulation.     

Apolipoprotein E Inhibits Cerebrovascular Pericyte Mobility through a RhoA Protein-mediated Pathway

Pericytes play a critical role in the cerebrovasculature within the CNS. These small contractile cells produce large quantities of apolipoprotein E (apoE) whose isoforms influence cerebrovascular functions and determine the genetic risk for Alzheimer disease. Despite extensive studies on astrocyte-secreted apoE, which supports synapses by transporting cholesterol to neurons, the biochemical properties and function of apoE secreted by pericytes are not clear. Because pericytes mediate important functions in the CNS, including the initiation of glial scar formation, angiogenesis, and maintenance of the blood-brain barrier, we investigated the potential role of apoE in pericyte mobility. We found that knockdown of apoE expression significantly accelerates pericyte migration, an effect that can be rescued by exogenous apoE3, but not apoE4, a risk factor for Alzheimer disease. ApoE-regulated migration of pericytes also requires the function of the low-density lipoprotein receptor-related protein 1 (LRP1), a major apoE receptor in the brain that is abundantly expressed in pericytes. Because apoE-knockdown also leads to enhanced cell adhesion, we investigated the role of apoE in the regulation of the actin cytoskeleton. Interestingly, we found that the levels of active RhoA are increased significantly in apoE knockdown pericytes and that RhoA inhibitors blocked pericyte migration. Taken together, our results suggest that apoE has an intrinsic role in pericyte mobility, which is vital in maintaining cerebrovascular function. These findings provide novel insights into the role of apoE in the cerebrovascular system.     

Transduction of Extracellular Cues into Cell Polarity: the Role of the Transmembrane Proteoglycan NG2

Resident progenitor cells expressing nerve/glial antigen 2 (NG2) such as oligodendrocyte precursor cells (OPC) and pericytes persist in the adult brain. The transmembrane proteoglycan NG2 regulates migration of both these cell types in response to growth factors or specific components of the extracellular matrix. This role of NG2 is linked to the control of cell polarity. The polarization of OPC toward an acute lesion in the brain is impaired in NG2-deficient mice, supporting this concept. A review of the signaling pathways impinged on by NG2 reveals key proteins of cell polarity: phosphatidylinositol 3-kinase, focal adhesion kinase, Rho GTPases, and polarity complex proteins. In the scope of cell migration, I discuss here how the interplay of NG2 with signaling transmitted by extracellular cues can control the establishment of cell polarity, and I propose a model to integrate the apparent opposite effects of NG2 on cellular dynamics.     

Sirtuin 3 is essential for hypertension‐induced cardiac fibrosis via mediating pericyte transition

Hypertension is the key factor for the development of cardiac fibrosis and diastolic dysfunction. Our previous study showed that knockout of sirtuin 3 (SIRT3) resulted in diastolic dysfunction in mice. In the present study, we explored the role of SIRT3 in angiotensin II (Ang‐II)–induced cardiac fibrosis and pericyte‐myofibroblast transition. NG2 tracing reporter NG2‐DsRed mouse was crossed with wild‐type (WT) mice and SIRT3KO mice. Cardiac function, cardiac fibrosis and reactive oxygen species (ROS) were measured. Mice infused with Ang‐II for 28 days showed a significant reduction of SIRT3 expression in the mouse hearts. Knockout of SIRT3 sensitized Ang‐II‐induced elevation of isovolumic relaxation time (IVRT) and reduction of ejection fraction (EF) and fractional shortening (FS). Ang‐II‐induced cardiac fibrosis, capillary rarefaction and hypertrophy were further enhanced by knockout of SIRT3. NG2 pericyte tracing reporter mice infused with Ang‐II had a significantly increased number of NG2‐DsRed pericyte in the heart. Knockout of SIRT3 further enhanced Ang‐II‐induced increase of pericytes. To examine pericyte‐myofibroblast/fibroblast transition, DsRed pericytes were co‐stained with FSP‐1 and α‐SMA. Ang‐II infusion led to a significant increase in numbers of DsRed⁺/FSP‐1⁺ and DsRed⁺/α‐SMA⁺ cells, while SIRT3KO further developed pericyte‐myofibroblast/fibroblast transition. In addition, knockout of SIRT3 promoted Ang‐II‐induced NADPH oxidase‐derived ROS formation together with increased expression of transforming growth factor beta 1 (TGF‐β1). We concluded that Ang‐II induced cardiac fibrosis partly by the mechanisms involving SIRT3‐mediated pericyte‐myofibroblast/fibroblast transition and ROS‐TGF‐β1 pathway.     

Inhibition of Transforming Growth Factor-<Emphasis Type="Bold">β</Emphasis> Production in Brain Pericytes Contributes to Cyclosporin A-Induced Dysfunction of the Blood-Brain Barrier

1. The present study was designed to clarify whether brain pericytes and pericyte-derived transforming growth factor-β1 (TGF-β1) participate in cyclosporin A (CsA)-induced dysfunction of the blood-brain barrier (BBB). 2. The presence of brain pericytes markedly aggravated CsA-increased permeability of MBEC4 cells to sodium fluorescein and accumulation of rhodamine 123 in MBEC4 cells. 3. Exposure to CsA significantly decreased the levels of TGF-β1 mRNA in brain pericytes in pericyte co-cultures. Treatment with TGF-β1 dose-dependently inhibited CsA-induced hyperpermeability and P-glycoprotein dysfunction of MBEC4 cells in pericyte co-cultures. 4. These findings suggest that an inhibition of brain pericyte-derived TGF-β1 contributes to the occurrence of CsA-induced dysfunction of the BBB.     

Peripheral nerve pericytes originating from the blood-nerve barrier expresses tight junctional molecules and transporters as barrier-forming cells

The objective of this study was to establish pure blood-nerve barrier (BNB)-derived peripheral nerve pericyte cell lines and to investigate their unique properties as barrier-forming cells. We isolated peripheral nerve, brain, and lung pericytes from transgenic rats harboring the temperature-sensitive simian virus 40 large T-antigen gene. These cell lines expressed several pericyte markers such as alpha-smooth muscle actin, NG2, osteopontin, and desmin, whereas they did not express endothelial cell markers such as vWF and PECAM. In addition, these cell lines expressed several tight junction molecules such as occludin, claudin-12, ZO-1, and ZO-2. In particular, the expression of occludin was detected in peripheral nerve and brain pericytes, although it was not detected in lung pericytes by a Western blot analysis. An immunocytochemical analysis confirmed that occludin and ZO-1 were localized at the cell-cell boundaries among the pericytes. Brain and peripheral nerve pericytes also showed significantly higher trans-pericyte electrical resistance values and lower inulin clearances than lung pericytes. We considered that occludin localized at the cell-cell boundaries among the pericytes might mechanically stabilize the microvessels of the BNB and the blood-brain barrier. Furthermore, we also showed that these cell lines expressed many barrier-related transporters. ABCG2, p-gp, MRP-1, and Glut-1 were detected by a Western blot analysis and were observed in the cytoplasm and outer membrane by an immunocytochemical analysis. These transporters on pericytes might facilitate the peripheral nerve-to-blood efflux and blood-to-peripheral nerve influx transport of substrates in cooperation with those on endothelial cells in order to maintain peripheral nerve homeostasis.     

In situ analysis of microvascular pericytes in hypertensive rat brains

We used immunofluorescence microscopy and isoactin-specific antibodies to characterize the pattern and prevalence of pericytes within the brain microcirculation. Blood pressures of normotensive, Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats were measured prior to sacrifice and pressure-perfusion fixation. WKY and SHR brains were subdivided into ten major regions prior to ultracryomicrotomy. Sections 0.3-0.5 micron wide were treated with 10-40 micrograms/ml affinity-purified antibodies to the muscle and non-muscle actin isoforms. These localization studies show that there are four times the number of pericyte-rich capillaries in the SHR motor cortex compared to WKY counterparts (59.9 vs. 15.3%). In contrast, the sensory cortex of both rat strains is deficient in muscle actin staining surrounding the capillaries. The most striking difference in pericyte presence and muscle actin antibody staining between the SHR and WKY was observed in the tegmentum of the brainstem. There is nearly a one-to-one coincidence observed in pericyte and capillary profiles present within thin, frozen sections of the SHR midbrain. SHR pons capillaries were also pericyte-enriched. WKY analyses of plastic embedded thin sections confirmed the presence of pericytes and their filament-enriched processes encircling the capillaries of the hypertensive brains. These results suggest that pericytes may play important roles in hypertension and cerebrovascular disease processes.     

The Angiopoietin-1 Variant COMP-Ang1 Enhances BMP2-Induced Bone Regeneration with Recruiting Pericytes in Critical Sized Calvarial Defects

Craniofacial bone defects are observed in a variety of clinical situations, and their reconstructions require coordinated coupling between angiogenesis and osteogenesis. In this study, we explored the effects of cartilage oligomeric matrix protein-angiopoietin 1 (COMP-Ang1), a synthetic and soluble variant of angiopoietin 1, on bone morphogenetic protein 2 (BMP2)-induced cranial bone regeneration, and recruitment and osteogenic differentiation of perivascular pericytes. A critical-size calvarial defect was created in the C57BL/6 mouse and COMP-Ang1 and/or BMP2 proteins were delivered into the defects with absorbable collagen sponges. After 3 weeks, bone regeneration was evaluated using micro-computed tomography and histologic examination. Pericyte recruitment into the defects was examined using immunofluorescence staining with anti-NG2 and anti-CD31 antibodies. In vitro recruitment and osteoblastic differentiation of pericyte cells were assessed with Boyden chamber assay, staining of calcified nodules, RT-PCR and Western blot analyses. Combined administration of COMP-Ang1 and BMP2 synergistically enhanced bone repair along with the increased population of CD31 (an endothelial cell marker) and NG2 (a specific marker of pericyte) positive cells. In vitro cultures of pericytes consistently showed that pericyte infiltration into the membrane pore of Boyden chamber was more enhanced by the combination treatment. In addition, the combination further increased the osteoblast-specific gene expression, including bone sialoprotein (BSP), osteocalcin (OCN) and osterix (OSX), phosphorylation of Smad/1/5/8, and mineralized nodule formation. COMP-Ang1 can enhance BMP2-induced cranial bone regeneration with increased pericyte recruitment. Combined delivery of the proteins might be a therapeutic strategy to repair cranial bone damage.     

Endogenous Brain Pericytes Are Widely Activated and Contribute to Mouse Glioma Microvasculature

Glioblastoma multiforme (GBM) is the most common brain tumor in adults. It presents an extremely challenging clinical problem, and treatment very frequently fails due to the infiltrative growth, facilitated by extensive angiogenesis and neovascularization. Pericytes constitute an important part of the GBM microvasculature. The contribution of endogenous brain pericytes to the tumor vasculature in GBM is, however, unclear. In this study, we determine the site of activation and the extent of contribution of endogenous brain pericytes to the GBM vasculature. GL261 mouse glioma was orthotopically implanted in mice expressing green fluorescent protein (GFP) under the pericyte marker regulator of G protein signaling 5 (RGS5). Host pericytes were not only activated within the glioma, but also in cortical areas overlying the tumor, the ipsilateral subventricular zone and within the hemisphere contralateral to the tumor. The host-derived activated pericytes that infiltrated the glioma were mainly localized to the tumor vessel wall. Infiltrating GFP positive pericytes co-expressed the pericyte markers platelet-derived growth factor receptor-β (PDGFR-β) and neuron-glial antigen 2. Interestingly, more than half of all PDGFR-β positive pericytes within the tumor were contributed by the host brain. We did not find any evidence that RGS5 positive pericytes adopt another phenotype within glioma in this paradigm. We conclude that endogenous pericytes become activated in widespread areas of the brain in response to an orthotopic mouse glioma. Host pericytes are recruited into the tumor and constitute a major part of the tumor pericyte population.     

FGF2-dependent neovascularization of subcutaneous Matrigel plugs is initiated by bone marrow-derived pericytes and macrophages

Vessel-like networks are quickly formed in subcutaneous FGF2-supplemented Matrigel plugs by two cell types: NG2(+) pericytes and F4/80(+) macrophages. Although not detected in these networks until 7 days after plug implantation, the appearance of CD31(+) endothelial cells marks the onset of vessel perfusion and the establishment of mature vessel morphology, with endothelial cells invested tightly by pericytes and more loosely by macrophages. Evidence that mature vessels develop from pericyte/macrophage networks comes from experiments in which 5-day plugs are transplanted into EGFP(+) recipients and allowed to mature. Fewer than 5% of pericytes in mature vessels are EGFP(+) in this paradigm, demonstrating their presence in the networks prior to plug transplantation. Endothelial cells represent the major vascular cell type recruited during later stages of vessel maturation. Bone marrow transplantation using EGFP(+) donors establishes that almost all macrophages and more than half of the pericytes in Matrigel vessels are derived from the bone marrow. By contrast, only 10% of endothelial cells exhibit a bone marrow origin. The vasculogenic, rather than angiogenic, nature of this neovascularization process is unique in that it is initiated by pericyte and macrophage progenitors, with endothelial cell recruitment occurring as a later step in the maturation process.     

中枢神经系统周细胞表达结蛋白desmin

目的:本研究着重探讨中枢神经系统周细胞是否表达desmin。方法:取3周龄雄性Wistar大鼠,取其大脑和脊髓组织,分离提取培养脑微血管周细胞(BMP)和脊髓微血管周细胞(SCMP)两种周细胞,用周细胞非特异性标记物神经胶质抗原2(NG2)和α-平滑肌肌动蛋白(α-SMA)双标鉴定周细胞,并用免疫荧光方法定性定量测定两种周细胞表达desmin阳性率,免疫印记分析实验定量测定两种周细胞表达desmin。结果:观察刚分离得到的脑微血管和脊髓微血管,前者在长度和密度上多于后者,培养至第3天时,观察到BMP更趋向于聚集,而SCMP则更分散。培养至第9天时,两种周细胞基本铺满了培养皿。经用周细胞非特异性标记物NG2和α-SMA双标鉴定,确定分离得到的细胞为周细胞,免疫荧光和western blot实验结果表明两种周细胞均表达desmin,且SCMP表达desmin阳性率显著多于BMP(P0.001),SCMP表达desmin量显著多于BMP(P0.05)。结论:中枢神经系统周细胞表达结蛋白desmin,表达量不同暗示了血脑屏障和血脊髓屏障之间存在差异。     

Nuclear Localization of the δ Subunit of Ca2+/Calmodulin-Dependent Protein Kinase II in Rat Cerebellar Granule Cells

Abstract : To examine the physiological roles of the δ subunit of Ca²⁺/calmodulin-dependent protein kinase ∥ (CaM kinase ∥δ) in brain, we examined the localization of CaM kinase ∥δ in the rat brain. A specific antibody to CaM kinase ∥δ1-δ4 isoforms was prepared by immunizing rabbits with a synthesized peptide corresponding to the unique carboxyl-terminal end of these isoforms. The prepared antibody did not recognize the α, β, and γ subunits, which were each overexpressed in NG108-15 cells. Immunoblot analysis on various regions and the nuclear fractions from rat brains suggested that some isoforms of CaM kinase ∥δ1-δ4 were abundant in the nucleus in the cerebellum. Total RNA from the cerebellum was analyzed by RT-PCR with a primer pair from variable domain 1 to variable domain 2. We detected the three PCR products δ3.1, δ3.4, and δ3 that contained the nuclear localization signal. These CaM kinase ∥δ3 isoforms were localized in the nuclei in transfected NG108-15 cells. Immunohistochemical study suggested the existence of these isoforms in the nuclei in cerebellar granule cells. These results suggest that CaM kinase ∥δ3 isoforms are involved in nuclear Ca²⁺ signaling in cerebellar granule cells.     

Skeletal muscle pericyte subtypes differ in their differentiation potential

Neural progenitor cells have been proposed as a therapy for central nervous system disorders, including neurodegenerative diseases and trauma injuries, however their accessibility is a major limitation. We recently isolated Tuj1 + cells from skeletal muscle culture of Nestin–GFP transgenic mice however whether they form functional neurons in the brain is not yet known. Additionally, their isolation from nontransgenic species and identification of their ancestors is unknown. This gap of knowledge precludes us from studying their role as a valuable alternative to neural progenitors. Here, we identified two pericyte subtypes, type-1 and type-2, using a double transgenic Nestin–GFP/NG2–DsRed mouse and demonstrated that Nestin–GFP +/Tuj1 + cells derive from type-2 Nestin–GFP +/NG2–DsRed +/CD146 + pericytes located in the skeletal muscle interstitium. These cells are bipotential as they generate either Tuj1 + cells when cultured with muscle cells or become “classical” α-SMA + pericytes when cultured alone. In contrast, type-1 Nestin–GFP ?/NG2–DsRed +/CD146 + pericytes generate α-SMA + pericytes but not Tuj1 + cells. Interestingly, type-2 pericyte derived Tuj1 + cells retain some pericytic markers (CD146 +/PDGFRβ +/NG2 +). Given the potential application of Nestin–GFP +/NG2–DsRed +/Tuj1 + cells for cell therapy, we found a surface marker, the nerve growth factor receptor, which is expressed exclusively in these cells and can be used to identify and isolate them from mixed cell populations in nontransgenic species for clinical purposes.