Immunomodulatory therapy with glatiramer acetate reduces endoplasmic reticulum stress and mitochondrial dysfunction in experimental autoimmune encephalomyelitis.

Endoplasmic reticulum (ER) stress and mitochondrial dysfunction are found in lesions of multiple sclerosis (MS) and animal models of MS such as experimental autoimmune encephalomyelitis (EAE), and may contribute to the neuronal loss that underlies permanent impairment. We investigated whether glatiramer acetate (GA) can reduce these changes in the spinal cords of chronic EAE mice by using routine histology, immunostaining, and electron microscopy. EAE spinal cord tissue exhibited increased inflammation, demyelination, mitochondrial dysfunction, ER stress, downregulation of NAD+ dependent pathways, and increased neuronal death. GA reversed these pathological changes, suggesting that immunomodulating therapy can indirectly induce neuroprotective effects in the CNS by mediating ER stress.

A subset of mobilized human hematopoietic stem cells express germ layer lineage genes which can be modulated by culture conditions.

BACKGROUND: Adult bone marrow contains stem cells that replenish the myeloid and lymphoid lineages. A subset of human and mouse CD34+ bone marrow stem cells can be propagated in culture to autonomously express embryonic stem cell genes and embryonic germ layer lineage genes. The current study was undertaken to determine whether these CD34+ stem cells could be obtained from human blood, whether gene expression could be modulated by culture conditions and whether the cells produce insulin. METHODS: Human peripheral blood buffy coat cells and mobilized CD34+ cells from human blood and from blood from C57Bl/6 J mice were cultured in hybridoma medium or neural stem cell induction medium supplemented with interleukin (IL)-3, IL-6, and stem cell factor (SCF). Changes in mRNA and protein expression were assessed by Western blot analysis and by immunohistochemistry. Mass spectrometry was used to assess insulin production. RESULTS: We were able to culture CD34+ cells expressing embryonic stem cell and embryonic germ layer lineage genes from adult human peripheral blood after standard mobilization procedures and from mouse peripheral blood. Gene expression could be modulated by culture conditions, and the cells produced insulin in culture. CONCLUSION: These results suggest a practical method for obtaining large numbers of CD34+ cells from humans to allow studies on their potential to differentiate into other cell types.

Salutary effects of glibenclamide during the chronic phase of murine experimental autoimmune encephalomyelitis.

BACKGROUND: In multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), inflammation is perpetuated by both infiltrating leukocytes and astrocytes. Recent work implicated SUR1-TRPM4 channels, expressed mostly by astrocytes, in murine EAE. We tested the hypothesis that pharmacological inhibition of SUR1 during the chronic phase of EAE would be beneficial. METHODS: EAE was induced in mice using myelin oligodendrocyte glycoprotein (MOG) 35-55. Glibenclamide (10 µg/day) was administered beginning 12 or 24 days later. The effects of treatment were determined by clinical scoring and tissue examination. Drug within EAE lesions was identified using bodipy-glibenclamide. The role of SUR1-TRPM4 in primary astrocytes was characterized using patch clamp and qPCR. Demyelinating lesions from MS patients were studied by immunolabeling and immunoFRET. RESULTS: Administering glibenclamide beginning 24 days after MOG35-55 immunization, well after clinical symptoms had plateaued, improved clinical scores, reduced myelin loss, inflammation (CD45, CD20, CD3, p65), and reactive astrocytosis, improved macrophage phenotype (CD163), and decreased expression of tumor necrosis factor (TNF), B-cell activating factor (BAFF), chemokine (C-C motif) ligand 2 (CCL2) and nitric oxide synthase 2 (NOS2) in lumbar spinal cord white matter. Glibenclamide accumulated within EAE lesions, and had no effect on leukocyte sequestration. In primary astrocyte cultures, activation by TNF plus IFNγ induced de novo expression of SUR1-TRPM4 channels and upregulated Tnf, Baff, Ccl2, and Nos2 mRNA, with glibenclamide blockade of SUR1-TRPM4 reducing these mRNA increases. In demyelinating lesions from MS patients, astrocytes co-expressed SUR1-TRPM4 and BAFF, CCL2, and NOS2. CONCLUSIONS: SUR1-TRPM4 may be a druggable target for disease modification in MS.

Cell-based delivery of brain-derived neurotrophic factor in experimental allergic encephalomyelitis.

Brain-derived neurotrophic factor (BDNF) is a pleiotropic cytokine with neuroprotective properties that has been identified as a potential therapeutic agent for diseases of the central nervous system (CNS). The use of BDNF has been limited by a short serum half-life and poor penetration of the blood-brain barrier. To address this limitation we have explored cell-based approaches to delivery. We have used experimental allergic encephalomyelitis (EAE), an inflammatory disease of the CNS, as a model system. We engineered hematopoietic stem cells to produce BDNF to determine the feasibility and effectiveness of cell-based delivery of BDNF into the CNS in EAE. We review those studies here.

Hematopoietic progenitors express myelin basic protein and ensheath axons in Shiverer brain.

Oligodendroglia are cells of the central nervous system (CNS) that form myelin sheath, which insulates neuronal axons. Neuropathologies of the CNS include dysmyelination of axons in multiple sclerosis and CNS trauma. Cell replacement is a promising but largely untested therapy for dysmyelination. Shiverer mouse, a genetic mutant that does not synthesize full-length myelin basic protein (MBP), a critical prerequisite protein in CNS myelin sheath formation, provides an unequivocal model for determining the potential of stem cells to become oligodendroglia. We demonstrate that adult wild-type mouse bone marrow stem cells can express MBP and ensheath axons when transplanted into Shiverer brain.

Adult hematopoietic progenitors are multipotent in chimeric mice.

Embryonic stem cells (ESCs) and adult somatic cells, induced to pluripotency (iPSCs), can differentiate into multiple cell lineages. We previously reported that adult mammalian bone marrow contains a sub-population of CD34+ cells that express genes of ESCs and genes required to generate iPSCs. They also express lineage genes of the three embryonic germ layers. Are these CD34+ cells multipotent? Here, CD34+ bone marrow stem cells from adult male ROSA mice, which carry two markers: the ß-galactosidase gene and the male Y chromosome, were transplanted into blastocysts of wildtype mice. Each female ROSA chimera generated had a distinct pattern of male-derived organs expressing ß-galactosidase; e.g., ectodermal brain, dorsal root ganglia and skin; mesodermal heart, bone and bone marrow; and endodermal pancreas, intestine, and liver. Thus, adult mammals carry cells that appear to exhibit a developmental potential reminiscent of ESCs and iPSCs suggesting they could be used for cell replacement therapy.

Use of engineered bone marrow stem cells to deliver brain derived neurotrophic factor under the control of a tetracycline sensitive response element in experimental allergic encephalomyelitis.

Brain derived neurotrophic factor (BDNF) has neuroprotective properties but its use has been limited by poor penetration of the blood brain barrier. Treatment using bone marrow stem cells (BMSC) or retroviruses as vectors reduces the clinical and pathological severity of experimental allergic encephalomyelitis (EAE). We have refined the BMSC based delivery system by introducing a tetracycline sensitive response element to control BDNF expression. We have now tested that construct in EAE and have shown a reduction in both the clinical and pathological severity of the disease. Further, we looked for changes in sirtuin1 and nicotinamide phosphoribosyltransferase expression that would be consistent with a neuroprotective effect.

Retinal neural progenitors express topographic map markers.

Transplantation of neural stem cells for replacing neurons after neurodegeneration requires that the transplanted stem cells accurately reestablish the lost neural circuits in order to restore function. Retinal ganglion cell axons project to visual centers of the brain forming circuits in precise topographic order. In chick, dorsal retinal neurons project to ventral optic tectum, ventral neurons to dorsal tectum, anterior neurons to posterior tectum and posterior neurons to anterior tectum; forming a continuous point-to-point map of retinal cell position in the tectal projection. We found that when stem cells derived from ventral retina were implanted in dorsal host retina, the stem cells that became ganglion cells projected to dorsal tectum, appropriate for their site of origin in retina but not appropriate for their site of implant in retina. This led us to ask if retinal progenitors exhibit topographic markers of cell position in retina. Indeed, retinal neural progenitors express topographic markers: dorsal stem cells expressed more Ephrin B2 than ventral stem cells and, conversely, ventral stem cells expressed more Pax-2 and Ventroptin than dorsal stem cells. The fact that neural progenitors express topographic markers has pertinent implications in using neural stem cells in cell replacement therapy for replacing projecting neurons that express topographic order, e.g., analogous neurons of the visual, auditory, somatosensory and motor systems.

Brain-derived neurotrophic factor gene delivery in an animal model of multiple sclerosis using bone marrow stem cells as a vehicle.

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, is neuroprotective in animal models of neurodegenerative diseases. However, BDNF has a short half-life and its efficacy in the central nervous system (CNS), when delivered peripherally, is limited due to the blood-brain barrier (BBB). We have developed a means of delivering BDNF into the CNS using genetically engineered bone marrow stem cells (BMSCs) as a vehicle, and have explored the clinical effects of BDNF on outcomes in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). BDNF-engineered-BMSCs were transplanted (i.v.) into irradiated 2-week-old SJL/J female mice. Eight weeks after transplantation, mice were immunized with a peptide of proteolipid protein (PLP(139-151)). Mice, which had received BDNFengineered BMSCs, showed a significant delay in EAE onset and a reduction in overall clinical severity compared to mice receiving BMSC transfected with an empty vector lacking the BDNF gene. In addition, pathological examination showed that BDNF delivery reduced demyelination and increased remyelination. Inhibition of pro-inflammatory cytokines TNF-alpha and IFN-gamma and enhanced expression of the antiinflammatory cytokines IL-4, IL-10, and IL-11 were found in the CNS tissues of the BDNF transplanted group. These results support the use of BMSCs as vehicles to deliver BDNF into the CNS of EAE animals. This is a potentially novel therapeutic approach that might be used to deliver BDNF gene or genes for other therapeutic proteins into the CNS in MS or in other diseases of the CNS in which accessibility of therapeutic proteins is limited due to the BBB.

Stem cell based delivery of IFN-beta reduces relapses in experimental autoimmune encephalomyelitis.

Interferon-beta (IFN-beta), an approved treatment of multiple sclerosis (MS), produces only partial clinical responses. IFN-beta therapy has been limited by its short serum half-life and limited ability to cross the blood brain barrier. We have developed a means of delivering the IFN-beta gene both systemically and into the central nervous system (CNS) using bone marrow stem cells (BMSCs) as a vehicle and examined the therapeutic efficacy of this approach in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. A retroviral expression vector (pLXSN-IFNbeta) was used to stably transfect virus producer PA317 cells to generate retrovirus containing the IFN-beta gene which then was used to transduce BMSCs. IFN-beta engineered BMSCs were transplanted (i.v.) into mice that then were immunized with proteolipoprotein (PLP) to initiate EAE. IFN-beta-engineered BMSCs transplanted mice showed a significant inhibition of EAE onset, and the overall clinical severity was less compared to control groups. IFN-beta delivery strongly reduced infiltration of mononuclear cells possibly by inhibiting cell adhesion molecules. Reduced demyelination and increased remyelination were also observed in the IFN-beta treated group. Furthermore, inhibition of the pro-inflammatory cytokines TNF-alpha, IFN-gamma and IL-12 and enhanced expression of the anti-inflammatory cytokines IL-10, IL-4 and TGF-beta was observed in CNS tissue. In addition, mice receiving IFN-beta had reduced apoptosis and increases in growth promoting factors including BDNF, CNTF, PDGF and VEGF. These results suggest that BMSCs can be used as vehicles to deliver the IFN-beta into the CNS. This is a potentially novel therapeutic approach which might be used in MS and other diseases of the CNS in which drug access is limited.

Brain derived neurotrophic factor treatment reduces inflammation and apoptosis in experimental allergic encephalomyelitis.

Multiple sclerosis is an inflammatory disease of the central nervous system (CNS) which includes a neurodegenerative component. Brain derived neurotrophic factor (BDNF) is a neuroprotective agent which might be useful in preventing neurodegeneration but its application has been limited because the blood brain barrier restricts its access to the CNS. We have developed a novel delivery system for BDNF using transformed bone marrow stem cells (BMSC) and undertook studies of EAE to determine whether the delivery of BDNF could reduce inflammation and apoptosis. Mice receiving BDNF producing BMSC had reduced clinical impairment compared to control mice receiving BMSC that did not produce BDNF. Pathological examination of brain and spinal cord showed a reduction in inflammatory infiltrating cells in treated compared to control mice. Apoptosis was reduced in brain and spinal cord based on TUNEL and cleaved Caspase-3 staining. Consistent with the known mechanism of action of BDNF on apoptosis, Bcl-2 and Akt were increased in treated mice. Further studies suggested that these increases could be mediated by inhibition of both caspase dependent and caspase independent pathways. These results suggest that the BDNF delivered by the transformed bone marrow stem cells reduced clinical severity, inflammation and apoptosis in this model.

Potassium channel blockers and openers as CNS neurologic therapeutic agents.

Potassium (K+) channels are the most heterogeneous and widely distributed class of ion channels. K(+) channels are dynamic pore-forming transmembrane proteins known to play important roles in all cell types underlying both normal and pathophysiological functions. Essential for such diverse physiological processes as nerve impulse propagation, muscle contraction, cellular activation and the secretion of biologically active molecules, various K(+) channels are recognized as potential therapeutic targets in the treatment of multiple sclerosis, Alzheimer's disease, Parkinson's disease, epilepsy, stroke, brain tumors, brain/spinal cord ischemia, pain and schizophrenia, migraine, as well as cardiac arrhythmias, pulmonary hypertension, diabetes, cervical cancer, urological diseases and sepsis. In addition to their importance as therapeutic targets, certain K(+) channels are gaining attention for their beneficial roles in anesthesia, neuroprotection and cardioprotection. The K(+) channel gene families (subdividing into multiple subfamilies) include voltage-gated (K(v): K(v)1-K(v)12 or KCNA-KCND, KCNF-KCNH, KCNQ, KCNS), calcium-activated (K(Ca): K(Ca)1-K(Ca)5 or KCNM-KCNN), inwardly rectifying (K(ir): K(ir)1-K(ir)7 or KCNJ) and background/leak or tandem 2-pore (K(2P): K(2P)1-K(2P)7, K(2P)9-K(2P)10, K(2P)12-K(2P)13, K(2P)15-K(2P)18 or KCNK) K(+) channels. Worldwide, the pharmaceutical industry is actively developing better strategies for targeting ion channels, in general, and K(+) channels, in particular, already generating over $6 billion in sales per annum from drugs designed to block or modulate ion channel function. This review provides an overview of recent patents on emerging K(+) channel blockers and activators (openers) with potential for development as new and improved nervous system therapeutic agents.

Hematopoietic progenitors express neural genes.

Bone marrow, or cells selected from bone marrow, were reported recently to give rise to cells with a neural phenotype after in vitro treatment with neural-inducing factors or after delivery into the brain. However, we showed previously that untreated bone marrow cells express products of the neural myelin basic protein gene, and we demonstrate here that a subset of ex vivo bone marrow cells expresses the neurogenic transcription factor Pax-6 as well as neuronal genes encoding neurofilament H, NeuN (neuronal nuclear protein), HuC/HuD (Hu-antigen C/Hu-antigen D), and GAD65 (glutamic acid decarboxylase 65), as well as the oligodendroglial gene encoding CNPase (2',3' cyclic nucleotide 3'-phosphohydrolase). In contrast, astroglial glial fibrillary acidic protein (GFAP) was not detected. These cells also were CD34+, a marker of hematopoietic stem cells. Cultures of these highly proliferative CD34+ cells, derived from adult mouse bone marrow, uniformly displayed a phenotype comparable with that of hematopoietic progenitor cells (CD45+, CD34+, Sca-1+, AA4.1+, cKit+, GATA-2+, and LMO-2+). The neuronal and oligodendroglial genes expressed in ex vivo bone marrow also were expressed in all cultured CD34+ cells, and GFAP was not observed. After CD34+ cell transplantation into adult brain, neuronal or oligodendroglial markers segregated into distinct nonoverlapping cell populations, whereas astroglial GFAP appeared, in the absence of other neural markers, in a separate set of implanted cells. Thus, neuronal and oligodendroglial gene products are present in a subset of bone marrow cells, and the expression of these genes can be regulated in brain. The fact that these CD34+ cells also express transcription factors (Rex-1 and Oct-4) that are found in early development elicits the hypothesis that they may be pluripotent embryonic-like stem cells.