WO2006138412A2 - Procedes permettant de traiter des troubles de demyelinisation - Google Patents

Procedes permettant de traiter des troubles de demyelinisation Download PDF

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WO2006138412A2
WO2006138412A2 PCT/US2006/023215 US2006023215W WO2006138412A2 WO 2006138412 A2 WO2006138412 A2 WO 2006138412A2 US 2006023215 W US2006023215 W US 2006023215W WO 2006138412 A2 WO2006138412 A2 WO 2006138412A2
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protein
mice
gene
animal
demyelination
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PCT/US2006/023215
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English (en)
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WO2006138412A3 (fr
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Brian Popko
Wensheng Lin
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University Of Chicago
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Priority claimed from US11/431,372 external-priority patent/US7423194B2/en
Priority claimed from US11/431,782 external-priority patent/US8053627B2/en
Priority claimed from US11/431,601 external-priority patent/US7884260B2/en
Application filed by University Of Chicago filed Critical University Of Chicago
Priority to CA002612374A priority Critical patent/CA2612374A1/fr
Priority to JP2008517080A priority patent/JP2008546704A/ja
Priority to EP20060773189 priority patent/EP1909562A4/fr
Publication of WO2006138412A2 publication Critical patent/WO2006138412A2/fr
Publication of WO2006138412A3 publication Critical patent/WO2006138412A3/fr
Priority to IL188127A priority patent/IL188127A/en

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Definitions

  • This invention is in the field of neurology. Specifically, the invention relates to the discovery and characterization of molecular components that play a role in neuronal demyelination or remyelination. In addition, the invention relates to the generation of an animal model that exhibits hypomyelination. The compositions and methods embodied in the present invention are particularly useful for drug screening and/or treatment of demyelination disorders.
  • Neuronal demyelination is a deleterious condition characterized by a reduction of myelin protein in the nervous system.
  • Myelin is a vital component of the central (CNS) and peripheral (PNS) nervous system, which encases the axons of neurons and forms an insulating layer known as the myelin sheath. The presence of the myelin sheath enhances the speed and integrity of nerve signal in form of electric potential propagating down the neural axon.
  • the loss of myelin sheath produces significant impairment in sensory, motor and other types of functioning as nerve signals reach their targets either too slowly, asynchronously (for example, when some axons in a nerve conduct faster than others), intermittently (for example, when conduction is impaired only at high frequencies), or not at all.
  • the myelin sheath is formed by the plasma membrane, or plasmalemma, of glial cells - oligodendrocytes in the CNS, and Schwann cells in the PNS.
  • each oligodendrocyte in the CNS must produce as much as approximately 5000 ⁇ m 2 of myelin surface area per day and approximately 10 5 myelin protein molecules per minute (Pfeiffer, et al. (1993) Trends Cell Biol. 3: 191-197).
  • Myelinating oligodendrocytes have been identified at demyelinated lesions, indicating that demyelinated axons may be repaired with the newly synthesized myelin.
  • Neuronal demyelination is manifested in a large number of hereditary and acquired disorders of the CNS and PNS. These disorders include Multiple Sclerosis (MS), Progressive Multifocal Leukoencephalopathy (PML), Encephalomyelitis, Central Pontine Myelolysis (CPM), Anti-MAG Disease, Leukodystrophies: Adrenoleukodystrophy (ALD), Alexander's Disease, Canavan Disease, Krabbe Disease, Metachromatic Leukodystrophy (MLD), Pelizaeus-Merzbacher Disease, Refsum Disease, Cockayne Syndrome, Van der Knapp Syndrome, and Zellweger Syndrome, Guillain-Barre Syndrome (GBS), chronic inflammatory demyelinating polyneuropathy (CIDP), multifocual motor neuropathy (MMN), Alzheimer's disease and progressive supernuclear palsy.
  • MS Multiple Sclerosis
  • PMM Progressive Multifocal Leukoencephalopathy
  • CPM Central Pontine Myelolysis
  • Multiple sclerosis is the most common demyelinating disease of the central nervous system, affecting approximately 1,000,000 people worldwide and some 250,000 to 350,000 people in the United States. l" Jt I,, '' 1 ⁇ ' Jd ⁇ lia ⁇ i&fchaii ⁇ teiy ⁇ ' llieribally by relapses and remissions, and leading eventually to chronic disability.
  • the earlier phase of multiple sclerosis is characterized by the autoimmune inflammatory strike against myelin sheath leading to paralysis, lack of coordination, sensory disturbances and visual impairment.
  • the subsequent chronic progressive phase of the disease is typically due to active degeneration of the myelin sheath and 5 inadequate remyelination of the demyelinated lesions (Franklin (2002) Nat. Rev. Neurosci. 3: 705-714; Bruck, et al. (2003) 7. Neurol. Sd. 206: 181-185; Compston, et al. (2002) Lancet 359: 1221-1231).
  • the precise etiology and pathogenesis of this disease remain unknown.
  • pathologic, genetic, and immunologic features have been identified which suggest that the disease involves inflammatory and autoimmune basis. See, for example Waksman, et al. (1984) Proc. Soc. Exp. Biol. Med.
  • IFN- ⁇ pleotropic cytokine interferon- ⁇
  • the present invention provides a method of developing a biologically active agent that reduces neuronal demyelination.
  • the method involves the steps of (a) contacting a candidate agent with a myelinating cell; (b) detecting an altered expression of a gene or gene product or an altered activity of said gene product relative to a control cell, said gene or gene product being correlated with endoplasmic reticulum (ER) stress; and (c) selecting said agent as a candidate if the level of expression of said gene or gene product, or the level of 0 activity of said gene product is modulated relative to said control cell.
  • ER endoplasmic reticulum
  • the present invention also provides a method of developing a biologically active agent that promotes neuronal remyelination.
  • the method comprises (a) contacting a candidate biologically active agent with a myelinating cell from a demyelinated lesion of a subject; and (b) detecting an altered expression of a gene or gene product or an altered activity of said gene product relative to a control cell, said gene or gene product being 5 correlated with endoplasmic reticulum (ER) stress; and (c) selecting said agent as a candidate if the level of expression of said gene or gene product, or the level of activity of said gene product is modulated relative to said control cell.
  • ER endoplasmic reticulum
  • the present invention further provides a method of testing for a biologically active agent that modulates a phenomenon associated with a demyelination disorder.
  • Such method involves (a) administering a 0 candidate agent to a non-human transgenic animal, wherein demyelination occurs in said animal upon expression , , - lJr L the effect of said agent upon a phenomenon associated with a demyelination disorder.
  • Also provided in the present invention is a method of testing for a biologically active agent that modulates a phenomenon associated with a demyelination disorder, by performing the following steps: (a) 5 contacting a candidate agent with a cell derived from a non-human transgenic animal; (b) detecting an altered expression of a gene or gene product or an altered activity of said gene product relative to a control cell, said gene or gene product being correlated with endoplasmic reticulum (ER) stress; and (c) selecting the agent as effective to modulate a phenomenon associated with demyelination disorder if the level of expression of said gene or gene product, or the level of activity of said gene product is modulated relative to said control cell.
  • ER endoplasmic reticulum
  • the present invention provides another method for testing for a biologically active agent that modulates a phenomenon associated with a demyelination disorder.
  • the method involves the steps of: (a) administering a candidate biologically active agent to a test animal generated by a method comprising (i) inducing neuronal demyelination in said test animal, and (ii) allowing said test animal to recover from the demyelination induction for a sufficient amount of time so that remyelination of a demyelinated lesion is exhibited; and (b) 5 determining the effect of said agent upon a phenomenon associated with a demyelination disorder.
  • the phenomenon associated with a demyelination disorder is characterized by a loss of oligodendrocytes in the central nervous system or Schwann cells in the peripheral nervous system.
  • the phenomenon associated with a demyelination disorder is characterized by a decrease in myelinated axons in the central nervous system or peripheral nervous system.
  • phenomenon associated with a demyelination disorder is characterized by a reduction in the levels oligodendrocytes or Schwann cell markers, preferably proteinaceous markes.
  • Non-limiting exemplary marker protein of a myelinating cell is selected from the group consisting of CCl, myelin basic protein (MBP), ceramide galactosyltransferase (CGT), myelin associated glycoprotein (MAG), myelin oligodendrocyte glycoprotein (MOG), oligodendrocyte-myelin glycoprotein (OMG), 5 cyclic nucleotide phosphodiesterase (CNP), NOGO, myelin protein zero (MPZ), peripheral myelin protein 22
  • PMP22 protein 2 (P2), galactocerebroside (GaIC), sulfatide and proteolipid protein (PLP).
  • MPZ, PMP22 and P2 are preferred markers for Schwann cells.
  • the demyelination disorder referred therein is multiple sclerosis.
  • the demyelination disorder is selected from the group consisting of Progressive Multifocal 0 Leukoencephalopathy (PML), Encephalomyelitis, Central Pontine Myelolysis (CPM), Anti-MAG Disease,
  • Leukodystrophies Adrenoleukodystrophy (ALD), Alexander's Disease, Canavan Disease, Krabbe Disease, Metachromatic Leukodystrophy (MLD), Pelizaeus-Merzbacher Disease, Refsum Disease, Cockayne Syndrome, Van der Knapp Syndrome, and Zellweger Syndrome, Guillain-Barre Syndrome (GBS), chronic inflammatory demyelinating polyneuropathy (CIDP), multifocual motor neuropathy (MMN), Alzheimer's disease and 5 progressive supernuclear palsy.
  • the biologically active agent employed in the cell-based assays may be selected from the group consisting of biological or chemical compound such as a simple or complex organic or inorganic molecule, peptide, peptide mimetic, protein (e.g. antibody), liposome, small interfering RNA, or a polynucleotide (e.g. anti-sense).
  • biological or chemical compound such as a simple or complex organic or inorganic molecule, peptide, peptide mimetic, protein (e.g. antibody), liposome, small interfering RNA, or a polynucleotide (e.g. anti-sense).
  • the present invention also provides a non-human transgenic animal having: (a) stably integrated into the genome of said animal a transgenic nucleotide sequence encoding interferon-gamma (INF- ⁇ ); and (b) an altered expression of at least one other gene; wherein upon expression of said INF-7, said animal exhibits a greater ' ir ' d ⁇ gySflHfeSy.elSH ' ⁇ iiiiSlali'li'to a transgenic animal having a stably integrated transgenic nucleotide sequence encoding interferon-gamma (ENF- ⁇ ) as in (a), but lacking said altered expression of said at least one other gene.
  • ENF- ⁇ interferon-gamma
  • the at least one other gene is correlated with endoplasmic reticulum stress.
  • genes include but are not limited to pancreatic ER kinase gene (p-PERK), eukaryotic translation initiation factor 2 alpha (eIF-2 ⁇ , eukaryotic translation initiation factor beta (eIF-2 ⁇ , inositol requiring 1 (IElEl), activating transcription factor 6 (ARTF6), CAATT enhancer-binding protein homologous protein (CHOP), binding- immunoglobulin protein (BIP), caspase-12, growth and DNA damage protein 34 (GADD34), CreP (a constitutive repressor of eIF2alpha phosphorylation), suppressor of cytokine signaling 1 (SOCSl), and X-box-binding protein- 1 (XBP-I).
  • p-PERK pancreatic ER kinase gene
  • eIF-2 ⁇ eukaryotic translation initiation factor 2 alpha
  • eIF-2 ⁇ eukaryotic translation initiation factor
  • the non-human transgenic animal comprises a heterozygous knock-out of pancreatic ER kinase gene (PERK), and stably integrated into the genome of said animal a transgenic nucleotide sequence comprising interferon-gamma (INF- ⁇ ).
  • PERK pancreatic ER kinase gene
  • the animal exhibits an increased vulnerability to INF- ⁇ -mediated neuronal demyelination relative to a wildtype animal.
  • Cells derived from such the subject transgenic animals are also provided.
  • Also included in the present invention is a method of inhibiting neuronal demyelination in a subject comprising administering to said subject an amount of biologically active agent effective to modulate stress level of endoplasmic reticulum (ER) in a myelinating cell, in the perheral or in the central nverous system.
  • the myelinating cell can be an oligodendrocyte or a Schwann cell.
  • the biologically active agent is effective to reduce a sustained stress level of endoplasmic reticulum (ER) in a myelinating cell.
  • the biologically active agent is an interferon-gamma (INF- ⁇ ) antagonist with the proviso that said interferon-gamma (INF- ⁇ ) antagonist is not an anti-INF- ⁇ antibody when applied after the onset of neuronal demyelination.
  • the biologically active agent is an interferon-gamma (INF- ⁇ ) or interferon-gamma (INF- ⁇ ) agonist administered prior to the onset of neuronal demyelination to yield a prophylactic effect.
  • the biologically active agent can be characterized by the ability to reduce a sustained stress level of ER, which in turn can be characterized by a decrease in the levels of proteins correlated with endoplasmic reticulum (ER) stress.
  • ER stress correlated proteins include but are not limited to phosphorylated pancreatic ER kinase gene (p-PERK), eukaryotic translation initiation factor 2 alpha (eIF-2 ⁇ ), eukaryotic translation initiation factor beta (eIF-2 ⁇ ), inositol requiring 1 (IREl), activating transcription factor 6 (ARTF6), CAATT enhancer-binding protein homologous protein (CHOP), binding-immunoglobulin protein
  • BIP caspase-12
  • GADD34 growth and DNA damage protein 34
  • CreP a constitutive repressor of eIF2alpha phosphorylation
  • XBP-I X-box-binding protein-1
  • a method of promoting remyelination of a neuron in a subject after an occurrence of neuronal demyelination comprising administering to said subject an amount of pharmaceutical agent effective to modulate stress level of endoplasmic reticulum (ER) in neuronal tissues undergoing remyelination.
  • the contemplated biologically active agent is an INF- ⁇ antagonist, including but not limited to anti- INF- ⁇ antibody or an antigen-binding fragment thereof.
  • the biologically active agent is effective to reduce a sustained stress level of endoplasmic reticulum (ER) in a myelinating cell.
  • the biologically active agent is effective to activate eIF-2 ⁇ pathway by increasing eIF-2 ⁇ kinase activity or increasing the level of phosphorylated eIF-2 ⁇ present in a cell. In yet another aspect, the biologically active agent is effective to activate eIF-2 ⁇ pathway by increasing PERK kinase activity or increasing the level of phosphorylated PERK or PERK dimer present in a cell. In still yet another aspect, the .
  • GADD34 pathway results in reduced GADD34 signaling.
  • the deactivation of GADD34 pathway results in a reduction of PPI (protein phosphatase 1) phosphatase activity or a reduction in the level of PPI present in a cell.
  • PPI protein phosphatase 1
  • the present invention further provides a method of ameliorating progression of a demyelination disorder in a subject in need for such treatment.
  • the method comprises reducing in said subject the level of interferon-gamma (INF- ⁇ ) present in said subject's neuronal tissues that are undergoing remyelination or INF- ⁇ signaling.
  • the reduction of the level of IKF- ⁇ is effected by delivering to a demyelinated lesion an amount of a pharmaceutical composition comprised of interferon-gamma (INF- ⁇ ) antagonist (e.g., an 0 anti-INF- 7 antibody or an antigen-binding fragment).
  • a reduction in INF- ⁇ signaling is effected by a reduction in the level of a downstream signaling molecule of INF- ⁇ or biological activity thereof.
  • the downstream signaling molecule of INF- ⁇ comprises SOCSl and/or Statl.
  • Figure 1 depicts the results of ELISA analysis of IFN- ⁇ expression pattern in double transgenic mice (GFAP/tTA, TRE/IFN- ⁇ ).
  • Figure 2 depicts the comparative results of immunostaining mature oligodendrocytes with anti- 0 CCl antibodies in the corpus callosum of double transgenic mice treated with cuprizone.
  • FIG. 3 depicts the results of immunohistochemical and electron microscopic analysis of the corpus callosum of both DOX+ and DOX-double transgenic mice.
  • B Percentage of remyelinated axons was calculated from 4 mice at 8 weeks, * p ⁇ 0.01
  • Figure 5 depicts the results of real-time PCR detecting the relative RNA levels of MBP, PLP and
  • Figure 6 depicts the immunostaining of NG2 positive OPCs in the corpus callosum of DOX+ and
  • Figure 8 shows the effect of INF- ⁇ on the CNS during recovery from EAE at PID50.
  • A MBP immunostaining in the lumbar spinal cord of DOX+ mice.
  • B MBP immunostaining in the lumbar spinal cord of DOX- mice that had been released from doxycycline at PID7.
  • C Toluidine blue staining in the lumbar spinal cord of DOX+ mice.
  • D Toluidine blue staining in the lumbar spinal cord of DOX- mice that had been released from doxycycline at PID7.
  • E CCl immunostaining in the lumbar spinal cord of DOX+ mice.
  • Figure 9 shows the inflammatory infiltration in demyelinating lesions in the CNS of mice with EAE. CNS delivery of INF- ⁇ at the recovery stage of EAE enhances inflammatory infiltration in demyelination lesions.
  • A CD3 immunostatining in the lumbar spinal cord of DOX+ mice.
  • B CD3 immunostatining in the lumbar spinal cord of DOX- mice that had been release from doxyxycline at PID7.
  • C CDl Ib immunostatining in the lumbar spinal cord of DOX+ mice.
  • D CDl Ib immunostatining in the lumbar spinal cord of DOX- mice that had been release from doxyxycline at PID7.
  • E Real-time PCR analysis of the expression of inflammatory markers in the spinal cord of DOX+ and DOX- mice at PID50. Experiments were done in triplicate; *p ⁇ 0.05,
  • Figure 10 depicts the effect of INF- ⁇ on the expression ER stress markers during remyelination.
  • DOX- mice that are wild type or are heterozygous for a mutation in the PERK enzyme (PERK+/-).
  • (B) Graph showing the percent remyelinated axons in 5 mice at week 9, *p ⁇ 0.01.
  • Figure 12 shows a comparison of the number of oligodendrocytes in the corpus callosum of DOX+ and DOX- mice that are wild type or are heterozygous for a mutation in the PERK enzyme (PERK+/-).
  • Figure 13 shows that IFN- ⁇ -induced apoptosis in cultured rat oligodendrocytes is associated with
  • ER stress (A) Untreated oligodendrocytes that underwent differentiation for 7 days. (B) Oligodendrocytes that 5 underwent differentiation for 5 days and treatment with 70 U/ml IFN-7 for 48 h, revealing cell shrinkage and aggregation of cell bodies (arrow). (C) TUNEL and CNP double labeling for untreated oligodendrocytes that underwent differentiation for 7 days. (D) TUNEL and CNP double labeling for oligodendrocytes that underwent differentiation for 5 days and treatment with 70 U/ml IFN- ⁇ for 48 h. (E) Quantitation of TUNEL and CNPase double positive cells, * p ⁇ 0.05.
  • Figure 15 shows hypersensitivity of PERK+/- mice to conditional mis-expression of IFN- ⁇ .
  • Mouse survival curve ⁇ n 40 for each group).
  • B and C p-eIF-2 ⁇ and CCl double labeling in the spinal cord of 14-d-old GFAPHTA; TREIWNA; PERK+/- mice that received doxycycline (B) or were released from doxycycline at E 14 (C).
  • Figure 16 shows that double transgenic mice with a PERK+/- background develop severe hypomyelination.
  • A MBP immunostaining in the spinal cord of 14-day-old double transgenic mice that received doxycycline.
  • B MBP immunostaining in the spinal cord of 14-day-old GFAPHTA; TRE/IFN- ⁇ PERK+/- mice that received doxycycline.
  • C MBP immunostaining in the spinal cord of 14-day-old double transgenic mice released from doxycycline at E 14.
  • D MBP immunostaining in the spinal cord of 14-day-old GFAPHTA;
  • FIG. 17 shows that double transgenic mice with a PERK+/- background develop severe hypomyelination.
  • A Ultrastructural examination showing normal myelination in the spinal cord of 14-day-old double transgenic mice that received doxycycline.
  • B Ultrastructural examination showing normal myelination in spinal cord of 14-day-old GFAPHTA; TRE/IFN-y; PERK+/- mice that received doxycycline.
  • C Ultrastructural examination showing minor hypomyelination in the spinal cord of 14-day-old double transgenic mice released . .
  • Figure 18 shows that the levels of MBP, PLP and CGT rnRNA were significantly decreased in the
  • Figure 19 shows that double transgenic mice with a PERK+/- background lose the majority of the oligodendrocytes in the CNS.
  • B TUNEL and CCl double labeling in spinal cord of 14-day-old double transgenic mice that received doxycycline.
  • C TUNEL and CCl double labeling in the spinal cord of 14-day-old GFAPHTA; TRE/IFN-y; PERK+/- mice that received doxycycline.
  • B BIP and CCl double immunostaining in the cerebellum of 10-week-old double transgenic mice that received doxycycline.
  • C BIP and CCl double immunostaining in the cerebellum of 10-week-old GFAPHTA; TRE/IFN-y; PERK+/- mice that received doxycycline.
  • D BIP and CCl double 5 immunostaining in the cerebellum of 10-week-old double transgenic mice released from doxycycline at 4 weeks of age.
  • Figure 21 shows a comparison of the onset and progression of EAE in DOX+ and DOX- mice that are wild type (DOX triple; PERK+/+) or are heterozygous for a mutation (DOX triple; PERK+/-) in the PERK enzyme.
  • A Changes in the mean clinical score for mice with and without EAE.
  • Figure 23 shows that CNS delivery of IFN- ⁇ at EAE onset protects against EAE-induced
  • Figure 24 shows that IFN- ⁇ protected against EAE-induced demyelination through its cytoprotecive effects on oligodendrocyte.
  • FIG. 25 depicts real-time PCR analysis for the expression pattern of cytokines in the spinal cord at the peak of disease.
  • CNS delivery of IFN- ⁇ did not significantly affect the expression of TNF- ⁇ .
  • CNS delivery of IFN- ⁇ decreased the expression of IL-2 in spinal cord of mice on a PERK+/+ background, but did not change IL- 12 expression in mice on a PERK+/- background.
  • D CNS delivery of IFN- ⁇ decreased the expression of IL-12 in spinal cord of mice 0 on a PERK+/+ background, but did not change IL-12 expression in mice on a PERK+/- background.
  • E CNS delivery of IFN- ⁇ decreased the expression of IL-23 in spinal cord of mice on a PERK+/+ background, but did not change IL-12 expression in mice on a PERK+/- background.
  • Figure 27 shows dual label immunohistochetnical analysis of the effect of the loss of function of 0 GADD34.
  • GADD34 was undetectable in oligodendrocytes from the spinal cord of 8-week old naive mice,
  • the arrow points to double labeling of CCl and ⁇ -eIF2 ⁇ , S'" i[ U " ifwliiMfflSyil'imodist.a'civalonibf eIF2 ⁇ in a few oligodendrocytes of the lumbar spinal cord of control mice with EAE at PID17.
  • CCl and p-e ⁇ F2 ⁇ ! double labeling showed the level of p-eIF2o: was increased in oligodendrocytes (arrow) in GADDH null mice at PID17.
  • Figure 28 shows immunostaining of MBP in sections of the lumbar spinal cord from GADD34-
  • mice and control mice show MBP immunostaining of the lumbar spinal cord tissue from
  • FIG. 30 shows the expression of Flag-SOCSl.
  • Figure 31 shows the colocalization of Flag-SOCSl and PLP in vivo. Dual immunostaining of wild-type (A-C, top row) and PLP/SOCS1 (D-F, bottom row) cerebellar tissue, harvested at postnatal day 21, was performed using anti-PLP/Cy3 (A, D, red) and anti-Flag/FITC (B, E, green) antibodies, and DAPI nuclear stain (C, F, blue). PLP positive structures of the wild-type samples (A, red) demonstrated no immunopositivity for anti-Flag (B) and no signal colocalization was established (C).
  • PLP positive oligodendrocytes in the wild-type culture demonstrated no immunopositivity for anti-Flag (B), and no signal 5 colocalization was established (C).
  • PLP positive oligodendrocytes (D) in the PLP/SOCS1 cultures expressed Flag-SOCSl (E), and strong colocalization between anti-PLP and anti-Flag signals was detected (F).
  • Figure 33 shows the differential inhibition of Statl nuclear translocation. Mixed primary
  • oligodendrocyte cultures from wild-type (A-D; top row) and PLP/SOCS1 (E-H; bottom row) mice were stimulated with IOOU IFN- ⁇ for 30 min, and dual immunostainings using anti-PLP/Cy3 (A, E; red), anti-Statl (B, F; green), and DAPI nuclear stain (D, H; blue) were performed and the fluorescent signals digitally overlayed (C and G, overlay between PLP/Cy3 and Statl/FITC signals; D and H, overlay between PLP/Cy3, Statl/FITC and DAPI signals).
  • Statl was colocalized with DAPI stained nuclei of all cells, including the
  • Figure 35 shows the SOCSl -mediated protection of oligodendrocytes and myelin.
  • the IFN- ⁇ expression (A), oligodendrocyte density (CCl cells/mm 2 ) (B), G ratio (C), and percent unmyelinated axons (D) were examined among littermates of three transgenic systems: 172 xPLP/SOCSl, 184/UQxPLP/SOCSI and 184/67 y -PLP/SOCSl at postnatal day 21 (see Results for complete description).
  • the relative amount of IFN- ⁇ expression differed among the systems but no statistical difference was found in the levels of expression between
  • Figure 37 shows SOCSl -mediated myelin protection.
  • Representative images of the quanitated areas from GFAP/tTA x TRE/IFN-y x PLP/SOCS1 (184/67 xPLP/SOCSl) mice at postnatal day 21: A. Wild-type; B. PLP/SOCS1; C. 184/67 and D; 184/67 xPLP/SOCSl. Electron micrographs of corpus callosum; Bar 500nm.
  • oligonucleotide are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown.
  • polynucleotides coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, nitrons, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • nucleotide • • ⁇ > l" tr CI " ir,sttdli£iinyp ⁇ bd ' iiSI
  • the sequence of nucleotides maybe interrupted by non-nucleotide components.
  • a polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
  • a “nucleotide probe” or “probe” refers to a polynucleotide used for detecting or identifying its
  • Hybridization refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues.
  • the hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner.
  • the complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a
  • a hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR, or the enzymatic cleavage of a polynucleotide by a ribozyme.
  • hybridized refers to the ability of the polynucleotide to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues.
  • the 15 hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner.
  • the complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these.
  • the hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.
  • expression refers to the process by which a polynucleotide is transcribed into mRNA and/or the process by which the transcribed mRNA (also referred to as “transcript”) is subsequently being translated into peptides, polypeptides, or proteins.
  • the transcripts and the encoded polypeptides are collectedly referred to as "gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.
  • “Signal transduction” is a process during which stimulatory or inhibitory signals are transmitted 0 into and within a cell to elicit an intracellular response. A molecule can mediate its signaling effect via direct or indirect interact with downstream molecules of the same pathway or related pathway(s).
  • INF ⁇ signaling can involve a host of downstream molecules including but not limted to one or more of the following proteins: PERK, eIF-2 ⁇ , SOCSl, and Statl.
  • polypeptide polypeptide
  • peptide protein
  • the terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to 5 polymers of amino acids of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.
  • amino acid refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical 0 isomers, and amino acid analogs and peptidomimetics.
  • myelinating cell refers to those cells capable of producing myelin which insulates axons in the nervous system.
  • exemplary myelinating cells are oligodendrocytes responsible for I"" L- 1 ⁇ r(Jkfe ⁇ fa
  • a "subject,” “individual” or “patient” is used interchangeably herein, which refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, 5 humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.
  • a "control" is an alternative subject or sample used in an experiment for comparison purpose.
  • a central aspect of the present invention is the discovery of the association of neuronal demyelination with endoplasmic reticulum (ER) stress level in cells that play a role in neuronal myelination.
  • ER endoplasmic reticulum
  • the present invention provides method of developing a biologically active agent that reduces neuronal demyelination.
  • the method involves the steps of (a) contacting a candidate agent with a myelinating cell; (b) detecting an altered expression of a gene or gene product or an altered activity of said gene product relative to a control cell, said gene or gene product being correlated with
  • the present invention provides a method of developing a biologically active agent that promotes neuronal remyelination.
  • the method comprises the steps of (a) contacting a candidate biologically active agent with a myelinating cell from a demyelinated lesion of a subject; and (b) detecting an 0 altered expression of a gene or gene product or an altered activity of said gene product relative to a control cell, said gene or gene product being correlated with endoplasmic reticulum (ER) stress; and (c) selecting said agent as a candidate if the level of expression of said gene or gene product, or the level of activity of said gene product is modulated relative to said control cell.
  • ER endoplasmic reticulum
  • the practice of the invention involves a comparison of the expression of a gene or gene product or 5 the activity of said gene product in a test myelinating cell (whether oligodendrocyte or Schwann cell) relative to a control cell.
  • the test myelinating cell used for this invention can be isolated from central or peripheral nervous systems, and includes cell culture derived therefrom and the progeny thereof, and section or smear prepared from the source, or any other samples of the brain that contain oligodendrocytes or Schwann cells or their progenitors. Where desired, one may choose to use enriched these cell cultures that are substantially free of other neuronal cell 0 types such as neurons, microglial cells, and astrocytes.
  • oligodendrocyte precursors in demyelinated lesions, including but not limited to lesions inflicted by pathogens or physical injuries, and lesions caused by toxic agents such as cuprizone.
  • myelinating cells that are directly exposed to IFN- ⁇ or that are derived from subjects whose nervous systems have been exposed to IFN- ⁇ . For instance, one may choose to employ oligodendrocytes derived from transgenic animals that are ectopically expressed IFN- ⁇ in the central 0 nervous systems.
  • Such myelinating cells may overexpress or underexpress ER-stress causing genes, or jp t
  • PERK p ancreat i c ER kinase gene
  • myelinating cells can be generated by introducing into the cell a genetic
  • Non-limiting exemplary viral expression vectors are vectors derived from RNA viruses such as retroviruses, and DNA viruses such as adenoviruses and adeno-associated viruses.
  • Non-viral expression vectors include but are not limited to plasmids, cosmids, and DNA/liposome complexes.
  • the genetic vehicles can be engineered to 0 carry regulatory sequences that direct tissue specific, cell specific, or even organelle specific expression of the exogenous genes carried therein.
  • tissue or cell specific regulatory sequences have been demonstrated applicable for expressing transgenes in the central nervous systems and peripheral nervous system.
  • An exemplary sequence is the transcriptional regulatory sequence of the glial fibrillary acidic gene (GFAP).
  • the regulatory sequence allows 5 ectopical expression of transgenes in the central nervous system and peripheral nervous system (e.g., in the
  • subcellular localization sequence can be any one of the following: (a) a signal sequence that directs secretion of the gene product outside of the cell; (b) a 0 membrane anchorage domain that allows attachment of the protein to the plasma membrane or other membraneous compartment of the cell; (c) a nuclear localization sequence that mediates the translocation of the encoded protein to the nucleus; (d) an endoplasmic reticulum retention sequence (e.g. KDEL sequence) that confines the encoded protein primarily to the ER; or (e) any other sequences that play a role in differential subcellular distribution of a encoded protein product.
  • a signal sequence that directs secretion of the gene product outside of the cell
  • a 0 membrane anchorage domain that allows attachment of the protein to the plasma membrane or other membraneous compartment of the cell
  • nuclear localization sequence that mediates the translocation of the encoded protein to the nucleus
  • an endoplasmic reticulum retention sequence e.g. KDEL sequence
  • the genetic vehicles can be inserted into a host cell (e.g., myelinating cells such as oligodendrocytes or Schwann cells) by any methods known in the art. Suitable methods may include transfection using calcium phosphate precipitation, DEAE-dextran, electroporation, or microinjection. [0085] The selection of an appropriate control cell or tissue is dependent on the test cell or tissue initially selected and its phenotypic or genotypic characteristic which is under investigation. Whereas the test myelinating 0 cell is derived from demyelinated lesions, one or more counterparts from non-demyelinated tissues can be used as control cells.
  • a host cell e.g., myelinating cells such as oligodendrocytes or Schwann cells
  • Suitable methods may include transfection using calcium phosphate precipitation, DEAE-dextran, electroporation, or microinjection.
  • the selection of an appropriate control cell or tissue is dependent on the test cell or tissue initially selected and its phen
  • a biologically active agent effective to modulate neuronal demyelination is intended to include, but not be limited to a biological or chemical compound such as a simple or 5 complex organic or inorganic molecule, peptide, peptide mimetic, protein (e.g. antibody), liposome, small interfering RNA, or a polynucleotide (e.g. anti-sense).
  • a class of preferred agents include those that block the downstream signaling effect of a target molecule.
  • This class of agents may include soluble ligand receptors or derivatives thereof that compete for the binding of the ligands with the native receptors, typically anchored on the cell, thereby preventing the ligands from mediating their downstream effect.
  • the methodology is known in the 0 art. See, e.g., Economides et al. (2003) Nat Med 9(l):47-52.
  • a vast array of compounds can be synthesized, for example polymers, such as polypeptides and polynucleotides, and synthetic organic compounds based on various core structures, and these are also l" lt C I e'o] ⁇ tli3 ⁇ f)fiyffiheir ⁇ lil.
  • various natural sources can provide compounds for screening, such as plant or animal extracts, and the like.
  • the active agent can be used alone or in combination with another modulator, having the same or different biological activity as the agents identified by the subject screening method.
  • a preferred class of agent is IFN- ⁇ antagonist. As is 5 understood by one skilled in the art, an antagonist inhibits the biological activity mediated by a target that it interacts.
  • the IFN- ⁇ antagonist of the present invention encompasses simple or complex organic or inorganic molecule, peptide, peptide mimetic, protein (e.g. antibody), liposome, small interfering RNA, or a 0 polynucleotide (e.g. anti-sense) that can reduce the deleterious effect of IFN- ⁇ on neuronal demyelination.
  • IFN- ⁇ or IFN- ⁇ agonist e.g., salubrinol
  • an agonist activates the biological activity mediated by a target that it interacts.
  • An agonist can assert its inhibitory effect by directly binding to or directly interacting with the target.
  • An agonist can also assert its stimulatory effect 5 indirectly by first interacting with a molecule in the same signaling pathway.
  • the IFN- ⁇ agonist of the present invention encompasses simple or complex organic or inorganic molecule, peptide, peptide mimetic, protein (e.g. antibody), liposome, small interfering RNA, or a polynucleotide (e.g.
  • the agent when the agent is a composition other than naked RNA, the agent may be directly added to the 0 cell culture or added to culture medium for addition. As is apparent to those skilled in the art, an "effective" amount must be added which can be empirically determined.
  • the agent when it is a polynucleotide, it may be introduced directly into a cell by transfection or electroporation. Alternatively, it may be inserted into the cell using a gene delivery vehicle or other methods as described above.
  • ER-stress related genes encompass all nucleic acids encoding proteins that 5 correlate with stress in the ER.
  • these proteins play a role in ER homeostasis.
  • stress whether endogenous or exogenous, that can be manifested in a cell; these include but are not limited to pathogenic infection, chemical insult, genetic mutation, nutrient deprivation, and even normal cellular differentiation.
  • disruption of the homeostasis and hence ER stress is evidenced by the accumulation of 0 unfolded or misfolded proteins in the ER lumen (Rutkowski, et al. (2004) Trends Cell Biol. 14: 20-28; Ma, et al.
  • the unfolded protein response may involve 1) transcriptional induction of ER chaperone proteins whose function is both to increase folding capacity of the ER and prevent protein aggregation; 2) translational attenuation to reduce protein overload and subsequent 5 accumulation of unfolded proteins; and 3) removal of misfolded proteins from the ER through retrograde transport coupled to their degradation by the 26S proteasome.
  • ER chaperone proteins whose function is both to increase folding capacity of the ER and prevent protein aggregation
  • translational attenuation to reduce protein overload and subsequent 5 accumulation of unfolded proteins
  • ER-stress related genes include pancreatic amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, and others.
  • ER-stress related genes include pancreatic amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids
  • ER kinase gene PERK
  • eukaryotic translation initiation factor 2 alpha eIF-2 ⁇
  • eukaryotic translation initiation factor beta eIF-2 ⁇
  • inositol requiring 1 IREl
  • activating transcription factor 6 ARTF6
  • CAATT enhancer- CHOP
  • binding-immunoglobulin protein BIP
  • CreP a constitutive repressor of eIF2alpha phosphorylation
  • XBP-I X-box- binding protein-1
  • An altered expression of an ER-stress related gene or gene product can be determined by assaying for a difference in the mRNA levels of the corresponding genes between the test myelinating cell and a control cell, when they are contacted with a candidate agent.
  • the differential expression of the ER-stress related gene is determined by detecting a difference in the level of the encoded polypeptide or gene product.
  • nucleic acid contained in a biological sample comprising myelinating cells is first extracted according to standard methods in the art.
  • mRNA can be isolated using various lytic enzymes or chemical solutions according to the procedures set forth in Sambrook et al. (1989), supra or extracted by nucleic- acid-binding resins following the accompanying instructions provided by the manufacturers.
  • the mRNA contained in the extracted nucleic acid sample is then detected by amplification procedures or conventional hybridization assays (e.g. Northern blot analysis) according to methods widely known in the art or based on the methods exemplified herein.
  • amplification means any method employing a primer and a polymerase capable of replicating a target sequence with reasonable fidelity.
  • Amplification may be carried out by natural or recombinant DNA polymerases such as TaqGoldTM, T7 DNA polymerase, Klenow fragment of E.coli DNA polymerase, and reverse transcriptase.
  • a preferred amplification method is PCR.
  • the isolated RNA can be subjected to a reverse transcription assay that is coupled with a quantitative polymerase chain reaction (RT-PCR) in order to quantify the expression level of an ER-stress related gene.
  • RT-PCR quantitative polymerase chain reaction
  • Detection of the gene expression level can be conducted in real time in an amplification assay.
  • the amplified products can be directly visualized with fluorescent DNA-binding agents including but not limited to DNA intercalators and DNA groove binders. Because the amount of the intercalators incorporated into the double-stranded DNA molecules is typically proportional to the amount of the amplified DNA products, one can conveniently determine the amount of the amplified products by quantifying the fluorescence of the intercalated dye using conventional optical systems in the art.
  • DNA-binding dye suitable for this application include SYBR green, SYBR blue, DAPI, propidium iodine, Hoeste, SYBR gold, ethidium bromide, acridines, proflavine, acridine orange, acriflavine, fluorcoumanin, ellipticine, daunomycin, chloroquine, distamycin D, chromomycin, homidium, mithramycin, ruthenium polypyridyls, anthramycin, and the like.
  • probe-based quantitative amplification relies on the sequence-specific detection of a desired amplified product. It utilizes fluorescent, target-specific probes (e.g., TaqMan® probes) resulting in increased specificity and sensitivity. Methods for performing probe-based quantitative amplification are well established in the art and are taught in
  • probes are allowed to form stable complexes with the target polynucleotides (e.g., ER-stress related genes) contained within the biological sample derived from the test subject in a hybridization reaction.
  • target polynucleotides e.g., ER-stress related genes
  • the target polynucleotides provided in the sample are chosen to be u .
  • GOnipllmeHtafy. ' tb ISkjueleeswrae antisense nucleic acids.
  • the target polynucleotide is selected to be complementary to sequences of the sense nucleic acid.
  • hybridization can be performed under conditions of various stringency. Suitable hybridization conditions for the practice of the present invention are such that the recognition interaction between the probe and target ER-stress related gene is both sufficiently specific and sufficiently stable.
  • hybridization assay can be formed using probes immobilized on any solid support, including but are not limited to nitrocellulose, glass, silicon, and a variety of gene arrays.
  • a preferred hybridization assay is conducted on high-density gene chips as described in U.S. Patent No. 5,445,934.
  • the nucleotide probes are conjugated to a detectable label.
  • Detectable labels suitable for use in the present invention include any composition detectable by photochemical, biochemical, spectroscopic, immunochemical, electrical, optical or chemical means.
  • a wide variety of appropriate detectable labels are known in the art, which include fluorescent or chemiluminescent labels, radioactive isotope labels, enzymatic or other ligands.
  • a fluorescent label or an enzyme tag such as digoxigenin, ⁇ -galactosidase, urease, alkaline phosphatase or peroxidase, avidin/biotin complex.
  • the detection methods used to detect or quantify the hybridization intensity will typically depend upon the label selected above.
  • radiolabels may be detected using photographic film or a phosphoimager.
  • Fluorescent markers may be detected and quantified using a photodetector to detect emitted light.
  • Enzymatic labels are typically detected by providing the enzyme with a substrate and measuring the reaction product produced by the action of the enzyme on the substrate; and finally colorimetric labels are detected by simply visualizing the colored label.
  • An agent-induced change in expression of ER-stress related genes can also be determined by examining the corresponding gene products.
  • Determining the protein level typically involves a) contacting the protein contained in a biological sample comprising myelinating cells with an agent that specifically bind to the ER-stress related protein; and (b) identifying any agentprotein complex so formed.
  • the agent that specifically binds an ER-stress related protein is an antibody, preferably a monoclonal antibody.
  • the reaction is performed by contacting the agent with a sample of the ER-stress related proteins derived from the test samples under conditions that will allow a complex to form between the agent and the ER- stress related proteins. The formation of the complex can be detected directly or indirectly according to standard procedures in the art.
  • the agents are supplied with a detectable label and unreacted agents may be removed from the complex; the amount of remaining label thereby indicating the amount of complex formed.
  • an indirect detection procedure requires the agent to contain a label introduced either chemically or enzymatically. A desirable label generally does not interfere with binding or the stability of the resulting agentpolypeptide complex. However, the label is typically designed to be accessible to an antibody for an effective binding and hence generating a detectable signal.
  • ⁇ po jjgj'g , j::j; Q g, A ' wi ⁇ &ESel/ E-" labels suitable for detecting protein levels are known in the art.
  • Non-limiting examples include radioisotopes, enzymes, colloidal metals, fluorescent compounds, bioluminescent compounds, and chemiluminescent compounds.
  • agentpolypeptide complexes formed during the binding reaction can be quantified by standard quantitative assays. As illustrated above, the formation of agentpolypeptide complex can be measured directly by the amount of label remained at the site of binding. In an alternative, the ER-stress related protein is tested for its ability to compete with a labeled analog for binding sites on the specific agent. In this competitive assay, the amount of label captured is inversely proportional to the amount of ER-stress related protein present in a test sample. [00105] A number of techniques for protein analysis based on the general principles outlined above are available in the art.
  • Radioimmunoassays include but are not limited to radioimmunoassays, ELISA (enzyme linked immunoradiometric assays), "sandwich” immunoassays, immunoradiometric assays, in situ immunoassays (using e.g., colloidal gold, enzyme or radioisotope labels), western blot analysis, immunoprecipitation assays, immunofluorescent assays, and SDS-PAGE.
  • Antibodies that specifically recognize or bind to ER-stress related proteins are preferable for conducting the aforementioned protein analyses. Where desired, antibodies that recognize a specific type of post- translational modifications (e.g., ER-stress inducible modifications) can be used.
  • Post-translational modifications include but are not limited to glycosylation, lipidation, acetylation, and phosphorylation. These antibodies may be purchased from commercial vendors. For example, anti-phosphotyrosine antibodies that specifically recognize tyrosine-phosphorylated proteins are available from a number of vendors including Invitrogen and Perkin Elmer.
  • Anti-phosphotyrosine antibodies are particularly useful in detecting proteins that are differentially phosphorylated on their tyrosine residues in response to an ER stress.
  • proteins include but are not limited to eukaryotic translation initiation factor 2 alpha (eIF-2q).
  • eIF-2q eukaryotic translation initiation factor 2 alpha
  • these antibodies can be generated using conventional polyclonal or monoclonal antibody technologies by immunizing a host animal or an antibody-producing cell with a target protein that exhibits the desired post-translational modification.
  • tissue-specific, cell-specific or subcellular structure specific antibodies capable of binding to protein markers that are preferentially expressed in certain tissues, cell types, or subcellular structures.
  • oligodendrocyte co-staining with one or more antibodies specific for oligodendrocyte markers can be used.
  • markers for oligodendrocyte include but are not limited to CCl, myelin basic protein (MBP), ceramide galactosyltransferase (CGT), myelin associated glycoprotein (MAG), myelin oligodendrocyte glycoprotein (MOG), oligodendrocyte-myelin glycoprotein (OMG), cyclic nucleotide phosphodiesterase (CNP), NOGO, myelin protein zero (MPZ), peripheral myelin protein 22 (PMP22), protein 2 (P2), galactocerebroside (GaIC), sulfatide and proteolipid protein (PLP).
  • MBP myelin basic protein
  • CCT myelin associated glycoprotein
  • MOG myelin oligodendrocyte glycoprotein
  • OMG oligodendrocyte-myelin glyco
  • ER-stress related protein To detect or quantify an ER-stress related protein localized in a specific subcellular structure, co-staining with one or more antibodies directed to antigens differentially present in such structure is preferably performed.
  • organelle specific antibodies A wide variety of organelle specific antibodies is available in the art. Non- limiting examples include endoplasmic reticulum (ER) specific antibodies directed to the ER resident protein BIP, plasma membrane specific antibodies reactive with cell surface receptors such as epidermal growth factor receptor (EGF receptor), Golgi specific antibody ⁇ -adaptin, and cytokeratin specific antibodies which differentiate ari ⁇ 2fOe3 cfelhlypes (e.g., between epithelial and stromal cells).
  • EGF receptor epidermal growth factor receptor
  • Golgi specific antibody ⁇ -adaptin Golgi specific antibody ⁇ -adaptin
  • cytokeratin specific antibodies which differentiate ari ⁇ 2fOe3 cfelhlypes (e.g., between epithelial and
  • An altered expression of an ER-stress related gene can also be determined by examining a change in activity of the gene product relative to a control cell.
  • the assay for an agent-induced change in the activity of an ER-stress related protein will dependent on the biological activity and/or the signal transduction pathway that is under investigation.
  • a change in its ability to phosphorylate the downstream substrate(s) can be determined by a variety of assays known in the art. Representative assays include but are not limited to immunoblotting and immunoprecipitation with antibodies such as anti-phosphotyrosine antibodies that recognize phosphorylated proteins.
  • kinase activity can be detected by high throughput chemiluminescent assays such as AlphaScreenTM (available from Perkin Elmer) and eTagTM assay (Chan-Hui, et al. (2003) Clinical Immunology 111: 162-174).
  • high throughput chemiluminescent assays such as AlphaScreenTM (available from Perkin Elmer) and eTagTM assay (Chan-Hui, et al. (2003) Clinical Immunology 111: 162-174).
  • the ER-stress related protein is part of a signaling cascade leading to a fluctuation of intracellular pH condition
  • pH sensitive molecules such as fluorescent pH dyes can be used as the reporter molecules.
  • the ER-stress related protein is an ion channel
  • fluctuations in membrane potential and/or intracellular ion concentration can be monitored.
  • a number of commercial kits and high- throughput devices are particularly suited for a rapid and robust screening for modulators of ion channels.
  • TM Representative instruments include FLIPR (Molecular Devices, Inc.) and VIPR (Aurora Biosciences). These instruments are capable of detecting reactions in over 1000 sample wells of a microplate simultaneously, and providing real-time measurement and functional data within a second or even a minisecond.
  • the ER-stress related protein is a protease
  • its activity in cleaving substrate proteins can be detected by analyzing the cleaved polypeptides.
  • Several methods for analyzing polypeptides are available in the art. Non-limiting exemplary methods are 2-D electrophoresis, mass spectrum analysis, and peptide sequencing.
  • the candidate agents identified by the subject method can be further characterized, in whole or in part, by their abilities to modulate neuronal demyelination that occurs in a wide variety of conditions. For instance, neuronal demyelination may occur in disorders inflicted by pathogens or physical injuries, disorders attributable to genetic predispositions, inflammation and/or autoimmune responses.
  • neuronal demyelination may occur upon bacterial or viral infection as in, e.g., HIV-vacuolar myelinopathy and HTLV. It may also result from direct contact with toxic substances or accumulation of toxic metabolites in the body as in, e.g., central pontine myelinolysis and vitamin deficiencies. Neuronal demyelination may also manifest in spinal cord injury, genetic disorders including but not limited to leukodystrophies, adrenoleukodystrophy, degenerative multi-system atrophy, Binswanger encephalopathy, tumors in the central nervous system, and multiple sclerosis.
  • neuronal demyelination can be characterized by a loss of oligodendrocytes in the central nervous system or Schwann cells in the peripheral nervous system. It can also be determined by a decrease in myelinated axons in the nervous system, or by a reduction in the levels of oligodendrocyte or Schwann cell markers.
  • Exemplary marker proteins of oligodendrocytes or Schwann cells include but are not limited to CCl, myelin basic protein (MBP), ceramide galactosyltransferase (CGT), myelin associated glycoprotein (MAG), myelin oligodendrocyte glycoprotein (MOG), oligodendrocyte-myelin glycoprotein (OMG), cyclic nucleotide phosphodiesterase (CNP), NOGO, myelin protein zero (MPZ), peripheral myelin protein 22
  • the % Se subject method encompass substances that can inhibit the deleterious morphological characteristics of neuronal demyelination.
  • Candidate agents identified by the subject method can be broadly categorized into the following two classes.
  • the first class encompasses agents that when administered into a cell or a subject, reduce the level of expression or activity of an ER-stress causing gene or protein.
  • the second class includes agents that augment the level of expression or activity of an ER-stress suppressing gene or protein (e.g., BIP and PERK).
  • the agent reduces the levels of proteins (e.g., major histocompatibility complex I) that are characteristic of an endoplasmic reticulum stress in remyelinating oligodendrocytes in the central nervous system or Schwann cells in the peripheral nervous system.
  • proteins e.g., major histocompatibility complex I
  • the development of a biologically active agent beneficial for a neuronal demyelination condition may also involve the use of animal models. Accordingly, the present invention provides a method of using animal models for testing a biologically active agent that modulates a phenomenon associated with a demyelination disorder.
  • the method comprises the steps of: (a) administering a candidate biologically active agent to a test animal generated by a method comprising (i) inducing neuronal demyelination in said test animal, and (ii) allowing said test animal to recover from the demyelination induction for a sufficient amount of time so that remyelination of a demyelinated lesion is exhibited; and (b) determining the effect of said agent upon a phenomenon associated with a demyelination disorder.
  • the animal models of the present invention encompass any non-human vertebrates that are amenable to procedures yielding a neuronal demyelination condition in the animal's nervous systems including the central and peripheral nervous system.
  • Preferred model organisms include but are not limited to mammals, primates, and rodents.
  • Non-limiting examples of the preferred models are rats, mice, guinea pigs, cats, dogs, rabbits, pigs, chimpanzees, and monkeys.
  • the test animals can be wildtype or transgenic.
  • the subject method employs a transgenic animal having stably integrated into the genome a transgenic nucleotide sequence encoding interferon-gamma (INF- ⁇ ).
  • the subject method involves a transgenic animal having an altered expression of at least one other gene, wherein upon expression of INF- ⁇ , the animal exhibits a greater degree of demyelination relative to a transgenic animal having a stably integrated transgenic nucleotide sequence encoding interferon-gamma (INF-7) alone.
  • the at least one other gene encodes an ER-stress related protein.
  • the test animal is a heterozygous knock-out of pancreatic ER kinase gene (PERK), having a stably integrated into the genome a transgenic nucleotide sequence encoding interferon-gamma (INF-7).
  • tissue specific and cell specific regulatory sequences are available for expressing transgenes in the central nervous systems.
  • An exemplary sequence is the transcriptional regulatory sequence of the glial fibrillary acidic gene (GFAP).
  • GFAP glial fibrillary acidic gene
  • the regulatory sequence allows ectopical expression of transgenes in the central nervous system and specifically in the astrocytes.
  • the transgene can be operably linked to the corresponding subcellular localization sequences by recombinant DNA techniques widely practiced in the art.
  • Exemplary subcellular localization sequences include but are not limited to (a) a signal sequence that directs secretion of the gene product outside of the cell; (b) a membrane anchorage domain that allows attachment of the protein to the plasma ⁇ p C " iria ⁇ MfiaOfitheiiiiiSi ⁇ iii ⁇ 'a ⁇ yufe compartment of the cell; (c) a nuclear localization sequence that mediates the translocation of the encoded protein to the nucleus; (d) an endoplasmic reticulum retention sequence (e.g. KDEL sequence) that confines the encoded protein primarily to the ER; or (e) any other sequences that play a role in differential subcellular distribution of a encoded protein product.
  • a signal sequence that directs secretion of the gene product outside of the cell
  • a membrane anchorage domain that allows attachment of the protein to the plasma ⁇ p C " iria ⁇ MfiaOfitheiiiiiSi ⁇ ii ⁇ 'a
  • a demyelination condition in the test animal generally refers to a decrease in myelinated axons in the nervous systems (e.g., the central or peripheral nervous system), or by a reduction in the levels of markers of myelinating cells, such as oligodendrocytes and Schwann cells.
  • Exemplary markers for identifying myelinating cells include but are not limited to CCl, myelin basic protein (MBP), ceramide galactosyltransferase (CGT), myelin associated glycoprotein (MAG), myelin oligodendrocyte glycoprotein (MOG), oligodendrocyte-myelin 0 glycoprotein (OMG), cyclic nucleotide phosphodiesterase (CNP), NOGO, myelin protein zero (MPZ), peripheral myelin protein 22 (PMP22), protein 2 (P2), galactocerebroside (GaIC), sulfatide and proteolipid protein (PLP). [00120] These phenomena can be observed by immunohistochemical means or protein analysis described herein.
  • sections of the test animal's brain can be stained with antibodies that specifically recognize an oligodendrocyte marker.
  • the expression levels of oligodendrocyte markers can be quantified 5 by immunoblotting, hybridization means, and amplification procedures, and any other methods that are well- established in the art and/or provided herein.
  • a number of methods for inducing demyelination in a test animal have been established. For instance, neuronal demyelination may be inflicted by pathogens or physical injuries, agents that induce inflammation and/or autoimmune responses in the test animal.
  • a preferred method employs demyelination- 0 induced agents including but not limited to IFN- ⁇ and cuprizone (bis-cyclohexanone oxaldihydrazone). The cuprizone-induced demyelination model is described in Matsushima, et al. (2001) Brain Pathol. 11 : 107-116.
  • test animals are typically fed with a diet containing cuprizone for a few weeks ranging from about 1 to about 10 weeks.
  • the animal is allowed to 5 recover for a sufficient amount of time to allow remyelination at or near the previously demyelinated lesions.
  • Remeylination can be ascertained by observing an increase in myelinated axons in the nervous systems (e.g., in the central or peripheral nervous system), or by detecting an increase in the levels of 0 marker proteins of a myelinating cell. The same methods of detecting demyelination can be employed to determine whether remyelination has occurred.
  • Determining the effect of the test agent upon a phenomenon associated with a demyelination may involve any suitable methods known in the art, including but not limited to those mentioned in the above cell- based assay section. In general, immunohistochemical and electron microscopic analysis can be performed to 5 visualize the effect of the test agent. In addition, procedures applicable for detecting differential expression of
  • ER-stress related genes or gene products can be employed. Techniques for measuring activities of ER-stress related proteins are also applicable.
  • ER-stress related genes include pancreatic ER kinase gene (PERK), eukaryotic translation initiation factor 2 alpha (eIF-2ot, eukaryotic translation initiation factor beta (eIF-2 ⁇ , inositol requiring 1 (IREl), activating transcription factor 6 (ARTF6), CAATT 0 enhancer-binding protein homologous protein (CHOP), binding-immunoglobulin protein (BIP), caspase-12, growth and DNA damage protein 34 (GADD34), CreP (a constitutive repressor of eIF2alpha phosphorylation), and X-box-binding protein- 1 (XBP-I).
  • PERK pancreatic ER kinase gene
  • eIF-2ot eukaryotic translation initiation factor 2 alpha
  • eIF-2 ⁇ eukaryotic translation initiation factor beta
  • the present invention provides a non-human transgenic animal suitable for elucidating the pathogenesis of neuronal demyelination conditions.
  • the transgenic animal is also useful for developing biologically active agent effective to inhibit neuronal demyelination or promote remyelination of demyelinated lesions.
  • the subject transgenic animal has (a) stably integrated into the genome of the 5 animal a transgenic nucleotide sequence encoding interferon-gamma (INF- ⁇ ); and (b) an altered expression of at least one other gene, wherein upon expression of said INF- ⁇ , the animal exhibits a greater degree of demyelination relative to a transgenic animal having a stably integrated transgenic nucleotide sequence encoding interferon-gamma (INF- ⁇ ) as in (a), but lacking said altered expression of said at least one other gene.
  • the present invention contemplates transgenic animals that carry one or more desired transgenes 0 in all their cells, as well as animals which carry the transgenes in some, but not all their cells, i.e., mosaic animals.
  • a desired transgene may be integrated as a single copy or in concatamers, e.g., head-to-head 5 tandems or head-to-tail tandems.
  • the desired transgene may also be selectively introduced into and activated in a particular tissue or cell type, preferably cells within the central nervous system.
  • the regulatory sequences required for such a cell- type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
  • the targeted cell types are located in the nervous systems, including the central and peripheral nervous systems.
  • gene targeting is preferred.
  • vectors containing some nucleotide sequences homologous to the endogenous counterpart are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous gene. 5 [00128] Advances in technologies for embryo micromanipulation now permit introduction of heterologous
  • totipotent or pluripotent stem cells can be transformed by microinjection, calcium phosphate mediated precipitation, liposome fusion, retroviral infection or other means.
  • the transformed cells are then introduced into the embryo, and the embryo will then develop into a transgenic animal.
  • developing embryos are infected with a viral vector containing a desired 0 transgene so that the transgenic animals expressing the transgene can be produced from the infected embryo.
  • a desired transgene is coinjected into the pronucleus or cytoplasm of the embryo, preferably at the single cell stage, and the embryo is allowed to develop into a mature transgenic animal.
  • transgenic animals of the present invention can be broadly categorized into two types:
  • knockouts and “knockins”.
  • a “knockout” has an alteration in the target gene via the introduction of transgenic sequences that results in a decrease of function of the target gene, preferably such that target gene expression is insignificant or undetectable.
  • a “knockin” is a transgenic animal having an alteration in a host cell genome that results in an augmented expression of a target gene, e.g., by introduction of an additional copy of the target gene, 0 or by operatively inserting a regulatory sequence that provides for enhanced expression of an endogenous copy of the target gene.
  • the knock-in or knock-out transgenic animals can be heterozygous or homozygous with respect to the target genes. Both knockouts and knockins can be "bigenic”.
  • Bigenic animals have at least two host cell " IF " .gefig'slEeSyillterdl! " M ⁇ $efM$k bigenic animal carries a transgene encoding IFN- ⁇ and another transgenic sequence that disrupts the function of at least one other gene.
  • the at least one other gene is an ER-stress related gene.
  • the other gene can be an exogenous gene, i.e., a gene that is not present in the host cell, or an endogenous gene, i.e., the introduced gene finds an endogenous counterpart native to the recipient animal.
  • ER-stress related gene may be selected from the group consisting of pancreatic ER kinase gene (PERK), eukaryotic translation initiation factor 2 alpha (eIF-2 ⁇ , eukaryotic translation initiation factor beta (eIF-2 ⁇ , inositol requiring 1 (IREl), activating transcription factor 6 (ARTF6), CAATT enhancer-binding protein homologous protein (CHOP), binding-immunoglobulin protein (BIP), caspase-12, growth and DNA damage protein 34 (GADD34), CreP (a constitutive repressor of eIF2alpha phosphorylation), and X-box-binding protein- 1
  • PERK pancreatic ER kinase gene
  • eIF-2 ⁇ eukaryotic translation initiation factor 2 alpha
  • eIF-2 ⁇ eukaryotic translation initiation factor beta
  • IREl activating transcription factor 6
  • ARTF6 activating transcription factor 6
  • a preferred non-human transgenic animal comprises a heterozygous knockout of pancreatic ER kinase gene (PERK), and has stably integrated into the genome a transgenic nucleotide sequence comprising interferon-gamrna (INF- ⁇ ).
  • PERK pancreatic ER kinase gene
  • INF- ⁇ interferon-gamrna
  • a preferred transgenic animal exhibits an increased vulnerability to INF- ⁇ -mediated neuronal demyelination relative to a wildtype animal.
  • cells of the subject non-human transgenic animal comprise stably integrated in the genome a transgene encoding INF- ⁇ and an alteration in at least one other gene encoding a protein correlated with ER stress.
  • the subject cells are from the central nervous system.
  • the subject cells are oligodendrocytes.
  • the subject cells are from the peripheral nervous system including but not limited to Schwann cells.
  • these cells are particularly useful for conducting cell-based assays for elucidating the molecular bases of neuronal demyelination conditions, and for developing agents effective for inhibiting neuronal demyelination or promoting remyelination.
  • compositions of the Present Invention may be used for the preparation of medicaments for treating neuronal demyelination disorders.
  • a preferred class of agents is effective to alleviate IFN- ⁇ elicited ER stress in oligodendrocytes.
  • the selected agent of this invention can be administered to treat neuronal demyelination inflicted by pathogens such as bacteria and viruses.
  • the selected agent can be used to treat neuronal demyelination caused by toxic substances or accumulation of toxic metabolites in the body as in, e.g., central pontine myelinolysis and vitamin deficiencies.
  • the agent can be used to treat demyelination caused by physical injury, such as spinal cord injury.
  • the agent can be administered to treat demyelination manifested in disorders having genetic attributes, genetic disorders including but not limited to leukodystrophies, adrenoleukodystrophy, degenerative multi-system atrophy, Binswanger encephalopathy, tumors in the central nervous system, and multiple sclerosis.
  • a biologically active agent of the invention e.g., encapsulation in liposomes, microparticles, microcapsules, expression by recombinant cells, receptor-mediated endocytosis (see, e.g., Wu and Wu, (1987), J. Biol. Chem. 262:4429-4432), construction of a therapeutic nucleic acid as part of a retroviral or other vector, etc.
  • Methods of delivery include but are not limited to intra-arterial, intra-muscular, intravenous, intranasal, and oral routes.
  • the agents are delivered to a subject's nerve systems, preferably the central nervous system. In another embodiment, the agents are administered to neuronal tissues undergoing remyelination.
  • Administration of the selected agent can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. 0 [00138]
  • the preparation of pharmaceutical compositions of this invention is conducted in accordance with generally accepted procedures for the preparation of pharmaceutical preparations. See, for example, Remington's Pharmaceutical Sciences 18th Edition (1990), E.W. Martin ed., Mack Publishing Co., PA.
  • processing may include mixing with appropriate non- 5 toxic and non-interfering components, sterilizing, dividing into dose units, and enclosing in a delivery device.
  • compositions for oral, intranasal, or topical administration can be supplied in solid, semi-solid or liquid forms, including tablets, capsules, powders, liquids, and suspensions.
  • Compositions for injection can be supplied as liquid solutions or suspensions, as emulsions, or as solid forms suitable for dissolution or suspension in liquid prior to injection.
  • a preferred composition is 0 one that provides a solid, powder, or aerosol when used with an appropriate aerosolizer device.
  • Liquid pharmaceutically acceptable compositions can, for example, be prepared by dissolving or dispersing a polypeptide embodied herein in a liquid excipient, such as water, saline, aqueous dextrose, glycerol, or ethanol.
  • the composition can also contain other medicinal agents, pharmaceutical agents, adjuvants, carriers, and auxiliary substances such as wetting or emulsifying agents, and pH buffering agents.
  • the pharmaceutical compositions can be formulated in slow release or sustained release forms, whereby a relatively consistent level of the active compound are provided over an extended period.
  • Another embodiment of the present invention is a method of promoting remyelination using stem cells in a subject.
  • cultured stem cells are typically transfected with a gene capable of 0 ameliorating ER stress in myelinating oligodendrocytes.
  • the genetically modified stem cells are then introduced into the CNS of a subject suffering from a neuronal demyelination condition. Any means effective to deliver the genetically modified stem cells are applicable.
  • the stem cells are directly injected into the nervous system of a subject. This methodology is described in detail in e.g., Morris, et al. (1997) J Biol Chem. 272(7): 4327-34, which is incorporated herein by reference.
  • Candidate genes to be introduced into stem cells include but 5 are not limited to BIP, PERK, and suppressor of cytokine signaling 1 (SOCSl).
  • BIP is required to protect cells from ER stress; overexpression of BIP permits continued translation of cellular mRNAs, and hence reduces ER stress.
  • SOCSl is known to block INF- ⁇ signal transduction.
  • stem cells overexpressing SOCSl and hence myelinating cells derived from such stem cells are expected to be less sensitive to the demyelinating effect mediate by INF- ⁇ . - ,
  • P C T..f6ijj$ ⁇ O & ⁇ ⁇ Ipipu ⁇ itiins ' ⁇ ofleuronal cells can be produced from differentiating cultures of embryonic stem cells (Li et al., (1998) Curr. Biol. 8: 971-974), and have been used in experimental models to correct various deficits in animal model systems (review, Svendsen and Smith, Trends in Neurosci. 22: 357-364).
  • neuronal cells can be derived from human embryonal carcinoma cells, and can be
  • pluripotent stem cells may be embryonic stem cells (ES) or embryonic germ cells (EG). Generation
  • 10 of neural progenitors from stem cells in vitro may serve as an unlimited source of cells for tissue reconstruction and for the delivery and expression of genes in the nervous system.
  • ES and EG lines may be cultured on feeder layers, and may be grown and maintained in an undifferentiated state in the presence of recombinant hormones such as Fibroblast Growth Factor and Leukemia Inhibitory Factor. Differentiation can be
  • tissue-specific reversible transformation can be used for establishing differentiated neuronal cell lines using stem cells as a starting material according to the method described in US Patent Application 20060068496, which is incorporated herein by reference.
  • US Patent Application 20060068496 discloses methods that employ tissue specific expression of a transforming gene, which
  • GFAP glial fibrillary acetic protein
  • Markers that can be used to identify a neuronal cell include but are not limited to GFAP and MPB.
  • the preferred cell for use in cell therapy is human embryonic stem cell.
  • the present invention provides an enriched preparation of undifferentiated human embryonic stem cells capable of expressing a modulator of one or more ER stress proteins and that can proliferate in vitro and differentiate into neural
  • neural progenitor cells are first derived from human ES cells, which are engineered to express one or more modulators of the ER stress pathway, and subsequently are differentiated into mature neuronal cells, and glial cells inlcuding oligodendrocyte and astrocyte cells.
  • the neuronal progenitor cells are first differentiated into mature neuronal cells that include oligodendrocytes and astrocytes, which are subsequently engineered to express
  • the modulators that can be induced to be expressed in the neural progenitor cells include modulators of protein folding and maturation, protein transport, protein synthesis and modification, Ca 2+ homeostasis, transcription factors, UPR target genes, and proteins that mediate apoptosis.
  • Modulators of protein folding and maturation include but are not limited to modulators of BIP/GRP78, protein disulfide-isomerase-
  • ERdj4 collagen binding protein 2
  • ORP150 oxygen regulated protein 150 kD
  • FK506-binding protein FKBP13
  • GRP94 protein disulfide-isomerase ERp70-like, protein disulfide-isomerase ERp60-like, proline 4-hydroxylase /3-subunit (P4HB), hsc70 (71-kD heat shock 1
  • !P C but are not limited to modulators of putative mitochondrial membrane protein import receptor (hTIM44), translocon-associated protein delta subunit (TRAP ⁇ ), and transmembrane protein rnp24.
  • Modulators of protein synthesis and modification include but are not limited to modulators of glycyl-tRNA synthase, alanyl-tRNA synathase, asparagine synthase, glutamine-fructose-6- 5 phosphate amidotransferase (GFAT), and integral membrane protein 1 (ITMl).
  • Modulators OfCa 2+ homeostasis include but are not limited to modulators of calreticulin, stanniocalcin 2, plasma membrane Ca 2+ pumping ATPase, calnexin, novel DNA-binding/EF-hand/leucine zipper protein (NEFA), and nucleonidin 1.
  • Modulators of transcription factors include but are not limited to modulators of CHOP, C/EBP-beta, TGF-j8-stimulated clone 22 (TSC22)-like, X-box-binding protein 1 (XBP-I), and Egr-1.
  • Modulators of UPR target genes nclude but are 0 not limited to modulators of HERP and SERP/RAMP4.
  • Modulators of apoptosis include but are not limited to modulators of Bbc3/PUMA, caspase 3, and caspase 12.
  • the modulators that are expressed in the undifferentiated or differentiated neuronal cells are modulators of the PERK pathway.
  • one or more modulators of the PERK pathway can be expressed in the undifferentiated or differentiated neural cells.
  • the neural progenitor cells can be 5 induced to express a synthetic PERK fusion enzyme that enhances the level of the phosphorylated eIF-2 ⁇ (p-eIF-
  • the present invention provides neural progenitor cells, neuronal cells and glial cells that express modulators of the PERK pathway and can used for cell therapy and gene therapy.
  • the route of delivery that is selected for the stem cells is crucial in that it helps to determine whether or not repair of 0 the damaged organ will occur. A high stem cell concentration near the damaged area increases the chances that sufficient stem cell localization and differentiation occurs in order to repair the organ. In many cases this involves the targeted and regional administration of stem cells.
  • Gene therapy is an alternative approach for promoting remyelination by modulating ER stress of myelinating cells.
  • a pharmaceutically 5 acceptable vector is preferred, such as a replication-incompetent retroviral or adenoviral vector.
  • Pharmaceutically acceptable vectors containing an ER-stress suppressing genes of this invention can be further modified for transient or stable expression of the inserted polynucleotide.
  • the term "pharmaceutically acceptable vector” includes, but is not limited to, a vector or delivery vehicle having the ability to selectively target and introduce an ER-stress suppressing gene into cells located in the nerve systems, and preferably 0 oligodendrocytes in the nerve systems.
  • a replication-incompetent retroviral vector is LNL6
  • RNAi RNA interference
  • RNAi may be used to create a pseudo "knockout", i.e. a system in which the expression of the product encoded by a gene or coding region of interest is reduced, resulting in an overall reduction of the activity of the encoded product in a system.
  • RNAi may be performed to target a nucleic T ⁇ ac ⁇ diiyiMiiSt. ⁇ )ril!agiiiii ⁇ t " y!ikriant thereof, to in turn reduce its expression and the level of activity of the product which it encodes.
  • a system may be used for functional studies of the product, as well as to treat disorders related to the activity of such a product.
  • RNAi is described in for example Hammond et al. (2001) Science 10;293(5532): 1146-50., Caplen et al. (2001) Proc Natl Acad Sd USA. 2001 Aug 14;98(17):9742-7, all of which are herein incorporated by reference.
  • RNAi short interfering RNAs
  • RNAi RNA-induced silencing complex
  • RNAi may be effected by the introduction of suitable in vitro synthesized siRNA or siRNA-like molecules into cells. RNAi may for example be performed using chemically-synthesized RNA. Alternatively, suitable expression vectors may be used to transcribe such RNA either in vitro or in vivo.
  • In vitro transcription of sense and antisense strands may be effected using for example T7 RNA polymerase, in which case the vector may comprise a suitable coding sequence operably-linked to a T7 promoter.
  • the in vitro-transcribed RNA may in embodiments be processed (e.g. using E. coli RNase III) in vitro to a size conducive to RNAi.
  • the sense and antisense transcripts are combined to form an RNA duplex which is introduced into a target cell of interest.
  • Other vectors may be used, which express small hairpin RNAs (shRNAs) which can be processed into siRNA-like molecules.
  • the expression of one or more ER-stress causing genes may be inhibited by introducing into or generating within a cell an siRNA or siRNA-like molecule corresponding to a nucleic acid encoding the ER stress-causing gene or fragment thereof, or to an nucleic acid homologous thereto.
  • siRNA-like molecule refers to a nucleic acid molecule similar to a siRNA (e.g. in size and structure) and capable of eliciting siRNA activity, i.e. to effect the RNAi-mediated inhibition of expression.
  • such a method may entail the direct administration of the siRNA or siRNA-like molecule into a cell, or use of the vector-based methods described above.
  • the siRNA or siRNA-like molecule is less than about 30 nucleotides in length. In a further embodiment, the siRNA or siRNA-like molecule is about 21- 23 nucleotides in length. In an embodiment, siRNA or siRNA-like molecule comprises a 19-21 bp duplex portion, each strand having a 2 nucleotide 3' overhang. In embodiments, the siRNA or siRNA-like molecule is substantially homologous to a nucleic acid encoding the ER stress-causing gene or a fragment or variant (or a fragment of a variant) thereof. Such a variant is capable of encoding a protein having ER stress-causing activity.
  • RNAi methods can be used to inhibit expression of IFN- ⁇ in, for example, the central nervous system.
  • ER stress-causing genes that may be inhibited by RNAi methods include, but are not limited to IFN- ⁇ , GADD34, protein phosphatase 1 (PPl), and one or more genes that mediate apoptosis e.g. genes that encode caspases.
  • IFN- ⁇ does not affect the initial phase demyelination, oligodendrocyte loss or reduction of myelin gene 0 expression
  • mice that allow for temporally regulated delivery of IFN- ⁇ using the tetracycline controllable system
  • the transgenic mice were generated by mating linel 10 GFAP/tTA mice on the C57BL/6 background with line 184 TRE/IFN-y on the C57BL/6 background to produce 5 GFAP/tTA; TRE/IFN-y double transgenic mice (Lin, et al. (2004) J. Neurosci. 24: 10074-10083).
  • Transcriptional activation of the TRE/IFN- ⁇ rransgene by tTA was repressed in the control (DOX+) mice by adding 0.05 mg/ml doxycycline to the drinking water which was provided ad libitum from the day of conception.
  • the DOX+ double transgenic mice were GFAP/tTA; TRE/IFN-ydo ⁇ ble transgenic animals fed cuprizone chow and never released from the doxycycline solution.
  • DOX- double transgenic mice were GFAP/tTA; TRE/IFN-ydouble transgenic 0 animals fed cuprizone chow and released from doxycycline to induce expression of IFN- ⁇ .
  • MHC-I MHC-I
  • oligodendrocytes loss of oligodendrocytes
  • myelin genes expression of myelin genes
  • PCR was performed with iQ supermix (Bio-Rad, Hercules CA) on a Bio-Rad iQ real-time PCR detection system p ., negligence, ,. an( j p ro bes (Integrated DNA Technologies Inc., Coralville, IA) for real-time PCR were as follows:
  • the probe was MHC-I 5 probe: TGCTGGGCCCTGGGCTTCTACC.
  • oligodendrocytes The effect of INF- ⁇ on the population of oligodendrocytes was evaluated by immunohistochemical analysis of oligodendrocytes using an anti -CCl antibody (APC7, 1:50; EMD Biosciences, Inc., La Jolla, CA) Immunohistochemistry was perfomed on brain sections obtained from mice that were first anesthetized mice and perfused through the left cardiac ventricle with 4% paraformaldehyde in 0.1M PBS. The brains were removed, 0 postfixed with paraformaldehyde, cryopreserved in 30% sucrose, embedded in OCT and frozen on dry ice. Frozen sections were cut in a cryostat at a thickness of lO ⁇ m.
  • Coronal sections at the fornix region of the corpus callosum corresponding to Sidman sections 241-251 were selected for use, and all comparative analyses were restricted to midline corpus callosum (Sidman, et al. (1971) Atlas of the Mouse Brain and Spinal Cord (Harvard Univ. Press, Cambridge, Massachusetts).
  • frozen sections were treated with -20°C acetone, 5 blocked with PBS containingl0% NGS and 0.1% Triton X-100 and incubated overnight with the primary antibody diluted in blocking solution.
  • Appropriate fluorochrome- or enzyme-labeled secondary antibodies were used for detection.
  • An antibody against CCl was used as a marker for mature oligodendrocytes.
  • Antibody against MBP (1:1000; Sternberger Monoclonals, Lutherville, MA) was used to verify the degree of myeli ⁇ ation.
  • Antibody 0 against active-caspase-3 (1:50, Cell signaling Technology, Beverly, MA) was used as a marker for apoptotic cells.
  • Fluorescent stained sections were mounted with Vectashield mounting medium with DAPI (Vector Laboratories) and visualized with a Zeiss Axioplan fluorescence microscope. Images were captured using a Photometries PXL CCD camera connected to an Apple Macintosh computer using the Open Lab software suite. Immunopositive cells were quantified by counting positive cells within the median of the corpus callosum, confined to an area of 5 0.04 mm 2 . Only those cells with nuclei observable by DAPI staining were counted. Each MBP immunostaining slide was scored on a scale of zero to four. A score of zero indicates complete demyelination, and a score of four indicates normal myelination in the corpus callosum of adult mice.
  • MBP sense primer GCTCCCTGCCCCAGAAGT
  • MBP antisense 0 primer TGTCACAATGTTCTTGAAGAAATGG
  • MBP probe AGCACGGCCGGACCCAAGATG
  • PLP sense primer CACTTACAACTTCGCCGTCCT
  • PLP antisense primer
  • CGT sense primer TTATCGGAAATTCACAAGGATCAA; and CGT antisense primer:
  • the degree of 5 demyelination was determined as the level of MBP was also determined by immunohistochemical analysis as described above and using an antibody against MBP (1: 1000; Sternberger Monoclonals, Lutherville, MA). Demyelination was also assessed by electron microscopy as follows. Mice were anesthetized and perfused with 0.1 M PBS containing 4% paraformaldehyde and 2.5% glutaraldehyde (PH 7.3).
  • Brains were sliced into 1-m ⁇ n sections, and the section corresponding to the region of the fornix was trimmed and processed for analysis and 0 oriented so that a cross-section of the corpus callosum was achieved. Thin sections were cut, stained with uranyl acetate and lead citrate and analyzed as previously described (Coetzee, et al. (1996) Cell 86: 209-219). The total percent of remyelinated axons was based on the analysis of a minimum of 300 fibers per mouse.
  • IFN- ⁇ suppresses remyelination in demyelinated lesions.
  • the effect of IFN- ⁇ on the remyelination in the cuprizone treated mice described in Example 1 was determined following withdrawal of cuprizone at week 5. The experimental methods used are the same as described in Example 1.
  • Figure IA and B show that the increased level of INF- ⁇ persisted in the DOX- mice even after withdrawal of cuprizone, and the increase in INF- ⁇ was accompanied by a sustained increase in MCH- 1.
  • Week 5 a significant number of oligodendrocytes were seen in the corpus callosum of the DOX+ mice, while the regeneration of oligodendrocytes in the DOX- was suppressed ( Figure 2A and B). Remyelination in the corpus callosum of DOX+ double transgenic mice began at week 6, and was evident 2 weeks after cuprizone was removed from the diet (week 8; Fig. 3).
  • IFN- ⁇ dramatically reduces repopulation of CCl positive oligodendrocytes in demyelinated lesions.
  • Remyelination occurs by the repopulation of demyelinated lesions by oligodendrocyte precursors
  • OPCs at the site of the lesion where they differentiate into oligodendrocytes
  • CCl positive oligodendrocytes are C et al 1998; Mason et al 2000)). Therefore, repopulation of a lesion may be evaluated by the presence of CCl and/or NG2 positive cells.
  • OPC OPC
  • the data indicate that the reduction in the number of CCl positive oligodendrocytes contributes to the poor remyelination of demyelinated lesions in the presence of IFN- ⁇ , and that IFN- ⁇ delays the recruitment of OPCs to the site of lesion without significantly affecting the number of OPCs that are recruited.
  • Double transgenic mice that allow for temporally regulated delivery of INF- ⁇ to the CNS were used to assess the role of INF- ⁇ in the pathogenesis of EAE.
  • GFAP/tTA; TRE/IFN-y double transgenic mice, as described in Example 1 were used to determine the effect of
  • mice were fed doxycycline from the day of conception to repress the expression of INF- ⁇ , and were immunized with myelin oligodendrocyte protein (MOG35-55) to induce EAE.
  • MOG35-55 myelin oligodendrocyte protein
  • the level of INF- ⁇ was determined in the control (D0X+) and the PID7 DOX- mice using an ELISA immunoassay as described in Example 1, and the severity of the disease was assessed as a function of a clinical score.
  • the spinal cord from DOX+ and DOX- animals with EAE was immunostained for MBP, and the remyelination of axons was evaluated.
  • the number of oligodendrocytes was determined in the spine from DOX+ and DOX- mice as the number of CCl positive cells.
  • Axonal damage was evaluted by immunostaining of non- phosphorylated neurofilament-H.
  • Antibodies to non-phosphorylated neurofilament-H were SMI32 diluted to
  • INF- ⁇ enhances the inflammatory response in EAE demyelinating lesions
  • INF- ⁇ is known to induce MHC antigens and activate macrophages and T lymphocytes.
  • the inflammatory effect of INF- ⁇ was studied in the CNS during remyelination by determining the infiltration of T 0 cells and macrophages, and measuring the expression of MHC-I, TNF-a, IL-2, IL-12 and IL-17.
  • MHC-I probe TGCTGGGCCCTGGGCTTCTACC; TNF- ⁇ sense primer
  • GGCAGGTTCTGTCCCTTTCA TNF- ⁇ antisense primer ACCGCCTGGAGTTCTGGA, TNF-a probe CCCAAGGCGCCACATCTCCCT;
  • IL-2 sense primer CTACAGCGGAAGCACAGCAG, IL-2 antisense primer ATTTGAAGGTGAGCATCCTGGG, IL-2 probe AGCAGCAGCAGCAGCAGCAGCA;
  • IL-12 sense primer 5 CTCTATGGTCAGCGTTCCAACA, IL-12 antiense primer GGAGGTAGCGTGATTGACACAT, IL-12 probe
  • IL-17 sense primer ATGCTGTTGCTGCTGCTGAG IL-17 antisense primer TTTGGACACGCTGAGCTTTGAG, IL-17 probe CGCTGCTGCCTTCACTGTAGCCGC
  • IL-23 sense primer CTTCTCCGTTCCAAGATCCTTCG IL-23 antisense primer GGCACTAAGGGCTCAGTCAGA
  • IL-23 probe TGCTGCTCCGTGGGCAAAGACCC inducible nitric oxide synthase (iNOs) sense primer 0 GCTGGGCTGTACAAACCTTCC, iNOs sense primer TTGAGGTCTAAAGGCTCCGG, iNOS probe
  • BIP antisense primer AGAGCGGAACAGGTCCATGT
  • CHOP sense primer CCACCACACCTGAAAGCAGAA
  • CHOP antisense primer AGGTGCCCCCAATTTCATCT; CHOP probe TGAGTCCCTGCCTTTCACCTTGGAGA.
  • the extracts (40 ⁇ g) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose.
  • the blots were incubated with primary antibody (see below), and the signal was revealed by chemiluminescence after reacting with horseradish peroxidase-conjugated second antibody.
  • primary antibodies were used: anti-eIF-2 ⁇ ; (1:500; Santa Cruz, Santa Cruz, CA), anti-p-eIF-2 ⁇ (1:1000, Cell signaling Technology), anti-CHOP (1:500; Santa Cruz) and anti-actin (1:1000; Sigma, St Louis, MO).
  • IK L I nig displayed increased levels of p-eIF-2 ⁇ (Fig. 1OD and 10E).
  • PERK modulates the severity of the reduction in remyelination induced by IFN- ⁇ .
  • the level of caspase-3 was determined by immunolu ' stochemical methods using anti capsase antibody (active-casoase-3 antibody, 1:50; CeI Signaling Technology) and according to the method described in Example 1.
  • Demyelination was induced in six-week-old GFAPHTA; TRE/IFN-y double transgenic mice on a PERK+/- background that had been maintained on doxycycline, by simultaneously treating the mice with 0.2% cuprizone and releasing them 5 from doxycycline (DOX- PERK +/-). Remyelination was allowed to occur by withdrawing cuprizone at week 6.
  • Apoptosis of oligodendrocytes that is induced by INF- ⁇ is associated with ER stress in vitro [00205] To test whether apoptosis of oligodendrocytes that is induced by INF- ⁇ affects ER stress, the effect on the morphology, the degree of apoptosis and the expression of ER markers was studied in a culture of 5 oligodendrocyte precursor cells (OPC). Oligodendrocyte progenitors were cultured from neonatal rat brains
  • a mixed glial culture was grown in flasks in medium containing 10% fetal bovine serum (FBS), and when the astrocyte layer became confluent (10-14 days), oligodendrocyte progenitors were separated from astrocytes and microglia using an orbital shaker.
  • FBS fetal bovine serum
  • oligodendrocyte progenitors were separated from astrocytes and microglia using an orbital shaker.
  • Cells greater than 95% of which were A2B5 positive, GFAP negative and CDl Ib negative, were cultured in 0.5% FBS 0 containing medium, which also contained PDGF (10 ng/ml) and FGF (5 ng/ml) (both from R&D Systems,
  • progenitor cells that had been cultured in differentaition medium for 5 or 7 days were treated with 2 ⁇ g/ml of tunicamycin (Sigma Aldrich) for 6 hours.
  • the level of apoptosis of the OPC cells was determined by double staining CNP (1:200; 0 Sternberger Monoclonals, Lutherville, MD) and TUNEL using the ApopTag Kit (Serologicals Corp., Norcross,
  • RNA expression of ER markers binding 5 immunoglobulin protein (BIP), CAAT enhancer binding protein homologous protein (CHOP), and caspase 12 was determined using real time PCR. RNA was isolated from cultured cells and mice brain using Trizol reagent (Invitrogen, Carlsbad, CA) and treated with DNAaseI (Invitrogen) to eliminate genomic DNA.
  • Reverse transcription was performed using Superscript First Strand Synthesis System for RT-PCR kit (Invitrogen). Realtime PCR was performed with iQ Supermix (Bio-Rad, Hercules, CA) on a Bio-Rad iQ real-time PCR detection 0 system (Bio-Rad).
  • mice CHOP sense primer CCACCACACCTGAAAGCAGAA mouse CHOP antisense primer AGGTGCCCCCAATTTCATCT; CHOP probe TGAGTCCCTGCCTTTCACCTTGGAGA; mouse BIP sense primer ACTCCGGCGTGAGGTAGAAA; mouse BIP antisense primer AGAGCGGAACAGGTCCATGT; BIP probe TTCTCAGAGACCCTTACTCGGGCCAAATT; 5 mouse caspase 12 sense primer ATGCTGACAGCTCCTCATGGA; and mouse caspase antisense primer TGAGAGCCAGACGTGTTCGT.
  • Tissues or cultured cells were rinsed in ice cold phosphate- buffered saline (PBS) and then immediately homogenized in 5 volumes of Triton X-100 buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM EDTA, 10 mM tetrasodium pyrophosphate, 100 r
  • oligodendrocyte progenitor cells were allowed to differentiate for five days in defined media, at which point approximately 40% of the cells expressed the myelin protein 2'3 '-cyclic nucleotide 0 3'-phosphodiesterase (CNP) and extended branched processes ( Figure 13A and 13C). These cells did not extend the flat membrane sheets that are characteristic of more mature oligodendrocyte cultures. When treated with 70 U/ml IFN- ⁇ for 48h these cells showed abnormal morphological changes; including cell shrinkage and aggregation of cell bodies, followed by detachment from the culture plate ( Figure 13A and 13B).
  • OPCs myelin protein 2'3 '-cyclic nucleotide 0 3'-phosphodiesterase
  • Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) and CNP double labeling revealed that 5 IFN- ⁇ induced apoptosis in a significant number of oligodendrocytes ( Figure 13C, 13D and 13E). Furthermore, the caspase-3 activity in the cell lysates of IFN- ⁇ -treated oligodendrocytes was markedly increased ( Figure 13F). Thus, 70 U/ml of IFN- ⁇ is able to induce apoptosis in oligodendrocytes that are actively synthesizing myelin components.
  • oligodendrocyte progenitor cells that had been allowed to differentiate for five days in defined media expressed the myelin protein 2'3'-cyclic nucleotide 3'- 5 phosphodiesterase (CNP) and extended branched processes ( Figure 13A and 13C). These cells did not extend the flat membrane sheets that are characteristic of more mature oligodendrocyte cultures. When treated with 70 U/ml IFN- ⁇ for 48h these cells showed abnormal morphological changes; including cell shrinkage and aggregation of cell bodies, followed by detachment from the culture plate ( Figure 13A and 13B).
  • Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) and CNP double labeling revealed that IFN- ⁇ induced 0 apoptosis in a significant number of oligodendrocytes ( Figure 13C, 13D and 13E). Furthermore, the caspase-3 activity in the cell lysates of IFN- ⁇ -treated oligodendrocytes was markedly increased ( Figure 13F). .
  • caspase-12 an ER-localized caspase, is activated by ER stress and can lead to cleavage of cas ⁇ ase-3 (Nakagawa, et al. (2000) Nature 403: 98-103; Lamkanfi, et al. (2004) Cell Death Differ. 11: 365-368).
  • the induction of caspase-12 was observed after treatment of oligodendrocytes with IFN- ⁇ ( Figure 13G).
  • the level of the active fragment of caspase-12 was strongly elevated after 48 h of IFN- ⁇ treatment ( Figure 13H).
  • EXAMPLE 9 Hypomyelination induced by ectopic expression of IFN- ⁇ is associated with ER stress. [00214] To study the effect of INF- ⁇ on the myelination of oligodendrocytes during mouse development, transgenic mice that allow for temporally regulated delivery of IFN- ⁇ to the CNS using the tetracycline (tet) controllable system (Lin, et al. (2004) J. Neurosci. 24: 10074-10083) were generated. To drive tTA expression in astrocytes, the transcriptional regulatory region of the glial fibrillary acidic protein (GFAP) gene was chosen (Brenner, et al. (1994) J. Neurosci. 14: 1030-1037).
  • GFAP glial fibrillary acidic protein
  • mice were mated with TRE/IFN-y mice to produce animals hemizygous for both transgenes.
  • DOX+ doxycycline
  • expression of the IFN- ⁇ transgene is repressed ( Figure 10A); when the double transgenic mice are released from doxycycline (DOX-) INF- ⁇ is expressed.
  • mice received doxycycline up to day 14 of development (E14), at which time the experimental animals (DOX-) stopped receiving doxycycline, while the control animals (DOX+) were continuosly fed the dug.
  • the mRNA for IFN- ⁇ could be detected in the DOX- mice as early as 10 days after birth (data not shown).
  • the primers for the PCR reactions for BIP, CHOP, and caspase 12 are given in example 1.
  • the sense and antisense primers for INF- ⁇ were GATATCTCGAGGAACTGGCAAAA and CTTCAAAGAGTCTGAGGTAGAAAGAGATAAT, respectively; and the sense and antisense primers for MHC- 1 were MHC-I sense primer: ATTCCCCAAAGGCCCATGT; and MHC-I antisense primer:
  • Coronal sections at the fornix region of the corpus callosum corresponding to Sidman sections 241-251 were selected for use, and all comparative analyses were restricted to midline corpus callosum (Sidman, et al. (1971) Atlas of the Mouse Brain and Spinal Cord (Harvard Univ. Press, Cambridge, Massachusetts).
  • frozen sections were treated with -20°C acetone, blocked with PBS containing 10% NGS and 0.1% Triton X-100 and incubated overnight with the primary antibody diluted in blocking solution.
  • Appropriate fluorochrome- or enzyme-labeled secondary antibodies were used for detection.
  • An antibody against CCl was used as a marker for mature oligodendrocytes.
  • Antibody against MBP (1:1000; Sternberger Monoclonals, Lutherville, MA) was used to verify the degree of myelination.
  • Antibody against active-caspase-3 (1:50, Cell signaling Technology,
  • IFN- ⁇ upregulated BIP and CHOP expression approximately 1.6 and 2 times the control levels and strongly enhanced caspase-12 expression in the CNS of these animals ( Figure 14A). More notably, the level of the active fragment of caspase-12 was also increased in the CNS of these animals ( Figure 14B). Furthermore, colocalization analysis with the CCl antibody revealed that oligodendrocytes increased expression of BEP ( Figure 14C and 14D), p-eIF-2 ⁇ ( Figure 14E and 14F) and caspase-12 ( Figure 14G and 14H).
  • PERK enzyme was evaluated using transgenic mice that are heterozygous for a loss of function of mutation in pancreatic ER kinase (PERK) (Harding, et al (2001) MoI. Cell 7: 1153-1163). The phenotype of the transgenic animals was analyzed as a function of the level of p-eIF-2oc was evaluated in the PERK +/- mice.
  • GFAPHTA and TRE/IFN-y double transgenic mice described in Example 1 were crossed with PERK+/- mice, and the resulting progeny were intercrossed to obtain double transgenic mice that were homozygous or heterozygous for the PERK ⁇ nutation
  • Double transgenic GFAP/tTA; TRE/IFN-ymice on a PERK+/+ background, released from doxycycline at E14 (E14 DOX- PERK+/+) showed the expected minor tremor and ataxia but good survival.
  • the double transgenic mice on a PERK+/- background (E14 DOX- PERK+/-) had a much more severe phenotype.
  • These animals were considerably smaller than IFN- ⁇ expressing PERK+/+ littermates or PERK+/- animals that did not inherit the combination of GFAP/tTA and TRE/IFN-y alleles, and showed severe tremor and ataxia, and approximately two-thirds of these mice experienced tonic seizures.
  • the level of phosphorylted eIF-2 ⁇ was determined severity of the phenotype of the mice was evaluated.
  • the level of ⁇ -eIF-2 ⁇ was significantly greater in the oligodendrocytes from PERK+/+ mice when compared to that measured in the PERK+/- mice (Fig 14E and F), whereas a modest increase in p-eIF-2 ⁇ was seen in the CNS of the PERK+/- mice ( Figure 15 B and C).
  • the loss of function mutation in pERK did not significantly affect the RNA level of BIP, CHOP and Caspase-12 ( Figure 15D).
  • EXAMPLE 12 Loss of oligodendrocytes following IFN- ⁇ mis-expression in PEKK+/- mice.
  • Oligodendrocytes in adult animals are less sensitive to IFN-7 than actively myelinating oligodendrocytes from younger animals.
  • oligodendrocytes in adult mice produce lower levels of membrane proteins and lipids, just enough to maintain homeostasis in the myelin structure (Morell, et al. (1999) Basic Neurochemistry: Molecular, Cellular, and Medical Aspects (Philadelphia, PA: Lippincott-Raven Publishers): 69-93).
  • the ER of oligodendrocytes in adult animals may thus have more spare capacity to process increased protein load and as such may be less sensitive to 5 disruptions of the protein secretory pathway.
  • double transgenic mice were allowed to develop to maturity, at which time IFN- ⁇ expression in the CNS was initiated.
  • Real-time PCR analysis showed that double transgenic animals released from doxycycline at 4 weeks of age started expressing IFN- ⁇ at approximately 6 weeks of age, and the levels of IFN- ⁇ mRNA and protein in the CNS were comparable with those in developing mice released from doxycycline at E 14 (data not shown).
  • IFN- ⁇ did not affect oligodendrocyte 0 survival in adult mice, even in mice on a PERK+/- background ( Figure 2OB, 2OC, 2OD and 20E).
  • ultrastructural examination revealed normal myelin in the CNS of 10-week-old double transgenic mice with a wild-type or a PERK+/- background that were released from doxycycline at 4 weeks of age ( Figure 2OF, 2OG, 2OH and 201).
  • a modest induction of BIP and CHOP by IFN- ⁇ was observed in the cerebellum of 10-week-old double transgenic mice released from doxycycline at 4 weeks of age ( Figure 20A).
  • colocalization 5 analysis showed that mature oligodendrocytes did not significantly increase BIP expression (Figure 2OB, 2OC,
  • Double transgenic mice that allow for temporally regulated delivery of ESEF- ⁇ to the CNS were used to assess the role of INF- ⁇ in the pathogenesis of EAE.
  • the control animals were continuously fed doxycycline (DOX+ double mice), while the experimental animals (DOX- double mice) were deprived of the antibiotic to allow for the 5 expression of INF- ⁇ . The effect of INF- ⁇ was monitored as follows.
  • DOX+ animals at PID 14 showed that INF- ⁇ increased the expression of iNOs, TNF-a, IL-2, IL-12 and IL-IO, but did not affect the expression of IL-2 and IL-5 ( Figure 22).
  • EXAMPLE 15 Administration of INF- ⁇ at the onset of EAE protects oligodendrocytes from demyelination through a cytoprotective mechanism and independently of its anti-inflammatory properties.
  • T cells are primed in peripheral immune system and enter the CNS well before the onset of clinical disease Hickey et al, (199I) J Neurosci Res 28 :254-260.
  • Hickey et al (199I) J Neurosci Res 28 :254-260.
  • the level of T-cell and monocyte infiltration was assessed and the expression of known inflammatory cytokines evaluated.
  • Oligodendrocyte death has been shown to modulate inflammatory infiltration in EAE lesions
  • PERK pathway Demyelination and oligodendrocyte loss are the hallmarks of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). It has long been known that low levels of stress that activate downstream signaling pathways without causing severe cell injury can protect against subsequent exposure to more-severe stressful events.
  • Pancreatic endoplasmic reticulum kinase encodes an ER stress-inducible kinase that phosphorylates eukaryotic translation initiation factor 2 ⁇ (eIF2 ⁇ ) and enhances the stress-induced expression of numerous cytoprotective genes (Harding et al, (1999) Nature397:27l-274; Lu et al, (2004) EMBO J 23:169-119; Harding et al, (2003) MoI Cell 11:619-633).
  • a genetics-based approach was used to examine the involvement of the PERK pathway in the protective effects of IFN- ⁇ on EAE.
  • Mice that are heterozygous for a loss of function mutation in PERK are phenotypically healthy but display evidence of haploid insufficiency (Harding et al, 2000; Lin et al, 2005).
  • TRE/IFN-yraice were crossed with PERK+/- mice, and their progeny was crossed with GFAP/tTA mice to obtain triple transgenic mice that were heterozygous for the PERK mutation.
  • GADD34 is a stress-inducible gene that encodes a regulatory subunit that engages protein phosphatase- 1 (PPl) and targets dephosphorylation of the eukaryotic translation elongation factor eIF2 ⁇ (Connor et al, 2001; Novoa, 2001; Novoa et al, 2003). GADD34-mill mice 5 are healthy, but a loss of GADD34 increases the level of phosphorylated eIF2 ⁇ (p-eIF2 ⁇ ) in stressed cells.
  • PPl protein phosphatase- 1
  • GADD34-nvll animals are markedly protected from cell death caused by ER stress (Jousse et al, 2003; Marciniak et al., 2005). Therefore, role of eIF2 ⁇ in the pathogenesis of EAE was tested using GADD34-m ⁇ l mice.
  • GADD34-mx ⁇ mice were generated. The mice have been backcrossed 6 times with C57BL/6 0 mice. EAE is induced as follows. Mice were given a subcutaneous injection in the flank and tail base of 200 ⁇ g
  • GADD34-null mice when compared to the control animals. Therefore, inhibition of GADD34 delays the onset of
  • Tissue damage in the CNS is determined by immunohistochemistry analysis and toluidine blue 0 staining as described by Lin et al. (submitted).
  • Spinal cord tissue is analyzed for tissue damage at the peak of disease severity.
  • An antibody against CCl (APC7) is used as a marker to identify mature oligodendrocytes; an antibody against MBP is used to determine the degree of demyelination; and an antibody against nonphosphorylated neurof ⁇ lament-H (SMI32) is used to detect axonal damage.
  • the severity of demyelination and axonal injury is examined by staining the tissue with toluidine blue.
  • real-time PCR is perfomed to determine the steady-state levels of mRNAs that encode for MBP, PLP, and ceramide galactosyltransferase (CGT) in the spinal cord.
  • CCT ceramide galactosyltransferase
  • EXAMPLE 18 Loss of GADD34 protects oligodendrocytes from aginst demyelination during EAE.
  • PERK-eIF2 ⁇ pathway is activated in oligodendricytes during EAE by the immune cytokine IFN- ⁇ .
  • Chakrabarty et al. (2004) have shown that activation of the PKR-eIF2 ⁇ pathway occurs in inflammatory cells in the CNS of animals with active EAE.
  • double immunostaining of CD3 and p-eIF2 ⁇ is performed to determine whether the level of p-eIF2 ⁇ is increased in T cells in the CNS of the GADD34-xmll mice when compared to the wild type animals.
  • the immune responses in the CNS and peripheral immune system in GADD34-mi ⁇ l and littermate control mice are analyzed as described in Lin et al. (submitted).
  • EXAMPLE 19 The role of GADD34 in demyelination: effects of the loss of function of GADD34 in vitro [00258]
  • experiments that use siRNA to block the expression of GADD34 are performed on purified rat oligodendrocytes in culture. The experimental protocol is described by Lin et al. (submitted).
  • Oligodendrocyte progenitors (OPCs) are cultured from neonatal rat brain as follows.
  • a mixed glial culture is grown in flasks in medium containing 10% FBS, and when the astrocyte layer becomes confluent (after 10-14 days), OPCs are separated from astrocytes and microglia using an orbital shaker. More than 95% of the cells are A2B5 positive,
  • siRNA Small interfering RNA
  • PDGF PDGF (10 ng/mL), and FGF (5 ng/mL)
  • FGF 5 ng/mL
  • Small interfering RNA is designed using siRNA Target Finder software (Ambion). and used to inhibit the expression of the rat GADD34.
  • the target sequence of the siRNA is used in conjuction with the ⁇ Silencer Expression Vectors Insert Design Tool (Ambion) to generate hairpin siRNA that encodes DNA oligonucleotide inserts that are derived from the siRNA target sequence.
  • the program adds the loop sequence and overhangs that are used for cloning.
  • the hairpin siRNA-encoding DNA oligonucleotide inserts are cloned into the pSilencer 4.1-CMV vector (Ambion) according to the manufacturer's instructions, and the pSilencer 4.1-CMV vectors encoding hairpin siRNAs that are specific for GADD34 will be transfected into OPCs using the Nucleofector kit (Amaxa) according to the protocol of the manufacturer.
  • Control oligodendrocytes are transfected with control vectors that are pSilencer 4.1-CMV vectors that lack GADD34 siRNA. After transfection, the cells !P IL of 10-14 days.
  • the transfected OPCs are analyzed for the presence GADD34 mKNA using Taqman real-time PCR and for the presence of GADD34 protein using Western blot analysis.
  • Salubrinol the protective effect of salubrinol (Sal) is tested to determine whether Sal protects oligodendrocytes from cytokines, oxidants, peroxynitrite donors, and glutamate.
  • Salubrinol has been shown to specifically inhibit the PP1-GADD34 phosphatase activity, which results in sustained eIF2 ⁇ phosphorylation in stressed cells (Boyce et al., 2005). Sal is combined with TNF- ⁇ , H 2 O 2 , the peroxynitrite donor SIN-I, and glutamate is added to the cells that have been allowed to differentiate for 7 days.
  • GADD34 deletion elevates the level of p- eIF2 ⁇ in oligodendrocytes and protects against EAE-induced demyelination and oligodendrocyte loss. It is expected that GADD34 siRNA transfection and Sal will protect oligodendrocytes from cytokine exposure, reactive oxidative/nitrative stress, and glutamate excitoxicity. Various aspects of the cytoprotective effects of Sal with those of GADD34 siRNA transfection are compared. The protective effects of Sal in mice with EAE and persons with MS are tested.
  • PERK pathway The activation of the PERK pathway is initiated by dimerization of PERK, which leads to trans- autophosphorylation and increased ability to phosphorylate its substrate, eIF2 ⁇ . Normally, this dimerization event is driven by the stress-sensing ER lumenal domain of PERK (Harding 1999). Other researchers have fused the eIF2 ⁇ kinase effector domain of PERK to a polypeptide containing 2 modified FK506 binding domains (Fv2E) to generate a fusion protein, Fv2E-PERK, and have demonstrated that the activity of this artificial eIF2 ⁇ kinase,
  • Fv2E-PERK is subordinate to the dimerizer AP20187 and is uncoupled from upstream signaling of ER stress (Lu et al., 2004).
  • oligodendrocyte precursor cells OPCs
  • the Fv2E-PERK construct is subcloned into the mammalian vector pcDNA3.1 to generate a mammalian expression plasmid that expresses a fusion protein Fv2E-PERK protein (pcDNA3.1-Fv2E-PERK).
  • Purified rat OPCs as described above are transfected with pcDNA3.1 -Fv2E-PERK using the Nucleofactor kit (Amaxa), and selected with G418 (400 ug/ml) for 10-14 days.
  • the OPCs are induced to differentiate for 7 days, and the activation of the PERK-eIF2 ⁇ pathway is evaluated in the presence of the dimerizer AP20187 alone, or in combination with one of TNF- ⁇ , H 2 O 2 , peroxynitrite donor SIN-I, or glutamate. Willi''" " " "'"'' encoding Fv2-PERK is verified using real time PCR, and the expression of Fv2-PERK protein is verified by Western blot analysis. The effect of the activation of the PERK-eIF2 ⁇ pathway is assessed by Western blot analysis for p-eIF2 ⁇ following addition of AP20187 to the transfected OPCs.
  • the viability of transfected oligodendrocytes is determined using the MTT assay, TUNEL assay, and caspase-3 activity assay, as described above following differentiation of OPCs for 7 days. The same analyses are made following administration of AP20187 to the OPCs in the presence of TNF-a, H2O2, peroxynitrite donor SIN-I, or glutamate. Oligodendrocyte viability is determined by MTT and TUNEL assays, and caspase activity. [00267] It is expected that AP20187 will protect the Fv2-PERK-expressing oligodendrocytes from effector(s) of the ER stress pathway.
  • RNA microarray analysis is used to identify the cytoprotective genes that are upregulated.
  • OPCs that have been transfected with pcDNA3.1 -Fv2E-PERK are differentiated for 7 days and treated with AP20187.
  • RNA is isolated from AP20187-treated transfected cells and control transfected cells by using Trizol reagent (Invitrogen), and DNAseI (Invitrogen) is added to eliminate genomic DNA. Fluorescent- labeled RNA probes are prepared and hybridized to Aff ⁇ metrix rat high-density oligonucleotide arrays.
  • EXAMPLE 22 Activation of the PERK-eIF2 ⁇ pathway provides cytopoprotection to oligodendrocytes during EAE [00271] To provide direct evidence that activation of the PERK-eIF2 ⁇ pathway protects against EAE- induced tissue damage through its cytoprotective effects on oligodendrocytes, the activity of PERK is modulated in mice that allow for controllable activation of the PERK pathway in oligodendrocytes.
  • Fv2E-PERK (PLP/Fv2E-PERK) is engineered, and transgene fragments are injected into fertilized C57BL/6 mouse oocytes.
  • mice that allow for modest activation of the PERK pathway in oligodendrocytes after administration of AP20187 are selected for EAE experiments.
  • the mice are immunized with MOG33-35 peptide as described in Example .
  • Fv2E-PERK in oligodendrocytes is activated by administering AP20187 before the onset of EAE onset.
  • the activation of eIF2 ⁇ in oligodendrocytes is verified by CCl and p-eIF2 ⁇ double immunostaining, and the clinical phenotype, the histopathologic findings in the CNS, and the immune response in the CNS are characterized as described in the Examples above.
  • PERK pathway in remyelinating oligodendrocytes promotes remyelination in EAE-induced demyelinated lesions
  • EAE is induced in PLP/Fv2 E-PER mice, and the role of PERK is evaluated in the presenc eand absence of AP201187.
  • the PLP/Fv2E-PERK mice are immunized with MOG33-35 peptide as described in Example , and Fv2E-PERK is activated in oligodendrocytes by administering AP20187 to the mice during the recovery stage of EAE.
  • the immune response in the CNS is assessed by immunostaining for CDl Ib and CD3 immunostaining, and by real-time PCR analyses for IFN- ⁇ , TNF- ⁇ , iNOs, IL-2, IL-4, IL-5, IL-10, IL- 12, IL-17, and IL-23.
  • the PERK-eIF-2 ⁇ pathway is activated in oligodendrocytes from lesions in patients with MS
  • CNS tissue of patients with MS is obtained from the Human Brain and Spinal Fluid Resource
  • RNA is isolated from the frozen samples using Trizol reagent (Invitrogen) and treated with DNAseI (Invitrogen) to eliminate genomic DNA. Reverse transcription is performed using the Superscript First ' " StrM'SyBffieMsf Steln%f RT-PCR kit (Invitrogen).
  • the expression of IFN- ⁇ and the ER stress markers BIP and CHOP is quantified.
  • the protein levels of PERK, eIF2 ⁇ , p-PERK, and p-eIF2 ⁇ are determined using Western blot analysis.
  • the remaining frozen tissue will be embedded in optimal cutting temperature compound.
  • Frozen sections (10 ⁇ m thick) of the tissue samples are prepared, and CCl immunostaining is used to determine the number of oligodendrocytes in the demyelinated lesions. MBP immunostaining is used to verify the degree of demyelination.
  • the immune response in the CNS is assessed by CDl Ib immunostaining and CD3 immunostaining.
  • CCl and p-PERK double immunostaining, or CCl and p-eIF2 ⁇ double immunostaining is performed to demonstrate the activation of the PERK-eIF2 ⁇ pathway in MS lesions.
  • demyelinated MS-induced lesions can be divided into the following 3 categories: active (acute), chronic active, and chronic inactive (Lassmann, 1998; Trapp et al., 1999).
  • active acute
  • chronic active chronic inactive
  • the activation of the PERK-eIF2 ⁇ pathway is expected to be detected in only some of the tissue samples.
  • GADD34 increases the level of p-eIF2 ⁇ in oligodendrocytes and protects against EAE-induced demyelination
  • Phosphorylation of eIF2 ⁇ is a highly conserved point of convergence among signaling pathways that adapt eukaryotic cells to diverse stressful conditions (Jousse et al (2003) J. Cell. Biol. 163:767-775; Proud CG (2005) Setnin. Cell. Dev. Biol. 16:3-12).
  • Four protein kinases are known to couple the otherwise unrelated stresses of protein malfolding in the ER (PERK), amino acid deprivation (GCN2), viral infection (PKR) and heme deficiency (HRI) to the phosphorylation of eIF2 ⁇ (Proud CG (2005) Semin. Cell. Dev. Biol. 16:3-12).
  • ISR integrated stress response
  • GADD34 is regulated by induction of the cytosolic transcription factor ATF4, the translation of which is upregulated in the presence of eIF2 ⁇ phosphorylation, thus creating a tight auto-feedback loop (Novoa et al (2003) EMBO J. 22: 1180-1187; Jiang et al (2004) MoI. Cell. Biol. 24: 1365-1377).
  • GADD34 deletion increases the level of p-eIF2 ⁇ in stressed cells and protects cells from stress (Jousse et al (2003) J. Cell.
  • EAE was induced in GADD34-nuU mice and control animals as described in Example 17.
  • GADD34- ⁇ mll mice were backcrossed with C57BL/6 mice at least 6 times.
  • EAE was induced in mice by administering to the mice a subcutaneous injection of 200 ⁇ g MOG 35-55 peptide emulsified in complete 5 Freund's adjuvant supplemented with 600 ⁇ g of Mycobacterium tuberculosis in the flank and tail base.
  • Two 400- ng intraperitoneal injections of pertussis toxin were given 24 and 72 h later.
  • GADD34 deletion dramatically reduced the axonal damage in the lumbar spinal cord of GADD34 null mice, compared with control mice ( Figure 28g and 28h). Taken together, these data indicate GADD34 deletion increases the level of p-eIF2 ⁇ in oligodendrocytes, thus prolongs in the integrated- stress response and protects against EAE-induced demyelination.
  • EAE induction including less severe demyelinaiton at the peak of disease.
  • agents that prolong eIF2o: phosphorylation might provide protection to oligodendrocytes against detrimental inflammatory factors the ⁇ "" "effelr'of sllutrmol'f Sal)' B ⁇ 't ⁇ t ⁇ presence of the immune cytokine IFN- ⁇ was tested in an in vitro model of myelination.
  • Hippocampal organotypic cultures (HOC), which have been shown to myelinate well in vitro.
  • HOCs were prepared and maintained as described previously (Kunkler and Kraig (1997) / Cereb Blood Flow Metab. 17:26-43), and were maintained in vitro for 7 days before use.
  • SAL at various concentrations was combined with 100U/ml IFN- ⁇ and added to the HOC cultures for 7 days.
  • Robust myelination was observed in untreated HOCs, as demonstrated by abundant levels of the myelin-specif ⁇ c protein myelin basic protein (MBP) in the cultures ( Figure 29).
  • MBP myelin-specif ⁇ c protein myelin basic protein
  • IFN- ⁇ has been shown to inhibit remyelination in demyelinated lesions (See Examples 2-7). Taken together, these data indicate that treatment with agents that promote or prolong eIF2 ⁇ phosphorlyation could promote myelin repair in immune-mediated demyelination diseases.
  • the transgenic mouse line PLP/SOCS1 was generated using a construct containing a PLP expression cassette and SOCSl cDNA ( Figure 30A).
  • the PLP expression cassette has been described elsewhere and has been used for oligodendrocyte-specific expression of a number of transgenes (Wight et al., 1993; Fuss et al., 2000; Doerflinger et al., 2003; Gonzales et al., 2005).
  • a SOCSl cDNA clone (Starr et al., 1998) was used as it contained a Flag-epitope sequence that served as a marker for SOCSl expression in polymerase chain reaction (PCR)-based or anti-Flag antibody-based detection methods (Einhauer and Jungbauer, 2001). Briefly, the Flag- SOCSl cDNA was excised from the original expression vector pEF-FLAG-I/m4A2 with Xbal. The fragment was
  • the resulting pNEB193/SOCSl vector was further digested with Ascl (partial digestion) and Pad to release the Flag-SOCSl fragment, which was subcloned into the polylinker region of the PLP expression cassette at the same restriction sites.
  • the PLP/SOCS1 vector was digested with Apal and Sacll (partial digestion) and a linear 15-kb transgene was isolated for microinjection into fertilized (C57BL/6J x DBA/2J) oocytes. Offspring positive for the transgene were identified by amplifying tail DNA by PCR using transgene-specific primers.
  • transgenic mice were subsequently bred with C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME) establishing a transgenic line.
  • C57BL/6J mice Jackson Laboratories, Bar Harbor, ME
  • the transgenic mice MBP/IFN-y (line 172) and GFAPhTA x TRE/IFN-y (lines 184/110 and
  • mice are transgenic animals in which IFN- ⁇ expression is driven by the myelin basic protein (MBP) transcriptional control region (Gao et al., 2000).
  • MBP myelin basic protein
  • the two TRE/IFN-y mouse lines, line 110 and line 67, used in the experiments produce different amounts of IFN- ⁇ when crossed to GFAPHTA mice (184/110 and 184/67) (Lin e al., * a tetracycline-off-inducible system in which the glial fibrilary acidic protein (GFAP) transcriptional control region drives the expression of tTA, which in turn, binds to the TRE (tet responsive element) and initiates the expression of IFN- ⁇ .
  • GFAP glial fibrilary acidic protein
  • Administration of doxycycline suppresses tTA DNA binding and IFN- ⁇ expression, and doxycycline removal allows for temporally-controlled induction of IFN- ⁇ expression (Gao et al., 1999).
  • IFN- ⁇ -overexpressing mice were crossed to the PLP/SOCS1 mice in double-transgenic
  • MBP/IFN-yx PLP/SOCS1 triple-transgenic (GFAP/tTA x TRE/IFN-y* PLP/SOCS1) mating systems.
  • MBP/IFN-y x PLP/SOCS1 172 x PLP/SOCS1 mating was performed according to a standard mating protocol.
  • the GFAP/tTA x TRE/IFN-y x PLP/SOCS1 matings were performed in a 2-step mating process: GFAP/tTA mice (line 184) were initially crossed to PLP/SOCSln ⁇ ce, and double-positive (184 x PLP/SOCS1) offspring were then crossed to the TRE/IFN-y lines 110 and 67, separately.
  • This second mating step was performed according to the previously described "tet-off" protocol.
  • Doxycycline 0.05mg/ml Sigma-Aldich was added to the water of impregnated female mice until embryonic day 14, after which the animals were switched back to normal water, thereby allowing initiation of IFN- ⁇ transcription, which peaks during the postnatal period (Lin et al., 2005).
  • PCR Qiagen PCR kit, Valencia, CA
  • transgene-specific screening primers Flag-SOCSl sense primer, 5'-
  • CCAGGACGACGATGACAAGA-3' and Flag-SOCSl anti-sense primer 5'-TCAGGGGTCCCCA ATAGAAG- 3'; MBP/IFN- ⁇ sense primer, 5'-ATGAGGAAGAGCTGCAAAGC-S', and MBP/IFN- ⁇ anti-sense primer, 5- GGTGACAGACTC CAAGCACA-3'; GFAP/tTA sense primer, 5'-TCGCTTTCCTCTGAACGCTTCTCG-S' and GFAP/tTA anti-sense primer, 5'-TCTGAACGCTGTGACTTGGAGTGTCC-S'; TRE/IFN- ⁇ sense primer 5'- CGAATTCGAGCTCGG TACCC-3' and TRMFN- ⁇ anti-sense primer 5'-CCATCCTTTGCCATTCCTCCAG-S' (Integrated DNA Technologies Inc.).
  • e) Northern blot analyses and Quantitative PCR methods 5'-TCAGGGGTCCCCA ATAGAAG- 3'; M
  • Northern blots were performed by separating 20 ⁇ g of total RNA in a 1.2% denaturing agarose gel. The samples were transferred to a nylon membrane and hybridized overnight with a SOCS 1 probe that had been randomly labeled by PCR (GenAm ⁇ 2400; Perkin-Elmer, Welleslay, MA) with [ ⁇ - 32 P] dCTP and [ ⁇ - 32 P] dATP (New England Nuclear/Perkin-Elmer, Welleslay, MA). Kodak film was exposed to the hybridized membrane at -8O 0 C for 48hrs and was developed using the M7B Kodak processor (Kodak, Rochester, NY).
  • RNAasel-treated (Invitrogen) total RNA was stripped and hybridized with a radiolabeled probe specific for the 28S ribosomal RNA (Baerwald et al., 1998). or real-time PCR) was performed by first reverse transcribing l ⁇ g of DNAasel-treated (Invitrogen) total RNA using oligo(dT)i 2- i 8 and Superscript II reverse transcriptase (Invitrogen RT-PCR kit).
  • lysates from brain and spleen of several PLP/SOCS1 mice and wild-type mice were obtained by tissue homogenization in RIPA buffer (Santa Cruz Biotechnology, Santa Cruz, CA). After incubation on ice for 15 min, lysates were centrifuged at 14,000 rpm for 30 min and the supernatants collected. Protein samples (50 ⁇ g) were electrophoresed on 15% SDS-polyacrylamide gels, transferred to PVDF membranes (Trans-blot SD apparatus, Bio-Rad Laboratories), incubated overnight with mouse anti-Flag antibody (M2, diluted to 1:1000) (Sigma-Aldrich, St.
  • mice and rabbit anti-Flag antibody (dilution, 1:100; Sigma-Aldrich), mouse anti-CCl antibody (dilution, 1:20; Oncogene), mouse MHC class I antibody (dilution, 1:100; Chemicon International, Temecula, CA), mouse anti- h Ii ,,, ,.
  • Oligodendrocyte cell density was assessed digitally using Axiovision software, at postnatal day
  • mice selected for electron microscopy studies were perfused with 4% paraformaladehyde and
  • the brains were sectioned sagittally through the corpus callosum dividing the brain in two symmetrical halves. Approximately 2mm 3 samples from the genu and the splenum of both halves of corpus callosum were obtained using a Wild M3C stereotype microscope. The orientation of the specimen in the resin blocks yielding axonal crosssections (well seen myelin rings) was chosen, and established by toluedine blue staining of a few sample sections. The resin blocks with the chosen orientation were processed for electron microscope examination.
  • the G ratio (axonal diameter/fiber diameter ratio) of myelinated axons was assessed by digitally selecting the area encircled by the inner and outer surfaces of the myelin sheath, obtaining the axonal (inner) and the fiber (outer) diameters, and dividing their corresponding values (axonal diameter/fiber diameter ratio).
  • i ⁇ h"miry iligUeri ⁇ rocytes cultures and STATl translocation assay
  • brain tissue was harvested from 2-3-day-old newborn pups of PLP/SOGS7 and C57B1/6J matings. Because the litters contained transgenic and wild-type pups, the brain of each animal was processed individually, cultured separately, and later genotype matched. Each brain was digested separately using 0.25% trypsin and lO ⁇ g/ml of DNAaseI (Invitrogen) in Dulbecco's modified Eagle's medium (DMEM) for 20 min at 37 0 C, and cells were cultured on separate poly-D-lysine-coated 75-mm 2 flasks (Sigma- Aldrich).
  • DMEM Dulbecco's modified Eagle's medium
  • the cultures were maintained with 10% fetal bovine serum DMEM at 37°C with 5% CO 2 for 12 days, and then switched to a defined medium containing 5 ⁇ Vml of insulin, 50/ ⁇ g/ml of transferrin, 3OnM of selenium, 1OnM of biotin, 1OnM of progesterone, 15nM of T3, 0.1% bovine serum albumin, and 1% ampicillin-streptomycin (Sigma-Aldrich).
  • the cultures were treated with lOOU/mL of IFN- ⁇ (Calbiochem, San Diego, CA) for 30 min. Dual immunostaining for anti-PLP and anti-Statl antibodies, and DAPI nuclear staining were performed as described above.
  • the Statl nuclear translocation assay was performed in six separate culture preparations. One hundred PLP positive cells were manually counted in both wild-type and PLP/SOCS1 cultures. The results were presented as mean ⁇ SD percent cells positive for Statl nuclear translocation. j) Statistical analysis
  • the PLP/SOCS1 transgenic mice which were generated as described in Example 25, were designed to express Flag epitope-tagged SOCSl in myelinating cells (Fig. 30A). These mice exhibit no phenotypic abnormalities, breed and produce transgenic progeny in a Mendelian fashion, and live a normal life span. Histological evaluation, including electron microscopy, performed at different time points up to 1 year of age revealed no significant differences in the myelination patterns or the number, density, or morphology of oligodendrocytes (see below) between transgenic and wild-type littermates.
  • Flag-SOCS 1 The expression of Flag-SOCS 1 in primary mixed oligodendrocyte cultures established from transgenic animals by dual immunostaining with anti-PLP and anti-Flag antibodies was also detected (Fig. 32). Expression of Flag-SOCSl was detected only in cultures from PLP/SOCS1 animals and only in cells expressing PLP. Virtually all PLP-positive cells were also positive for Flag-SOCSl. The colocalization between anti-Flag and anti-PLP immunoreactivity appeared to involve both the cell body and cell processes (Fig. 32F).
  • MHC class I molecule expression was neither detectable in control wild-type mice nor PLP/SOSC1 mice (Fig. 34A-D). Consistent with previous reports, MBP/IFN-ymice exhibited upregulated expression of the MHC class I molecule, with diffuse protein localization along the myelin sheath (Fig. 34E, F) (Corbin et al., 1996). The double-transgenic mice (MBP/IFN-y x PLP/SOCS1), however, displayed a differential pattern of MHC class I molecule expression (Fig.
  • oligodendrocytes and myelin positive for Flag-tagged SOCSl did not express detectable levels of MHC class I molecule, whereas, cells negative for transgene expression, and in close proximity to the SOCSl -positive cells, demonstrated strong immunoreactivity (Fig. 34G-J). Similar differential upregulation of MHC class I molecule expression was observed following the direct administration of IFN- ⁇ in the brain of PLP/SOCS1 mice (data not shown). Together, these data indicate that oligodendrocytes from
  • PLP/SOCS1 mice display diminished responsiveness to IFN- ⁇ . . _
  • mice The litter (Fl generation) of each mating system was divided into four study groups depending on their genotype: wild-type/single transgenic controls, mice expressing SOCSl only, mice expressing IFN- ⁇ only, and mice expressing both IFN- ⁇ and SOCSl.
  • a total of 40 animals per mating system (10 animals per each study group) were collected and examined clinically and histologically at postnatal day 21.
  • Phenotypic comparisons of littermates were performed from birth to postnatal day 21 and evaluation consisted of behavioral observation and challenged ladder walking to elicit tremor.
  • MB PIIFN-y (line 172) mice express low levels of IFN- ⁇ in the CNS and displayed no behavioral abnormalities, in accordance with findings reported elsewhere (Corbin et al., 1996).
  • mice 184/67 xPLPISOCSl were stratified according to their genotype into four groups: Wild-type/control mice, mice expressing SOCSl only, mice expressing IFN- ⁇ only, and mice expressing both IFN- ⁇ and SOCSl. Ten mice per group were clinically followed during the first three postnatal weeks and the incidence of tremor recorded. [00317] The phenotypes of wild-type mice and single-transgenic control mice (GFAPItTA [184], TREIIFN- y [67 and HO]), and PLPISOCSl mice were clinically normal.
  • the tremoring phenotype which varied in severity, was identified in almost all double-transgenic GFAPItTA x TREIIFN-y mice overexpressing IFN- ⁇ : 80% (8/10) of 184/110 mice and 100% (10/10) of 184/67 mice.
  • Triple-transgenic GFAPItTA x TREIIFN-y x PLPISOCSl mice overexpressing both IFN- ⁇ and SOCSl appeared to be protected, because only 10% (1/10) of 184/110 x PLPISOCSl mice, and 30% (3/10) of 184/67 x PLPISOCSl mice developed tremor (Table 2).
  • IFN- ⁇ expression was detected in M£P//iW- ⁇ single-transgenic and MBP/IFN-y x PLP/SOCS1 double-transgenic littermates of the 172 xPLP/SOCSl transgenic system, in GFAP/tTA x TRE/IFN-y double-transgenic and GFAP/tTA x TRE/IFN-y x PLP/SOCS1 triple-transgenic littermates of thel84/110 x PLP/SOCS transgenic system, and in GFAP/tTA x TRE/IFN-y double-transgenic and GFAP/tTA 5 x TRE/IFN-y x PLP/SOCS 1 triple-transgenic littermates of the 184/67 x PLP /SOCl transgenic system.
  • IFN- ⁇ expression Two characteristics of IFN- ⁇ expression were observed. First, the three mating systems differed in their expression levels; MBP/IFN-y x PLP/SOCS1 (172 x PLP/SOCS1) expressed the lowest, and GFAP/tTA x TRE/IFN-y x PLP/SOCS1 (184/67 x PLP/SOC1) expressed the highest IFN- ⁇ levels. Secondly, the littermates of the same mating system, expressing IFN- ⁇ only or both IFN- ⁇ and SOCSl, did not differ in their expression levels. No 0 detectible levels of IFN- ⁇ were found in the wild-type, the GFAP/tTA and TRE/IFN-y single-transgenic, and the
  • mice from the Fl generation differed depending on genotype (Fig. 35B and Fig. 36). Wild-type mice and the 0 single-transgenic mice (PLP/SOCS1, GFAP/tTA, and TRE/IFN-y) had comparable oligodendrocyte densities.
  • oligodendrocyte loss in the GFAP/tTA x TRE/IFN-y 'mice overexpressing IFN- ⁇ compared with the wild-type and single-transgenic littermates: approximately 20% of oligodendrocytes were lost in 184/110 mice (from 146 ⁇ 6 CCl (+) cells/mm 2 in the wild-type mice to 115 ⁇ 8 CCl (+) cells/mm 2 in the IFN- ⁇ mice), and approximately 40% were lost in 184/67 mice (from 144 ⁇ 5 CCl (+) cells/mm 2 in the wild-type mice to 5 79 ⁇ 9 CCl (+) cells/mm 2 in the IFN- ⁇ overexpressing mice).
  • PLP/SOCS1 (172 x PLP/SOCS1) mating system.
  • ⁇ GFAP/tTA x TRE/IFN-y x PLP/SOCS1 significant differences in G ratios were found among the Fl generation mice, depending on genotype (Fig. 35C and Fig. 36).
  • G ratios among the wild-type and the GFAP/tTA, TRE/IFN-y, and PLP/SOCS1 single-transgenic littermates were similar.
  • IFN- ⁇ -overexpressing GFAP/tTA x TRE/IFN-y littermates displayed significantly increased G ratios indicating hypomyelination (defined as a G ratio >0.8): 0.89 ⁇ 0.02 for 184/110 mice and 0.95 ⁇ 0.04 for the 184/67 mice (Fig. 35C).
  • the myelin abnormalities were further quantified by determining the percentage of unmyelinated axons in the various transgenic genotypes (Fig. 35D). We found no significant difference in the percentage of unmyelinated axons (less than 9%) among the Fl generation littermates of the MBP/IFN-y x PLP/SOCS1 (172 x
  • T-cell-derived cytokine IFN- ⁇ within the CNS is believed to play a critical role in the pathogenesis of immune-mediated demyelinating disorders (Panitch et al, 1987; Galbinski et al., 1999; Tran et al., 2000; Vartanian et al., 1996; Horwitz et al. 1997; Steinman, 2001). Nevertheless, the cellular target of the cytokine's effect remains unresolved. In this report we describe the generation of transgenic mice in which the oligodendrocytes display a significantly reduced capacity to respond to IFN- ⁇ .
  • mice are protected from the injurious effect of ectopic expression of IFN- ⁇ within the CNS, suggesting that a direct deleterious effect of IFN- ⁇ on oligodendrocytes contributes to immune-mediated disease pathogenesis. As discussed below, the work described has significant clinical implications. [00323] Transgenic animals that ectopically express IFN- ⁇ in the CNS during postnatal development are hypomyelinated and contain reduced numbers of oligodendrocytes (Corbin et al., 1996; LeFerla et al., 2000; Lin et al., 2005).
  • IFN- ⁇ has also been shown to have harmful effects on oligodendrocytes and their progenitors in vitro. There is considerable evidence to suggest that at least part of the injurious effect of this cytokine is mediated through the activation of microglial cells. IFN- ⁇ -treated microglia release cytotoxic agents, including nitric oxide and tumor necrosis factor alpha, which are known to be damaging to oligodendrocytes (Merrill et al.,
  • oligodendrocytes have a direct, harmful effect on oligodendrocytes (Torres et al., 1995; Agresti et al., 1996; Baerwald et al., 1998; Andrews et al., 1998; Lin et al., 2005).
  • IFN- ⁇ has been shown to inhibit cell cycle exit of oligodendroglial progenitor cells, which may predispose these cells to apoptotic death (Chew et al., 2005).
  • IFN- ⁇ has been shown to be a very powerful apoptosis-inducing agent for developing oligodendrocytes (Baerwald et al., 1998, 2000; Lin et al., 2005). Oligodendrocytes that have been allowed to mature oligodendroglial markers are less sensitive to the presence of the cytokine, although they do eventually succumb to necrosis (Baerwald et al., 1998).
  • transgenic mice that exhibited diminished oligodendrocyte-specific responsiveness to IFN- ⁇ .
  • Transgenic mice expressing either the dominant-negative form of IFN- ⁇ receptor subunit 1 (IFNGRl) or the suppressor of cytokine signaling 1 (SOCSl) have been previously described (Flodstrom et al., 2001; Gonzales at al., 2005, Hindinger et al., 2005).
  • IFNGRl IFN- ⁇ receptor subunit 1
  • SOCSl suppressor of cytokine signaling 1
  • SOCSl is an intracellular protein that blocks IFN- ⁇ mediated Statl activation (i.e., phosphorylation) by Jak kinases (Starr et al., 1997; Song and Shuai 1998; Sakamoto et al., 2000; Yasukawa et al., 2000; Kubo et al.,
  • Such low constitutive expression may limit the oligodendrocyte capacity for effective downregulation of IFN- ⁇ /Jak/Statl signaling, resulting in enhanced IFN- ⁇ cellular effects.
  • the rescuing effect of SOCSl overexpression in oligodendrocytes that was observed in our experimental system supports this possibility.
  • IFN- ⁇ plays a deleterious role in the immune-mediated demyelinating disorder multiple sclerosis (Popko et al., 1997, Steinman 2001). IFN- ⁇ is found in demyelinated lesions and its levels in cerebrospinal fluid correlate with disease severity (Vartanian et al., 1996;
  • Stem cell therapy is rapidly gaining interest as a potential therapeutic approach to demyelinating disorders such as multiple sclerosis and adrenoleukodystrophy (review in Keirstead 2005).
  • Stem cells engineered to be resistant to the harmful cytokines present in the extracellular milieu of the breached CNS is expected to stand a better chance of surviving and accomplishing remyelination. It is, therefore, of therapeutic interest to identify signaling pathways that play differential roles in oligodendrocyte injury and development.
  • Our results describing inhibition of IFN-7-mediated oligodendrocyte injury without induction of any observable oligodendrocyte or myelin abnormalities provide support for such an approach.
  • Socsl is a critical inhibitor of interferon-gamma signaling and prevents the potentially fatal action of this cytokine. Cell 98:598-608.
  • a myelin proteolipid protein-lacZ fusion protein is developmentally regulated and targeted to the myelin membrane in transgenic mice. J Cell Biol 123:443-454.

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  • Neurosurgery (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Cell Biology (AREA)
  • Urology & Nephrology (AREA)
  • Epidemiology (AREA)
  • Physics & Mathematics (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Animal Husbandry (AREA)
  • General Physics & Mathematics (AREA)

Abstract

L'invention concerne le champ de la neurologie. Plus spécifiquement, l'invention concerne la découverte et la caractérisation de composants moléculaires qui jouent un rôle dans la démyélinisation ou la remyélinisation neuronales. En outre l'invention concerne la production d'un animal modèle qui présente une hypomyélinisation. Les compositions et procédés de cette invention sont particulièrement utiles pour le criblage de médicaments et/ou le traitement de troubles de démyélinisation.
PCT/US2006/023215 2005-06-14 2006-06-14 Procedes permettant de traiter des troubles de demyelinisation WO2006138412A2 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
CA002612374A CA2612374A1 (fr) 2005-06-14 2006-06-14 Procedes permettant de traiter des troubles de demyelinisation
JP2008517080A JP2008546704A (ja) 2005-06-14 2006-06-14 脱髄障害を処置するための方法
EP20060773189 EP1909562A4 (fr) 2005-06-14 2006-06-14 Procedes permettant de traiter des troubles de demyelinisation
IL188127A IL188127A (en) 2005-06-14 2007-12-13 Method of developing a biologically active agent that modulates demyelination disorder, uses thereof and a method of testing the same

Applications Claiming Priority (12)

Application Number Priority Date Filing Date Title
US69069105P 2005-06-14 2005-06-14
US60/690,691 2005-06-14
US74482606P 2006-04-13 2006-04-13
US60/744,826 2006-04-13
US79200706P 2006-04-14 2006-04-14
US60/792,007 2006-04-14
US11/431,372 US7423194B2 (en) 2005-06-14 2006-05-09 Animal models for demyelination disorders
US11/431,782 2006-05-09
US11/431,601 2006-05-09
US11/431,372 2006-05-09
US11/431,782 US8053627B2 (en) 2005-06-14 2006-05-09 Methods for treating demyelination disorders
US11/431,601 US7884260B2 (en) 2005-06-14 2006-05-09 Cell-based screen for agents useful for reducing neuronal demyelination or promoting neuronal remyelination

Publications (2)

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WO2006138412A2 true WO2006138412A2 (fr) 2006-12-28
WO2006138412A3 WO2006138412A3 (fr) 2007-08-02

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EP (1) EP1909562A4 (fr)
JP (2) JP2008546704A (fr)
CA (1) CA2612374A1 (fr)
IL (1) IL188127A (fr)
WO (1) WO2006138412A2 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008241704A (ja) * 2007-02-27 2008-10-09 Prima Meat Packers Ltd 新規ストレスバイオマーカー及びその用途
WO2008154644A1 (fr) * 2007-06-12 2008-12-18 Case Western Reserve University Mort cellulaire ciblée
JP2010101631A (ja) * 2008-10-21 2010-05-06 Japan Science & Technology Agency 組織内の脂質抗原の免疫染色方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of EP1909562A4 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008241704A (ja) * 2007-02-27 2008-10-09 Prima Meat Packers Ltd 新規ストレスバイオマーカー及びその用途
WO2008154644A1 (fr) * 2007-06-12 2008-12-18 Case Western Reserve University Mort cellulaire ciblée
US8124749B2 (en) 2007-06-12 2012-02-28 Case Western Reserve University Targeted cell death
US8704037B2 (en) 2007-06-12 2014-04-22 Case Western Reserve University Targeted cell death
JP2010101631A (ja) * 2008-10-21 2010-05-06 Japan Science & Technology Agency 組織内の脂質抗原の免疫染色方法

Also Published As

Publication number Publication date
CA2612374A1 (fr) 2006-12-28
WO2006138412A3 (fr) 2007-08-02
JP2012176984A (ja) 2012-09-13
EP1909562A4 (fr) 2015-04-08
IL188127A (en) 2012-03-29
EP1909562A2 (fr) 2008-04-16
JP2008546704A (ja) 2008-12-25
IL188127A0 (en) 2008-03-20

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