Paul E. Polak

Noradrenaline deficiency in brain increases β-amyloid plaque burden in an animal model of Alzheimer's disease

Loss of Locus coeruleus (LC) noradrenergic (NA) neurons occurs in several neurodegenerative conditions including Alzheimers disease (AD). In vitro and in vivo studies have shown that NA influences several features of AD disease including inflammation, neurodegeneration, and cognitive function. In the current study we tested if LC loss influenced beta amyloid (Abeta) plaque deposition. LC neuronal degeneration was induced in transgenic mice expressing mutant V717F human amyloid precursor protein (APP) by treatment with the selective neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine DSP4 (5mg/kg every 2 weeks beginning at age 3 months). At 9 months of age, when control mice show low amyloid load, DSP4-treated mice showed an approximately 5-fold increase in the average number of Abeta plaques. This was accompanied by an increase in the levels of APP C-terminal cleavage fragments. DSP4-treatment increased both microglial and astroglial activation. In vivo, DSP4-treatment decreased expression and activity of the Abeta degrading enzyme neprilysin, while in vitro NA increased phagocytosis of Abeta1-42 by microglia. These findings suggest that noradrenergic innervation from LC are needed to maintain adequate Abeta clearance, and therefore that LC degeneration could contribute to AD pathogenesis.

Protective effects of a peroxisome proliferator-activated receptor-β/δ agonist in experimental autoimmune encephalomyelitis

Agonists of the peroxisome proliferator-activated receptor gamma (PPARg) exert anti-inflammatory and anti-proliferative effects which led to testing of these drugs in experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. In contrast, the effect of PPARdelta (PPARy) agonists in EAE is not yet known. We show that oral administration of the selective PPARy agonist GW0742 reduced clinical symptoms in C57BL/6 mice that had been immunized with encephalitogenic myelin oligodendrocyte glycoprotein (MOG) peptide. In contrast to previous results with PPARg agonists, GW0742 only modestly attenuated clinical symptoms when the drug was provided simultaneously with immunization, but a greater reduction was observed if administered during disease progression. Reduced clinical symptoms were accompanied by a reduction in the appearance of new cortical lesions, however cerebellar lesion load was not reduced. Treatment of T-cells with GW0742 either in vivo or in vitro did not reduce IFNg production; however GW0742 reduced astroglial and microglial inflammatory activation and IL-1h levels in EAE brain. RTPCR analysis showed that GW0742 increased expression of some myelin genes. These data demonstrate that PPARy agonists, like other PPAR ligands, can exert protective actions in an autoimmune model of demyelinating disease. D 2005 Elsevier B.V. All rights reserved.

Astrocyte-Derived MCP-1 Mediates Neuroprotective Effects of Noradrenaline

The neurotransmitter noradrenaline (NA) can provide neuroprotection against insults including inflammatory stimuli and excitotoxicity, which may involve paracrine effects of neighboring glial cells. Astrocytes express and secrete a variety of inflammatory and anti-inflammatory molecules; however, the effects of NA on astrocyte chemokine expression have not been well characterized. In primary astrocytes, NA increased expression of chemokine CCL2 (MCP-1) at the mRNA and protein levels. NA increased activation of an MCP-1 promoter driving luciferase expression, which was replicated by β-adrenergic receptor agonists and a cAMP analog, and blocked by a specific β2-adrenergic receptor antagonist. In primary neurons, addition of MCP-1 reduced NMDA-dependent glutamate release as well as glutamate-dependent Ca2+ entry. Similarly, conditioned media from NA-treated astrocytes reduced glutamate release, an effect that was blocked by neutralizing antibody to MCP-1, whereas MCP-1 dose-dependently reduced neuronal damage attributable to NMDA or to glutamate. MCP-1 significantly reduced lactate dehydrogenase release from neurons after oxygen–glucose deprivation (OGD) and prevented the loss of ATP levels that occurred after OGD or treatment with glutamate. Incubation of neurons with astrocytes separated by a membrane to prevent physical contact showed that NA induced astrocyte release of sufficient MCP-1 to reduce neuronal damage attributable to OGD. These findings indicate that the neuroprotective effects of NA are mediated, at least in part, by induction and release of astrocyte MCP-1.

The anti-inflammatory effects of dimethyl fumarate in astrocytes involve glutathione and haem oxygenase-1

DMF (dimethyl fumarate) exerts anti-inflammatory and pro-metabolic effects in a variety of cell types, and a formulation (BG-12) is being evaluated for monotherapy in multiple sclerosis patients. DMF modifies glutathione (GSH) levels that can induce expression of the anti-inflammatory protein HO-1 (haem oxygenase-1). In primary astrocytes and C6 glioma cells, BG-12 dose-dependently suppressed nitrite production induced by either LI [LPS (lipopolysaccharide) at 1 μg/ml plus IFNγ (interferon γ) at 20 units/ml] or a mixture of pro-inflammatory cytokines, with greater efficacy in C6 cells. BG-12 reduced NOS2 (nitric oxide synthase 2) mRNA levels and activation of a NOS2 promoter, reduced nuclear levels of NF-κB (nuclear factor κB) p65 subunit and attenuated loss of IκBα (inhibitory κBα) in both cell types, although with greater effects in astrocytes. In astrocytes, LI decreased mRNA levels for GSHr (GSH reductase) and GCL (c-glutamylcysteine synthetase), and slightly suppressed GSHs (GSH synthetase) mRNAs. Co-treatment with BG-12 prevented those decreased and increased levels above control values. In contrast, LI reduced GSHp (GSH peroxidase) and GCL in C6 cells, and BG-12 had no effect on those levels. BG-12 increased nuclear levels of Nrf2 (nuclear factor-erythroid 2 p45 subunit-related factor 2), an inducer of GSH-related enzymes, in astrocytes but not C6 cells. In astrocytes, GSH was decreased by BG-12 at 2 h and increased at 24 h. Prior depletion of GSH using buthionine-sulfoximine increased the ability of BG-12 to reduce nitrites. In astrocytes, BG-12 increased HO-1 mRNA levels and effects on nitrite levels were blocked by an HO-1 inhibitor. These results demonstrate that BG-12 suppresses inflammatory activation in astrocytes and C6 glioma cells, but with distinct mechanisms, different dependence on GSH and different effects on transcription factor activation.

The Elongation Domain of ELL Is Dispensable but Its ELL-Associated Factor 1 Interaction Domain Is Essential for MLL-ELL-Induced Leukemogenesis

ABSTRACT The MLL-ELL chimeric gene is the product of the (11;19)(q23p13.1) translocation associated with de novo and therapy-related acute myeloid leukemias (AML). ELL is an RNA polymerase II elongation factor that interacts with the recently identified EAF1 (ELL associated factor 1) protein. EAF1 contains a limited region of homology with the transcriptional activation domains of three other genes fused to MLL in leukemias, AF4, LAF4, and AF5q31. Using an in vitro transformation assay of retrovirally transduced myeloid progenitors, we conducted a structure-function analysis of MLL-ELL. Whereas the elongation domain of ELL was dispensable, the EAF1 interaction domain of ELL was critical to the immortalizing properties of MLL-ELL in vitro. To confirm these results in vivo, we transplanted mice with bone marrow transduced with MLL fused to the minimal EAF1 interaction domain of ELL. These mice all developed AML, with a longer latency than mice transplanted with the wild-type MLL-ELL fusion. Based on these results, we generated a heterologous MLL-EAF1 fusion gene and analyzed its transforming potential. Strikingly, we found that MLL-EAF1 immortalized myeloid progenitors in the same manner as that of MLL-ELL. Furthermore, transplantation of bone marrow transduced with MLL-EAF1 induced AML with a shorter latency than mice transplanted with the MLL-ELL fusion. Taken together, these results indicate that the leukemic activity of MLL-ELL requires the EAF1 interaction domain of ELL, suggesting that the recruitment by MLL of a transactivation domain similar to that in EAF1 or the AF4/LAF4/AF5q31 family may be a critical common feature of multiple 11q23 translocations. In addition, these studies support a critical role for MLL partner genes and their protein-protein interactions in 11q23 leukemogenesis.

The noradrenaline precursor L-DOPS reduces pathology in a mouse model of Alzheimer's disease

Damage to noradrenergic neurons in the locus coeruleus (LC) is a hallmark of Alzheimers disease (AD) and may contribute to disease progression. In 5xFAD transgenic mice, which accumulate amyloid burden at early ages, the LC undergoes stress as evidenced by increased astrocyte activation, neuronal hypertrophy, reduced levels of LC-enriched messenger RNAs (mRNAs), and increased inflammatory gene expression. Central nervous system (CNS) noradrenaline (NA) levels in 5-month-old male 5xFAD mice were increased using the NA precursor L-threo-3,4-dihydroxyphenylserine (L-DOPS). After 1 month, L-DOPS treatment improved learning in the Morris water maze test compared with vehicle-treated mice. L-DOPS increased CNS NA levels, and average latency times in the water maze test were inversely correlated to NA levels. L-DOPS reduced astrocyte activation and Thioflavin-S staining; increased mRNA levels of neprilysin and insulin degrading enzyme, and of several neurotrophins; and increased brain-derived neurotrophic factor protein levels. These data demonstrate the presence of LC stress in a robust mouse model of AD, and suggest that raising CNS NA levels could provide benefit in AD.

The heat-shock protein 90 inhibitor 17-allylamino-17-demethoxygeldanamycin suppresses glial inflammatory responses and ameliorates experimental autoimmune encephalomyelitis

The heat‐shock response (HSR), a highly conserved cellular response, is characterized by rapid expression of heat‐shock proteins (HSPs), and inhibition of other synthetic activities. The HSR can attenuate inflammatory responses, via suppression of transcription factor activation. A HSR can be induced pharmacologically by HSP90 inhibitors, through activation of the transcription factor Heat Shock Factor 1 (HSF1). In the present study we characterized the effects of 17‐allylamino‐17‐demethoxygeldanamycin (17‐AAG), a less toxic derivative of the naturally occurring HSP90 inhibitor geldanamycin, on glial inflammatory responses and the development of experimental autoimmune encephalomyelitis. In primary enriched glial cultures, 17‐AAG dose dependently reduced lipopolysaccharide‐dependent expression and activity of inducible nitric oxide synthase, attenuated interleukin (IL)‐1β expression and release, increased inhibitor of κB protein levels, and induced HSP70 expression. 17‐AAG administration to mice immunized with myelin oligodendrocyte glycoprotein peptide prevented disease onset when given at an early time, and reduced clinical symptoms when given during ongoing disease. T cells from treated mice showed a reduced response to immunogen re‐stimulation, and 17‐AAG reduced CD3‐ and CD28‐dependent IL‐2 production. Together, these data suggest that HSP90 inhibitors could represent a new approach for therapeutic intervention in autoimmune diseases such as multiple sclerosis.

Mature Oligodendrocytes Cultured in the Absence of Neurons Recapitulate the Ontogenic Development of Myelin Membranes

We have maintained isolated oligodendrocytes in culture for up to 5 months. Over time cultured cells acquire conspicuous membranous profiles that either emanate from cell processes and perikarya or are apposed to them. These membranous profiles have biochemical and ultrastructural features characteristic of myelin. Because the cells used in this study are post-myelination (cells are isolated from white matter of mature brains), we argue that this is a case of resynthesis of myelin membranes. Because this process takes place in the absence of neurons, we have termed it myelin palingenesis in order to distinguish it from remyelination, a term commonly used to designate reensheathment of axons. Myelin palingenesis may occur seemingly without wrapping and spiraling, but membranes wrapped around oligodendrocyte processes and soma are also observed. Irrespective of the modes of formation, those membranes that are compact have the fine structure of normal myelin. Thus, long-term cultures of mature oligodendrocytes should be a good model system in helping to decipher the molecular and cellular mechanisms involved in myelination and in remyelination.

A novel fluorescent probe that is brain permeable and selectively binds to myelin.

Myelin is a multilayered glial cell membrane that forms segmented sheaths around large-caliber axons of both the central nervous system (CNS) and peripheral nervous system (PNS). Myelin covering insures rapid and efficient transmission of nerve impulses. Direct visual assessment of local changes of myelin content in vivo could greatly facilitate diagnosis and therapeutic treatments of myelin-related diseases. Current histologic probes for the visualization of myelin are based on antibodies or charged histochemical reagents that do not enter the brain. We have developed a series of chemical compounds including (E,E)-1,4-bis(4′-aminostyryl)-2-dimethoxy-benzene termed BDB and the subject of this report, which readily penetrates the blood-brain barrier and selectively binds to the myelin sheath in brain. BDB selectively stains intact myelinated regions in wild-type mouse brain, which allows for delineation of cuprizone-induced demyelinating lesions in mouse brain. BDB can be injected IV into the brain and selectively detect demyelinating lesions in cuprizone-treated mice in situ. These studies justified further investigation of BDB as a potential myelin-imaging probe to monitor myelin pathology in vivo.

Increasing CNS Noradrenaline Reduces EAE Severity

The endogenous neurotransmitter noradrenaline (NA) is known to exert potent anti-inflammatory effects in glial cells, as well as provide neuroprotection against excitatory and inflammatory stimuli. These properties raise the possibility that increasing levels of NA in the central nervous system (CNS) could provide benefit in neurological diseases and conditions containing an inflammatory component. In the current study, we tested this possibility by examining the consequences of selectively modulating CNS NA levels on the development of clinical signs in experimental autoimmune encephalomyelitis (EAE). In mice immunized with myelin oligodendrocyte glycoprotein peptide to develop a chronic disease, pretreatment to selectively deplete CNS NA levels exacerbated clinical scores. Elevation of NA levels using the selective NA reuptake inhibitor atomoxetine did not affect clinical scores, while treatment of immunized mice with the synthetic NA precursor l-threo-3,4-dihydroxyphenylserine (l-DOPS) prevented further worsening. In contrast, treatment of mice with a combination of atomoxetine and l-DOPS led to significant improvement in clinical scores as compared to the control group. The combined treatment reduced astrocyte activation in the molecular layer of the cerebellum as assessed by staining for glial fibrillary protein but did not affect Th1 or Th17 type cytokine production from splenic T cells. These data suggest that selective elevation of CNS NA levels could provide benefit in EAE and multiple sclerosis without influencing peripheral immune responses.