Lucia Dumitrescu-Ozimek

Peroxisome proliferator-activated receptor-γ agonists prevent experimental autoimmune encephalomyelitis

The development of clinical symptoms in multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE) involves T‐cell activation and migration into the central nervous system, production of glial‐derived inflammatory molecules, and demyelination and axonal damage. Ligands of the peroxisome proliferator‐activated receptor (PPAR) exert anti‐inflammatory effects on glial cells, reduce proliferation and activation of T cells, and induce myelin gene expression. We demonstrate in two models of EAE that orally administered PPARγ ligand pioglitazone reduced the incidence and severity of monophasic, chronic disease in C57BL/6 mice immunized with myelin oligodendrocyte glycoprotein peptide and of relapsing disease in B10.Pl mice immunized with myelin basic protein. Pioglitazone also reduced clinical signs when it was provided after disease onset. Clinical symptoms were reduced by two other PPARγ agonists, suggesting a role for PPARγ activation in protective effects. The suppression of clinical signs was paralleled by decreased lymphocyte infiltration, lessened demyelination, reduced chemokine and cytokine expression, and increased inhibitor of kappa B (IkB) expression in the brain. Pioglitazone also reduced the antigen‐dependent interferon‐γ production from EAE‐derived T cells. These results suggest that orally administered PPARγ agonists could provide therapeutic benefit in demyelinating disease.

Locus ceruleus controls Alzheimer's disease pathology by modulating microglial functions through norepinephrine

Locus ceruleus (LC)-supplied norepinephrine (NE) suppresses neuroinflammation in the brain. To elucidate the effect of LC degeneration and subsequent NE deficiency on Alzheimers disease pathology, we evaluated NE effects on microglial key functions. NE stimulation of mouse microglia suppressed Aβ-induced cytokine and chemokine production and increased microglial migration and phagocytosis of Aβ. Induced degeneration of the locus ceruleus increased expression of inflammatory mediators in APP-transgenic mice and resulted in elevated Aβ deposition. In vivo laser microscopy confirmed a reduced recruitment of microglia to Aβ plaque sites and impaired microglial Aβ phagocytosis in NE-depleted APP-transgenic mice. Supplying the mice the norepinephrine precursor L-threo-DOPS restored microglial functions in NE-depleted mice. This indicates that decrease of NE in locus ceruleus projection areas facilitates the inflammatory reaction of microglial cells in AD and impairs microglial migration and phagocytosis, thereby contributing to reduced Aβ clearance. Consequently, therapies targeting microglial phagocytosis should be tested under NE depletion.

Focal glial activation coincides with increased BACE1 activation and precedes amyloid plaque deposition in APP[V717I] transgenic mice

BackgroundInflammation is suspected to contribute to the progression and severity of neurodegeneration in Alzheimers disease (AD). Transgenic mice overexpressing the london mutant of amyloid precursor protein, APP [V717I], robustly recapitulate the amyloid pathology of AD.MethodsEarly and late, temporal and spatial characteristics of inflammation were studied in APP [V717I] mice at 3 and 16 month of age. Glial activation and expression of inflammatory markers were determined by immunohistochemistry and RT-PCR. Amyloid deposition was assessed by immunohistochemistry, thioflavine S staining and western blot experiments. BACE1 activity was detected in brain lysates and in situ using the BACE1 activity kit from R&D Systems, Wiesbaden, Germany.ResultsFoci of activated micro- and astroglia were already detected at age 3 months, before any amyloid deposition. Inflammation parameters comprised increased mRNA levels coding for interleukin-1β, interleukin-6, major histocompatibility complex II and macrophage-colony-stimulating-factor-receptor. Foci of CD11b-positive microglia expressed these cytokines and were neighbored by activated astrocytes. Remarkably, β-secretase (BACE1) mRNA, neuronal BACE1 protein at sites of focal inflammation and total BACE1 enzyme activity were increased in 3 month old APP transgenic mice, relative to age-matched non-transgenic mice. In aged APP transgenic mice, the mRNA of all inflammatory markers analysed was increased, accompanied by astroglial iNOS expression and NO-dependent peroxynitrite release, and with glial activation near almost all diffuse and senile Aβ deposits.ConclusionThe early and focal glial activation, in conjunction with upregulated BACE1 mRNA, protein and activity in the presence of its substrate APP, is proposed to represent the earliest sites of amyloid deposition, likely evolving into amyloid plaques.

Systemic inflammation induces apoptosis with variable vulnerability of different brain regions

During severe sepsis several immunological defence mechanisms initiate a cascade of inflammatory events leading to multi-organ failure including septic encephalopathy and ultimately death. To assess the reaction and participation of parenchymal brain cells during endotoxaemia, the present study evaluates micro- and astroglial activation, expression of the inducible nitric oxide synthase (iNOS) pro- and antiapoptotic protein levels Bax and Bcl-2, and apoptosis. Male Wistar rats received 10 mg/kg lipopolysaccharide (LPS) or vehicle intraperitoneally and were sacrificed for brain collection at 4, 8 or 24 h after induction of experimental sepsis. One group of animals received 10 mg/kg of the NOS inhibitor N-monomethyl-L-arginine (L-NMMA) intraperitoneally 1 day before and during the experiment. Immunohistochemical evaluation revealed a sepsis-induced, time-dependent increase in the immunoreactivity of iNOS, glial fibrillary acidic protein (GFAP) and activated microglia (ED-1), paralleled by a time-dependent increase of apoptotic brain cells marked by terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL), an increase of Bax-positive cells and a decrease of Bcl-2-positive cells. Evaluation of different brain regions revealed that the hippocampus is the most vulnerable region during experimental sepsis. iNOS-inhibition with L-NMMA significantly reduced the number of apoptotic cells in hippocampus, midbrain and cerebellum. In addition, it reduced the increase of the proapoptotic protein Bax in all examined brain regions and reduced the decrease of Bcl-2-positive cells in the hippocampus. We therefore conclude, that peripheral inflammation leads to a profound glial activation, the generation of nitric oxide and changes of Bax and Bcl-2 protein regulation critical for apoptosis.

Locus Ceruleus Degeneration Promotes Alzheimer Pathogenesis in Amyloid Precursor Protein 23 Transgenic Mice

Locus ceruleus (LC) degeneration and loss of cortical noradrenergic innervation occur early in Alzheimer’s disease (AD). Although this has been known for several decades, the contribution of LC degeneration to AD pathogenesis remains unclear. We induced LC degeneration with N-(2-chloroethyl)-N-ethyl-bromo-benzylamine (dsp4) in amyloid precursor protein 23 (APP23) transgenic mice with a low amyloid load. Then 6 months later the LC projection areas showed a robust elevation of glial inflammation along with augmented amyloid plaque deposits. Moreover, neurodegeneration and neuronal loss significantly increased. Importantly, the paraventricular thalamus, a nonprojection area, remained unaffected. Radial arm maze and social partner recognition tests revealed increased memory deficits while high-resolution magnetic resonance imaging-guided micro-positron emission tomography demonstrated reduced cerebral glucose metabolism, disturbed neuronal integrity, and attenuated acetylcholinesterase activity. Nontransgenic mice with LC degeneration were devoid of these alterations. Our data demonstrate that the degeneration of LC affects morphology, metabolism, and function of amyloid plaque-containing higher brain regions in APP23 mice. We postulate that LC degeneration substantially contributes to AD development.

The Oral Antidiabetic Pioglitazone Protects from Neurodegeneration and Amyotrophic Lateral Sclerosis-Like Symptoms in Superoxide Dismutase-G93A Transgenic Mice

Amyotrophic lateral sclerosis (ALS) represents a fatal neurodegenerative disorder characterized by progressive death of the upper and lower motor neurons. Because accompanying inflammation may interact with and promote neurodegeneration, anti-inflammatory treatment strategies are being evaluated. Because peroxisome proliferator-activated receptor γ (PPARγ) agonists act as potent anti-inflammatory drugs, we tested whether superoxide dismutase (SOD1)-G93A transgenic mice, a mouse model of ALS, benefit from oral treatment with the PPARγ agonist pioglitazone (Pio). Pio-treated transgenic mice revealed improved muscle strength and body weight, exhibited a delayed disease onset, and survived significantly longer than nontreated SOD1-G93A mice. Quantification of motor neurons of the spinal cord at day 90 revealed complete neuroprotection by Pio, whereas nontreated SOD1-G93A mice had lost 30% of motor neurons. This was paralleled by preservation of the median fiber diameter of the quadriceps muscle, indicating not only morphological but also functional protection of motor neurons by Pio. Activated microglia were significantly reduced at sites of neurodegeneration in Pio-treated SOD1-G93A mice, as were the protein levels of cyclooxygenase 2 and inducible nitric oxide synthase. Interestingly, mRNA levels of the suppressor of cytokine signaling 1 and 3 genes were increased by Pio, whereas both the mRNA and protein levels of endogenous mouse SOD1 and of transgenic human SOD1 remained unaffected.

Norepinephrine modulates salutary microglial functions in transgenic models of Alzheimer's disease

Background: Loss of locus ceruleus neurons, degeneration of noradrenergic projections and decrease of norepinephrine (NE) in projection areas, such as the neocortex and hippocampus, represent early features of Alzheimer’s disease. To elucidate the effect of NE-depletion on AD pathology, we evaluated NE effects on microglial functions in vitro and in vivo. Methods: To elucidate the effect of NE-depletion on AD pathology, we evaluated NE effects on microglial functions in vitro and in vivo using primary microglia and APPV717I transgenic mice. Results: NE stimulation of mouse microglia suppressed amyloid beta-induced cytokine and chemokine production in a selective and concentration-dependent manner. NE also increased microglial migration and phagocytosis of fibrillar amyloid-beta. Similarly, locus ceruleus degeneration induced by the noradrenergic neurotoxin DSP4 increased mRNA and protein levels of inflammatory mediators and enzyme systems in the affected CNS areas of APPV717I-transgenic mice. NE-depleted APP V717-transgenic mice showed more beta-amyloid deposits within the hippocampus and frontal cortex, compared to APPV717-transgenic mice with intact NE innervation. Confocal microscopy revealed that the number of Ab-containing microglia was higher in control compared to NE-depleted APP-transgenic mice, suggesting that normal NE levels support beta-amyloid removal. Conclusions: These data indicate that early LC degeneration and concomitant decrease of NE concentration in LC projection areas, such as the neocortex and hippocampus, facilitate the inflammatory reaction of microglial cells in the AD brain. At the same time, NE deficiency impairs microglial key functions, such as migration and phagocytosis, thereby contributing to reduced beta-amyloid clearance. Consequently, therapies targeting microglial phagocytosis should be tested under NE-depletion to mimic AD conditions. In addition, the b-adrenergic system may represent a target for pharmacological intervention.