Magdalena Sastre

Contribution of inflammatory processes to Alzheimer's disease: molecular mechanisms

There is compelling evidence that Alzheimers disease (AD) amyloid‐β (Aβ) deposition is associated with a local inflammatory response, which is initiated by the activation of microglia and the recruitment of astrocytes. These cells secrete a number of cytokines and neurotoxic products that may contribute to neuronal degeneration and cell death. It has been documented that long‐term intake of non‐steroidal anti‐inflammatory drugs (NSAIDs) decrease the risk for developing AD and delay the onset of the disease. The mechanism behind these NSAIDs is still controversial and several hypotheses have been raised, including changes in the amyloid precursor protein (APP) metabolism, in Aβ aggregation and a decrease in inflammatory mediators. Recently, it was proposed that some NSAIDs might activate the peroxisome proliferator‐activated receptor‐γ (PPAR‐γ). PPAR‐γ belongs to a family of nuclear receptors that are able to regulate the transcription of pro‐inflammatory molecules, such as iNOS. The activation of PPAR‐γ has been recently reported to reduce Aβ levels in cell culture and AD animal models. The implication of PPAR‐γ in the control of Aβ‐induced inflammation suggests a new target for AD therapy and emphasize the contribution of neuroinflammatory mechanisms to the pathogenesis of AD.

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.

Development of α-synuclein immunoreactive astrocytes in the forebrain parallels stages of intraneuronal pathology in sporadic Parkinson’s disease

Astrocytic α-synuclein-immunoreactive inclusions have recently been noted to develop in sporadic Parkinson’s disease (PD). Here, the presence of immunoreactive astrocytes is reported in 14 autopsy cases with clinically diagnosed PD and a neuropathological stage of 4 or higher. The labeled astrocytes occur preferentially in prosencephalic regions (amygdala, thalamus, septum, striatum, claustrum, and cerebral cortex). They appear first in layers V–VI of the temporal mesocortex, then in the striatum and in thalamic nuclei that project to the cortex. The topographical distribution pattern of these astrocytes closely parallels that of the cortical intraneuronal Lewy neurites and Lewy bodies, which, from their foothold in the mesocortex, gradually encroach upon neocortical association areas and even the primary fields. Thus, labeling of astrocytes appears to accompany the formation of neuronal inclusion bodies. Relatively small immunoreactive cortical pyramidal neurons in layers V–VI probably project to nearby destinations, such as the striatum and thalamus. Inasmuch as the projection neurons of both the striatum and the dorsal thalamus do not develop Lewy bodies, it is suggested that the most likely cause of the astrocytic reaction may be a slightly altered α-synuclein molecule that escapes from terminal axons of affected cortico-striatal or cortico-thalamic neurons and is taken up by astrocytes. Other aggregated proteins known to co-occur with PD-associated intraneuronal lesions, e.g., Aβ protein or neurofibrillary changes of the Alzheimer type, do not appear to influence the development of the α-synuclein immunoreactive astrocytes.

Proinflammatory Stimulation and Pioglitazone Treatment Regulate Peroxisome Proliferator-Activated Receptor γ Levels in Peripheral Blood Mononuclear Cells from Healthy Controls and Multiple Sclerosis Patients

The peroxisome proliferator-activated receptor γ (PPAR-γ) belongs to a receptor superfamily of ligand-activated transcription factors involved in the regulation of metabolism and inflammation. Oral administration of PPAR-γ agonists ameliorates the clinical course and histopathological features in experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis (MS), and PPAR-γ agonist treatment of PBMCs from MS patients suppresses PHA-induced cell proliferation and cytokine secretion. These effects are pronounced when cells are preincubated with the PPAR-γ agonists and reexposed at the time of stimulation, indicating a sensitizing effect. To characterize the mechanisms underlying this sensitizing effect, we analyzed PPAR-γ expression in PMBCs of MS patients and healthy controls. Surprisingly, MS patients exhibited decreased PPAR-γ levels compared with controls. PHA stimulation of PBMCs from healthy controls resulted in a significant loss of PPAR-γ, which was prevented by in vitro preincubation of the cells or in vivo by long-term oral medication with the PPAR-γ agonist pioglitazone. Differences in PPAR-γ expression were accompanied by changes in PPAR-γ DNA-binding activity, as preincubation with pioglitazone increased DNA binding of PPAR-γ. Additionally, preincubation decreased NF-κB DNA-binding activity to control levels, whereas the inhibitory protein IκBα was increased. In MS patients, pioglitazone-induced increase in PPAR-γ DNA-binding activity and decrease in NF-κB DNA-binding activity was only observed in the absence of an acute MS relapse. These results suggest that the sensitizing effect observed in the preincubation experiments is mediated by prevention of inflammation-induced suppression of PPAR-γ expression with consecutive increase in PPAR-γ DNA-binding activity.

Anti-inflammatory and antiproliferative actions of PPAR-γ agonists on T lymphocytes derived from MS patients

Peroxisomal proliferator‐activated receptors (PPARs) belong to a nuclear receptor superfamily of ligand‐activated transcription factors. The PPAR‐γ isoform is expressed in human T lymphocytes, and oral administration of PPAR‐γ agonists ameliorates the clinical course and histopathological features in experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis, suggesting a potential role for PPAR‐γ agonists in the treatment of autoimmune diseases. To assess a potential therapeutic role of PPAR‐γ agonists in multiple sclerosis, we compared the immunomodulatory effects of the thiazolidinedione (TZD) drugs pioglitazone (PIO) and ciglitazone and the non‐TZD PPAR‐γ agonist GW347845 on human T leukemia cells (Jurkat cells) and phytohemagglutinin (PHA)‐stimulated peripheral blood mononuclear cells (PBMCs) derived from 21 multiple sclerosis patients and 12 healthy donors. PIO, ciglitazone, and GW347845 suppressed PHA‐induced T cell proliferation by 40–50% and secretion of interferon‐γ and tumor necrosis factor α, by 30–50%. Inhibition of proliferation was increased to ∼80% and that of proinflammatory cytokine secretion, to 80–90% when PBMCs were first preincubated with PPAR‐γ agonists and re‐exposed at the time of PHA stimulation, indicating a sensitizing effect of PPAR‐γ agonists. Inhibition of proliferation was also observed in the tetanus toxoid‐specific T cell line KHS.TT2, albeit to a lesser extent. The antiproliferative effects of PIO and GW347845 were accompanied by a decrease of cell viability. Electron microscopy and Western blot analysis revealed DNA condensation and down‐regulation of bcl‐2, suggesting the induction of apoptosis in activated T lymphocytes. In summary, the data support the potential use of PPAR‐γ agonists as immunomodulatory, therapeutic agents for autoimmune diseases.

Inhibition of in Vivo Glioma Growth and Invasion by Peroxisome Proliferator-Activated Receptor γ Agonist Treatment

The peroxisome proliferator-activated receptor γ (PPARγ), a member of the nuclear hormone receptor family, represents a possible new target in glioma therapy. Because PPARγ plays a crucial role in regulation of insulin sensitivity, synthetic agonists are already in clinical use for type II diabetes treatment. Beyond these metabolic effects, PPARγ agonists exhibit antineoplastic effects. In this study, we investigated the antineoplastic effects of the PPARγ agonist pioglitazone in glioma cells. Pioglitazone reduced cellular viability of rat, human, and PPARγ-overexpressing glioma cells in vitro in a time- and concentration-dependent manner. No antineoplastic effects were induced by pioglitazone in glioma cells overexpressing a PPARγ mutant. Furthermore, proliferation was reduced by pioglitazone, as measured by Ki-67 immunoreactivity, in vitro. Continuous intracerebral infusion of pioglitazone into gliomas induced by intrastriatal injection of C6 cells reduced tumor volumes by 83%. Oral administration of pioglitazone reduced tumor volumes by 76.9%. Subsequent brain tissue analysis revealed induction of apoptotic cell death. Ki-67 expression and BrdU incorporation revealed a reduction of proliferation in vivo. Reduced invasion of C6 cells and lower matrix metalloproteinase 9 levels in vivo indicate pioglitazone-mediated reduction of invasion. Together, these data indicate that pioglitazone may be of potential use in treatment of malignant gliomas.

Noradrenaline induces expression of peroxisome proliferator activated receptor gamma (PPARγ) in murine primary astrocytes and neurons

Cerebral inflammatory events play an important part in the pathogenesis of Alzheimers disease (AD). Agonists of the peroxisome proliferator‐activated receptor gamma (PPARγ), a nuclear hormone receptor that mediates anti‐inflammatory actions of non‐steroidal anti‐inflammatory drugs (NSAIDs) and thiazolidinediones, have been therefore proposed as a potential treatment of AD. Experimental evidence suggests that cortical noradrenaline (NA) depletion due to degeneration of the locus ceruleus (LC) – a pathological hallmark of AD – plays a permissive role in the development of inflammation in AD. To study a possible relationship between NA depletion and PPARγ‐mediated suppression of inflammation we investigated the influence of NA on PPARγ expression in murine primary cortical astrocytes and neurons. Incubation of astrocytes and neurons with 100 µm NA resulted in an increase of PPARγ mRNA as well as PPARγ protein levels in both cell types. These effects were blocked by the β‐adrenergic antagonist propranolol but not by the α‐adrenergic antagonist phentolamine, suggesting that they might be mediated by β‐adrenergic receptors. Our results indicate for the first time that PPARγ expression can be modulated by the cAMP signalling pathway, and suggest that the anti‐inflammatory effects of NA on brain cells may be partly mediated by increasing PPARγ levels. Conversely, decreased NA due to LC cell death in AD may reduce endogenous PPARγ expression and therefore potentiate neuroinflammatory processes.

P4-011 : Expression of GGA1 is altered in Alzheimer’s disease and regulates the generation of amyloid β-peptide

elevated at presynaptic areas using an immunoelectron microscopic technique with anti-oligomeric A antibody. Next, the long-term potentiation (LTP) recorded in the dentate gyrus and in the CA1 area of the hippocampus was significantly suppressed in the crossbred mice, compared with APP transgenic mice. The LTP in the dentate gyrus was more strongly suppressed than that in the CA1 area of the hippocampus, reflecting an increase in quantity of A oligomers. Finally, the crossbred mice displayed obvious cognitive abnormalities consistently in different learning and memory paradigms. These results indicate that a reduction in neprilysin activity causes synaptic and cognitive impairment through a local increase of oligomeric A in the synaptic sites of the brain before the onset of amyloid plaque formation. Thus, reduced neprilysin activity appears to be a causative event that is at least partly responsible for the memoryassociated symptoms of AD.