p38α-MAPK-deficient myeloid cells ameliorate symptoms and pathology of APP-transgenic Alzheimer's disease mice.

Alzheimer's disease (AD), the most common cause of dementia in the elderly, is pathologically characterized by extracellular deposition of amyloid-ß peptides (Aß) and microglia-dominated inflammatory activation in the brain. p38α-MAPK is activated in both neurons and microglia. How p38α-MAPK in microglia contributes to AD pathogenesis remains unclear. In this study, we conditionally knocked out p38α-MAPK in all myeloid cells or specifically in microglia of APP-transgenic mice, and examined animals for AD-associated pathologies (i.e., cognitive deficits, Aß pathology, and neuroinflammation) and individual microglia for their inflammatory activation and Aß internalization at different disease stages (e.g., at 4 and 9 months of age). Our experiments showed that p38α-MAPK-deficient myeloid cells were more effective than p38α-MAPK-deficient microglia in reducing cerebral Aß and neuronal impairment in APP-transgenic mice. Deficiency of p38α-MAPK in myeloid cells inhibited inflammatory activation of individual microglia at 4 months but enhanced it at 9 months. Inflammatory activation promoted microglial internalization of Aß. Interestingly, p38α-MAPK-deficient myeloid cells reduced IL-17a-expressing CD4-positive lymphocytes in 9 but not 4-month-old APP-transgenic mice. By cross-breeding APP-transgenic mice with Il-17a-knockout mice, we observed that IL-17a deficiency potentially activated microglia and reduced Aß deposition in the brain as shown in 9-month-old myeloid p38α-MAPK-deficient AD mice. Thus, p38α-MAPK deficiency in all myeloid cells, but not only in microglia, prevents AD progression. IL-17a-expressing lymphocytes may partially mediate the pathogenic role of p38α-MAPK in peripheral myeloid cells. Our study supports p38α-MAPK as a therapeutic target for AD patients.

Magnetic resonance imaging of cerebrospinal fluid outflow after low-rate lateral ventricle infusion in mice.

The anatomical routes for the clearance of cerebrospinal fluid (CSF) remain incompletely understood. However, recent evidence has given strong support for routes leading to lymphatic vessels. A current debate centers upon the routes through which CSF can access lymphatics, with evidence emerging for either direct routes to meningeal lymphatics or along cranial nerves to reach lymphatics outside the skull. Here, a method was established to infuse contrast agent into the ventricles using indwelling cannulae during imaging of mice at 2 and 12 months of age by magnetic resonance imaging. As expected, a substantial decline in overall CSF turnover was found with aging. Quantifications demonstrated that the bulk of the contrast agent flowed from the ventricles to the subarachnoid space in the basal cisterns. Comparatively little contrast agent signal was found at the dorsal aspect of the skull. The imaging dynamics from the 2 cohorts revealed that the contrast agent was cleared from the cranium through the cribriform plate to the nasopharyngeal lymphatics. On decalcified sections, we confirmed that fluorescently labeled ovalbumin drained through the cribriform plate and could be found within lymphatics surrounding the nasopharynx. In conclusion, routes leading to nasopharyngeal lymphatics appear to be a major efflux pathway for cranial CSF.

Decreased pH in the aging brain and Alzheimer's disease.

Using publicly available data sets, we compared pH in the human brain and the cerebrospinal fluid (CSF) of postmortem control and Alzheimer's disease cases. We further investigated the effects of long-term acidosis in vivo in the APP-PS1 mouse model of Alzheimer's disease. We finally examined in vitro whether low pH exposure could modulate the release of proinflammatory cytokines and the uptake of amyloid beta by microglia. In the human brain, pH decreased with aging. Similarly, we observed a reduction of pH in the brain of C57BL/6 mice with age. In addition, independent database analyses revealed that postmortem brain and CSF pH is further reduced in Alzheimer's disease cases compared with controls. Moreover, in vivo experiments showed that low pH CSF infusion increased amyloid beta plaque load in APP-PS1 mice. We further observed that mild acidosis reduced the amyloid beta 42-induced release of tumor necrosis factor-alpha by microglia and their capacity to uptake this peptide. Brain acidosis is associated with aging and might affect pathophysiological processes such as amyloid beta aggregation or inflammation in Alzheimer's disease.

Normal brain aging and Alzheimer's disease are associated with lower cerebral pH: an in vivo histidine 1H-MR spectroscopy study.

It is unclear whether alterations in cerebral pH underlie Alzheimer's disease (AD) and other dementias. We performed proton spectroscopy after oral administration of histidine in healthy young and elderly persons and in patients with mild cognitive impairment and dementia (total N = 147). We measured cerebral tissue pH and ratios of common brain metabolites in relation to phosphocreatine and creatine (Cr) in spectra acquired from the hippocampus, the white matter (WM) of the centrum semiovale, and the cerebellum. Hippocampal pH was inversely associated with age in healthy participants but did not differ between patients and controls. WM pH was low in AD and, to a lesser extent, mild cognitive impairment but not in frontotemporal dementia spectrum disorders and pure vascular dementia. Furthermore, WM pH provided incremental diagnostic value in addition to N-acetylaspartate to Cr ratio. Our study suggests that in vivo assessment of pH may be a useful marker for the differentiation between AD and other types of dementia.

Lymphatic outflow of cerebrospinal fluid is reduced in glioma.

Glioblastoma is a malignant brain tumor with mean overall survival of less than 15 months. Blood vessel leakage and peritumoral edema lead to increased intracranial pressure and augment neurological deficits which profoundly decrease the quality of life of glioblastoma patients. It is unknown how the dynamics of cerebrospinal fluid (CSF) turnover are affected during this process. By monitoring the transport of CSF tracers to the systemic blood circulation after infusion into the cisterna magna, we demonstrate that the outflow of CSF is dramatically reduced in glioma-bearing mice. Using a combination of magnetic resonance imaging (MRI) and near-infrared (NIR) imaging, we found that the circulation of CSF tracers was hindered after cisterna magna injection with reduced signals along the exiting cranial nerves and downstream lymph nodes, which represent the major CSF outflow route in mice. Due to blockage of the normal routes of CSF bulk flow within and from the cranial cavity, CSF tracers were redirected into the spinal space. In some mice, impaired CSF clearance from the cranium was compensated by a lymphatic outflow from the sacral spine.

Clearance of cerebrospinal fluid from the sacral spine through lymphatic vessels.

The pathways of circulation and clearance of cerebrospinal fluid (CSF) in the spine have yet to be elucidated. We have recently shown with dynamic in vivo imaging that routes of outflow of CSF in mice occur along cranial nerves to extracranial lymphatic vessels. Here, we use near-infrared and magnetic resonance imaging to demonstrate the flow of CSF tracers within the spinal column and reveal the major spinal pathways for outflow to lymphatic vessels in mice. We found that after intraventricular injection, a spread of CSF tracers occurs within both the central canal and the spinal subarachnoid space toward the caudal end of the spine. Outflow of CSF tracers from the spinal subarachnoid space occurred predominantly from intravertebral regions of the sacral spine to lymphatic vessels, leading to sacral and iliac LNs. Clearance of CSF from the spine to lymphatic vessels may have significance for many conditions, including multiple sclerosis and spinal cord injury.

Rapid lymphatic efflux limits cerebrospinal fluid flow to the brain.

The relationships between cerebrospinal fluid (CSF) and brain interstitial fluid are still being elucidated. It has been proposed that CSF within the subarachnoid space will enter paravascular spaces along arteries to flush through the parenchyma of the brain. However, CSF also directly exits the subarachnoid space through the cribriform plate and other perineural routes to reach the lymphatic system. In this study, we aimed to elucidate the functional relationship between CSF efflux through lymphatics and the potential influx into the brain by assessment of the distribution of CSF-infused tracers in awake and anesthetized mice. Using near-infrared fluorescence imaging, we showed that tracers quickly exited the subarachnoid space by transport through the lymphatic system to the systemic circulation in awake mice, significantly limiting their spread to the paravascular spaces of the brain. Magnetic resonance imaging and fluorescence microscopy through the skull under anesthetized conditions indicated that tracers remained confined to paravascular spaces on the surface of the brain. Immediately after death, a substantial influx of tracers occurred along paravascular spaces extending into the brain parenchyma. We conclude that under normal conditions a rapid CSF turnover through lymphatics precludes significant bulk flow into the brain.

Analysis of the vasculature by immunohistochemistry in paraffin-embedded brains.

The brain vasculature can be investigated in different ways ranging from in vivo to biochemical analysis. Immunohistochemistry is a simple and powerful technique that can also be applied to archival tissues. However, staining of brain vessels on paraffin sections has been challenging. In this study, we developed an optimized method that can be used in paraffin-embedded mouse and human brain tissues derived from healthy controls and neurological disorders such as Alzheimer's disease. We subsequently showed that this method is fully compatible with the detection of glial cells and key markers of Alzheimer's disease including amyloid beta and phosphorylated Tau protein. Furthermore, we observed that the length of microvasculature in hippocampus of TgCRND8 Alzheimer's disease mouse model is reduced, which is correlated with the decreased blood flow in hippocampus as determined by arterial spin labeling perfusion magnetic resonance imaging. Finally, we determined that the microvasculature length in two other Alzheimer's disease mouse models, APP and PS1 double-transgenic mice and P301S Tau-transgenic mice, is also shortened in the dentate gyrus. Thus, we have established a new, simple and robust method to characterize the brain vasculature in the mouse and human brain.

Blockade of Experimental Multiple Sclerosis by Inhibition of the Acid Sphingomyelinase/Ceramide System.

BACKGROUND: Multiple sclerosis (MS) is a severe and common autoimmune disorder of the central nervous system. Despite the availability of several novel treatment options, the disease is still poorly controlled, since the pathophysiological mechanisms are not fully understood. METHODS: We tested the role of the acid sphingomyelinase/ceramide system in a model of MS, i.e. experimental autoimmune encephalomyelitis (EAE). Mice were immunized with myelin-oligodendrocyte glycoprotein and the development of the disease was analyzed by histology, immunological tests and clinical assessment in wildtype and acid sphingomyelinase (Asm)-deficient mice. RESULTS: Genetic deficiency of acid sphingomyelinase (Asm) protected against clinical symptoms in EAE and markedly attenuated the characteristic detrimental neuroinflammatory response. T lymphocyte adhesion, integrity of tight junctions, blood-brain barrier disruption and subsequent intracerebral infiltration of inflammatory cells were blocked in Asm-deficient mice after immunization. This resulted in an almost complete block of the development of disease symptoms in these mice, while wildtype mice showed severe neurological symptoms typical for EAE. CONCLUSION: Activation of the Asm/ceramide system is a central step for the development of EAE. Our findings may serve to identify novel therapeutic strategies for MS patients.

Stimulation of TLR4 Attenuates Alzheimer's Disease-Related Symptoms and Pathology in Tau-Transgenic Mice.

Alzheimer's disease (AD) is characterized by intracellular neurofibrillary tangles. The primary component, hyperphosphorylated Tau (p-Tau), contributes to neuronal death. Recent studies have shown that autophagy efficiently degrades p-Tau, but the mechanisms modulating autophagy and subsequent p-Tau clearance in AD remain unclear. In our study, we first analyzed the relationship between the inflammatory activation and autophagy in brains derived from aged mice and LPS-injected inflammatory mouse models. We found that inflammatory activation was essential for activation of autophagy in the brain, which was neuronal ATG5-dependent. Next, we found that autophagy in cultured neurons was enhanced by LPS treatment of cocultured macrophages. In further experiments designed to provoke chronic mild stimulation of TLR4 without inducing obvious neuroinflammation, we gave repeated LPS injections (i.p., 0.15 mg/kg, weekly for 3 mo) to transgenic mice overexpressing human Tau mutant (P301S) in neurons. We observed significant enhancement of neuronal autophagy, which was associated with a reduction of cerebral p-Tau proteins and improved cognitive function. In summary, these results show that neuroinflammation promotes neuronal autophagy and that chronic mild TLR4 stimulation attenuates AD-related tauopathy, likely by activating neuronal autophagy. Our study displays the beneficial face of neuroinflammation and suggests a possible role in the treatment of AD patients.

Deficiency of IκB Kinase ß in Myeloid Cells Reduces Severity of Experimental Autoimmune Encephalomyelitis.

In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), peripherally developed myelin-reactive T lymphocytes stimulate myeloid cells (ie, microglia and infiltrated macrophages) to trigger an inflammatory reaction in the central nervous system, resulting in demyelination and neurodegeneration. IκB kinase ß (IKKß) is a kinase that modulates transcription of inflammatory genes. To investigate the pathogenic role of IKKß in MS, we developed strains in which IKKß was conditionally ablated in myeloid cells and established active or passive EAE in these animals. Deficiency of IKKß in myeloid cells ameliorated EAE symptoms and suppressed neuroinflammation, as shown by decreased infiltration of T lymphocytes and macrophages and reduced inflammatory gene transcription in the spinal cord at the peak or end stage of EAE. Myeloid deficiency of IKKß also reduced the transcription of Rorc or Il17 genes in T lymphocytes isolated from lymph nodes, spleen, and spinal cord of EAE mice. Moreover, cultured splenocytes isolated from myeloid IKKß-deficient EAE mice released less IL-17, interferon-γ, and granulocyte-macrophage colony-stimulating factor after treatment with myelin peptide than splenocytes from IKKß wild-type EAE mice. Thus, deficiency of myeloid IKKß attenuates the severity of EAE by inhibiting both the neuroinflammatory activity and the activation of encephalitogenic T lymphocytes. These results suggest IKKß may be a potential target for MS patients, especially when neuroinflammation is the primary problem.

Abnormal galactosylation of immunoglobulin G in cerebrospinal fluid of multiple sclerosis patients.

BACKGROUND: Glycosylation alterations have been associated with the development of several human diseases and their animal models, including multiple sclerosis. OBJECTIVES: We aimed to determine whether immunoglobulin G galactosylation might be changed in multiple sclerosis. METHODS: Immunoglobulin G was isolated from serum and cerebrospinal fluid of patients with multiple sclerosis or viral meningitis and control patients without history of inflammatory or autoimmune disease. A lectin-based assay was used to investigate potential galactosylation modifications of immunoglobulin G. RESULTS AND CONCLUSION: Galactosylation of immunoglobulin G isolated from cerebrospinal fluid of control patients was found to be age- and gender-dependent. In addition, immunoglobulin G galactosylation was significantly altered in cerebrospinal fluid but not in serum of multiple sclerosis patients. Furthermore, this modification was correlated with an active progression of multiple sclerosis. Finally, the loss of galactosyl moieties was not simply associated with inflammation as no such change was detected in viral meningitis patients characterized by brain inflammation.

Long-term treatment with Ginkgo biloba extract EGb 761 improves symptoms and pathology in a transgenic mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by extracellular deposits of amyloid ß peptide (Aß) and microglia-dominated neuroinflammation. The therapeutic options for AD are currently limited. In this study, we investigated the antiinflammatory effects and the underlying molecular mechanisms of Ginkgo biloba extract EGb 761 when administered to TgCRND8 AD mice, which overexpress human Alzheimer's amyloid precursor protein (APP) specifically in neurons. We gave APP-transgenic mice EGb 761 as a dietary supplement for 2 or 5months. Plasma concentrations of EGb 761 components in mice were in the same range as such concentrations in humans taking EGb 761 at the recommended dose (240mg daily). Treatment with EGb 761 for 5months significantly improved the cognitive function of the mice as measured by the Barnes Maze test. It also attenuated the loss of synaptic structure proteins, such as PSD-95, Munc18-1, and SNAP25. Treatment with EGb 761 for 5months inhibited microglial inflammatory activation in the brain. The effects of treatment with EGb 761 for 2months were weak and not statistically significant. Moreover, EGb 761 activated autophagy in microglia. Treatment with EGb 761 decreased Aß-induced microglial secretion of TNF-α and IL-1ß and activation of caspase-1, both of which were abolished by the inhibition of autophagy. Treatment with EGb 761 also reduced the concentrations of NLRP3 protein that colocalized with LC3-positive autophagosomes or autolysosomes in microglia. Additionally, long-term treatment with EGb 761 may reduce cerebral Aß pathology by inhibiting ß-secretase activity and Aß aggregation. Therefore, long-term treatment with G. biloba extract EGb 761, a clinically available and well-tolerated herbal medication, ameliorates AD pathology by antiinflammatory and Aß-directed mechanisms.

IKKß deficiency in myeloid cells ameliorates Alzheimer's disease-related symptoms and pathology.

Alzheimer's disease (AD) is characterized by extracellular amyloid-ß (Aß) deposits and microglia-dominated inflammatory activation. Innate immune signaling controls microglial inflammatory activities and Aß clearance. However, studies examining innate immunity in Aß pathology and neuronal degeneration have produced conflicting results. In this study, we investigated the pathogenic role of innate immunity in AD by ablating a key signaling molecule, IKKß, specifically in the myeloid cells of TgCRND8 APP-transgenic mice. Deficiency of IKKß in myeloid cells, especially microglia, simultaneously reduced inflammatory activation and Aß load in the brain and these effects were associated with reduction of cognitive deficits and preservation of synaptic structure proteins. IKKß deficiency enhanced microglial recruitment to Aß deposits and facilitated Aß internalization, perhaps by inhibiting TGF-ß-SMAD2/3 signaling, but did not affect Aß production and efflux. Therefore, inhibition of IKKß signaling in myeloid cells improves cognitive functions in AD mice by reducing inflammatory activation and enhancing Aß clearance. These results contribute to a better understanding of AD pathogenesis and could offer a new therapeutic option for delaying AD progression.

Role of BMPs in controlling the spatial and temporal origin of GFAP astrocytes in the embryonic spinal cord.

In the vertebrate central nervous system (CNS), astrocytes are the most abundant and functionally diverse glial cell population. However, the mechanisms underlying their specification and differentiation are still poorly understood. In this study, we have defined spatially and temporally the origin of astrocytes and studied the role of BMPs in astrocyte development in the embryonic chick spinal cord. Using explant cultures, we show that astrocyte precursors started migrating out of the neuroepithelium in the mantle layer from E5, and that the dorsal-most level of the neuroepithelium, from the roof plate to the dl3 level, did not generate GFAP-positive astrocytes. Using a variety of early astrocyte markers together with functional analyses, we show that dorsal-most progenitors displayed a potential for astrocyte production but that dorsally-derived BMP signalling, possibly mediated through BMP receptor 1B, promoted neuronal specification instead. BMP treatment completely prevented astrocyte development from intermediate spinal cord explants at E5, whereas it promoted it at E6. Such an abrupt change in the response of this tissue to BMP signalling could be correlated to the onset of new foci of BMP activity and enhanced expression of BMP receptor 1A, suggesting that BMP signalling could promote astrocyte development in this region.

Lipoxin A4 inhibits IL-1beta-induced IL-8 and ICAM-1 expression in 1321N1 human astrocytoma cells.

There is a growing appreciation that endogenously produced mediators may actively promote the resolution of inflammation. Lipoxins (LX) are a group of recently discovered lipid mediators that have been shown to exert anti-inflammatory and proresolution effects on cells of myeloid and nonmyeloid origin. LXs mediate a number of processes, including regression of pro-inflammatory cytokine production, inhibition of cell proliferation, and stimulation of phagocytosis of apoptotic leukocytes by macrophages. Lipoxin A(4) (LXA(4)) is one of the principal LXs formed by mammalian cells. Recently, a G protein-coupled receptor that binds LXA(4,) the lipoxin A(4) receptor, was identified in astrocytes and microglia, suggesting that these cells may be a target for LX action in the brain. In this study, we have investigated the potential of LXA(4) to modify inflammatory responses of astrocytes, using the 1321N1 human astrocytoma cell line as a model system. As shown by quantitative RT-PCR, LXA(4) (10 nM) significantly inhibited (P < 0.05) the IL-1beta-induced stimulation of IL-8 and ICAM-1 expression in these cells. Furthermore, LXA(4) (10 nM) decreased the expression of IL-1beta-induced IL-8 protein levels (P < 0.05). LXA(4) (10 nM) was found to inhibit IL-1beta-induced degradation of IkappaBalpha (P < 0.05), and the activation of an NFkappaB regulated reporter gene construct (P < 0.05). Overall, these data suggest that LXA(4) exerts anti-inflammatory effects in 1321N1 astrocytoma cells at least in part via an NFkappaB-dependent mechanism. It is concluded that LXA(4) may represent a potentially novel therapeutic approach to acute or chronic inflammation in the brain.