Pharmacological modulation of TSPO in microglia/macrophages and neurons in a chronic neurodegenerative model of prion disease.

Neuroinflammation is an important component of many neurodegenerative diseases, whether as a primary cause or a secondary outcome. For that reason, either as diagnostic tools or to monitor progression and/or pharmacological interventions, there is a need for robust biomarkers of neuroinflammation in the brain. Mitochondrial TSPO (18 kDa Translocator protein) is one of few available biomarkers of neuroinflammation for which there are clinically available PET imaging agents. In this study, we further characterised neuroinflammation in a mouse model of prion-induced chronic neurodegeneration (ME7) including a pharmacological intervention via a CSF1R inhibitor. This was achieved by autoradiographic binding of the second-generation TSPO tracer, [3H]PBR28, along with a more comprehensive examination of the cellular contributors to the TSPO signal changes by immunohistochemistry. We observed regional increases of TSPO in the ME7 mouse brains, particularly in the hippocampus, cortex and thalamus. This increased TSPO signal was detected in the cells of microglia/macrophage lineage as well as in astrocytes, endothelial cells and neurons. Importantly, we show that the selective CSF1R inhibitor, JNJ-40346527 (JNJ527), attenuated the disease-dependent increase in TSPO signal, particularly in the dentate gyrus of the hippocampus, where JNJ527 attenuated the number of Iba1+ microglia and neurons, but not GFAP+ astrocytes or endothelial cells. These findings suggest that [3H]PBR28 quantitative autoradiography in combination with immunohistochemistry are important translational tools for detecting and quantifying neuroinflammation, and its treatments, in neurodegenerative disease. Furthermore, we demonstrate that although TSPO overexpression in the ME7 brains was driven by various cell types, the therapeutic effect of the CSF1R inhibitor was primarily to modulate TSPO expression in microglia and neurons, which identifies an important route of biological action of this particular CSF1R inhibitor and provides an example of a cell-specific effect of this type of therapeutic agent on the neuroinflammatory process.

Inhibition of IL-34 Unveils Tissue-Selectivity and Is Sufficient to Reduce Microglial Proliferation in a Model of Chronic Neurodegeneration.

The proliferation and activation of microglia, the resident macrophages in the brain, is a hallmark of many neurodegenerative diseases such as Alzheimer's disease (AD) and prion disease. Colony stimulating factor 1 receptor (CSF1R) is critically involved in regulating microglial proliferation, and CSF1R blocking strategies have been recently used to modulate microglia in neurodegenerative diseases. However, CSF1R is broadly expressed by many cell types and the impact of its inhibition on the innate immune system is still unclear. CSF1R can be activated by two independent ligands, CSF-1 and interleukin 34 (IL-34). Recently, it has been reported that microglia development and maintenance depend on IL-34 signaling. In this study, we evaluate the inhibition of IL-34 as a novel strategy to reduce microglial proliferation in the ME7 model of prion disease. Selective inhibition of IL-34 showed no effects on peripheral macrophage populations in healthy mice, avoiding the side effects observed after CSF1R inhibition on the systemic compartment. However, we observed a reduction in microglial proliferation after IL-34 inhibition in prion-diseased mice, indicating that microglia could be more specifically targeted by reducing IL-34. Overall, our results highlight the challenges of targeting the CSF1R/IL34 axis in the systemic and central compartments, important for framing any therapeutic effort to tackle microglia/macrophage numbers during brain disease.

Achieving Biocompatible SABRE: An in†vitro Cytotoxicity Study.

Production of a biocompatible hyperpolarized bolus for signal amplification by reversible exchange (SABRE) could open the door to simple clinical diagnosis via magnetic resonance imaging. Essential to successful progression to preclinical/clinical applications is the determination of the toxicology profile of the SABRE reaction mixture. Herein, we exemplify the cytotoxicity of the SABRE approach using in vitro cell assays. We conclude that the main cause of the observed toxicity is due to the SABRE catalyst. We therefore illustrate two catalyst removal methods: one involving deactivation and ion-exchange chromatography, and the second using biphasic catalysis. These routes produce a bolus suitable for future in vivo study.

Coupled Proliferation and Apoptosis Maintain the Rapid Turnover of Microglia in the Adult Brain.

Microglia play key roles in brain development, homeostasis, and function, and it is widely assumed that the adult population is long lived and maintained by self-renewal. However, the precise temporal and spatial dynamics of the microglial population are unknown. We show in mice and humans that the turnover of microglia is remarkably fast, allowing the whole population to be renewed several times during a lifetime. The number of microglial cells remains steady from late postnatal stages until aging and is maintained by the spatial and temporal coupling of proliferation and apoptosis, as shown by pulse-chase studies, chronic in vivo imaging of microglia, and the use of mouse models of dysregulated apoptosis. Our results reveal that the microglial population is constantly and rapidly remodeled, expanding our understanding of its role in the maintenance of brain homeostasis.

CSF1R blockade slows the progression of amyotrophic lateral sclerosis by reducing microgliosis and invasion of macrophages into peripheral nerves.

Inflammation is a common neuropathological feature in several neurological disorders, including amyotrophic lateral sclerosis (ALS). We have studied the contribution of CSF1R signalling to inflammation in ALS, as a pathway previously reported to control the expansion and activation of microglial cells. We found that microglial cell proliferation in the spinal cord of SOD1(G93A) transgenic mice correlates with the expression of CSF1R and its ligand CSF1. Administration of GW2580, a selective CSF1R inhibitor, reduced microglial cell proliferation in SOD1(G93A) mice, indicating the importance of CSF1-CSF1R signalling in microgliosis in ALS. Moreover, GW2580 treatment slowed disease progression, attenuated motoneuron cell death and extended survival of SOD1(G93A) mice. Electrophysiological assessment revealed that GW2580 treatment protected skeletal muscle from denervation prior to its effects on microglial cells. We found that macrophages invaded the peripheral nerve of ALS mice before CSF1R-induced microgliosis occurred. Interestingly, treatment with GW2580 attenuated the influx of macrophages into the nerve, which was partly caused by the monocytopenia induced by CSF1R inhibition. Overall, our findings provide evidence that CSF1R signalling regulates inflammation in the central and peripheral nervous system in ALS, supporting therapeutic targeting of CSF1R in this disease.

Etanercept in Alzheimer disease: A randomized, placebo-controlled, double-blind, phase 2 trial.

OBJECTIVES: To determine whether the tumor necrosis factor α inhibitor etanercept is well tolerated and obtain preliminary data on its safety in Alzheimer disease dementia. METHODS: In a double-blind study, patients with mild to moderate Alzheimer disease dementia were randomized (1:1) to subcutaneous etanercept (50 mg) once weekly or identical placebo over a 24-week period. Tolerability and safety of this medication was recorded including secondary outcomes of cognition, global function, behavior, and systemic cytokine levels at baseline, 12 weeks, 24 weeks, and following a 4-week washout period. This trial is registered with EudraCT (2009-013400-31) and ClinicalTrials.gov (NCT01068353). RESULTS: Forty-one participants (mean age 72.4 years; 61% men) were randomized to etanercept (n = 20) or placebo (n = 21). Etanercept was well tolerated; 90% of participants (18/20) completed the study compared with 71% (15/21) in the placebo group. Although infections were more common in the etanercept group, there were no serious adverse events or new safety concerns. While there were some interesting trends that favored etanercept, there were no statistically significant changes in cognition, behavior, or global function. CONCLUSIONS: This study showed that subcutaneous etanercept (50 mg/wk) was well tolerated in this small group of patients with Alzheimer disease dementia, but a larger more heterogeneous group needs to be tested before recommending its use for broader groups of patients. CLASSIFICATION OF EVIDENCE: This study shows Class I evidence that weekly subcutaneous etanercept is well tolerated in Alzheimer disease dementia.

Reduced expression of the presynaptic co-chaperone cysteine string protein alpha (CSPα) does not exacerbate experimentally-induced ME7 prion disease.

Infection of mice with the ME7 prion agent results in well-characterised neuropathological changes, which includes vacuolation, neurodegeneration and synaptic degeneration. Presynaptic dysfunction and degeneration is apparent through the progressive reduction in synaptic vesicle proteins and eventual loss of synapses. Cysteine string protein alpha (CSPα), which regulates refolding pathways at the synapse, exhibits an early decline during chronic neurodegeneration implicating it as a mediator of disease mechanisms. CSPα null mice develop a progressive neuronal dysfunction through disruption of the integrity of presynaptic function. In this study, we investigated whether reduced expression of CSPα would exacerbate ME7 prion disease. Wild type (+/+) and heterozygous (+/-) mice, which express about a ∼50% reduction in CSPα, were used as a distinct genetic background on which to impose prion disease. +/+ and +/ - mice were inoculated with brain homogenate from either a normal mouse brain (NBH) or from the brain of a mouse which displayed clinical signs of prion disease (ME7). Behavioural tests, western blotting and immunohistochemistry, which resolve key elements of synaptic dysfunction, were used to assess the effect of reduced CSPα on disease. Behavioural tests revealed no change in the progression of disease in ME7-CSPα +/- animals compared to ME7-CSPα +/+ animals. In addition, the accumulation of misfolded PrP(Sc), the diseased associated gliosis or synaptic loss were not different. Thus, the misfolding events that generate synaptic dysfunction and lead to synaptic loss are unlikely to be mediated by a disease associated decrease in the refolding pathways associated with CSPα.

In-vivo RGB marking and multicolour single-cell tracking in the adult brain.

In neuroscience it is a technical challenge to identify and follow the temporal and spatial distribution of cells as they differentiate. We hypothesised that RGB marking, the tagging of individual cells with unique hues resulting from simultaneous expression of the three basic colours red, green and blue, provides a convenient toolbox for the study of the CNS anatomy at the single-cell level. Using γ-retroviral and lentiviral vector sets we describe for the first time the in-vivo multicolour RGB marking of neurons in the adult brain. RGB marking also enabled us to track the spatial and temporal fate of neural stem cells in the adult brain. The application of different viral envelopes and promoters provided a useful approach to track the generation of neurons vs. glial cells at the neurogenic niche, allowing the identification of the prominent generation of new astrocytes to the striatum. Multicolour RGB marking could serve as a universal and reproducible method to study and manipulate the CNS at the single-cell level, in both health and disease.

Temporal dynamics of hippocampal neurogenesis in chronic neurodegeneration.

The study of neurogenesis during chronic neurodegeneration is crucial in order to understand the intrinsic repair mechanisms of the brain, and key to designing therapeutic strategies. In this study, using an experimental model of progressive chronic neurodegeneration, murine prion disease, we define the temporal dynamics of the generation, maturation and integration of new neurons in the hippocampal dentate gyrus, using dual pulse-chase, multicolour γ-retroviral tracing, transmission electron microscopy and patch-clamp. We found increased neurogenesis during the progression of prion disease, which partially counteracts the effects of chronic neurodegeneration, as evidenced by blocking neurogenesis with cytosine arabinoside, and helps to preserve the hippocampal function. Evidence obtained from human post-mortem samples, of both variant Creutzfeldt-Jakob disease and Alzheimer's disease patients, also suggests increased neurogenic activity. These results open a new avenue into the exploration of the effects and regulation of neurogenesis during chronic neurodegeneration, and offer a new model to reproduce the changes observed in human neurodegenerative diseases.

Differential role of CCR2 in the dynamics of microglia and perivascular macrophages during prion disease.

The expansion of the microglial population is one of the hallmarks of numerous brain disorders. The addition of circulating progenitors to the pool of brain macrophages can contribute to the progression of brain disease and needs to be precisely defined to better understand the evolution of the glial and inflammatory reactions in the brain. We have analyzed the degree of infiltration/recruitment of circulating monocytes to the microglial pool, in a prion disease model of chronic neurodegeneration. Our results indicate a minimal/absent level of CCR2-dependent recruitment of circulating monocytes, local proliferation of microglia is the main driving force maintaining the amplification of the population. A deficiency in CCR2, and thus the absence of recruitment of circulating monocytes, does not impact microglial dynamics, the inflammatory profile or the temporal behavioral course of prion disease. However, the lack of CCR2 has unexpected effects including the failure to recruit perivascular macrophages in diseased but not healthy CNS and a small reduction in microglia proliferation. These data define the composition of the CNS-resident macrophage populations in prion disease and will help to understand the dynamics of the CNS innate immune response during chronic neurodegeneration.

Analysis of the hippocampal proteome in ME7 prion disease reveals a predominant astrocytic signature and highlights the brain-restricted production of clusterin in chronic neurodegeneration.

Prion diseases are characterized by accumulation of misfolded protein, gliosis, synaptic dysfunction, and ultimately neuronal loss. This sequence, mirroring key features of Alzheimer disease, is modeled well in ME7 prion disease. We used iTRAQ(TM)/mass spectrometry to compare the hippocampal proteome in control and late-stage ME7 animals. The observed changes associated with reactive glia highlighted some specific proteins that dominate the proteome in late-stage disease. Four of the up-regulated proteins (GFAP, high affinity glutamate transporter (EAAT-2), apo-J (Clusterin), and peroxiredoxin-6) are selectively expressed in astrocytes, but astrocyte proliferation does not contribute to their up-regulation. The known functional role of these proteins suggests this response acts against protein misfolding, excitotoxicity, and neurotoxic reactive oxygen species. A recent convergence of genome-wide association studies and the peripheral measurement of circulating levels of acute phase proteins have focused attention on Clusterin as a modifier of late-stage Alzheimer disease and a biomarker for advanced neurodegeneration. Since ME7 animals allow independent measurement of acute phase proteins in the brain and circulation, we extended our investigation to address whether changes in the brain proteome are detectable in blood. We found no difference in the circulating levels of Clusterin in late-stage prion disease when animals will show behavioral decline, accumulation of misfolded protein, and dramatic synaptic and neuronal loss. This does not preclude an important role of Clusterin in late-stage disease, but it cautions against the assumption that brain levels provide a surrogate peripheral measure for the progression of brain degeneration.

Microglia and macrophages of the central nervous system: the contribution of microglia priming and systemic inflammation to chronic neurodegeneration.

Microglia, the resident immune cells of the central nervous system (CNS), play an important role in CNS homeostasis during development, adulthood and ageing. Their phenotype and function have been widely studied, but most studies have focused on their local interactions in the CNS. Microglia are derived from a particular developmental niche, are long-lived, locally replaced and form a significant part of the communication route between the peripheral immune system and the CNS; all these components of microglia biology contribute to maintaining homeostasis. Microglia function is tightly regulated by the CNS microenvironment, and increasing evidence suggests that disturbances, such as neurodegeneration and ageing, can have profound consequences for microglial phenotype and function. We describe the possible biological mechanisms underlying the altered threshold for microglial activation, also known as 'microglial priming', seen in CNS disease and ageing and consider how priming may contribute to turning immune-to-brain communication from a homeostatic pathway into a maladaptive response that contributes to symptoms and progression of diseases of the CNS.

Regulation of microglial proliferation during chronic neurodegeneration.

An important component of chronic neurodegenerative diseases is the generation of an innate inflammatory response within the CNS. Microglial and astroglial cells play a key role in the development and maintenance of this inflammatory response, showing enhanced proliferation and activation. We studied the time course and regulation of microglial proliferation, using a mouse model of prion disease. Our results show that the proliferation of resident microglial cells accounts for the expansion of the population during the development of the disease. We identify the pathway regulated by the activation of CSF1R and the transcription factors PU.1 and C/EBPα as the molecular regulators of the proliferative response, correlating with the chronic human neurodegenerative conditions variant Creutzfeldt-Jakob disease and Alzheimer's disease. We show that targeting the activity of CSF1R inhibits microglial proliferation and slows neuronal damage and disease progression. Our results demonstrate that microglial proliferation is a major component in the evolution of chronic neurodegeneration, with direct implications for understanding the contribution of the CNS innate immune response to disease progression.

Innate inflammation in Parkinson's disease.

The resident macrophages of the brain--the microglia--are morphologically activated during the progression of Parkinson's disease. Observational studies in human postmortem material and studies in animal models seek to define the contribution that this innate immune response might make to the pathogenesis and rate of progression of Parkinson's disease. We review here some of the key conceptual issues that need to be considered when performing these studies. We highlight the fact that most postmortem studies have not given due consideration to common comorbidities present in patients with Parkinson's disease and also the limitations of attempting to extrapolate from animal models to a chronic progressive neurodegenerative disease in humans that lasts for many years.

Docosahexaenoic acid, but not eicosapentaenoic acid, reduces the early inflammatory response following compression spinal cord injury in the rat.

Docosahexaenoic acid (DHA, 22 : 6) and eicosapentaenoic acid (EPA, 20 : 5) are omega-3 polyunsaturated fatty acids (n-3 PUFAs) with distinct anti-inflammatory properties. Both have neuroprotective effects acutely following spinal cord injury (SCI). We examined the effect of intravenous DHA and EPA on early inflammatory events after SCI. Saline, DHA or EPA (both 250 nmol/kg) were administered 30 min after T12 compression SCI, to female Sprague-Dawley rats. DHA significantly reduced the number of neutrophils to some areas of the injured epicentre at 4 h and 24 h. DHA also reduced C-reactive protein plasma levels, whereas EPA did not significantly reduce neutrophils or C-reactive protein. Laminectomy and SCI elicited a sustained inflammatory response in the liver, which was not reversed by the PUFAs. The chemokine KC/GRO/CINC and the cytokine IL-6 provide gradients for chemotaxis of neutrophils to the epicentre. At 4 h after injury, there was a significant increase in IL-6, KC/GRO/CINC, IL-1ß and tumour necrosis factor-α in the epicentre, with a return to baseline at 24 h. Neither DHA nor EPA returned their levels to control values. These results indicate that the acute neuroprotective effects of n-3 PUFAs in rat compression SCI may be only partly attributed to reduction of some of the early inflammatory events occurring after injury.

The intrathecal CD163-haptoglobin-hemoglobin scavenging system in subarachnoid hemorrhage.

Delayed cerebral ischemia resulting from extracellular hemoglobin is an important determinant of outcome in subarachnoid hemorrhage. Hemoglobin is scavenged by the CD163-haptoglobin system in the circulation, but little is known about this scavenging pathway in the human CNS. The components of this system were analyzed in normal cerebrospinal fluid and after subarachnoid hemorrhage. The intrathecal presence of the CD163-haptoglobin-hemoglobin scavenging system was unequivocally demonstrated. The resting capacity of the CD163-haptoglobin-hemoglobin system in the normal CNS was 50 000-fold lower than that of the circulation. After subarachnoid hemorrhage, the intrathecal CD163-haptoglobin-hemoglobin system was saturated, as shown by the presence of extracellular hemoglobin despite detectable haptoglobin. Hemoglobin efflux from the CNS was evident, enabling rescue hemoglobin scavenging by the systemic circulation. Therefore, the CNS is not capable of dealing with significant intrathecal hemolysis. Potential therapeutic options to prevent delayed cerebral ischemia ought to concentrate on augmenting the capacity of the intrathecal CD163-haptoglobin-hemoglobin scavenging system and strategies to encourage hemoglobin efflux from the brain.

The role of microglia in synaptic stripping and synaptic degeneration: a revised perspective.

Chronic neurodegenerative diseases of the CNS (central nervous system) are characterized by the loss of neurons. There is, however, growing evidence to show that an early stage of this process involves degeneration of presynaptic terminals prior to the loss of the cell body. Synaptic plasticity in CNS pathology has been associated with microglia and the phenomenon of synaptic stripping. We review here the evidence for the involvement of microglia in synaptic stripping and synapse degeneration and we conclude that this is a case of guilt by association. In disease models of chronic neurodegeneration, there is no evidence that microglia play an active role in either synaptic stripping or synapse degeneration, but the degeneration of the synapse and the envelopment of a degenerating terminal appears to be a neuron autonomous event. We highlight here some of the gaps in our understanding of synapse degeneration in chronic neurodegenerative disease.

Microglial physiology: unique stimuli, specialized responses.

Microglia, the macrophages of the central nervous system parenchyma, have in the normal healthy brain a distinct phenotype induced by molecules expressed on or secreted by adjacent neurons and astrocytes, and this phenotype is maintained in part by virtue of the blood-brain barrier's exclusion of serum components. Microglia are continually active, their processes palpating and surveying their local microenvironment. The microglia rapidly change their phenotype in response to any disturbance of nervous system homeostasis and are commonly referred to as activated on the basis of the changes in their morphology or expression of cell surface antigens. A wealth of data now demonstrate that the microglia have very diverse effector functions, in line with macrophage populations in other organs. The term activated microglia needs to be qualified to reflect the distinct and very different states of activation-associated effector functions in different disease states. Manipulating the effector functions of microglia has the potential to modify the outcome of diverse neurological diseases.

Systemic inflammation induces acute behavioral and cognitive changes and accelerates neurodegenerative disease.

BACKGROUND: Chronic neurodegeneration results in microglial activation, but the contribution of inflammation to the progress of neurodegeneration remains unclear. We have shown that microglia express low levels of proinflammatory cytokines during chronic neurodegeneration but are "primed" to produce a more proinflammatory profile after systemic challenge with bacterial endotoxin (lipopolysaccharide [LPS]). METHODS: Here, we investigated whether intraperitoneal (IP) challenge with LPS, to mimic systemic infection, in the early stages of prion disease can 1) produce exaggerated acute behavioral (n = 9) and central nervous system (CNS) inflammatory (n = 4) responses in diseased animals compared with control animals, and 2) whether a single LPS challenge can accelerate disease progression (n = 34-35). RESULTS: Injection of LPS (100 microg/kg), at 12 weeks postinoculation (PI), resulted in heightened CNS interleukin-1 beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and interferon-beta (IFN-beta) transcription and microglial IL-1beta translation in prion-diseased animals relative to control animals. This inflammation caused exaggerated impairments in burrowing and locomotor activity, and induced hypothermia and cognitive changes in prion-diseased animals that were absent in LPS-treated control animals. At 15 weeks PI, LPS (500 microg/kg) acutely impaired motor coordination and muscle strength in prion-diseased but not in control animals. After recovery, these animals also showed earlier onset of disease-associated impairments on these parameters. CONCLUSIONS: These data demonstrate that transient systemic inflammation superimposed on neurodegenerative disease acutely exacerbates cognitive and motor symptoms of disease and accelerates disease progression. These deleterious effects of systemic inflammation have implications for the treatment of chronic neurodegeneration and associated delirium.