Copper ions, prion protein and Aß modulate Ca levels in central nervous system myelin in an NMDA receptor-dependent manner.

As in neurons, CNS myelin expresses N-Methyl-D-Aspartate Receptors (NMDARs) that subserve physiological roles, but have the potential to induce injury to this vital element. Using 2-photon imaging of myelinic Ca in live ex vivo mouse optic nerves, we show that Cu ions potently modulate Ca levels in an NMDAR-dependent manner. Chelating Cu in the perfusate induced a substantial increase in Ca levels, and also caused significant axo-myelinic injury. Myelinic NMDARs are shown to be regulated by cellular prion protein; only in prion protein KO optic nerves does application of NMDA + D-serine induce a large Ca increase, consistent with strong desensitization of these receptors in the presence of prion protein limiting Ca overload. Aß1-42 peptide induced a large Ca increase that was also Cu-dependent, and was blocked by NMDAR antagonism. Our results indicate that like in neurons, myelinic NMDARs permeate potentially injurious amounts of Ca, and are also potently regulated by micromolar Cu and activated by Aß1-42 peptides. These findings shed mechanistic light on the important primary white matter injury frequently observed in Alzheimer's brain.

Diagnosing Alzheimer's Disease from Circulating Blood Leukocytes Using a Fluorescent Amyloid Probe.

BACKGROUND: Toxic amyloid-ß (Aß) peptides aggregate into higher molecular weight assemblies and accumulate not only in the extracellular space, but also in the walls of blood vessels in the brain, increasing their permeability, and promoting immune cell migration and activation. Given the prominent role of the immune system, phagocytic blood cells may contact pathological brain materials. OBJECTIVE: To develop a novel method for early Alzheimer's disease (AD) detection, we used blood leukocytes, that could act as "sentinels" after trafficking through the brain microvasculature, to detect pathological amyloid by labelling with a conformationally-sensitive fluorescent amyloid probe and imaging with confocal spectral microscopy. METHODS: Formalin-fixed peripheral blood mononuclear cells (PBMCs) from cognitively healthy control (HC) subjects, mild cognitive impairment (MCI) and AD patients were stained with the fluorescent amyloid probe K114, and imaged. Results were validated against cerebrospinal fluid (CSF) biomarkers and clinical diagnosis. RESULTS: K114-labeled leukocytes exhibited distinctive fluorescent spectral signatures in MCI/AD subjects. Comparing subjects with single CSF biomarker-positive AD/MCI to negative controls, our technique yielded modest AUCs, which improved to the 0.90 range when only MCI subjects were included in order to measure performance in an early disease state. Combining CSF Aß42 and t-Tau metrics further improved the AUC to 0.93. CONCLUSION: Our method holds promise for sensitive detection of AD-related protein misfolding in circulating leukocytes, particularly in the early stages of disease.

Recent advances in understanding multiple sclerosis.

Emerging data point to important contributions of both autoimmune inflammation and progressive degeneration in the pathophysiology of multiple sclerosis (MS). Unfortunately, after decades of intensive investigation, the fundamental cause remains unknown. A large body of research on the immunobiology of MS has resulted in a variety of anti-inflammatory therapies that are highly effective at reducing brain inflammation and clinical/radiological relapses. However, despite potent suppression of inflammation, benefit in the more important and disabling progressive phase is extremely limited; thus, progressive MS has emerged as the greatest challenge for the MS research and clinical communities. Data obtained over the years point to a complex interplay between environment (e.g., the near-absolute requirement of Epstein-Barr virus exposure), immunogenetics (strong associations with a large number of immune genes), and an ever more convincing role of an underlying degenerative process resulting in demyelination (in both white and grey matter regions), axonal and neuro-synaptic injury, and a persistent innate inflammatory response with a seemingly diminishing role of T cell-mediated autoimmunity as the disease progresses. Together, these observations point toward a primary degenerative process, one whose cause remains unknown but one that entrains a nearly ubiquitous secondary autoimmune response, as a likely sequence of events underpinning this disease. Here, we briefly review what is known about the potential pathophysiological mechanisms, focus on progressive MS, and discuss the two main hypotheses of MS pathogenesis that are the topic of vigorous debate in the field: whether primary autoimmunity or degeneration lies at the foundation. Unravelling this controversy will be critically important for developing effective new therapies for the most disabling later phases of this disease.

Biochemically altered myelin triggers autoimmune demyelination.

Although immune attack against central nervous system (CNS) myelin is a central feature of multiple sclerosis (MS), its root cause is unresolved. In this report, we provide direct evidence that subtle biochemical modifications to brain myelin elicit pathological immune responses with radiological and histological properties similar to MS lesions. A subtle myelinopathy induced by abbreviated cuprizone treatment, coupled with subsequent immune stimulation, resulted in lesions of inflammatory demyelination. The degree of myelin injury dictated the resulting immune response; biochemical damage that was too limited or too extensive failed to trigger overt pathology. An inhibitor of peptidyl arginine deiminases (PADs), enzymes that alter myelin structure and correlate with MS lesion severity, mitigated pathology even when administered only during the myelin-altering phase. Moreover, cultured splenocytes were reactive against donor myelin isolates, a response that was substantially muted when splenocytes were exposed to myelin from donors treated with PAD inhibitors. By showing that a primary biochemical myelinopathy can trigger secondary pathological inflammation, "cuprizone autoimmune encephalitis" potentially reconciles conflicting theories about MS pathogenesis and provides a strong rationale for investigating myelin as a primary target for early, preventative therapy.

Axonal and myelinic pathology in 5xFAD Alzheimer's mouse spinal cord.

As an extension of the brain, the spinal cord has unique properties which could allow us to gain a better understanding of CNS pathology. The brain and cord share the same cellular components, yet the latter is simpler in cytoarchitecture and connectivity. In Alzheimer's research, virtually all focus is on brain pathology, however it has been shown that transgenic Alzheimer's mouse models accumulate beta amyloid plaques in spinal cord, suggesting that the cord possesses the same molecular machinery and conditions for plaque formation. Here we report a spatial-temporal map of plaque load in 5xFAD mouse spinal cord. We found that plaques started to appear at 11 weeks, then exhibited a time dependent increase and differential distribution along the cord. More plaques were found in cervical than other spinal levels at all time points examined. Despite heavy plaque load at 6 months, the number of cervical motor neurons in 5xFAD mice is comparable to wild type littermates. On detailed microscopic examination, fine beta amyloid-containing and beta sheet-rich thread-like structures were found in the peri-axonal space of many axons. Importantly, these novel structures appear before any plaque deposits are visible in young mice spinal cord and they co-localize with axonal swellings at later stages, suggesting that these thread-like structures might represent the initial stages of plaque formation, and could play a role in axonal damage. Additionally, we were able to demonstrate increasing myelinopathy in aged 5xFAD mouse spinal cord using the lipid probe Nile Red with high resolution. Collectively, we found significant amyloid pathology in grey and white matter of the 5xFAD mouse spinal cord which indicates that this structure maybe a useful platform to study mechanisms of Alzheimer's pathology and disease progression.

Multi-target-directed phenol-triazole ligands as therapeutic agents for Alzheimer's disease.

Alzheimer's disease (AD) is a multifactorial disease that is characterized by the formation of intracellular neurofibrillary tangles and extracellular amyloid-ß (Aß) plaque deposits. Increased oxidative stress, metal ion dysregulation, and the formation of toxic Aß peptide oligomers are all considered to contribute to the etiology of AD. In this work we have developed a series of ligands that are multi-target-directed in order to address several disease properties. 2-(1-(3-Hydroxypropyl)-1H-1,2,3-triazol-4-yl)phenol (POH), 2-(1-(2-morpholinoethyl)-1H-1,2,3-triazol-4-yl)phenol (PMorph), and 2-(1-(2-thiomorpholinoethyl)-1H-1,2,3-triazol-4-yl)phenol (PTMorph) have been synthesized and screened for their antioxidant capacity, Cu-binding affinity, interaction with the Aß peptide and modulation of Aß peptide aggregation, and the ability to limit Aß1-42-induced neurotoxicity in human neuronal culture. The synthetic protocol and structural variance incorporated via click chemistry, highlights the influence of R-group modification on ligand-Aß interactions and neuroprotective effects. Overall, this study demonstrates that the phenol-triazole ligand scaffold can target multiple factors associated with AD, thus warranting further therapeutic development.

Unique spectral signatures of the nucleic acid dye acridine orange can distinguish cell death by apoptosis and necroptosis.

Cellular injury and death are ubiquitous features of disease, yet tools to detect them are limited and insensitive to subtle pathological changes. Acridine orange (AO), a nucleic acid dye with unique spectral properties, enables real-time measurement of RNA and DNA as proxies for cell viability during exposure to various noxious stimuli. This tool illuminates spectral signatures unique to various modes of cell death, such as cells undergoing apoptosis versus necrosis/necroptosis. This new approach also shows that cellular RNA decreases during necrotic, necroptotic, and apoptotic cell death caused by demyelinating, ischemic, and traumatic injuries, implying its involvement in a wide spectrum of tissue pathologies. Furthermore, cells with pathologically low levels of cytoplasmic RNA are detected earlier and in higher numbers than with standard markers including TdT-mediated dUTP biotin nick-end labeling and cleaved caspase 3 immunofluorescence. Our technique highlights AO-labeled cytoplasmic RNA as an important early marker of cellular injury and a sensitive indicator of various modes of cell death in a range of experimental models.

Fluorescent Phosphorus Dendrimer as a Spectral Nanosensor for Macrophage Polarization and Fate Tracking in Spinal Cord Injury.

Dendrimers and dendriplexes, highly branched synthetic macromolecules, have gained popularity as new tools for a variety of nanomedicine strategies due to their unique structure and properties. We show that fluorescent phosphorus dendrimers are well retained by bone marrow-derived macrophages and exhibit robust spectral shift in its emission in response to polarization conditions. Fluorescence properties of this marker can also assist in identifying macrophage presence and phenotype status at different time points after spinal cord injury. Potential use of a single dendrimer compound as a drug/siRNA carrier and phenotype-specific cell tracer offers new avenues for enhanced cell therapies combined with monitoring of cell fate and function in spinal cord injury.

Cellular prion protein and NMDA receptor modulation: protecting against excitotoxicity.

Although it is well established that misfolding of the cellular prion protein (PrP(C)) into the ß-sheet-rich, aggregated scrapie conformation (PrP(Sc)) causes a variety of transmissible spongiform encephalopathies (TSEs), the physiological roles of PrP(C) are still incompletely understood. There is accumulating evidence describing the roles of PrP(C) in neurodegeneration and neuroinflammation. Recently, we identified a functional regulation of NMDA receptors by PrP(C) that involves formation of a physical protein complex between these proteins. Excessive NMDA receptor activity during conditions such as ischemia mediates enhanced Ca(2+) entry into cells and contributes to excitotoxic neuronal death. In addition, NMDA receptors and/or PrP(C) play critical roles in neuroinflammation and glial cell toxicity. Inhibition of NMDA receptor activity protects against PrP(Sc)-induced neuronal death. Moreover, in mice lacking PrP(C), infarct size is increased after focal cerebral ischemia, and absence of PrP(C) increases susceptibility of neurons to NMDA receptor-dependent death. Recently, PrP(C) was found to be a receptor for oligomeric beta-amyloid (Aß) peptides, suggesting a role for PrP(C) in Alzheimer's disease (AD). Our recent findings suggest that Aß peptides enhance NMDA receptor current by perturbing the normal copper- and PrP(C)-dependent regulation of these receptors. Here, we review evidence highlighting a role for PrP(C) in preventing NMDA receptor-mediated excitotoxicity and inflammation. There is a need for more detailed molecular characterization of PrP(C)-mediated regulation of NMDA receptors, such as determining which NMDA receptor subunits mediate pathogenic effects upon loss of PrP(C)-mediated regulation and identifying PrP(C) binding site(s) on the receptor. This knowledge will allow development of novel therapeutic interventions for not only TSEs, but also for AD and other neurodegenerative disorders involving dysfunction of PrP(C).

Metabolic injury to axons and myelin.

CNS white matter, the collection of axons and supporting glia of the mammalian CNS, makes up close to 50% of the human brain by volume. Interruption of vital interconnects within this tissue, even over a short segment, often leads to serious morbidity in a broad range of neurological disorders. Axons, glia and myelin express a complex array of conventional voltage gated ion channels, intracellular Ca(2+) release channels, neurotransmitter uptake and release mechanisms, together with matching transmitter receptors. Dysregulation of ion homeostasis induced by injury or energy failure leads to depolarization and intracellular Na(+) accumulation, which in turn triggers inappropriate ion translocation (i.e. Ca(2+) influx) and transmitter release; together these events further promote more Ca(2+) influx, while at the same time triggering even more toxic Ca(2+) release from intracellular Ca(2+) stores. Uncontrolled intracellular Ca(2+) increases overactivate a variety of Ca(2+)-sensitive enzyme systems culminating in permanent injury to axon, myelin and glia.

Aß neurotoxicity depends on interactions between copper ions, prion protein, and N-methyl-D-aspartate receptors.

N-methyl-d-aspartate receptors (NMDARs) mediate critical CNS functions, whereas excessive activity contributes to neuronal damage. At physiological glycine concentrations, NMDAR currents recorded from cultured rodent hippocampal neurons exhibited strong desensitization in the continued presence of NMDA, thus protecting neurons from calcium overload. Reducing copper availability by specific chelators (bathocuproine disulfonate, cuprizone) induced nondesensitizing NMDAR currents even at physiologically low glycine concentrations. This effect was mimicked by, and was not additive with, genetic ablation of cellular prion protein (PrP(C)), a key copper-binding protein in the CNS. Acute ablation of PrP(C) by enzymatically cleaving its cell-surface GPI anchor yielded similar effects. Biochemical studies and electrophysiological measurements revealed that PrP(C) interacts with the NMDAR complex in a copper-dependent manner to allosterically reduce glycine affinity for the receptor. Synthetic human Aß(1-42) (10 nM-5 µM) produced an identical effect that could be mitigated by addition of excess copper ions or NMDAR blockers. Taken together, Aß(1-42), copper chelators, or PrP(C) inactivation all enhance the activity of glycine at the NMDAR, giving rise to pathologically large nondesensitizing steady-state NMDAR currents and neurotoxicity. We propose a physiological role for PrP(C), one that limits excessive NMDAR activity that might otherwise promote neuronal damage. In addition, we provide a unifying molecular mechanism whereby toxic species of Aß(1-42) might mediate neuronal and synaptic injury, at least in part, by disrupting the normal copper-mediated, PrP(C)-dependent inhibition of excessive activity of this highly calcium-permeable glutamate receptor.

Prion protein expression level alters regional copper, iron and zinc content in the mouse brain.

The central role of the prion protein (PrP) in a family of fatal neurodegenerate diseases has garnered considerable research interest over the past two decades. Moreover, the role of PrP in neuronal development, as well as its apparent role in metal homeostasis, is increasingly of interest. The host-encoded form of the prion protein (PrP(C)) binds multiple copper atoms via its N-terminal domain and can influence brain copper and iron levels. The importance of PrP(C) to the regulation of brain metal homeostasis and metal distribution, however, is not fully understood. We therefore employed synchrotron-based X-ray fluorescence imaging to map the level and distributions of several key metals in the brains of mice that express different levels of PrP(C). Brain sections from wild-type, prion gene knockout (Prnp(-/-)) and PrP(C) over-expressing mice revealed striking variation in the levels of iron, copper, and even zinc in specific brain regions as a function of PrP(C) expression. Our results indicate that one important function of PrP(C) may be to regulate the amount and distribution of specific metals within the central nervous system. This raises the possibility that PrP(C) levels, or its activity, might regulate the progression of diseases in which altered metal homeostasis is thought to play a pathogenic role such as Alzheimer's, Parkinson's and Wilson's diseases and disorders such as hemochromatosis.

Early life activation of toll-like receptor 4 reprograms neural anti-inflammatory pathways.

A single postnatal exposure to the bacterial endotoxin, lipopolysaccharide (LPS), reduces the neuroimmune response to a subsequent LPS exposure in the adult rat. The attenuated fever and proinflammatory response is caused by a paradoxical, amplified, early corticosterone response to LPS. Here we identify the mechanisms underlying the heightened corticosterone response to LPS in adults after early life exposure to LPS. In postnatal LPS-treated rats, hypothalamic corticotrophin-releasing hormone mRNA, pituitary proopiomelanocortin mRNA, and circulating adrenocorticotrophic hormone were all increased after adult exposure to LPS without significant modification to hippocampal or hypothalamic glucocorticoid receptor mRNA or protein or vagally mediated afferent signaling to the brain. Postnatal LPS administration did cause a persistent upregulation of the LPS Toll-like receptor-4 (TLR4) mRNA in liver and spleen, but not in brain, pituitary, or adrenal gland. In addition, cyclooxygenase-2 (COX-2), which is a prostaglandin biosynthetic enzyme and is normally undetectable in most peripheral tissue, was constitutively expressed in the liver. Adult immune activation of the upregulated TLR4 and COX-2 caused a rapid, amplified rise in circulating, but not brain, prostaglandin E(2) that induced an early, enhanced activation of the hypothalamic-pituitary-adrenal (HPA) axis. Thus, postnatal LPS reprograms the neuroimmune axis by priming peripheral tissues to create a novel, prostaglandin-mediated activation of the HPA axis brought about by increased constitutive expression of TLR4 and COX-2.

Early life exposure to lipopolysaccharide suppresses experimental autoimmune encephalomyelitis by promoting tolerogenic dendritic cells and regulatory T cells.

The rising incidence of autoimmune diseases such as multiple sclerosis (MS) in developed countries might be due to a more hygienic environment, particularly during early life. To investigate this concept, we developed a model of neonatal exposure to a common pathogen-associated molecular pattern, LPS, and determined its impact on experimental autoimmune encephalomyelitis (EAE). Mice exposed to LPS at 2 wk of age showed a delayed onset and diminished severity of myelin oligodendrocyte glycoprotein (MOG)-induced EAE, induced at 12 wk, compared with vehicle-exposed animals. Spinal cord transcript levels of CD3epsilon and F4/80 were lower in LPS- compared with PBS-exposed EAE animals with increased IL-10 levels in the LPS-exposed group. Splenic CD11c(+) cells from LPS-exposed animals exhibited reduced MHC class II and CD83 expression but increased levels of CD80 and CD86 both before and during EAE. MOG-treated APC from LPS-exposed animals stimulated less T lymphocyte proliferation but increased expansion of CD4(+)FoxP3(+) T cells compared with APC from PBS-exposed animals. Neuropathological studies disclosed reduced myelin and axonal loss in spinal cords from LPS-exposed compared with PBS-exposed animals with EAE, and this neuroprotective effect was associated with an increased number of CD3(+)FoxP3(+) immunoreactive cells. Analyses of human brain tissue revealed that FoxP3 expression was detected in lymphocytes, albeit reduced in MS compared with non-MS patients' brains. These findings support the concept of early-life microbial exposure influencing the generation of neuroprotective regulatory T cells and may provide insights into new immunotherapeutic strategies for MS.

Absence of the cellular prion protein exacerbates and prolongs neuroinflammation in experimental autoimmune encephalomyelitis.

Although the physiological roles of the cellular prion protein (PrP C) remain to be fully elucidated, PrP C has been proposed to represent a potential regulator of cellular immunity. To test this hypothesis, we evaluated the consequences of PrP C deficiency on the course of experimental autoimmune encephalomyelitis induced by immunization with myelin oligodendrocyte glycoprotein peptide. Consistent with augmented proliferative responses and increased cytokine gene expression by myelin oligodendrocyte glycoprotein-primed Prnp-/- T cells, PrP C-deficient mice demonstrated more aggressive disease onset and a lack of clinical improvement during the chronic phase of experimental autoimmune encephalomyelitis. Acutely, Prnp-/- spinal cord, cerebellum, and forebrain exhibited higher levels of leukocytic infiltrates and pro-inflammatory cytokine gene expression, as well as increased spinal cord myelin basic protein and axonal loss. During the chronic phase, a remarkable persistence of leukocytic infiltrates was present in the forebrain and cerebellum, accompanied by an increase in interferon-gamma and interleukin-17 transcripts. Attenuation of T cell-dependent neuroinflammation thus represents a potential novel function of PrP C.

Prion protein attenuates excitotoxicity by inhibiting NMDA receptors.

It is well established that misfolded forms of cellular prion protein (PrP [PrPC]) are crucial in the genesis and progression of transmissible spongiform encephalitis, whereas the function of native PrPC remains incompletely understood. To determine the physiological role of PrPC, we examine the neurophysiological properties of hippocampal neurons isolated from PrP-null mice. We show that PrP-null mouse neurons exhibit enhanced and drastically prolonged N-methyl-D-aspartate (NMDA)-evoked currents as a result of a functional upregulation of NMDA receptors (NMDARs) containing NR2D subunits. These effects are phenocopied by RNA interference and are rescued upon the overexpression of exogenous PrPC. The enhanced NMDAR activity results in an increase in neuronal excitability as well as enhanced glutamate excitotoxicity both in vitro and in vivo. Thus, native PrPC mediates an important neuroprotective role by virtue of its ability to inhibit NR2D subunits.

Prion protein attenuates excitotoxicity by inhibiting NMDA receptors.

It is well established that misfolded forms of cellular prion protein (PrP [PrP(C)]) are crucial in the genesis and progression of transmissible spongiform encephalitis, whereas the function of native PrP(C) remains incompletely understood. To determine the physiological role of PrP(C), we examine the neurophysiological properties of hippocampal neurons isolated from PrP-null mice. We show that PrP-null mouse neurons exhibit enhanced and drastically prolonged N-methyl-d-aspartate (NMDA)-evoked currents as a result of a functional upregulation of NMDA receptors (NMDARs) containing NR2D subunits. These effects are phenocopied by RNA interference and are rescued upon the overexpression of exogenous PrP(C). The enhanced NMDAR activity results in an increase in neuronal excitability as well as enhanced glutamate excitotoxicity both in vitro and in vivo. Thus, native PrP(C) mediates an important neuroprotective role by virtue of its ability to inhibit NR2D subunits.

Antigen-induced Pten gene deletion in T cells exacerbates neuropathology in experimental autoimmune encephalomyelitis.

The Pten tumor suppressor gene is critical for normal intrathymic development of T cells; however, its role in mature antigen-activated T cells is less well defined. A genetically crossed mouse line, Pten(fl/fl) GBC, in which Pten gene deletions could be primarily confined to antigen-activated CD8+ T cells, enabled us to evaluate the consequences of Pten loss on the course of experimental autoimmune encephalomyelitis. Compared with Pten(fl/fl) controls, myelin oligodendrocyte glycoprotein (MOG) peptide-immunized Pten(fl/fl) GBC mice developed more severe and protracted disease. This was accompanied by increased spinal cord white matter myelin basic protein depletion and axonal damage, as well as a striking persistence of macrophage and granzyme B-expressing cellular neuroinfiltrates in the chronic phase of the disease. This persistence may be explained by the observation that anti-CD3 activated Pten(fl/fl) GBC T cells were more resistant to proapoptotic stimuli. Consistent with the predicted consequences of Pten loss, purified CD8+ T cells from Pten(fl/fl) GBC mice displayed augmented proliferative responses to anti-T-cell receptor stimulation, and MOG-primed Pten(fl/fl) GBC T cells exhibited a reduced activation threshold to MOG peptide. Pten(fl/fl) GBC mice also developed atypical central nervous system disease, manifested by prominent cervical cord and forebrain involvement. Collectively, our findings indicate that the phosphatidylinositol 3-kinase signaling pathway is an essential regulator of CD8+ T-cell effector function in experimental autoimmune encephalomyelitis.