Dense-core and diffuse Abeta plaques in TgCRND8 mice studied with synchrotron FTIR microspectroscopy

Plaques composed of the Abeta peptide are the main pathological feature of Alzheimer's disease. Dense-core plaques are fibrillar deposits of Abeta, showing all the classical properties of amyloid including beta-sheet secondary structure, while diffuse plaques are amorphous deposits. We studied both plaque types, using synchrotron infrared (IR) microspectroscopy, a technique that allows the chemical composition and average protein secondary structure to be investigated in situ. We examined plaques in hippocampal, cortical and caudal tissue from 5- to 21-month-old TgCRND8 mice, a transgenic model expressing doubly mutant amyloid precursor protein, and displaying impaired hippocampal function and robust pathology from an early age. Spectral analysis confirmed that the congophilic plaque cores were composed of protein in a beta-sheet conformation. The amide I maximum of plaque cores was at 1623 cm(-1), and unlike for in vitro Abeta fibrils, the high-frequency (1680-1690 cm(-1)) component attributed to antiparallel beta-sheet was not observed. A significant elevation in phospholipids was found around dense-core plaques in TgCRND8 mice ranging in age from 5 to 21 months. In contrast, diffuse plaques were not associated with IR detectable changes in protein secondary structure or relative concentrations of any other tissue components.     

Up‐regulation of the type 3 ryanodine receptor is neuroprotective in the TgCRND8 mouse model of Alzheimer’s disease

The cellular pathology of Alzheimer’s disease is progressive and protracted leading eventually to considerable neuronal death. The underlying mechanisms of the pathology are complex but changes in the control of intracellular Ca²⁺ are believed to contribute to the demise of neurons. In this study, we investigated the functional consequences of an increase in the expression of the type 3 isoform of the ryanodine receptor (RyR3). We found that although cortical neurons from TgCRND8 mice secreted significantly more amyloid beta protein and showed significantly increased RyR3 expression, they were no more sensitive to cell stress than non‐transgenic neurons. Furthermore, despite increased intracellular Ca²⁺ release in response to ryanodine, we found that basal Ca²⁺, K⁺‐evoked Ca²⁺ responses, and capacitative Ca²⁺ entry were no different in TgCRND8 neurons compared with non‐transgenic neurons. Therefore, as RyR3 up‐regulation did not affect neuronal health or global Ca²⁺ homeostasis, we investigated the effect of reducing RyR3 expression using small interfering RNA. Surprisingly, a reduction of RyR3 expression in TgCRND8, but not in non‐transgenic, neurons increased neuronal death. These data reveal a new role for RyR3 and indicate a novel potential therapeutic target to delay or prevent the progression of Alzheimer’s disease.     

Demonstration of aluminum in amyloid fibers in the cores of senile plaques in the brains of patients with Alzheimer’s disease

Aluminum (Al) exposure has been reported to be a risk factor for Alzheimer’s disease (senile dementia of Alzheimer type), although the role of Al in the etiology of Alzheimer’s disease remains controversial. We examined the presence of Al in the Alzheimer’s brain using energy-dispersive X-ray spectroscopy combined with transmission electron microscopy (TEM-EDX). TEM-EDX analysis allows simultaneous imaging of subcellular structures with high spatial resolution and analysis of small quantities of elements contained in the same subcellular structures. We identified senile plaques by observation using TEM and detected Al in amyloid fibers in the cores of senile plaques located in the hippocampus and the temporal lobe by EDX. Phosphorus and calcium were also present in the amyloid fibers. No Al could be detected in the extracellular space in senile plaques or in the cytoplasm of nerve cells. In this study, we demonstrated colocalization of Al and beta-amyloid (Abeta) peptides in amyloid fibers in the cores of senile plaques. The results support the following possibilities in the brains of patients with Alzheimer’s disease: Al could be involved in the aggregation of Abeta peptides to form toxic fibrils; Al might induce Abeta peptides into the beta-sheet structure; and Al might facilitate iron-mediated oxidative reactions, which cause severe damage to brain tissues.     

Synthesis and biological evaluation of radioiodinated 2,5-diphenyl-1,3,4-oxadiazoles for detecting β-amyloid plaques in the brain

This paper describes the synthesis and biological evaluation of a new series of 2,5-diphenyl-1,3,4-oxadiazole (1,3,4-DPOD) derivatives for detecting β-amyloid plaques in Alzheimer’s brains. The affinity for β-amyloid plaques was assessed by an in vitro binding assay using pre-formed synthetic Aβ42 aggregates. The new series of 1,3,4-DPOD derivatives showed affinity for Aβ42 aggregates with K_i values ranging from 20 to 349 nM. The 1,3,4-DPOD derivatives clearly stained β-amyloid plaques in an animal model of Alzheimer’s disease, reflecting the affinity for Aβ42 aggregates in vitro. Compared to 3,5-diphenyl-1,2,4-oxadiazole (1,2,4-DPOD) derivatives, they displayed good penetration of and fast washout from the brain in biodistribution experiments using normal mice. The novel radioiodinated 1,3,4-DPOD derivatives may be useful probes for detecting β-amyloid plaques in the Alzheimer’s brain.     

Insulin receptor mutation results in insulin resistance and hyperinsulinemia but does not exacerbate Alzheimer’s-like phenotypes in mice

Obesity is a risk factor for Alzheimer’s disease (AD), which is characterized by amyloid β depositions and cognitive dysfunction. Although insulin resistance is one of the phenotypes of obesity, its deleterious effects on AD progression remain to be fully elucidated. We previously reported that the suppression of insulin signaling in a mouse with a heterozygous mutation (P1195L) in the gene for the insulin receptor showed insulin resistance and hyperinsulinemia but did not develop diabetes mellitus [15]. Here, we generated a novel AD mouse model carrying the same insulin receptor mutation and showed that the combination of insulin resistance and hyperinsulinemia did not accelerate plaque formation or memory abnormalities in these mice. Interestingly, the insulin receptor mutation reduced oxidative damage in the brains of the AD mice. These findings suggest that insulin resistance is not always involved in the pathogenesis of AD.     

Somatostatin-immunoreactive fiber projections into the brain stem and the spinal cord of the rat

Summary By use of the PAP-immunohistochemical staining technique with serial sections, somatostatin-immunoreactive fiber projections into the brain stem and the spinal cord are described. These projections originate in the periventricular somatostatin-immunoreactive perikarya of the hypothalamus and form three main pathways: (1) along the stria medullaris thalami and the fasciculus retroflexus into the interpeduncular nucleus; (2) along the medial forebrain bundle into the mammillary body; and (3) via the periventricular gray and the bundle of Schütz into the midbrain tegmentum. Densely arranged immunoreactive fibers and/or basket-like fiber terminals are observed within the following afferent systems: somatic afferent systems (nucleus spinalis nervi trigemini, substantia gelatinosa dorsalis of the entire spinal cord), and visceral afferent systems (nucleus solitarius, regio intermediolateralis and substantia gelatinosa of the sacral spinal cord). These projections form terminals around the perikarya of the second afferent neuron. Perikarya of the third afferent neuron are influenced by somatostatin-immunoreactive projections into the auditory system (nucleus dorsalis lemnisci lateralis, nucleus corporis trapezoidei). Furthermore, a somatostatin-immunoreactive fiber projection is found in the ventral part of the medial accessory olivary nucleus, in nuclei of the limbic system (nucleus habenularis medialis, nuclei supramamillaris and mamillaris lateralis) and in the formatio reticularis (nucleus Darkschewitsch, nuclei tegmenti lateralis and centralis, nucleus parabrachialis lateralis, as well as individual perikarya of the reticular formation). Targets of these projections are interneurons within interlocking neuronal chains.Supported by the Deutsche Forschungsgemeinschaft (Grant Nr. Kr 569/3) and Stiftung Volkswagenwerk     

The 21-aminosteroid U-74389F increases the number of glial fibrillary acidic protein-expressing astrocytes in the spinal cord of control and wobbler mice

Summary 1. Wobbler mice suffer an autosomal recessive mutation producing severe motoneuron degeneration and dense astrogliosis, with increased levels of glial fibrillary acidic protein (GFAP) in the spinal cord and brain stem. They have been considered animal models of amyotrophic lateral sclerosis and infantile spinal muscular atrophy. 2. Using Wobbler mice and normal littermates, we investigated the effects of the membrane-active steroid Lazaroid U-74389F on the number of GFAP-expressing astrocytes and glucocorticoid receptors (GR). Lazaroids are inhibitors of oxygen radical-induced lipid peroxidation, and proved beneficial in cases of CNS injury and ischemia. 3. Four days after pellet implantation of U-74389F into Wobbler mice, hyperplasia and hypertophy of GFAP-expressing astrocytes were apparent in the spinal cord ventral and dorsal horn, areas showing already intense astrogliosis in untreated Wobbler mice. In control mice, U-74389F also produced astrocyte hyperplasia and hypertophy in the dorsal horn and hyperplasia in the ventral-lateral funiculi of the cord. 4. Givenin vivo U-74389F did not change GR in spinal cord of Wobbler or control mice, in line with the concept that it is active in membranes but does not bind to GR. Besides, U-74390F did not compete for [³H]dexamethasone binding when addedin vitro. 5. The results suggest that stimulation of proliferation and size of GFAP-expressing astrocytes by U-74389F may be a novel mechanism of action of this compound. The Wobbler mouse may be a valuable animal model for further pharmacological testing of glucocorticoid and nonglucocorticoid steroids in neurodegenerative diseases.     

Rosuvastatin reduces microglia in the brain of wild type and ApoE knockout mice on a high cholesterol diet; Implications for prevention of stroke and AD

We have previously shown that a high cholesterol (HC) diet results in increases in microglia load and levels of the pro-inflammatory cytokine interleukin-6 (IL-6) in the brains of wild type (WT) and apolipoprotein E knockout (ApoE−/−) mice. In the present investigation, we analyzed whether treatment with rosuvastatin, an inhibitor of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, would prevent the increases in inflammatory microglia and IL-6 levels in the brain and plasma of WT and ApoE−/− mice. We report that a HC diet resulted in an increased microglia load in the brains of WT and ApoE−/− mice, in support of our previous study. Treatment with rosuvastatin significantly decreased the microglia load in the brains of WT and ApoE−/− mice on a HC diet. Rosuvastatin treatment resulted in lowered plasma IL-6 levels in WT mice on a HC diet. However, in the present study the number of IL-6 positive cells in the brain was not significantly affected by a HC diet. A recent clinical study has shown that rosuvastatin reduces risk of ischemic stroke in patients with high plasma levels of the inflammatory marker C-reactive protein by 50%. The results from our study show that rosuvastatin reduces inflammatory cells in the brain. This finding is essential for furthering the prevention and treatment of neurodegenerative diseases such as Alzheimer’s disease (AD) and stroke.     

Analysis of interaction between etoricoxib and tramadol against mechanical hyperalgesia of spinal cord injury in rats

Drug combinations have the potential advantage of greater analgesia over monotherapy. The present study was aimed to assess any possible interaction (additive or potentiation) in the antinociceptive effects of etoricoxib; a novel cyclooxygenase-2 inhibitor, and tramadol; a typical opioid agonist when administered in combination against mechanical hyperalgesia induced by spinal cord injury in rats. The nature of interaction was analyzed using surface of synergistic interaction (SSI) analysis and an isobolographic analysis. Etoricoxib or tramadol when administered alone to rats, exhibited different antihyperalgesic potencies (ED50 etoricoxib: 0.58+/-0.19 mg/kg, po; ED50 tramadol: 9.85+/-0.57 mg/kg, po). However, both the drugs were found to be long acting against this model of hyperalgesia. Further, etoricoxib and tramadol were co-administered in fixed ratios of ED50 fractions. One combination (0.29/4.79 mg/kg, po: etoricoxib/tramadol) exhibited additivity and other three combinations (0.15/2.39, 0.08/1.19, and 0.04/0.59 mg/kg, po: etoricoxib/tramadol) resulted in potentiation when analyzed by SSI. The SSI was calculated from the total antihyperalgesic effect produced by the combination after the subtraction of the antihyperalgesic effect produced by each of the individual drug. In the isobolographic analysis, the experimental ED50 was found to be far below the line of additivity also indicating a significant (P < 0.05) synergistic antihyperalgesic effect when etoricoxib and tramadol was co-administered to rats. The synergistic antihyperalgesic effect of etoricoxib and tramadol combination suggests that these combinations may have clinical utility in mechanical hyperalgesia associated with spinal injury.     

Synthesis and evaluation of indolinyl- and indolylphenylacetylenes as PET imaging agents for beta-amyloid plaques

Two new phenylacetylene derivatives, 5-((4-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)phenyl)ethynyl)indoline 8 and 5-((4-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)phenyl)ethynyl)-1H-indole 14, targeting β-amyloid (Aβ) plaques have been prepared. In vitro binding carried out in tissue homogenates prepared from postmortem AD brains with [¹²⁵I]IMPY (6-iodo-2-(4′-dimethylamino-)phenyl-imidazo[1,2-a]pyridine) as the radioligand indicated good binding affinities (K_i = 4.0 and 1.5 nM for 8 and 14, respectively). Brain penetration of the corresponding radiofluorinated ligands, evaluated in the normal mice, showed good initial brain penetration (4.50 and 2.43% ID/g (injected dose/gram) for [¹⁸F]8 and [¹⁸F]14 at 2 min after injection) with moderate to low washout rates from the brain (1.71% ID/g at 2 h and 2.10% ID/g at 3 h, respectively). Autoradiography and homogenate binding studies demonstrated the high specific binding of [¹⁸F]14 to the Aβ plaques; however, [¹⁸F]8 showed low specific binding. These preliminary results identified that indolylphenylacetylene, 14, may be a good lead for further structural modification to develop a useful Aβ plaque imaging agent.     

Release in vivo of Met-enkephalin and encrypted forms of Met-enkephalin from brain and spinal cord of the anesthetized cat

Processing of the proenkephalin molecule will result in peptide fragments in which the pentapeptide YGGFM is included. We have employed a molecular sieve (2 kDa) separation, enzyme hydrolysis radioimmunoassay (RIA) treatment sequence which permits concurrent measurement of Met-enkephalin (Enk) and several enkephalin-encrypting (X-Enk) peptides in a single sample. Using this protocol, the release of Enk and X-Enk (total Enk - Enk) greater and less than 2 kDa from spinal cord and the mesencephalic aqueductal grey was assessed under resting conditions and during stimulation of the sciatic nerve in the chloralose-urethane anesthetized cat. Under resting conditions measurable levels of Enk (10.5±4.7; 9.1±2.1 pg/min) and X-Enk (47.8±19.7; 45.7±12.3 pg/min) are found in aqueductal and spinal superfusates, respectively. The X-Enk measured under resting and evoked conditions in aqueductal and spinal perfusates is associated almost exclusively (>90–95%) with fragments >2 kDa in size. These results, showing the relative absence of detectable levels of X-Enk forms <2 kDa, were confirmed by reverse phase chromatography. During sciatic nerve stimulation, the levels of both Enk and X-Enk were mildly elevated in spinal and ventricular perfusates. With the addition of thiorphan (10⁻⁵ M), though there was no effect on the resting release of either Enk or X-Enk, the levels of Enk measured under evoked conditions were significantly augmented in both ventricular and spinal perfusates.     

NIMA-related kinase 7 amplifies NLRP3 inflammasome pro-inflammatory signaling in microglia/macrophages and mice models of spinal cord injury

BackgroundNIMA-related kinase-7 (NEK7) is a serine/threonine kinase that drives cell-cycle dynamics by modulating mitotic spindle formation and cytokinesis. It is also a crucial modulator of the pro-inflammatory effects of NOD-like receptor 3 (NLRP3) inflammasome. However, the role of NEK7 in microglia/macrophages post-spinal cord injury (SCI) is not well defined.MethodsIn this study, we performed both in vivo and in vitro experiments. Using an in vivo mouse SCI model, NEK7 siRNAs were administered intraspinally. For in vitro analysis, BV-2 microglia cells with NEK7-siRNA were stimulated with 1 μg/ml lipopolysaccharide (LPS) and 2 mM Adenosine triphosphate (ATP).ResultsHere, we found that the mRNA and protein levels of NEK7 and NLRP3 inflammasomes were upregulated in spinal cord tissues of injured mice and BV-2 microglia cells exposed to Lipopolysaccharide (LPS) and Adenosine triphosphate (ATP). Further experiments established that NEK7 and NLRP3 interacted in BV-2 microglia cells, an effect that was eliminated following NEK7 ablation. Moreover, NEK7 ablation suppressed the activation of NLRP3 inflammasomes. Although NEK7 inhibition did not significantly improve motor function post-SCI in mice, it was found to attenuate local inflammatory response and inhibit the activation of NLRP3 inflammasome in microglia/macrophages of the injured spinal cord.ConclusionNEK7 amplifies NLRP3 inflammasome pro-inflammatory signaling in BV-2 microglia cells and mice models of SCI. Therefore, agents targeting the NEK7/NLRP3 signaling offers great promise in the treatment of inflammatory response post-SCI.     

Transplantation of mature adipocyte-derived dedifferentiated fat cells promotes locomotor functional recovery by remyelination and glial scar reduction after spinal cord injury in mice

Mature adipocyte-derived dedifferentiated fat cells (DFAT) have a potential to be useful as new cell-source for cell-based therapy for spinal cord injury (SCI), but the mechanisms remain unclear. The objective of this study was to examine whether DFAT-induced functional recovery is achieved through remyelination and/or glial scar reduction in a mice model of SCI. To accomplish this we subjected adult female mice (n = 22) to SCI. On the 8th day post-injury locomotor tests were performed, and the mice were randomly divided into two groups (control and DFAT). The DFAT group received stereotaxic injection of DFAT, while the controls received DMEM medium. Functional tests were conducted at repeated intervals, until the 36th day, and immunohistochemistry or staining was performed on the spinal cord sections. DFAT transplantation significantly improved locomotor function of their hindlimbs, and promoted remyelination and glial scar reduction, when compared to the controls. There were significant and positive correlations between promotion of remyelination or/and reduction of glial scar, and recovery of locomotor function. Furthermore, transplanted DFAT expressed markers for neuron, astrocyte, and oligodendrocyte, along with neurotrophic factors, within the injured spinal cord. In conclusion, DFAT-induced functional recovery in mice after SCI is probably mediated by both cell-autonomous and cell-non-autonomous effects on remyelination of the injured spinal cord.     

Wnt/BMP signal integration regulates the balance between proliferation and differentiation of neuroepithelial cells in the dorsal spinal cord

Multiple signaling pathways regulate proliferation and differentiation of neural progenitor cells during early development of the central nervous system (CNS). In the spinal cord, dorsal signaling by bone morphogenic protein (BMP) acts primarily as a patterning signal, while canonical Wnt signaling promotes cell cycle progression in stem and progenitor cells. However, overexpression of Wnt factors or, as shown here, stabilization of the Wnt signaling component beta-catenin has a more prominent effect in the ventral than in the dorsal spinal cord, revealing local differences in signal interpretation. Intriguingly, Wnt signaling is associated with BMP signal activation in the dorsal spinal cord. This points to a spatially restricted interaction between these pathways. Indeed, BMP counteracts proliferation promoted by Wnt in spinal cord neuroepithelial cells. Conversely, Wnt antagonizes BMP-dependent neuronal differentiation. Thus, a mutually inhibitory crosstalk between Wnt and BMP signaling controls the balance between proliferation and differentiation. A model emerges in which dorsal Wnt/BMP signal integration links growth and patterning, thereby maintaining undifferentiated and slow-cycling neural progenitors that form the dorsal confines of the developing spinal cord.     

Imaging and spatial distribution of beta-amyloid peptide and metal ions in Alzheimer's plaques by laser ablation-inductively coupled plasma-mass spectrometry

Laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) has been developed as a new strategy for detection and imaging of β-amyloid protein in immunohistochemical sections from the brains of a transgenic mouse model of Alzheimer’s disease. The distribution of β-amyloid deposits in tissue was based on measurement of Eu- and Ni-coupled antibodies. The laser-based methodologies (spot ablation, single line raster, and two-dimensional imaging) were also used to detect and map trace element distributions and thus provide a novel probe for both elemental and protein data. We also report the combination of laser capture microdissection with LA-ICP-MS as an alternative strategy for microanalysis of immunohistochemical sections.     

Discovery and optimization of a novel, selective and brain penetrant M1 positive allosteric modulator (PAM): the development of ML169, an MLPCN probe

This Letter describes a chemical lead optimization campaign directed at VU0108370, a weak M₁ PAM hit with a novel chemical scaffold from a functional HTS screen within the MLPCN. An iterative parallel synthesis approach rapidly established SAR for this series and afforded VU0405652 (ML169), a potent, selective and brain penetrant M₁ PAM with an in vitro profile comparable to the prototypical M₁ PAM, BQCA, but with an improved brain to plasma ratio.     

Guanosine reduces apoptosis and inflammation associated with restoration of function in rats with acute spinal cord injury

Spinal cord injury results in progressive waves of secondary injuries, cascades of noxious pathological mechanisms that substantially exacerbate the primary injury and the resultant permanent functional deficits. Secondary injuries are associated with inflammation, excessive cytokine release, and cell apoptosis. The purine nucleoside guanosine has significant trophic effects and is neuroprotective, antiapoptotic in vitro, and stimulates nerve regeneration. Therefore, we determined whether systemic administration of guanosine could protect rats from some of the secondary effects of spinal cord injury, thereby reducing neurological deficits. Systemic administration of guanosine (8 mg/kg per day, i.p.) for 14 consecutive days, starting 4 h after moderate spinal cord injury in rats, significantly improved not only motor and sensory functions, but also recovery of bladder function. These improvements were associated with reduction in the inflammatory response to injury, reduction of apoptotic cell death, increased sparing of axons, and preservation of myelin. Our data indicate that the therapeutic action of guanosine probably results from reducing inflammation resulting in the protection of axons, oligodendrocytes, and neurons and from inhibiting apoptotic cell death. These data raise the intriguing possibility that guanosine may also be able to reduce secondary pathological events and thus improve functional outcome after traumatic spinal cord injury in humans.     

Heat shock proteins as biomarkers for the rapid detection of brain and spinal cord ischemia: a review and comparison to other methods of detection in thoracic aneurysm repair

The heat shock proteins (HSPs) are members of highly conserved families of molecular chaperones that have multiple roles in vivo. We discuss the HSPs in general, and Hsp70 and Hsp27 in particular, and their rapid induction by severe stress in the context of tissue and organ expression in physiology and disease. We describe the current state of knowledge of the relationship and interactions between extra- and intracellular HSPs and describe mechanisms and significance of extracellular expression of HSPs. We focus on the role of the heat shock proteins as biomarkers of central nervous system (CNS) ischemia and other severe stressors and discuss recent and novel technologies for rapid measurement of proteins in vivo and ex vivo. The HSPs are compared to other proposed small molecule biomarkers for detection of CNS injury and to other methods of detecting brain and spinal cord ischemia in real time. While other biomarkers may be of use in prognosis and in design of appropriate therapies, none appears to be as rapid as the HSPs; therefore, no other measurement appears to be of use in the immediate detection of ongoing severe ischemia with the intention to immediately intervene to reduce the severity or risk of permanent damage.