Huntingtin Decreases Susceptibility to a Spontaneous Seizure Disorder in FVN/B Mice.

Huntington disease (HD) is an adult-onset neurodegenerative disorder that is caused by a trinucleotide CAG repeat expansion in the HTT gene that codes for the protein huntingtin (HTT in humans or Htt in mice). HTT is a multi-functional, ubiquitously expressed protein that is essential for embryonic survival, normal neurodevelopment, and adult brain function. The ability of wild-type HTT to protect neurons against various forms of death raises the possibility that loss of normal HTT function may worsen disease progression in HD. Huntingtin-lowering therapeutics are being evaluated in clinical trials for HD, but concerns have been raised that decreasing wild-type HTT levels may have adverse effects. Here we show that Htt levels modulate the occurrence of an idiopathic seizure disorder that spontaneously occurs in approximately 28% of FVB/N mice, which we have called FVB/N Seizure Disorder with SUDEP (FSDS). These abnormal FVB/N mice demonstrate the cardinal features of mouse models of epilepsy including spontaneous seizures, astrocytosis, neuronal hypertrophy, upregulation of brain-derived neurotrophic factor (BDNF), and sudden seizure-related death. Interestingly, mice heterozygous for the targeted inactivation of Htt (Htt+/- mice) exhibit an increased frequency of this disorder (71% FSDS phenotype), while over-expression of either full length wild-type HTT in YAC18 mice or full length mutant HTT in YAC128 mice completely prevents it (0% FSDS phenotype). Examination of the mechanism underlying huntingtin's ability to modulate the frequency of this seizure disorder indicated that over-expression of full length HTT can promote neuronal survival following seizures. Overall, our results demonstrate a protective role for huntingtin in this form of epilepsy and provide a plausible explanation for the observation of seizures in the juvenile form of HD, Lopes-Maciel-Rodan syndrome, and Wolf-Hirschhorn syndrome. Adverse effects caused by decreasing huntingtin levels have ramifications for huntingtin-lowering therapies that are being developed to treat HD.

Impact of wet-lab protocols on quality of whole-genome short-read sequences from foodborne microbial pathogens.

For successful elucidation of a food-borne infection chain, the availability of high-quality sequencing data from suspected microbial contaminants is a prerequisite. Commonly, those investigations are a joint effort undertaken by different laboratories and institutes. To analyze the extent of variability introduced by differing wet-lab procedures on the quality of the sequence data we conducted an interlaboratory study, involving four bacterial pathogens, which account for the majority of food-related bacterial infections: Campylobacter spp., Shiga toxin-producing Escherichia coli, Listeria monocytogenes, and Salmonella enterica. The participants, ranging from German federal research institutes, federal state laboratories to universities and companies, were asked to follow their routine in-house protocols for short-read sequencing of 10 cultures and one isolated bacterial DNA per species. Sequence and assembly quality were then analyzed centrally. Variations within isolate samples were detected with SNP and cgMLST calling. Overall, we found that the quality of Illumina raw sequence data was high with little overall variability, with one exception, attributed to a specific library preparation kit. The variability of Ion Torrent data was higher, independent of the investigated species. For cgMLST and SNP analysis results, we found that technological sequencing artefacts could be reduced by the use of filters, and that SNP analysis was more suited than cgMLST to compare data of different contributors. Regarding the four species, a minority of Campylobacter isolate data showed the in comparison highest divergence with regard to sequence type and cgMLST analysis. We additionally compared the assembler SPAdes and SKESA for their performance on the Illumina data sets of the different species and library preparation methods and found overall similar assembly quality metrics and cgMLST statistics.

Acidic fibroblast growth factor (FGF) potentiates glial-mediated neurotoxicity by activating FGFR2 IIIb protein.

Previous studies indicate that astrocytes are the brain cells that express acidic fibroblast growth factor (aFGF) and that the expression is increased upon activation. However, there has been no study investigating the significance of this phenomenon. Here we report that aFGF treatment of IFNγ-stimulated human astrocytes, and LPS/IFNγ-stimulated human microglia, enhances their secretion of inflammatory cytokines and other materials toxic to human neuroblastoma SH-SY5Y cells. The mechanism of aFGF enhancement involves stimulation of the receptor FGFR2 IIIb. We show by RT-PCR that this receptor, but not other FGF receptors, is robustly expressed by astrocytes and microglia. We establish by Western blotting, and immunohistochemistry on postmortem human brain tissue that the FGFR2 IIIb protein is expressed by both of these glial cell types. We blocked the inflammatory stimulant action of aFGF by transfecting microglia and astrocytes with a small inhibitory RNA (siRNA) to FGFR2 IIIb as well as by removal of aFGF using an anti-aFGF antibody. Treatment with bFGF in combination with the stimulants was without effect, but together with aFGF, it partially counteracted the action of aFGF, indicating that it may be a weak antagonist of FGFR2 IIIb. The inflammatory effect was also attenuated by treatment with inhibitors of protein kinase C, Src tyrosine kinase, and MEK-1/2 indicating the involvement of these intracellular pathways. Our data suggest that inhibition of expression or release of aFGF could have therapeutic potential by inhibiting inflammation in neurodegenerative diseases such as Alzheimer disease where many neuroinflammatory molecules are prominently expressed.

Inflammation in transgenic mouse models of neurodegenerative disorders.

Much evidence is available that inflammation contributes to the development of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and Huntington's disease. Our review investigates how well current mouse models reflect this aspect of the pathogenesis. Transgenic models of AD have been available for several years and are the most extensively studied. Modulation of cytokine levels, activation of microglia and, to a lesser extent, activation of the complement system have been reported. Mouse models of PD and HD so far show less evidence for the involvement of inflammation. An increasing number of transgenic mouse strains is being created to model human neurodegenerative diseases. A perfect model should reflect all aspects of a disease. It is important to evaluate continuously the models for their match with the human disease and reevaluate them in light of new findings in human patients. Although none of the transgenic mouse models recapitulates all aspects of the human disorder they represent, all models have provided valuable information on basic molecular pathways. In particular, the mouse models of Alzheimer disease have also led to the development of new therapeutic strategies such as vaccination and modulation of microglial activity.

Astrocytes are GABAergic cells that modulate microglial activity.

GABA is assumed to function in brain only as an inhibitory neurotransmitter. Here we report a much broader CNS role. We show that human astrocytes are GABAergic cells, and that human microglia are GABAceptive cells. We show that in adult human brain tissue, astrocytes immunostain for the GABA synthesizing enzyme GAD 67, the GABA metabolizing enzyme GABA-T and the GABA(A) and GABA(B) receptors. The intensity of staining is comparable or greater to that observed for known inhibitory neurons. We show that cultured human astrocytes strongly express the mRNA and protein for GAD 67, as well as GABA-T, and the GABA(A) and GABA(B) receptors. We further show that cultured human microglia express the mRNA and protein for GABA-T, in addition to the GABA(A) and GABA(B) receptors characterizing them as GABAceptive cells. We demonstrate that GABA suppresses the reactive response of both astrocytes and microglia to the inflammatory stimulants lipopolysaccharide (LPS) and interferon-γ by inhibiting induction of inflammatory pathways mediated by NFκB and P38 MAP kinase. This results in a reduced release of the inflammatory cytokines TNFα and IL-6 and an attenuation of conditioned medium neurotoxicity toward neuroblastoma SH-SY5Y cells. These inhibitory reactions are partially mimicked by the GABA(A) receptor agonist muscimol and the GABA(B) receptor agonist baclofen, indicating that GABA can stimulate both types of receptors in astrocytes as well as microglia. We conclude that the antiinflammatory actions of GABA offer new therapeutic opportunities since agonists should enhance the effectiveness of other antiinflammatory agents that operate through non-GABA pathways.

Distinct glutaminyl cyclase expression in Edinger-Westphal nucleus, locus coeruleus and nucleus basalis Meynert contributes to pGlu-Abeta pathology in Alzheimer's disease.

Glutaminyl cyclase (QC) was discovered recently as the enzyme catalyzing the pyroglutamate (pGlu or pE) modification of N-terminally truncated Alzheimer's disease (AD) Abeta peptides in vivo. This modification confers resistance to proteolysis, rapid aggregation and neurotoxicity and can be prevented by QC inhibitors in vitro and in vivo, as shown in transgenic animal models. However, in mouse brain QC is only expressed by a relatively low proportion of neurons in most neocortical and hippocampal subregions. Here, we demonstrate that QC is highly abundant in subcortical brain nuclei severely affected in AD. In particular, QC is expressed by virtually all urocortin-1-positive, but not by cholinergic neurons of the Edinger-Westphal nucleus, by noradrenergic locus coeruleus and by cholinergic nucleus basalis magnocellularis neurons in mouse brain. In human brain, QC is expressed by both, urocortin-1 and cholinergic Edinger-Westphal neurons and by locus coeruleus and nucleus basalis Meynert neurons. In brains from AD patients, these neuronal populations displayed intraneuronal pE-Abeta immunoreactivity and morphological signs of degeneration as well as extracellular pE-Abeta deposits. Adjacent AD brain structures lacking QC expression and brains from control subjects were devoid of such aggregates. This is the first demonstration of QC expression and pE-Abeta formation in subcortical brain regions affected in AD. Our results may explain the high vulnerability of defined subcortical neuronal populations and their central target areas in AD as a consequence of QC expression and pE-Abeta formation.

TDP-43 pathology in familial British dementia.

Trans-activation-responsive DNA-binding protein 43 (TDP-43) is a component of pathological inclusions in amyotrophic lateral sclerosis and several forms of sporadic and familial frontotemporal lobar degeneration. This has suggested defining a new class of diseases known as TDP-43 proteinopathies. However, it has been reported more recently that TDP-43 positive inclusions occur in other neurodegenerative disorders such as Alzheimer's disease, Dementia with Lewy Bodies and Parkinsonism dementia complex of Guam. Here we report the occurrence of TDP-43 inclusions in one other neurodegenerative disorder: familial British dementia. Using a variety of antibodies against phosphorylated and non-phosphorylated TDP-43 epitopes, we found intense accumulation occurred in the form of dystrophic neurites, neuronal cytoplasmic inclusions and was also occasionally associated with neurofibrillary tangles. Double immunostaining revealed that TDP-43 and tau aggregates were rarely directly colocalized, but co-existed in the same neurons as separate inclusions. Double staining with ubiquitin showed a direct colocalization with TDP-43. The phosphorylation-dependent TDP-43 antibodies proved superior to phosphorylation-independent antibodies in revealing pathological inclusions since the former did not stain non-phosphorylated TDP-43 in normal nuclei. Our results support the concept that TDP-43 pathology is not narrowly restricted, but is involved in the etiology of many neurodegenerative disorders.

Colocalization of transactivation-responsive DNA-binding protein 43 and huntingtin in inclusions of Huntington disease.

Transactivation-responsive DNA-binding protein 43 (TDP-43) is a component of pathological inclusions in amyotrophic lateral sclerosis and several forms of sporadic and familial frontotemporal lobar degeneration. Transactivation-responsive DNA-binding protein 43-immunostained inclusions have also been found in other neurodegenerative disorders including Alzheimer disease, dementia with Lewy bodies, and parkinsonism dementia complex of Guam. Here, we analyzed the occurrence of TDP-43 immunostaining in Huntington disease, which is characterized by inclusions containing mutated huntingtin. In all Huntington disease cases studied, TDP-43 was frequently colocalized with huntingtin in dystrophic neurites and various intracellular inclusions, but not in intranuclear inclusions; the latter were only stained with huntingtin and anti-ubiquitin antibodies. Two phosphorylation-dependent TDP-43 antibodies proved to be superior for detecting pathological inclusions because they did not stain nonphosphorylated TDP-43 in normal nuclei; staining of normal nuclei with phosphorylation-independent antibodies obscured the inclusions. Our results further add to the hypothesis that TDP-43 may be involved in the pathology of a variety of neurodegenerative disorders.

Inflammatory aspects of Alzheimer disease and other neurodegenerative disorders.

Alzheimer and a number of other neurodegenerative diseases are characterized by the presence of reactive microglia and reactive astrocytes in association with the lesions. The classic view that microglia exist primarily in either a resting or activated state needs to be broadened in view of recent results. Resting microglia are in constant activity sampling their surround. Activated microglia may be pro-inflammatory, releasing inflammatory cytokines and other inflammatory mediators, or anti-inflammatory, promoting the healing process. There is evidence that microglial phagocytosis is more powerful in the anti-inflammatory state. Activated astrocytes also have pro-inflammatory and anti-inflammatory properties. In the pro-inflammatory state they release inflammatory cytokines. In the anti-inflammatory state they release various growth factors. In AD and other neurodegenerative diseases, both microglia and astrocytes are in a pro-inflammatory state. From a therapeutic point of view it is desirable to find methods of tipping the balance towards an anti-inflammatory state for both types of glia.

Adhesion of exogenous human microglia and THP-1 cells to amyloid plaques of postmortem Alzheimer's disease brain.

Microglial phagocytosis of amyloid-beta (Abeta) deposits is involved in Abeta clearance in vivo. To explore the ability of microglia to phagocytose beta, we cultured human microglia or human monocytic THP-1 cells directly on unfixed frontal cortex sections of an Alzheimer disease (AD) case. We found that when these cells were activated by lipopolysaccharide (LPS) plus interferon (IFN)-gamma, they developed ameboid morphology and formed clusters around and attaching to amyloid plaques in the tissue. Some cells adhering to these plaques internalized Abeta and some appeared to be degraded. Nevertheless, no significant reduction of the overall Abeta burden was observed. If the cells were not stimulated, they adhered poorly to the sections. We quantified THP-1 cell adhesion to an AD brain section compared with a normal brain section and found it to be significantly increased. If a brain section was rinsed with phosphate buffered saline containing 0.1% Triton X-100, most LPS/IFN-gamma-activated THP-1 cells failed to adhere. However, in co-culture with human astrocytes, the number of adherent THP-1 cells was significantly increased. These results suggest that human microglial cells are capable of adhering to and phagocytosing post mortem AD plaque material but activation may be necessary. Astrocytes may further enhance the process.

GAD65, GAD67, and GABAT immunostaining in human brain and apparent GAD65 loss in Alzheimer's disease.

The GABAergic system is the main inhibitory neurotransmitter system in the vertebrate brain. Although it is well established that the GABAergic system is affected in neuropsychiatric disorders, in Alzheimer's disease (AD) it has been considered to be relatively spared. In this study we describe the immunohistochemical localization of the main enzymes of the GABAergic system; glutamate decarboxylase 65 (GAD65), GAD67, and GABA transferase (GABAT) in human brain. In neocortex, hippocampus, basal ganglia, and cerebellum, GAD65 and GAD67 immunoreactivity were found in neuropil granules, possibly axonal boutons or terminals, and in a subset of small to midsized neurons. GAD65 preferentially stained neuropil granules, while GAD67 preferentially stained neuronal cell bodies. GABAT intensely labeled many types of neurons and glia cells. While GAD65 and GAD67 stained the cytoplasm of cells homogeneously, GABAT labeling appeared irregular and granular. GAD65 immunoreactivity of neurons and neuropil was severely reduced in AD middle temporal gyrus, hippocampus, and putamen as determined by fluorescence and light microscopic immunohistochemistry. Western blotting revealed a similar reduction of GAD65, but not GAD67, protein levels in the middle temporal gyrus of AD. Our results suggest that the GABAergic system is more severely affected in AD than previously reported. This deficit may contribute to AD pathogenesis by loss of GABAergic inhibitory activity.

Upregulation and expression patterns of the angiogenic transcription factor ets-1 in Alzheimer's disease brain.

Immunohistochemical staining has been used to determine expression patterns of the angiogenic transcription factor, Ets-1, in the brains of Alzheimer's disease (AD) individuals. Brain tissue from non-demented controls showed little expression of Ets-1 whereas in AD brain tissue, Ets-1 was ubiquitously expressed in cortex and hippocampus. Double immunostaining with von Willerbrand factor demonstrated prominent Ets-1 intravascular immunoreactivity (ir) in AD cortical microvessels. In addition, Ets-1 also exhibited extravascular expression characterized by a diffuse pattern of Ets-1 ir in AD brain. Double staining also showed Ets-1 colocalization in microvasculature with the potent angiogenic agent, vascular endothelial growth factor (VEGF). Cell-associated tumor necrosis factor-α (TNF-α), a pro-inflammatory cytokine with pro-angiogenic activity, was primarily associated with diffuse extravascular Ets-1 ir. Clusters of HLA-DR positive microglia, resident immune cells of brain which release TNF-α, were also localized with diffuse Ets-1. Intravascular Ets-1 ir was maximally co-localized with soluble amyloid-ß peptide (Aß), Aß1-40, in microvasculature whereas diffuse extravascular Ets-1 ir appeared in proximity to Aß plaques in brain parenchyma. Similar overall results were obtained for patterns of Ets-1 staining in AD hippocampal tissue. This work provides novel findings on expression of the angiogenic transcription factor, Ets-1, in vascular remodeling and its association with pro-angiogenic factors, reactive microglia, and Aß deposition in AD brain.

Differential pathways for interleukin-1ß production activated by chromogranin A and amyloid ß in microglia.

Although chromogranin A (CGA) is frequently present in Alzheimer's disease (AD), senile plaques associated with microglial activation, little is known about basic difference between CGA and fibrillar amyloid-ß (fAß) as neuroinflammatory factors. Here we have compared the interleukin-1ß (IL-1ß) production pathways by CGA and fAß in microglia. In cultured microglia, production of IL-1ß was induced by CGA, but not by fAß. CGA activated both nuclear factor-κB (NF-κB) and pro-caspase-1, whereas fAß activated pro-caspase-1 only. For the activation of pro-caspase-1, both CGA and fAß needed the enzymatic activity of cathepsin B (CatB), but only fAß required cytosolic leakage of CatB and the NLRP3 inflammasome activation. In contrast, fAß induced the IL-1ß secretion from microglia isolated from the aged mouse brain. In AD brain, highly activated microglia, which showed intense immunoreactivity for CatB and IL-1ß, surrounded CGA-positive plaques more frequently than Aß-positive plaques. These observations indicate differential pathways for the microglial IL-1ß production by CGA and fAß, which may aid in better understanding of the pathological significance of neuroinflammation in AD.

Angiopoietin 2 mediates microvascular and hemodynamic alterations in sepsis.

Septic shock is characterized by increased vascular permeability and hypotension despite increased cardiac output. Numerous vasoactive cytokines are upregulated during sepsis, including angiopoietin 2 (ANG2), which increases vascular permeability. Here we report that mice engineered to inducibly overexpress ANG2 in the endothelium developed sepsis-like hemodynamic alterations, including systemic hypotension, increased cardiac output, and dilatory cardiomyopathy. Conversely, mice with cardiomyocyte-restricted ANG2 overexpression failed to develop hemodynamic alterations. Interestingly, the hemodynamic alterations associated with endothelial-specific overexpression of ANG2 and the loss of capillary-associated pericytes were reversed by intravenous injections of adeno-associated viruses (AAVs) transducing cDNA for angiopoietin 1, a TIE2 ligand that antagonizes ANG2, or AAVs encoding PDGFB, a chemoattractant for pericytes. To confirm the role of ANG2 in sepsis, we i.p. injected LPS into C57BL/6J mice, which rapidly developed hypotension, acute pericyte loss, and increased vascular permeability. Importantly, ANG2 antibody treatment attenuated LPS-induced hemodynamic alterations and reduced the mortality rate at 36 hours from 95% to 61%. These data indicate that ANG2-mediated microvascular disintegration contributes to septic shock and that inhibition of the ANG2/TIE2 interaction during sepsis is a potential therapeutic target.

Optineurin in Huntington's disease intranuclear inclusions.

Optineurin mutations cause adult-onset primary open-angle glaucoma and have been associated with some familial forms of amyotrophic lateral sclerosis (ALS). Optineurin is involved in many cellular processes and interacts with a variety of proteins, among them huntingtin (htt). Here we report that in Huntington's disease (HD) cortex, optineurin frequently occurs in neuronal intranuclear inclusions, and to a lesser extent, in inclusions in the neuropil and in perikarya. Most intranuclear optineurin-positive inclusions were co-labeled for ubiquitin, but they were only occasionally and more weakly co-labeled for htt. Optineurin-labeled neuropil and perikaryal inclusions were commonly co-labeled for ubiquitin and htt. Although these inclusions were common in cortex, they were rare in striatum. Our results show that in HD optineurin is present in intranuclear, neuropil and perikaryal inclusions. It is not clear whether this indicates a primary involvement in the disease process. In HD, the known interaction of htt and optineurin may suggest that a different process takes place as compared to other neurodegenerative disorders.

Selective inhibition of the membrane attack complex of complement by low molecular weight components of the aurin tricarboxylic acid synthetic complex.

Complement plays a vital role in both the innate and adaptive immune systems. It recognizes a target, opsonizes it, generates anaphylatoxins, and directly kills cells through the membrane attack complex (MAC). This final function, which assembles C5b-9(n) on viable cell surfaces, can kill host cells through bystander lysis. Here we identify for the first time compounds that can inhibit bystander lysis while not interfering with the other essential functions of complement. We show that aurin tricarboxylic acid (ATA), aurin quadracarboxylic acid (AQA), and aurin hexacarboxylic acid (AHA), block the addition of C9 to C5b-8 so that the MAC cannot form. These molecules inhibit hemolysis of human, rat, and mouse red cells with a half maximal inhibitory concentration (IC(50)) in the nanomolar range. When given orally to Alzheimer disease type B6SJL-Tg mice, they inhibit MAC formation in serum and improve memory retention. On autopsy, they show no evidence of harm to any organ. Aurin tricarboxylic acid, aurin quadracarboxylic acid, and aurin hexacarboxylic acid may be effective therapeutic agents in Alzheimer disease and other degenerative disorders where self damage from the MAC occurs.

Comparison of Vascular Perturbations in an Aß-Injected Animal Model and in AD Brain.

The validity of amyloid-ß peptide (Aß(1-42)) intrahippocampal injection, as an animal model of Alzheimer's disease (AD), has previously been considered in terms of inflammatory reactivity and neuronal damage. In this work, we have extended the testing of the animal model to vasculature by comparison of selected properties of microvessels in vivo with those in human AD brain tissue. The injection of Aß(1-42), relative to control PBS (phosphate buffered saline), increased the mean number of microvessels and diminished the mean length of microvessels in the molecular layer of dentate gyrus. The animal model showed Aß(1-42), but not PBS, injection was associated with abnormalities in morphology of microvessels which were characterized as looping, fragmented, knob-like, uneven, and constricted. In particular, numbers of constricted microvessels, defined as vessels with diameters less than 3 µm, were considerably enhanced for Aß(1-42), compared to PBS, injection. In comparison, human AD brain demonstrated an elevated number of microvessels with a diminished mean length relative to nondemented (ND) brain. Additionally, microvessel perturbations in AD brain showed a similar pattern of morphological abnormalities to those observed in Aß(1-42)-injected rat hippocampus. Constricted microvessels were a prominent feature of AD brain but were rarely observed in ND tissue. These results provide the first evidence that a peptide-injection animal model exhibits a commonality in perturbations of microvessels compared with those evident in AD brain.

Differential expression of interferon-gamma receptor on human glial cells in vivo and in vitro.

Although significant effects of interferon-gamma (IFN-gamma) on glial cells are well documented, information on the expression level and localization of glial IFN-gamma receptors (IFN-gamma-R) in the human central nervous system (CNS) is sparse. To examine the glial expression of IFN-gamma-R in the human CNS, immunohistochemistry and quantitative analyses were performed on Alzheimer disease hippocampus, Parkinson disease substantia nigra, amyotrophic lateral sclerosis spinal cord and corresponding areas from non-neurological cases. Almost all IFN-gamma-R-positive (IFN-gamma-R(+)) cells corresponded to GFAP-positive (GFAP(+)) astrocytes, while none of IFN-gamma-R(+) cells corresponded to IBA1-positive (IBA1(+)) microglia or MBP-positive (MBP(+)) oligodendrocytes in these neurological cases. We observed a similar pattern of glial IFN-gamma-R expression in non-neurological cases. Also, we quantitatively analyzed the IFN-gamma-R expression by cultured human glial cells using immunocytochemistry and reverse transcription polymerase chain reaction (RT-PCR). In contrast to in vivo results, almost all IFN-gamma-R(+) cells were IBA1(+) in microglial cultures, GFAP(+) in astrocytic cultures and MBP(+) in oligodendrocytic cultures. Moreover, no significant difference in IFN-gamma-R mRNA expression was found for these glial cell types by RT-PCR. These results suggest that the microglial and oligodendrocytic expression levels of IFN-gamma-R are much lower than the astrocytic expression levels in the human CNS in vivo, whereas all three types of glial cells constitutively express IFN-gamma-R when cultured in vitro.