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Neurodegenerative disorders are characterised by the activation of brain-resident microglia cells and by the infiltration of peripheral T cells. However, their interplay in disease has not been clarified yet. It is difficult to investigate complex cellular dynamics in living animals, and simple two-dimensional (2D) cell culture models do not resemble the soft 3D structure of brain tissue. Therefore, we developed a biomimetic 3D in vitro culture system for co-cultivation of microglia and T cells. As the activation and/or migration of immune cells in the brain might be affected by components of the extracellular matrix, defined 3D fibrillar collagen I-based matrices were constructed and modified with hyaluronan and/or chondroitin sulphate, resembling aspects of brain extracellular matrix. Murine microglia and spleen-derived T cells were cultured alone or in co-culture on the constructed matrices. Microglia exhibited in vivo-like morphology and T cells showed enhanced survival when co-cultured with microglia or to a minor degree in the presence of glia-conditioned medium. The open and porous fibrillar structure of the matrix allowed for cell invasion and direct cell-cell interaction, with stronger invasion of T cells. Both cell types showed no dependence on the matrix modifications. Microglia could be activated on the matrices by lipopolysaccharide resulting in interleukin-6 and tumour necrosis factor-α secretion. The findings herein indicate that biomimetic 3D matrices allow for co-cultivation and activation of primary microglia and T cells and provide useful tools to study their interaction in vitro.

The nonproteinogenic amino acid β-methylamino alarelevant example for environmental hazards are nonnine (BMAA) is a neurotoxin and represents a potential risk factor for neurodegenerative diseases. Despite intense research over the last years, the pathological mechanism of BMAA is still unclear. One of the main open questions is whether BMAA can be misincorporated into proteins, especially as a substitute for serine, and whether this has structural and functional consequences for the afflicted proteins leading to early onset neurodegeneration. In this study, we hypothesize that BMAA was indeed incorporated into Aβ

Background: Circadian rhythm disturbance is commonly observed in Alzheimer’s disease (AD). In mammals, these rhythms are orchestrated by the superchiasmatic nucleus (SCN). Our previous study in the Tg2576 AD mouse model suggests that inflammatory responses, most likely manifested by low GABA production, may be one of the underlying perpetrators for the changes in circadian rhythmicity and sleep disturbance in AD. However, the mechanistic connections between SCN dysfunction, GABA modulation, and inflammation in AD is not fully understood. Objective: To reveal influences of amyloid pathology in Tg2576 mouse brain on metabolism in SCN and to identify key metabolic sensors that couple SCN dysfunction with GABA modulation and inflammation. Methods: High resolution magic angle spinning (HR-MAS) NMR in conjunction with multivariate analysis was applied for metabolic profiling in SCN of control and Tg2576 female mice. Immunohistochemical analysis was used to detect neurons, astrocytes, expression of GABA transporter 1 (GAT1) and Bmal1. Results: Metabolic profiling revealed significant metabolic deficits in SCN of Tg2576 mice. Reductions in glucose, glutamate, GABA, and glutamine provide hints toward an impaired GABAergic glucose oxidation and neurotransmitter cycling in SCN of AD mice. In addition, decreased redox co-factor NADPH and glutathione support a redox disbalance. Immunohistochemical examinations showed low expression of the core clock protein, Bmal1, especially in activated astrocytes. Moreover, decreased expression of GAT1 in astrocytes indicates low GABA recycling in this cell type. Conclusion: Our results suggest that redox disbalance and compromised GABA signaling are important denominators and connectors between neuroinflammation and clock dysfunction in AD.

In Alzheimer's disease (AD), disturbances in the circadian rhythm and sleep-wake cycle are frequently observed. Both are controlled by the master clock: the suprachiasmatic nucleus (SCN), which was reported in postmortem studies of AD subjects to be compromised. However, the influence of age and gender on the biophysical integrity and subtle microstructural changes of SCN and mechanistic connections between SCN dysfunction and AD progression in vivo remain to be explored. In the present study, we utilized state-of-the-art in vivo magnetic resonance relaxation measurements in combination with immunohistochemistry to follow microstructural changes in SCN of the Tg2576 mouse model of AD. Longitudinal monitoring of in vivo T2 relaxation with age shows significant shortening of T2 values in the SCN of transgenic mice and more substantially in female transgenic than aged-matched controls. Multiexponential T2 analysis detected a unique long T2 component in SCN of transgenic mice which was absent in wild-type mice. Immunohistochemical examination revealed significantly elevated numbers of activated astrocytes and an increase in the astrocyte to neuron ratio in SCN of transgenic compared to wild-type mice. This increase was more substantial in female than in male transgenic mice. In addition, low GABA production in SCN of transgenic mice was detected. Our results offer a brief appraisal of SCN dysfunction in AD and demonstrate that inflammatory responses may be an underlying perpetrator for the changes in circadian rhythmicity and sleep disturbance in AD and could also be at the root of marked sex disparities observed in AD subjects.

Transgenic Tg2576 mice expressing human amyloid precursor protein (hAPP) with the Swedish mutation are among the most frequently used animal models to study the amyloid pathology related to Alzheimer's disease (AD). The transgene expression in this model is considered to be neuron‐specific. Using a novel hAPP‐specific antibody in combination with cell type‐specific markers for double immunofluorescent labelings and laser scanning microscopy, we here report that—in addition to neurons throughout the brain—astrocytes in the corpus callosum and to a lesser extent in neocortex express hAPP. This astrocytic hAPP expression is already detectable in young Tg2576 mice before the onset of amyloid pathology and still present in aged Tg2576 mice with robust amyloid pathology in neocortex, hippocampus, and corpus callosum. Surprisingly, hAPP immunoreactivity in cortex is restricted to resting astrocytes distant from amyloid plaques but absent from reactive astrocytes in close proximity to amyloid plaques. In contrast, neither microglial cells nor oligodendrocytes of young or aged Tg2576 mice display hAPP labeling. The astrocytic expression of hAPP is substantiated by the analyses of hAPP mRNA and protein expression in primary cultures derived from Tg2576 offspring. We conclude that astrocytes, in particular in corpus callosum, may contribute to amyloid pathology in Tg2576 mice and thus mimic this aspect of AD pathology.

Summary Alzheimer's disease (AD) is histopathologically characterized by neurodegeneration, the formation of intracellular neurofibrillary tangles and extracellular Aβ deposits that derive from proteolytic processing of the amyloid precursor protein (APP). As rodents do not normally develop Aβ pathology, various transgenic animal models of AD were designed to overexpress human APP with mutations favouring its amyloidogenic processing. However, these mouse models display tremendous differences in the spatial and temporal appearance of Aβ deposits, synaptic dysfunction, neurodegeneration and the manifestation of learning deficits which may be caused by age‐related and brain region‐specific differences in APP transgene levels. Consequentially, a comparative temporal and regional analysis of the pathological effects of Aβ in mouse brains is difficult complicating the validation of therapeutic AD treatment strategies in different mouse models. To date, no antibodies are available that properly discriminate endogenous rodent and transgenic human APP in brains of APP‐transgenic animals. Here, we developed and characterized rat monoclonal antibodies by immunohistochemistry and Western blot that detect human but not murine APP in brains of three APP‐transgenic mouse and one APP‐transgenic rat model. We observed remarkable differences in expression levels and brain region‐specific expression of human APP among the investigated transgenic mouse lines. This may explain the differences between APP‐transgenic models mentioned above. Furthermore, we provide compelling evidence that our new antibodies specifically detect endogenous human APP in immunocytochemistry, FACS and immunoprecipitation. Hence, we propose these antibodies as standard tool for monitoring expression of endogenous or transfected APP in human cells and APP expression in transgenic animals.

Prolyl oligopeptidase (PREP) has been implicated in neurodegeneration and neuroinflammation and has been considered a drug target to enhance memory in dementia. However, the true physiological role of PREP is not yet understood. In this paper, we report the phenotyping of a mouse line where the PREP gene has been knocked out. This work indicates that the lack of PREP in mice causes reduced anxiety but also hyperactivity. The cortical volumes of PREP knockout mice were smaller than those of wild type littermates. Additionally, we found increased expression of diazepam binding inhibitor protein in the cortex and of the somatostatin receptor-2 in the hippocampus of PREP knockout mice. Furthermore, immunohistochemistry and tail suspension test revealed lack of response of PREP knockout mice to lipopolysaccharide insult. Further analysis revealed significantly increased levels of polysialylated-neural cell adhesion molecule in PREP deficient mice. These findings might be explained as possible alteration in brain plasticity caused by PREP deficiency, which in turn affect behaviour and brain development.

INTRODUCTION: In Alzheimer's disease (AD), pathologic amyloid-beta (Aβ) is synaptotoxic and impairs neuronal function at the microscale, influencing brain networks at the macroscale before Aβ deposition. The latter can be detected noninvasively, in vivo, using resting-state functional MRI (rsfMRI), a technique used to assess brain functional connectivity (FC). METHODS: RsfMRI was performed longitudinally in TG2576 and PDAPP mice, starting before Aβ deposition to determine the earliest FC changes. Additionally, the role of pathologic Aβ on early FC alterations was investigated by treating TG2576 mice with the 3D6 anti-Aβ-antibody. RESULTS: Both transgenic models showed hypersynchronized FC before Aβ deposition and hyposynchronized FC at later stages. Early anti-Aβ treatment in TG2576 mice prevented hypersynchronous FC and the associated synaptic impairments and excitatory/inhibitory disbalances. DISCUSSION: Hypersynchrony of FC may be used as a new noninvasive read out of early AD and can be recovered by anti-Aβ treatment, encouraging preventive treatment strategies in familial AD.

The corpus callosum is the largest commissural fiber connecting left and right hemisphere of the brain. Emerging evidence suggests that a variety of abnormalities detected in the microstructure of this white matter fiber can be an early event in Alzheimer's disease (AD) pathology. However, little is known about tissue characteristics of these abnormalities and how these abnormalities evolve during AD progression. In this study, we measured in vivo magnetic resonance transverse relaxation times (T2) to longitudinally monitor changes in tissue integrity and abnormalities related to myelination and demyelination processes in corpus callosum of AD mouse models. The most striking finding of our study was a significant elongation of T2 values in the corpus callosum at 10, 14, 16 and 18 months of age compared to age-matched wild-type mice. In contrast, the gray matter regions surrounding the corpus callosum, such as the cortex and hippocampus, showed a significant T2 decrease compared to wild-type mice. Histological analyses clearly revealed demyelination, gliosis and amyloid-plaque deposition in the corpus callosum. Our results suggest that demyelinating and inflammatory pathology may result in prolonged relaxation time during AD progression. To our knowledge, this is the first in vivo T2 study assessing the microstructural changes with age in the corpus callosum of the Tg2576 mouse model and it demonstrates the application of T2 measurement to noninvasively detect tissue integrity of the corpus callosum, which can be an early event in disease progression.

The pyruvate-derived acetyl-CoA is a principal direct precursor substrate for bulk energy synthesis in the brain. Deficits of pyruvate dehydrogenase in the neocortex are common features of Alzheimer's disease and other age-related encephalopathies in humans. Therefore, amyloid-β overload in brains of diverse transgenic Alzheimer's disease model animals was investigated as one of neurotoxic compounds responsible for pyruvate dehydrogenase inhibition yielding deficits of cholinergic neurotransmission and cognitive functions. Brains of aged, 14-16-month-old Tg2576 mice contained 0.6 μmol/kg levels of amyloid-β1 - 42. Activities of pyruvate dehydrogenase complex, choline acetyltransferase, and several enzymes of acetyl-CoA and energy metabolism were found to be unchanged in both forebrain mitochondria and synaptosomes of Tg2576 mice, indicating preservation of structural integrity at least in cholinergic neuronal cells. However, in transgenic brain synaptosomes, pyruvate utilization, mitochondrial levels, and cytoplasmic acetyl-CoA levels, as well as acetylcholine content and its quantal release, were all found to be decreased by 25-40% . On the contrary, activation of pyruvate utilization was detected and no alterations in acetyl-CoA content and citrate or α-ketoglutarate accumulation were observed in transgenic whole brain mitochondria. These data indicate that amyloid-β evoked deficits in acetyl-CoA are confined to mitochondrial and cytoplasmic compartments of Tg2576 nerve terminals, becoming early primary signals paving the path for further stages of neurodegeneration. On the other hand, acetyl-CoA synthesis in mitochondrial compartments of glial cells seems to be activated despite amyloid-β accumulated in transgenic brains.

Antiretroviral therapy (ART) has yielded major advances in fighting the HIV pandemic by restoring protective immunity. However, a significant proportion of HIV patients co-infected with the opportunistic fungal pathogen Cryptococcus neoformans paradoxically develops a life-threatening immune reconstitution inflammatory syndrome (IRIS) during antiretroviral therapy. Despite several clinical studies, the underlying pathomecha-nisms are poorly understood. Here, we present the first mouse model of cryptococcal IRIS that allows for a detailed analysis of disease development. Lymphocyte-deficient RAG-1(-/-) mice are infected with C. neoformans and 4 weeks later adoptively transferred with purified CD4(+) T cells. Reconstitution of CD4(+) T cells is sufficient to induce a severe inflammatory disease similar to clinical IRIS in C. neoformans-infected RAG-1(-/-) mice of different genetic backgrounds and immunological phenotypes (i.e. C57BL/6 and BALB/c). Multiorgan inflammation is accompanied by a systemic release of distinct proinflammatory cytokines, i.e. IFN-γ, IL-6, and TNF-α. IRIS development is characterized by infection-dependent activation of donor CD4(+) T cells, which are the source of IFN-γ. Interestingly, IFN-γ-mediated effects are not required for disease induction. Taken together, this novel mouse model of cryptococcal IRIS provides a useful tool to verify potential mechanisms of pathogenesis, revealing targets for diagnosis and therapeutic interventions.

Age and sex are risk factors of Alzheimer's disease (AD). Among the neurotransmitter systems, gamma-aminobutyric acid (GABA) has been implicated in AD pathogenesis but the relevance of sex-specific GABAergic dysfunction during AD progression remains unknown. In the present study, we utilized state-of-the-art high-resolution magic angle spinning nuclear magnetic resonance to systematically monitor the brain region-, age-, and sex-specific modulation of GABA levels in wild-type and Tg2576 mice with amyloid pathology. In addition, we followed the possible role of reactive astrocytes in sex-specific GABA modulation. In female Tg2576 mice, hippocampal GABA levels were significantly elevated, along with higher number of reactive astrocytes and amyloid deposition. The elevated GABA was found to be produced via the monoamine oxidase-B route from putrescine in reactive astrocytes, more substantially in female than male mice, thus suggesting a role of astrocytes in memory impairment and sex-related differences in AD. Our results paint a coherent model of memory impairment in AD and signify that dynamic changes in regional GABA may be at the root of marked sex disparities observed in AD.

The morphology, structure, and dynamics of mature amyloid β (Aβ) fibrils formed by the Aβ variant, which is truncated at residue 11 and chemically modified by enzymatic pyroglutamate formation (pGlu11 -Aβ(11-40)), was studied along with the investigation of the toxicity of these Aβ variants to neurons and astrocytes. The fibrils of pGlu11 -Aβ (11-40) were more toxic than wildtype Aβ (1-40) and the longer pGlu3-Aβ (3-40) especially at higher concentration, whereas the overall morphology was quite similar. The secondary structure of pGlu11 -Aβ (11-40) fibrils shows the typical two β-strands connected by a short turn as known for mature fibrils of Aβ (1-40) and also pGlu3 -Aβ (3-40). Further insights into tertiary contacts exhibit some similarities of pGlu11 -Aβ (11-40) fibrils with wildtype Aβ (1-40), but also a so far not described contact between Gly25 and Ile31 . This highlights the biological importance of chemical modifications on the molecular structure of Aβ.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disease in the Western world and is characterized by a progressive loss of cognitive functions leading to dementia. One major histopathological hallmark of AD is the formation of amyloid-beta plaques, which is reproduced in numerous transgenic animal models overexpressing pathogenic forms of amyloid precursor protein (APP). In human AD and in transgenic amyloid plaque mouse models, several studies report altered rates of adult neurogenesis, i.e. the formation of new neurons from neural stem and progenitor cells, and impaired neurogenesis has also been attributed to contribute to the cognitive decline in AD. So far, changes in neurogenesis have largely been considered to be a consequence of the plaque pathology. Therefore, possible alterations in neurogenesis before plaque formation or in prodromal AD have been largely ignored. Here, we analysed adult hippocampal neurogenesis in amyloidogenic mouse models of AD at different points before and during plaque progression. We found prominent alterations of hippocampal neurogenesis before plaque formation. Survival of newly generated cells and the production of new neurons were already compromised at this stage. Moreover and surprisingly, proliferation of doublecortin (DCX) expressing neuroblasts was significantly and specifically elevated during the pre-plaque stage in the APP-PS1 model, while the Nestin-expressing stem cell population was unaffected. In summary, changes in neurogenesis are evident already before plaque deposition and might contribute to well-known early hippocampal dysfunctions in prodromal AD such as hippocampal overactivity.

Antiretroviral therapy (ART) has yielded major advances in fighting the HIV pandemic by restoring protective immunity. However, a significant proportion of HIV patients co-infected with the opportunistic fungal pathogen Cryptococcus neoformans paradoxically develops a life-threatening immune reconstitution inflammatory syndrome (IRIS) during antiretroviral therapy. Despite several clinical studies, the underlying pathomecha-nisms are poorly understood. Here, we present the first mouse model of cryptococcal IRIS that allows for a detailed analysis of disease development. Lymphocyte-deficient RAG-1(-/-) mice are infected with C. neoformans and 4 weeks later adoptively transferred with purified CD4(+) T cells. Reconstitution of CD4(+) T cells is sufficient to induce a severe inflammatory disease similar to clinical IRIS in C. neoformans-infected RAG-1(-/-) mice of different genetic backgrounds and immunological phenotypes (i.e. C57BL/6 and BALB/c). Multiorgan inflammation is accompanied by a systemic release of distinct proinflammatory cytokines, i.e. IFN-γ, IL-6, and TNF-α. IRIS development is characterized by infection-dependent activation of donor CD4(+) T cells, which are the source of IFN-γ. Interestingly, IFN-γ-mediated effects are not required for disease induction. Taken together, this novel mouse model of cryptococcal IRIS provides a useful tool to verify potential mechanisms of pathogenesis, revealing targets for diagnosis and therapeutic interventions.

Glutaminyl cyclases (QCs) catalyze the formation of pyroglutamate (pGlu) from glutamine precursors at the N-terminus of a number of peptide hormones, neuropeptides and chemokines. This post-translational modification stabilizes these peptides, protects them from proteolytical degradation or is important for their biological activity. However, QC is also involved in a pathogenic pGlu modification of peptides accumulating in protein aggregation disorders such as Alzheimer's disease and familial Danish and familial British dementia. Its isoenzyme (isoQC) was shown to contribute to aspects of inflammation by pGlu-modifying and thereby stabilizing the monocyte chemoattractant protein CCL2. For the generation of respective animal models and for pharmacological treatment studies the characterization of the mouse strain and brain region-specific expression of QC and isoQC is indispensible. In order to address this issue, we used enzymatic activity assays and specific antibodies to detect both QC variants by immunohistochemistry in nine different mouse strains. Comparing different brain regions, the highest enzymatic QC/isoQC activity was detected in ventral brain, followed by cortex and hippocampus. Immunohistochemical stainings revealed that QC/isoQC activity in cortex mostly arises from isoQC expression. For most brain regions, the highest QC/isoQC activity was detected in C3H and FVB mice, whereas low QC/isoQC activity was present in CD1, SJL and C57 mice. Quantification of QC- and isoQC-immunoreactive cells by unbiased stereology revealed a higher abundance of isoQC- than of QC-immunoreactive neurons in Edinger–Westphal nucleus and in substantia nigra. In the locus coeruleus, however, there were comparable densities of QC- and of isoQC-immunoreactive neurons. These observations are of considerable importance with regard to the selection of appropriate mouse strains for the study of QC/isoQC relevance in mouse models of neurodegeneration and neuroinflammation and for the testing of therapeutical interventions in these models.

Recently, Aβ peptide variants with an N-terminal truncation and pyroglutamate modification were identified and shown to be highly neurotoxic and prone to aggregation. This modification of Aβ is catalyzed by glutaminyl cyclase (QC) and pharmacological inhibition of QC diminishes Aβ deposition and accompanying gliosis and ameliorates memory impairment in transgenic mouse models of Alzheimer's disease (AD). QC expression was initially described in the hypothalamus, where thyrotropin-releasing hormone (TRH) is one of its physiological substrates. In addition to its hormonal role, a novel neuroprotective function of TRH following excitotoxicity and Aβ-mediated neurotoxicity has been reported in the hippocampus. Functionally matching this finding, we recently demonstrated QC expression by hippocampal interneurons in mouse brain.Here, we detected neuronal co-expression of QC and TRH in the hippocampus of young adult wild type mice using double immunofluorescence labeling. This provides evidence for TRH being a physiological QC substrate in hippocampus. Additionally, in neocortex of aged but not of young mice transgenic for amyloid precursor protein an increase of QC mRNA levels was found compared to wild type littermates. This phenomenon was not observed in hippocampus, which is later affected by Aβ pathology. However, in hippocampus of transgenic — but not of wild type mice — a correlation between QC and TRH mRNA levels was revealed. This co-regulation of the enzyme QC and its substrate TRH was reflected by a co-induction of both proteins in reactive astrocytes in proximity of Aβ deposits. Also, in primary mouse astrocytes a co-induction of QC and TRH was demonstrated upon Aβ stimulation.