Corinna Höfling

Early pathologic amyloid induces hypersynchrony of BOLD resting-state networks in transgenic mice and provides an early therapeutic window before amyloid plaque deposition.

In Alzheimers 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).

Early Changes in Hippocampal Neurogenesis in Transgenic Mouse Models for Alzheimer’s Disease

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.

Deficiency of prolyl oligopeptidase in mice disturbs synaptic plasticity and reduces anxiety-like behaviour, body weight, and brain volume.

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.

A novel experimental model of Cryptococcus neoformans-related immune reconstitution inflammatory syndrome (IRIS) provides insights into pathogenesis

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.

Mouse strain and brain region-specific expression of the glutaminyl cyclases QC and isoQC

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 Alzheimers 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.

Differential transgene expression patterns in Alzheimer mouse models revealed by novel human amyloid precursor protein-specific antibodies.

Alzheimers 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.

AβPP-Transgenic 2576 Mice Mimic Cell Type-Specific Aspects of Acetyl-CoA-Linked Metabolic Deficits in Alzheimer’s Disease

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 Alzheimers disease and other age-related encephalopathies in humans. Therefore, amyloid-β overload in brains of diverse transgenic Alzheimers 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.

Identification of thyrotropin-releasing hormone as hippocampal glutaminyl cyclase substrate in neurons and reactive astrocytes

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 Alzheimers 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.

Sex- and age-specific modulation of brain GABA levels in a mouse model of Alzheimer's disease

Age and sex are risk factors of Alzheimers 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.

Pyroglutamate modified Amyloid ß (11- 40) Fibrils are more Toxic than Wildtype Fibrils but Structurally Very Similar

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.