Tpr Misregulation in Hippocampal Neural Stem Cells in Mouse Models of Alzheimer's Disease.

Nuclear pore complexes (NPCs) are highly dynamic macromolecular protein structures that facilitate molecular exchange across the nuclear envelope. Aberrant NPC functioning has been implicated in neurodegeneration. The translocated promoter region (Tpr) is a critical scaffolding nucleoporin (Nup) of the nuclear basket, facing the interior of the NPC. However, the role of Tpr in adult neural stem/precursor cells (NSPCs) in Alzheimer's disease (AD) is unknown. Using super-resolution (SR) and electron microscopy, we defined the different subcellular localizations of Tpr and phospho-Tpr (P-Tpr) in NSPCs in vitro and in vivo. Elevated Tpr expression and reduced P-Tpr nuclear localization accompany NSPC differentiation along the neurogenic lineage. In 5xFAD mice, an animal model of AD, increased Tpr expression in DCX+ hippocampal neuroblasts precedes increased neurogenesis at an early stage, before the onset of amyloid-ß plaque formation. Whereas nuclear basket Tpr interacts with chromatin modifiers and NSPC-related transcription factors, P-Tpr interacts and co-localizes with cyclin-dependent kinase 1 (Cdk1) at the nuclear chromatin of NSPCs. In hippocampal NSPCs in a mouse model of AD, aberrant Tpr expression was correlated with altered NPC morphology and counts, and Tpr was aberrantly expressed in postmortem human brain samples from patients with AD. Thus, we propose that altered levels and subcellular localization of Tpr in CNS disease affect Tpr functionality, which in turn regulates the architecture and number of NSPC NPCs, possibly leading to aberrant neurogenesis.

Seed-induced Aß deposits in the corpus callosum disrupt white matter integrity in a mouse model of Alzheimer's disease.

Neuropathologically, Alzheimer's disease (AD) is characterized by the accumulation of amyloid-beta peptide (Aß) and subsequent formation of the so-called Aß plaques. Along with neuronal loss, previous studies report white matter anomalies and corpus callosum (CC) atrophy in AD patients. Notably, perturbations in the white matter can be observed years before expected disease onset, suggesting that early stages of disease progression play a role in AD-associated loss of myelin integrity. Through seed-induced deposition of Aß, we are able to examine alterations of central nervous system (CNS) integrity during the initial stages of plaque formation. In this study, we investigate the impact of Aß seeding in the CC utilizing various imaging techniques as well as quantitative gene expression analysis and demonstrate that Aß deposits result in an imbalance of glial cells in the CC. We found increased amounts of phagocytic microglia and reactive astrocytes, while oligodendrocyte progenitor cell (OPC) numbers were reduced. Moreover, white matter aberrations adjacent to the Aß seeding were observed together with an overall decline in callosal myelination. This data indicate that the initial stages of plaque formation induce oligodendrocyte dysfunction, which might ultimately lead to myelin loss.

Seed-induced Aß deposition alters neuronal function and impairs olfaction in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is characterized by the accumulation of amyloid-ß (Aß) which ultimately forms plaques. These Aß deposits can be induced in APP transgenic mouse models by prion-like seeding. It has been widely accepted that anosmia and hyposmia occur during the early stages of AD, even before cognitive deficits are present. In order to determine the impact of seed-induced Aß deposits on olfaction, we performed intracerebral injections of seed-competent brain homogenate into the olfactory bulb of young pre-depositing APP transgenic mice. Remarkably, we observed a dramatic olfactory impairment in those mice. Furthermore, the number of newborn neurons as well as the activity of cells in the mitral cell layer was decreased. Notably, exposure to an enriched environment reduced Aß seeding, vivified neurogenesis and most importantly reversed olfactory deficits. Based on our findings, we conclude that altered neuronal function as a result of induced Aß pathology might contribute to olfactory dysfunction in AD.

Distinct Aß pathology in the olfactory bulb and olfactory deficits in a mouse model of Aß and α-syn co-pathology.

Several degenerative brain disorders such as Alzheimer's disease (AD), Parkinson's disease (PD) and Dementia with Lewy bodies (DLB) are characterized by the simultaneous appearance of amyloid-ß (Aß) and α-synuclein (α-syn) pathologies and symptoms that are similar, making it difficult to differentiate between these diseases. Until now, an accurate diagnosis can only be made by postmortem analysis. Furthermore, the role of α-syn in Aß aggregation and the arising characteristic olfactory impairments observed during the progression of these diseases is still not well understood. Therefore, we assessed Aß load in olfactory bulbs of APP-transgenic mice expressing APP695KM670/671NL and PSEN1L166P under the control of the neuron-specific Thy-1 promoter (referred to here as APPPS1) and APPPS1 mice co-expressing SNCAA30P (referred to here as APPPS1 × [A30P]aSYN). Furthermore, the olfactory capacity of these mice was evaluated in the buried food and olfactory avoidance test. Our results demonstrate an age-dependent increase in Aß load in the olfactory bulb of APP-transgenic mice that go along with exacerbated olfactory performance. Our study provides clear evidence that the presence of α-syn significantly diminished the endogenous and seed-induced Aß deposits and significantly ameliorated olfactory dysfunction in APPPS1 × [A30P]aSYN mice.

Microglia contribute to the propagation of Aß into unaffected brain tissue.

Microglia appear activated in the vicinity of amyloid beta (Aß) plaques, but whether microglia contribute to Aß propagation into unaffected brain regions remains unknown. Using transplantation of wild-type (WT) neurons, we show that Aß enters WT grafts, and that this is accompanied by microglia infiltration. Manipulation of microglia function reduced Aß deposition within grafts. Furthermore, in vivo imaging identified microglia as carriers of Aß pathology in previously unaffected tissue. Our data thus argue for a hitherto unexplored mechanism of Aß propagation.

Meclofenamate causes loss of cellular tethering and decoupling of functional networks in glioblastoma.

BACKGROUND: Glioblastoma cells assemble to a syncytial communicating network based on tumor microtubes (TMs) as ultra-long membrane protrusions. The relationship between network architecture and transcriptional profile remains poorly investigated. Drugs that interfere with this syncytial connectivity such as meclofenamate (MFA) may be highly attractive for glioblastoma therapy. METHODS: In a human neocortical slice model using glioblastoma cell populations of different transcriptional signatures, three-dimensional tumor networks were reconstructed, and TM-based intercellular connectivity was mapped on the basis of two-photon imaging data. MFA was used to modulate morphological and functional connectivity; downstream effects of MFA treatment were investigated by RNA sequencing and fluorescence-activated cell sorting (FACS) analysis. RESULTS: TM-based network morphology strongly differed between the transcriptional cellular subtypes of glioblastoma and was dependent on axon guidance molecule expression. MFA revealed both a functional and morphological demolishment of glioblastoma network architectures which was reflected by a reduction of TM-mediated intercellular cytosolic traffic as well as a breakdown of TM length. RNA sequencing confirmed a downregulation of NCAM and axon guidance molecule signaling upon MFA treatment. Loss of glioblastoma communicating networks was accompanied by a failure in the upregulation of genes that are required for DNA repair in response to temozolomide (TMZ) treatment and culminated in profound treatment response to TMZ-mediated toxicity. CONCLUSION: The capacity of TM formation reflects transcriptional cellular heterogeneity. MFA effectively demolishes functional and morphological TM-based syncytial network architectures. These findings might pave the way to a clinical implementation of MFA as a TM-targeted therapeutic approach.

Detection of Synaptic Proteins in Microglia by Flow Cytometry.

A growing body of evidence indicates that microglia actively remove synapses in vivo, thereby playing a key role in synaptic refinement and modulation of brain connectivity. This phenomenon was mainly investigated in immunofluorescence staining and confocal microscopy. However, a quantification of synaptic material in microglia using these techniques is extremely time-consuming and labor-intensive. To address this issue, we aimed to quantify synaptic proteins in microglia using flow cytometry. With this approach, we first showed that microglia from the healthy adult mouse brain contain a detectable level of VGLUT1 protein. Next, we found more than two-fold increased VGLUT1 immunoreactivity in microglia from the developing brain (P15) as compared to adult microglia. These data indicate that microglia-mediated synaptic pruning mostly occurs during the brain developmental period. We then quantified the VGLUT1 staining in microglia in two transgenic models characterized by pathological microglia-mediated synaptic pruning. In the 5xFAD mouse model of Alzheimer's disease (AD) microglia exhibited a significant increase in VGLUT1 immunoreactivity before the onset of amyloid pathology. Moreover, conditional deletion of TDP-43 in microglia, which causes a hyper-phagocytic phenotype associated with synaptic loss, also resulted in increased VGLUT1 immunoreactivity within microglia. This work provides a quantitative assessment of synaptic proteins in microglia, under homeostasis, and in mouse models of disease.

Mechanisms of Pathogenic Tau and Aß Protein Spreading in Alzheimer's Disease.

Alzheimer's disease (AD) is pathologically defined by extracellular accumulation of amyloid-ß (Aß) peptides generated by the cleavage of amyloid precursor protein (APP), strings of hyperphosphorylated Tau proteins accumulating inside neurons known as neurofibrillary tangles (NFTs) and neuronal loss. The association between the two hallmarks and cognitive decline has been known since the beginning of the 20th century when the first description of the disease was carried out by Alois Alzheimer. Today, more than 40 million people worldwide are affected by AD that represents the most common cause of dementia and there is still no effective treatment available to cure the disease. In general, the aggregation of Aß is considered an essential trigger in AD pathogenesis that gives rise to NFTs, neuronal dysfunction and dementia. During the process leading to AD, tau and Aß first misfold and form aggregates in one brain region, from where they spread to interconnected areas of the brain thereby inducing its gradual morphological and functional deterioration. In this mini-review article, we present an overview of the current literature on the spreading mechanisms of Aß and tau pathology in AD since a more profound understanding is necessary to design therapeutic approaches aimed at preventing or halting disease progression.

Author Correction: Novel Hexb-based tools for studying microglia in the CNS.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Different effects of constitutive and induced microbiota modulation on microglia in a mouse model of Alzheimer's disease.

It was recently revealed that gut microbiota promote amyloid-beta (Aß) burden in mouse models of Alzheimer's disease (AD). However, the underlying mechanisms when using either germ-free (GF) housing conditions or treatments with antibiotics (ABX) remained unknown. In this study, we show that GF and ABX-treated 5x familial AD (5xFAD) mice developed attenuated hippocampal Aß pathology and associated neuronal loss, and thereby delayed disease-related memory deficits. While Aß production remained unaffected in both GF and ABX-treated 5xFAD mice, we noticed in GF 5xFAD mice enhanced microglial Aß uptake at early stages of the disease compared to ABX-treated 5xFAD mice. Furthermore, RNA-sequencing of hippocampal microglia from SPF, GF and ABX-treated 5xFAD mice revealed distinct microbiota-dependent gene expression profiles associated with phagocytosis and altered microglial activation states. Taken together, we observed that constitutive or induced microbiota modulation in 5xFAD mice differentially controls microglial Aß clearance mechanisms preventing neurodegeneration and cognitive deficits.

Novel Hexb-based tools for studying microglia in the CNS.

Microglia and central nervous system (CNS)-associated macrophages (CAMs), such as perivascular and meningeal macrophages, are implicated in virtually all diseases of the CNS. However, little is known about their cell-type-specific roles in the absence of suitable tools that would allow for functional discrimination between the ontogenetically closely related microglia and CAMs. To develop a new microglia gene targeting model, we first applied massively parallel single-cell analyses to compare microglia and CAM signatures during homeostasis and disease and identified hexosaminidase subunit beta (Hexb) as a stably expressed microglia core gene, whereas other microglia core genes were substantially downregulated during pathologies. Next, we generated HexbtdTomato mice to stably monitor microglia behavior in vivo. Finally, the Hexb locus was employed for tamoxifen-inducible Cre-mediated gene manipulation in microglia and for fate mapping of microglia but not CAMs. In sum, we provide valuable new genetic tools to specifically study microglia functions in the CNS.

Aß oligomers trigger and accelerate Aß seeding.

Aggregation of amyloid-ß (Aß) that leads to the formation of plaques in Alzheimer's disease (AD) occurs through the stepwise formation of oligomers and fibrils. An earlier onset of aggregation is obtained upon intracerebral injection of Aß-containing brain homogenate into human APP transgenic mice that follows a prion-like seeding mechanism. Immunoprecipitation of these brain extracts with anti-Aß oligomer antibodies or passive immunization of the recipient animals abrogated the observed seeding activity, although induced Aß deposition was still evident. Here, we establish that, together with Aß monomers, Aß oligomers trigger the initial phase of Aß seeding and that the depletion of oligomeric Aß delays the aggregation process, leading to a transient reduction of seed-induced Aß deposits. This work extends the current knowledge about the role of Aß oligomers beyond its cytotoxic nature by pointing to a role in the initiation of Aß aggregation in vivo. We conclude that Aß oligomers are important for the early initiation phase of the seeding process.

Aß Seeding as a Tool to Study Cerebral Amyloidosis and Associated Pathology.

Misfolded proteins can form aggregates and induce a self-perpetuating process leading to the amplification and spreading of pathological protein assemblies. These misfolded protein assemblies act as seeds of aggregation. In an in vivo exogenous seeding model, both the features of seeds and the position at which seeding originates are precisely defined. Ample evidence from studies on intracerebal injection of amyloid-beta (Aß)-rich brain extracts suggests that Aß aggregation can be initiated by prion-like seeding. In this mini-review article, we will summarize the past and current literature on Aß seeding in mouse models of AD and discuss its implementation as a tool to study cerebral amyloidosis and associated pathology.

Human organotypic brain slice culture: a novel framework for environmental research in neuro-oncology.

When it comes to the human brain, models that closely mimic in vivo conditions are lacking. Living neuronal tissue is the closest representation of the in vivo human brain outside of a living person. Here, we present a method that can be used to maintain therapeutically resected healthy neuronal tissue for prolonged periods without any discernible changes in tissue vitality, evidenced by immunohistochemistry, genetic expression, and electrophysiology. This method was then used to assess glioblastoma (GBM) progression in its natural environment by microinjection of patient-derived tumor cells into cultured sections. The result closely resembles the pattern of de novo tumor growth and invasion, drug therapy response, and cytokine environment. Reactive transformation of astrocytes, as an example of the cellular nonmalignant tumor environment, can be accurately simulated with transcriptional differences similar to those of astrocytes isolated from acute GBM specimens. In a nutshell, we present a simple method to study GBM in its physiological environment, from which valuable insights can be gained. This technique can lead to further advancements in neuroscience, neuro-oncology, and pharmacotherapy.

A Subset of Skin Macrophages Contributes to the Surveillance and Regeneration of Local Nerves.

The skin comprises tissue macrophages as the most abundant resident immune cell type. Their diverse tasks including resistance against invading pathogens, attraction of bypassing immune cells from vessels, and tissue repair require dynamic specification. Here, we delineated the postnatal development of dermal macrophages and their differentiation into subsets by adapting single-cell transcriptomics, fate mapping, and imaging. Thereby we identified a phenotypically and transcriptionally distinct subset of prenatally seeded dermal macrophages that self-maintained with very low postnatal exchange by hematopoietic stem cells. These macrophages specifically interacted with sensory nerves and surveilled and trimmed the myelin sheath. Overall, resident dermal macrophages contributed to axon sprouting after mechanical injury. In summary, our data show long-lasting functional specification of macrophages in the dermis that is driven by stepwise adaptation to guiding structures and ensures codevelopment of ontogenetically distinct cells within the same compartment.

Loss of TREM2 function increases amyloid seeding but reduces plaque-associated ApoE.

Coding variants in the triggering receptor expressed on myeloid cells 2 (TREM2) are associated with late-onset Alzheimer's disease (AD). We demonstrate that amyloid plaque seeding is increased in the absence of functional Trem2. Increased seeding is accompanied by decreased microglial clustering around newly seeded plaques and reduced plaque-associated apolipoprotein E (ApoE). Reduced ApoE deposition in plaques is also observed in brains of AD patients carrying TREM2 coding variants. Proteomic analyses and microglia depletion experiments revealed microglia as one origin of plaque-associated ApoE. Longitudinal amyloid small animal positron emission tomography demonstrates accelerated amyloidogenesis in Trem2 loss-of-function mutants at early stages, which progressed at a lower rate with aging. These findings suggest that in the absence of functional Trem2, early amyloidogenesis is accelerated due to reduced phagocytic clearance of amyloid seeds despite reduced plaque-associated ApoE.

Environmental enrichment reverses Aß pathology during pregnancy in a mouse model of Alzheimer's disease.

Several studies suggest that women have a higher risk to develop Alzheimer's disease (AD) than men. In particular, the number of pregnancies was shown to be a risk factor for AD and women with several pregnancies on average had an earlier onset of the disease, thus making childbearing a risk factor. However, the impact of being pregnant on Aß plaque pathology and adult neurogenesis still remains elusive. Postmortem analysis revealed that pregnant 5xFAD transgenic mice had significantly more Aß plaques in the hippocampus from G10 onwards and that the number of Ki67 and DCX positive cells dramatically decreased during the postpartum period. Furthermore, 5 months old 5xFAD transgenic mice that also nursed their offsprings for 4 weeks had a similar Aß plaque load than merely pregnant mice, indicating that pregnancy alone is sufficient to elevate Aß plaque levels. Interestingly, housing in an enriched environment reduced the Aß plaque load and vivified neurogenesis. Our results suggest that pregnancy alters Aß plaque deposition in 5xFAD transgenic mice and diminishes the generation of newborn neurons. We conclude that pregnancy alone is sufficient to induce this phenotype that can be reversed upon environmental enrichment.

Histone Deacetylases 1 and 2 Regulate Microglia Function during Development, Homeostasis, and Neurodegeneration in a Context-Dependent Manner.

Microglia as tissue macrophages contribute to the defense and maintenance of central nervous system (CNS) homeostasis. Little is known about the epigenetic signals controlling microglia function in vivo. We employed constitutive and inducible mutagenesis in microglia to delete two class I histone deacetylases, Hdac1 and Hdac2. Prenatal ablation of Hdac1 and Hdac2 impaired microglial development. Mechanistically, the promoters of pro-apoptotic and cell cycle genes were hyperacetylated in absence of Hdac1 and Hdac2, leading to increased apoptosis and reduced survival. In contrast, Hdac1 and Hdac2 were not required for adult microglia survival during homeostasis. In a mouse model of Alzheimer's disease, deletion of Hdac1 and Hdac2 in microglia, but not in neuroectodermal cells, resulted in a decrease in amyloid load and improved cognitive impairment by enhancing microglial amyloid phagocytosis. Collectively, we report a role for epigenetic factors that differentially affect microglia development, homeostasis, and disease that could potentially be utilized therapeutically.

Seed-induced Aß deposition is modulated by microglia under environmental enrichment in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is characterized by severe neuronal loss as well as the accumulation of amyloid-ß (Aß), which ultimately leads to plaque formation. Although there is now a general agreement that the aggregation of Aß can be initiated by prion-like seeding, the impact and functional consequences of induced Aß deposits (Aß seeding) on neurons still remain open questions. Here, we find that Aß seeding, representing early stages of plaque formation, leads to a dramatic decrease in proliferation and neurogenesis in two APP transgenic mouse models. We further demonstrate that neuronal cell death occurs primarily in the vicinity of induced Aß deposits culminating in electrophysiological abnormalities. Notably, environmental enrichment and voluntary exercise not only revives adult neurogenesis and reverses memory deficits but, most importantly, prevents Aß seeding by activated, phagocytic microglia cells. Our work expands the current knowledge regarding Aß seeding and the consequences thereof and attributes microglia an important role in diminishing Aß seeding by environmental enrichment.

The Role of Glial Cells and Synapse Loss in Mouse Models of Alzheimer's Disease.

Synapse loss has detrimental effects on cellular communication, leading to network disruptions within the central nervous system (CNS) such as in Alzheimer's disease (AD). AD is characterized by a progressive decline of memory function, cognition, neuronal and synapse loss. The two main neuropathological hallmarks are amyloid-ß (Aß) plaques and neurofibrillary tangles. In the brain of AD patients and in mouse models of AD several morphological and functional changes, such as microgliosis and astrogliosis around Aß plaques, as well as dendritic and synaptic alterations, are associated with these lesions. In this review article, we will summarize the current literature on synapse loss in mouse models of AD and discuss current and prospective treatments for AD.