LINE-1 retrotransposons contribute to mouse PV interneuron development.

Retrotransposons are mobile DNA sequences duplicated via transcription and reverse transcription of an RNA intermediate. Cis-regulatory elements encoded by retrotransposons can also promote the transcription of adjacent genes. Somatic LINE-1 (L1) retrotransposon insertions have been detected in mammalian neurons. It is, however, unclear whether L1 sequences are mobile in only some neuronal lineages or therein promote neurodevelopmental gene expression. Here we report programmed L1 activation by SOX6, a transcription factor critical for parvalbumin (PV) interneuron development. Mouse PV interneurons permit L1 mobilization in vitro and in vivo, harbor unmethylated L1 promoters and express full-length L1 mRNAs and proteins. Using nanopore long-read sequencing, we identify unmethylated L1s proximal to PV interneuron genes, including a novel L1 promoter-driven Caps2 transcript isoform that enhances neuron morphological complexity in vitro. These data highlight the contribution made by L1 cis-regulatory elements to PV interneuron development and transcriptome diversity, uncovered due to L1 mobility in this milieu.

Scanning ultrasound-mediated memory and functional improvements do not require amyloid-ß reduction.

A prevalent view in treating age-dependent disorders including Alzheimer's disease (AD) is that the underlying amyloid plaque pathology must be targeted for cognitive improvements. In contrast, we report here that repeated scanning ultrasound (SUS) treatment at 1 MHz frequency can ameliorate memory deficits in the APP23 mouse model of AD without reducing amyloid-ß (Aß) burden. Different from previous studies that had shown Aß clearance as a consequence of blood-brain barrier (BBB) opening, here, the BBB was not opened as no microbubbles were used. Quantitative SWATH proteomics and functional magnetic resonance imaging revealed that ultrasound induced long-lasting functional changes that correlate with the improvement in memory. Intriguingly, the treatment was more effective at a higher frequency (1 MHz) than at a frequency within the range currently explored in clinical trials in AD patients (286 kHz). Together, our data suggest frequency-dependent bio-effects of ultrasound and a dissociation of cognitive improvement and Aß clearance, with important implications for the design of trials for AD therapies.

Reversible expansion of tissue macrophages in response to macrophage colony-stimulating factor (CSF1) transforms systemic lipid and carbohydrate metabolism.

Macrophages regulate metabolic homeostasis in health and disease. Macrophage colony-stimulating factor (CSF1)-dependent macrophages contribute to homeostatic control of the size of the liver. This study aimed to determine the systemic metabolic consequences of elevating circulating CSF1. Acute administration of a CSF1-Fc fusion protein to mice led to monocytosis, increased resident tissue macrophages in the liver and all major organs, and liver growth. These effects were associated with increased hepatic glucose uptake and extensive mobilization of body fat. The impacts of CSF1 on macrophage abundance, liver size, and body composition were rapidly reversed to restore homeostasis. The effects of CSF1 on metabolism were independent of several known endocrine regulators and did not impact the physiological fasting response. Analysis using implantable telemetry in metabolic cages revealed progressively reduced body temperature and physical activity with no change in diurnal food intake. These results demonstrate the existence of a dynamic equilibrium between CSF1, the mononuclear phagocyte system, and control of liver-to-body weight ratio, which in turn controls systemic metabolic homeostasis. This novel macrophage regulatory axis has the potential to promote fat mobilization, without changes in appetence, which may have novel implications for managing metabolic syndrome.NEW & NOTEWORTHY CSF1 administration expands tissue macrophages, which transforms systemic metabolism. CSF1 drives fat mobilization and glucose uptake to support liver growth. The effects of CSF1 are independent of normal hormonal metabolic regulation. The effects of CSF1 are rapidly reversible, restoring homeostatic body composition. CSF1-dependent macrophages and liver size are coupled in a dynamic equilibrium.

Three methods for examining the de novo proteome of microglia using BONCAT bioorthogonal labeling and FUNCAT click chemistry.

Bioorthogonal labeling and click chemistry techniques allow the detailed examination of cellular physiology through tagging and visualizing newly synthesized proteins. Here, we describe three methods applying bioorthogonal non-canonical amino acid tagging and fluorescent non-canonical amino acid tagging to quantify protein synthesis in microglia. We describe steps for cell seeding and labeling. We then detail microscopy, flow cytometry, and Western blotting techniques. These methods can be easily adapted for other cell types to explore cellular physiology in health and disease. For complete details on the use and execution of this protocol, please refer to Evans et al. (2021).1.

Transcriptional signature in microglia isolated from an Alzheimer's disease mouse model treated with scanning ultrasound.

Transcranial scanning ultrasound combined with intravenously injected microbubbles (SUS+MB) has been shown to transiently open the blood-brain barrier and reduce the amyloid-ß (Aß) pathology in the APP23 mouse model of Alzheimer's disease (AD). This has been accomplished through the activation of microglial cells; however, their response to the SUS treatment is incompletely understood. Here, wild-type (WT) and APP23 mice were subjected to SUS+MB, using nonsonicated mice as sham controls. After 48 h, the APP23 mice were injected with methoxy-XO4 to label Aß aggregates, followed by microglial isolation into XO4+ and XO4- populations using flow cytometry. Both XO4+ and XO4- cells were subjected to RNA sequencing and transcriptome profiling. The analysis of the microglial cells revealed a clear segregation depending on genotype (AD model vs. WT mice) and Aß internalization (XO4+ vs. XO4- microglia), but interestingly, no differences were found between SUS+MB and sham in WT mice. Differential gene expression analysis in APP23 mice detected 278 genes that were significantly changed by SUS+MB in the XO4+ cells (248 up/30 down) and 242 in XO- cells (225 up/17 down). Pathway analysis highlighted differential expression of genes related to the phagosome pathway and marked upregulation of cell cycle-related transcripts in XO4+ and XO4- microglia isolated from SUS+MB-treated APP23 mice. Together, this highlights the complexity of the microglial response to transcranial ultrasound, with potential applications for the treatment of AD.

Isolation and culture of pure adult mouse microglia and astrocytes for in vitro characterization and analyses.

Microglia and astrocytes are implicated in aging and age-related diseases. Here, we present a protocol to isolate and culture these glia cells from the murine brain. The protocol consists of two parts: magnetic sorting of adult microglia and mechanical/magnetic sorting of adult microglia and astrocytes. We then describe the characterization of these glial cells by flow cytometry and immunohistochemistry. Microglia isolated from aged mice maintain age-related phenotype during culture. These purified glia cells can be applied in ex vivo studies.

An early proinflammatory transcriptional response to tau pathology is age-specific and foreshadows reduced tau burden.

Age is one of the strongest risk factors for the development of neurodegenerative diseases, the majority of which involve misfolded protein aggregates in the brain. These protein aggregates are thought to drive pathology and are attractive targets for the development of new therapies. However, it is unclear how age influences the onset of pathology and the accompanying molecular response. To address this knowledge gap, we used a model of seeded tau pathology to profile the transcriptomic changes in 3 and 12 month old mice in response to developing tau hyperphosphorylation and aggregation. First, we found the burden of hyperphosphorylated tau pathology in mice injected at 12 months of age was moderately reduced compared to animals injected at 3 months. On a molecular level, we found an inflammation-related subset of genes, including C3 and the disease-associated microglia genes Ctsd, Cst7, and Clec7a, were more expressed early in disease in 12 but not 3 month old mice. These findings provide evidence of an early, age-specific response to tau pathology, which could serve as a marker for the severity of downstream pathology.

Altered ribosomal function and protein synthesis caused by tau.

The synthesis of new proteins is a fundamental aspect of cellular life and is required for many neurological processes, including the formation, updating and extinction of long-term memories. Protein synthesis is impaired in neurodegenerative diseases including tauopathies, in which pathology is caused by aberrant changes to the microtubule-associated protein tau. We recently showed that both global de novo protein synthesis and the synthesis of select ribosomal proteins (RPs) are decreased in mouse models of frontotemporal dementia (FTD) which express mutant forms of tau. However, a comprehensive analysis of the effect of FTD-mutant tau on ribosomes is lacking. Here we used polysome profiling, de novo protein labelling and mass spectrometry-based proteomics to examine how ribosomes are altered in models of FTD. We identified 10 RPs which were decreased in abundance in primary neurons taken from the K3 mouse model of FTD. We further demonstrate that expression of human tau (hTau) decreases both protein synthesis and biogenesis of the 60S ribosomal subunit, with these effects being exacerbated in the presence of FTD-associated tau mutations. Lastly, we demonstrate that expression of the amino-terminal projection domain of hTau is sufficient to reduce protein synthesis and ribosomal biogenesis. Together, these data reinforce a role for tau in impairing ribosomal function.

Tau antibody isotype induces differential effects following passive immunisation of tau transgenic mice.

One of the main pathological hallmarks of Alzheimer's disease (AD) is the intraneuronal accumulation of hyperphosphorylated tau. Passive immunotherapy is a promising strategy for the treatment of AD and there are currently a number of tau-specific monoclonal antibodies in clinical trials. A proposed mechanism of action is to engage and clear extracellular, pathogenic forms of tau. This process has been shown in vitro to be facilitated by microglial phagocytosis through interactions between the antibody-tau complex and microglial Fc-receptors. As this interaction is mediated by the conformation of the antibody's Fc domain, this suggests that the antibody isotype may affect the microglial phagocytosis and clearance of tau, and hence, the overall efficacy of tau antibodies. We therefore aimed to directly compare the efficacy of the tau-specific antibody, RN2N, cloned into a murine IgG1/κ framework, which has low affinity Fc-receptor binding, to that cloned into a murine IgG2a/κ framework, which has high affinity Fc-receptor binding. Our results demonstrate, for RN2N, that although enhanced microglial activation via the IgG2a/κ isotype increased extracellular tau phagocytosis in vitro, the IgG1/κ isoform demonstrated enhanced ability to reduce tau pathology and microgliosis following passive immunisation of the P301L tau transgenic pR5 mouse model.

De novo proteomic methods for examining the molecular mechanisms underpinning long-term memory.

Memory formation is a fundamental function of the nervous system that enables the experience-based adaptation of behaviour. The formation, recall and updating of long-term memory (LTM) requires new protein synthesis through its direct involvement in neuronal processes, such as long-term potentiation (LTP), long-term depression (LTD) and synaptic scaling. We discuss the advantages and limitations of several emerging techniques which enable the tagging of newly synthesised proteins, including stable isotope labelling with amino acids in cell culture (SILAC), puromycin labelling, and non-canonical amino acid (NCAA) labelling. We further present how these methods allow for the identification and visualisation of proteins which are newly synthesised during different stages of memory formation. These emerging techniques will continue to expand our understanding of how memories are formed, consolidated and retrieved.

Delivery of Antibodies into the Brain Using Focused Scanning Ultrasound.

Only a small fraction of therapeutic antibodies targeting brain diseases are taken up by the brain. Focused ultrasound offers a possibility to increase uptake of antibodies and engagement through transient opening of the blood-brain barrier (BBB). In our laboratory, we are developing therapeutic approaches for neurodegenerative diseases in which an antibody in various formats is delivered across the BBB using microbubbles, concomitant with focused ultrasound application through the skull targeting multiple spots, an approach we refer to as scanning ultrasound (SUS). The mechanical effects of microbubbles and ultrasound on blood vessels increases paracellular transport across the BBB by transiently separating tight junctions and enhances vesicle- mediated transcytosis, allowing antibodies and therapeutic agents to effectively cross. Moreover, ultrasound also facilitates the uptake of antibodies from the interstitial brain into brain cells such as neurons where the antibody distributes throughout the cell body and even into neuritic processes. In our studies, fluorescently labeled antibodies are prepared, mixed with in-house prepared lipid-based microbubbles and injected into mice immediately before SUS is applied to the brain. The increased antibody concentration in the brain is then quantified. To account for alterations in normal brain homeostasis, microglial phagocytosis can be used as a cellular marker. The generated data suggest that ultrasound delivery of antibodies is an attractive approach to treat neurodegenerative diseases.

PTEN activation contributes to neuronal and synaptic engulfment by microglia in tauopathy.

Phosphatase and tensin homolog (PTEN) regulates synaptic density in development; however, whether PTEN also regulates synapse loss in a neurodegenerative disorder such as frontotemporal lobar degeneration with Tau deposition (FTLD-Tau) has not been explored. Here, we found that pathological Tau promotes early activation of PTEN, which precedes apoptotic caspase-3 cleavage in the rTg4510 mouse model of FTLD-Tau. We further demonstrate increased synaptic and neuronal exposure of the apoptotic signal phosphatidylserine that tags neuronal structures for microglial uptake, thereby linking PTEN activation to synaptic and neuronal structure elimination. By applying pharmacological inhibition of PTEN's protein phosphatase activity, we observed that microglial uptake can be decreased in Tau transgenic mice. Finally, we reveal a dichotomous relationship between PTEN activation and age in FTLD-Tau patients and healthy controls. Together, our findings suggest that in tauopathy, PTEN has a role in the synaptotoxicity of pathological Tau and promotes microglial removal of affected neuronal structures.

Cell-specific non-canonical amino acid labelling identifies changes in the de novo proteome during memory formation.

The formation of spatial long-term memory (LTM) requires the de novo synthesis of distinct sets of proteins; however, a non-biased examination of the de novo proteome in this process is lacking. Here, we generated a novel mouse strain, which enables cell-type-specific labelling of newly synthesised proteins with non-canonical amino acids (NCAAs) by genetically restricting the expression of the mutant tRNA synthetase, NLL-MetRS, to hippocampal neurons. By combining this labelling technique with an accelerated version of the active place avoidance task and bio-orthogonal non-canonical amino acid tagging (BONCAT) followed by SWATH quantitative mass spectrometry, we identified 156 proteins that were altered in synthesis in hippocampal neurons during spatial memory formation. In addition to observing increased synthesis of known proteins important in memory-related processes, such as glutamate receptor recycling, we also identified altered synthesis of proteins associated with mRNA splicing as a potential mechanism involved in spatial LTM formation.

Decreased synthesis of ribosomal proteins in tauopathy revealed by non-canonical amino acid labelling.

Tau is a scaffolding protein that serves multiple cellular functions that are perturbed in neurodegenerative diseases, including Alzheimer's disease (AD) and frontotemporal dementia (FTD). We have recently shown that amyloid-ß, the second hallmark of AD, induces de novo protein synthesis of tau. Importantly, this activation was found to be tau-dependent, raising the question of whether FTD-tau by itself affects protein synthesis. We therefore applied non-canonical amino acid labelling to visualise and identify newly synthesised proteins in the K369I tau transgenic K3 mouse model of FTD. This revealed massively decreased protein synthesis in neurons containing pathologically phosphorylated tau, a finding confirmed in P301L mutant tau transgenic rTg4510 mice. Using quantitative SWATH-MS proteomics, we identified changes in 247 proteins of the de novo proteome of K3 mice. These included decreased synthesis of the ribosomal proteins RPL23, RPLP0, RPL19 and RPS16, a finding that was validated in both K3 and rTg4510 mice. Together, our findings present a potential pathomechanism by which pathological tau interferes with cellular functions through the dysregulation of ribosomal protein synthesis.

TREM2 triggers microglial density and age-related neuronal loss.

The microglial triggering receptor expressed on myeloid cells 2 (TREM2) signals via the activatory membrane adaptor molecule TYROBP. Genetic variants or mutations of TREM2 or TYROBP have been linked to inflammatory neurodegenerative diseases associated with aging. The typical aging process goes along with microglial changes and mild neuronal loss, but the exact contribution of TREM2 is still unclear. Aged TREM2 knock-out mice showed decreased age-related neuronal loss in the substantia nigra and the hippocampus. Transcriptomic analysis of the brains of 24 months old TREM2 knock-out mice revealed 211 differentially expressed genes mostly downregulated and associated with complement activation and oxidative stress response pathways. Consistently, 24 months old TREM2 knock-out mice showed lower transcription of microglial (Aif1 and Tmem119), oxidative stress markers (Inos, Cyba, and Cybb) and complement components (C1qa, C1qb, C1qc, C3, C4b, Itgam, and Itgb2), decreased microglial numbers and expression of the microglial activation marker Cd68, as well as accumulation of oxidized lipids. Cultured microglia of TREM2 knock-out mice showed reduced phagocytosis and oxidative burst. Thus, microglial TREM2 contributes to age-related microglial changes, phagocytic oxidative burst, and loss of neurons with possible detrimental effects during physiological aging.

Rodent models for Alzheimer disease.

Animal models are indispensable tools for Alzheimer disease (AD) research. Over the course of more than two decades, an increasing number of complementary rodent models has been generated. These models have facilitated testing hypotheses about the aetiology and progression of AD, dissecting the associated pathomechanisms and validating therapeutic interventions, thereby providing guidance for the design of human clinical trials. However, the lack of success in translating rodent data into therapeutic outcomes may challenge the validity of the current models. This Review critically evaluates the genetic and non-genetic strategies used in AD modelling, discussing their strengths and limitations, as well as new opportunities for the development of better models for the disease.

Amyloid-ß and tau complexity - towards improved biomarkers and targeted therapies.

Most neurodegenerative diseases are proteinopathies, which are characterized by the aggregation of misfolded proteins. Although many proteins have an intrinsic propensity to aggregate, particularly when cellular clearance systems start to fail in the context of ageing, only a few form fibrillar aggregates. In Alzheimer disease, the peptide amyloid-ß (Aß) and the protein tau aggregate to form plaques and tangles, respectively, which comprise the histopathological hallmarks of this disease. This Review discusses the complexity of Aß biogenesis, trafficking, post-translational modifications and aggregation states. Tau and its various isoforms, which are subject to a vast array of post-translational modifications, are also explored. The methodological advances that revealed this complexity are described. Finally, the toxic effects of distinct species of tau and Aß are discussed, as well as the concept of protein 'strains', and how this knowledge can facilitate the development of early disease biomarkers for stratifying patients and validating new therapies. By targeting distinct species of Aß and tau for therapeutic intervention, the way might be paved for personalized medicine and more-targeted treatment strategies.

Accelerated aging exacerbates a pre-existing pathology in a tau transgenic mouse model.

Age is a critical factor in the prevalence of tauopathies, including Alzheimer's disease. To observe how an aging phenotype interacts with and affects the pathological intracellular accumulation of hyperphosphorylated tau, the tauopathy mouse model pR5 (expressing P301L mutant human tau) was back-crossed more than ten times onto a senescence-accelerated SAMP8 background to establish the new strain, SApT. Unlike SAMP8 mice, pR5 mice are characterized by a robust tau pathology particularly in the amygdala and hippocampus. Analysis of age-matched SApT mice revealed that pathological tau phosphorylation was increased in these brain regions compared to those in the parental pR5 strain. Moreover, as revealed by immunohistochemistry, phosphorylation of critical tau phospho-epitopes (P-Ser202/P-Ser205 and P-Ser235) was significantly increased in the amygdala of SApT mice in an age-dependent manner, suggesting an age-associated effect of tau phosphorylation. Anxiety tests revealed that the older cohort of SApT mice (10 months vs. 8 months) exhibited a behavioural pattern similar to that observed for age-matched tau transgenic pR5 mice and not the SAMP8 parental mice. Learning and memory, however, appeared to be governed by the accelerated aging background of the SAMP8 strain, as at both ages investigated, SAMP8 and SApT mice showed a decreased learning capacity compared to pR5 mice. We therefore conclude that accelerated aging exacerbates pathological tau phosphorylation, leading to changes in normal behaviour. These findings further suggest that SApT mice may be a useful novel model in which to study the role of a complex geriatric phenotype in tauopathy.

Tau physiology and pathomechanisms in frontotemporal lobar degeneration.

Frontotemporal lobar degeneration (FTLD) has been associated with toxic intracellular aggregates of hyperphosphorylated tau (FTLD-tau). Moreover, genetic studies identified mutations in the MAPT gene encoding tau in familial cases of the disease. In this review, we cover a range of aspects of tau function, both in the healthy and diseased brain, discussing several in vitro and in vivo models. Tau structure and function in the healthy brain is presented, accentuating its distinct compartmentalization in neurons and its role in microtubule stabilization and axonal transport. Furthermore, tau-driven pathology is discussed, introducing current concepts and the underlying experimental evidence. Different aspects of pathological tau phosphorylation, the protein's genomic and domain organization as well as its spreading in disease, together with MAPT-associated mutations and their respective models are presented. Dysfunction related to other post-transcriptional modifications and their effect on normal neuronal functions such as cell cycle, epigenetics and synapse dynamics are also discussed, providing a mechanistic explanation for the observations made in FTLD-tau cases, with the possibility for therapeutic intervention. In this review, we cover aspects of tau function, both in the healthy and diseased brain, referring to different in vitro and in vivo models. In healthy neurons, tau is compartmentalized, with higher concentrations found in the distal part of the axon. Cargo molecules are sensitive to this gradient. A disturbed tau distribution, as found in frontotemporal lobar degeneration (FTLD-tau), has severe consequences for cellular physiology: tau accumulates in the neuronal soma and dendrites, leading among others to microtubule depolymerization and impaired axonal transport. Tau forms insoluble aggregates that sequester additional molecules stalling cellular physiology. Neuronal communication is gradually lost as toxic tau accumulates in dendritic spines with subsequent degeneration of synapses and synaptic loss. Thus, by providing a mechanistic explanation for the observations made in FTLD-tau cases, arises a possibility for therapeutic interventions. This article is part of the Frontotemporal Dementia special issue.