Medin co-aggregates with vascular amyloid-ß in Alzheimer's disease.

Aggregates of medin amyloid (a fragment of the protein MFG-E8, also known as lactadherin) are found in the vasculature of almost all humans over 50 years of age1,2, making it the most common amyloid currently known. We recently reported that medin also aggregates in blood vessels of ageing wild-type mice, causing cerebrovascular dysfunction3. Here we demonstrate in amyloid-ß precursor protein (APP) transgenic mice and in patients with Alzheimer's disease that medin co-localizes with vascular amyloid-ß deposits, and that in mice, medin deficiency reduces vascular amyloid-ß deposition by half. Moreover, in both the mouse and human brain, MFG-E8 is highly enriched in the vasculature and both MFG-E8 and medin levels increase with the severity of vascular amyloid-ß burden. Additionally, analysing data from 566 individuals in the ROSMAP cohort, we find that patients with Alzheimer's disease have higher MFGE8 expression levels, which are attributable to vascular cells and are associated with increased measures of cognitive decline, independent of plaque and tau pathology. Mechanistically, we demonstrate that medin interacts directly with amyloid-ß to promote its aggregation, as medin forms heterologous fibrils with amyloid-ß, affects amyloid-ß fibril structure, and cross-seeds amyloid-ß aggregation both in vitro and in vivo. Thus, medin could be a therapeutic target for prevention of vascular damage and cognitive decline resulting from amyloid-ß deposition in the blood vessels of the brain.

Innate immune memory in the brain shapes neurological disease hallmarks.

Innate immune memory is a vital mechanism of myeloid cell plasticity that occurs in response to environmental stimuli and alters subsequent immune responses. Two types of immunological imprinting can be distinguished-training and tolerance. These are epigenetically mediated and enhance or suppress subsequent inflammation, respectively. Whether immune memory occurs in tissue-resident macrophages in vivo and how it may affect pathology remains largely unknown. Here we demonstrate that peripherally applied inflammatory stimuli induce acute immune training and tolerance in the brain and lead to differential epigenetic reprogramming of brain-resident macrophages (microglia) that persists for at least six months. Strikingly, in a mouse model of Alzheimer's pathology, immune training exacerbates cerebral ß-amyloidosis and immune tolerance alleviates it; similarly, peripheral immune stimulation modifies pathological features after stroke. Our results identify immune memory in the brain as an important modifier of neuropathology.

Medin aggregation causes cerebrovascular dysfunction in aging wild-type mice.

Medin is the most common amyloid known in humans, as it can be found in blood vessels of the upper body in virtually everybody over 50 years of age. However, it remains unknown whether deposition of Medin plays a causal role in age-related vascular dysfunction. We now report that aggregates of Medin also develop in the aorta and brain vasculature of wild-type mice in an age-dependent manner. Strikingly, genetic deficiency of the Medin precursor protein, MFG-E8, eliminates not only vascular aggregates but also prevents age-associated decline of cerebrovascular function in mice. Given the prevalence of Medin aggregates in the general population and its role in vascular dysfunction with aging, targeting Medin may become a novel approach to sustain healthy aging.

Second Harmonic Generation Microscopy as a Novel Intraoperative Assessment of Rat Median Nerve Injury.

PURPOSE: Nerves that are functionally injured but appear macroscopically intact pose the biggest clinical dilemma. Second Harmonic Generation (SHG) Microscopy may provide a real-time assessment of nerve damage, with the ultimate goal of allowing surgeons to accurately quantify the degree of nerve damage present. The aim of this study was to demonstrate the utility of SHG microscopy to detect nerve damage in vivo in an animal model. METHODS: Ten Sprague-Dawley rats were anesthetized and prepared for surgery. After surgical exposure and using a custom-made stretch applicator, the right median nerves were stretched by 20%, corresponding to a high strain injury, and held for 5 minutes. The left median nerve served as a sham control (SC), only being placed in the applicator for 5 minutes with no stretch. A nerve stimulator was used to assess the amount of stimulation required to induce a flicker and contraction of the paw. Nerves were then imaged using a multiphoton laser scanning microscope. RESULTS: Immediately after injury (day 0), SHG images of SC median nerves exhibited parallel collagen fibers with linear, organized alignment. In comparison with SC nerves, high strain nerves demonstrated artifacts indicative of nerve damage consisting of wavy, undulating fibers with crossing fibers and tears, as well as a decrease in the linear organization, which correlated with an increase in the mean stimulation required to induce a flicker and contraction of the paw. CONCLUSIONS: Second Harmonic Generation microscopy may provide the ability to detect an acute neural stretch injury in the rat median nerve. Epineurial collagen disorganization correlated with the stimulation required for nerve function. CLINICAL RELEVANCE: In the future, SHG may provide the ability to visualize nerve damage intraoperatively, allowing for better clinical decision-making. However, this is currently a research tool and requires further validation before translating to the clinical setting.

Early Aß reduction prevents progression of cerebral amyloid angiopathy.

OBJECTIVE: Clinical trials targeting ß-amyloid peptides (Aß) for Alzheimer disease (AD) failed for arguable reasons that include selecting the wrong stages of AD pathophysiology or Aß being the wrong target. Targeting Aß to prevent cerebral amyloid angiopathy (CAA) has not been rigorously followed, although the causal role of Aß for CAA and related hemorrhages is undisputed. CAA occurs with normal aging and to various degrees in AD, where its impact and treatment is confounded by the presence of parenchymal Aß deposition. METHODS: APPDutch mice develop CAA in the absence of parenchymal amyloid, mimicking hereditary cerebral hemorrhage with amyloidosis Dutch type (HCHWA-D). Mice were treated with a ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitor. We used 3-dimensional ultramicroscopy and immunoassays for visualizing CAA and assessing Aß in cerebrospinal fluid (CSF) and brain. RESULTS: CAA onset in mice was at 22 to 24 months, first in frontal leptomeningeal and superficial cortical vessels followed by vessels penetrating the cortical layers. CSF Aß increased with aging followed by a decrease of both Aß40 and Aß42 upon CAA onset, supporting the idea that combined reduction of CSF Aß40 and Aß42 is a specific biomarker for vascular amyloid. BACE1 inhibitor treatment starting at CAA onset and continuing for 4 months revealed a 90% Aß reduction in CSF and largely prevented CAA progression and associated pathologies. INTERPRETATION: This is the first study showing that Aß reduction at early disease time points largely prevents CAA in the absence of parenchymal amyloid. Our observation provides a preclinical basis for Aß-reducing treatments in patients at risk of CAA and in presymptomatic HCHWA-D. ANN NEUROL 2019;86:561-571.

Functional and transcriptomic insights into pathogenesis of R9C phospholamban mutation using human induced pluripotent stem cell-derived cardiomyocytes.

Dilated cardiomyopathy (DCM) can be caused by mutations in the cardiac protein phospholamban (PLN). We used CRISPR/Cas9 to insert the R9C PLN mutation at its endogenous locus into a human induced pluripotent stem cell (hiPSC) line from an individual with no cardiovascular disease. R9C PLN hiPSC-CMs display a blunted ß-agonist response and defective calcium handling. In 3D human engineered cardiac tissues (hECTs), a blunted lusitropic response to ß-adrenergic stimulation was observed with R9C PLN. hiPSC-CMs harboring the R9C PLN mutation showed activation of a hypertrophic phenotype, as evidenced by expression of hypertrophic markers and increased cell size and capacitance of cardiomyocytes. RNA-seq suggests that R9C PLN results in an altered metabolic state and profibrotic signaling, which was confirmed by gene expression analysis and picrosirius staining of R9C PLN hECTs. The expression of several miRNAs involved in fibrosis, hypertrophy, and cardiac metabolism were also perturbed in R9C PLN hiPSC-CMs. This study contributes to better understanding of the pathogenic mechanisms of the hereditary R9C PLN mutation in the context of human cardiomyocytes.

Conversion of Synthetic Aß to In Vivo Active Seeds and Amyloid Plaque Formation in a Hippocampal Slice Culture Model.

UNLABELLED: The aggregation of amyloid-ß peptide (Aß) in brain is an early event and hallmark of Alzheimer's disease (AD). We combined the advantages of in vitro and in vivo approaches to study cerebral ß-amyloidosis by establishing a long-term hippocampal slice culture (HSC) model. While no Aß deposition was noted in untreated HSCs of postnatal Aß precursor protein transgenic (APP tg) mice, Aß deposition emerged in HSCs when cultures were treated once with brain extract from aged APP tg mice and the culture medium was continuously supplemented with synthetic Aß. Seeded Aß deposition was also observed under the same conditions in HSCs derived from wild-type or App-null mice but in no comparable way when HSCs were fixed before cultivation. Both the nature of the brain extract and the synthetic Aß species determined the conformational characteristics of HSC Aß deposition. HSC Aß deposits induced a microglia response, spine loss, and neuritic dystrophy but no obvious neuron loss. Remarkably, in contrast to in vitro aggregated synthetic Aß, homogenates of Aß deposits containing HSCs induced cerebral ß-amyloidosis upon intracerebral inoculation into young APP tg mice. Our results demonstrate that a living cellular environment promotes the seeded conversion of synthetic Aß into a potent in vivo seeding-active form. SIGNIFICANCE STATEMENT: In this study, we report the seeded induction of Aß aggregation and deposition in long-term hippocampal slice cultures. Remarkably, we find that the biological activities of the largely synthetic Aß aggregates in the culture are very similar to those observed in vivo This observation is the first to show that potent in vivo seeding-active Aß aggregates can be obtained by seeded conversion of synthetic Aß in a living (wild-type) cellular environment.

Spine loss in primary somatosensory cortex during trace eyeblink conditioning.

Classical conditioning that involves mnemonic processing, that is, a "trace" period between conditioned and unconditioned stimulus, requires awareness of the association to be formed and is considered a simple model paradigm for declarative learning. Barrel cortex, the whisker representation of primary somatosensory cortex, is required for the learning of a tactile variant of trace eyeblink conditioning (TTEBC) and undergoes distinct map plasticity during learning. To investigate the cellular mechanism underpinning TTEBC and concurrent map plasticity, we used two-photon imaging of dendritic spines in barrel cortex of awake mice while being conditioned. Monitoring layer 5 neurons' apical dendrites in layer 1, we show that one cellular expression of barrel cortex plasticity is a substantial spine count reduction of ∼15% of the dendritic spines present before learning. The number of eliminated spines and their time of elimination are tightly related to the learning success. Moreover, spine plasticity is highly specific for the principal barrel column receiving the main signals from the stimulated vibrissa. Spines located in other columns, even those directly adjacent to the principal column, are unaffected. Because layer 1 spines integrate signals from associative thalamocortical circuits, their column-specific elimination suggests that this spine plasticity may be the result of an association of top-down signals relevant for declarative learning and spatially precise ascending tactile signals.

Multiple factors contribute to the peripheral induction of cerebral ß-amyloidosis.

Deposition of aggregated amyloid-ß (Aß) peptide in brain is an early event and hallmark pathology of Alzheimer's disease and cerebral Aß angiopathy. Experimental evidence supports the concept that Aß multimers can act as seeds and structurally corrupt other Aß peptides by a self-propagating mechanism. Here we compare the induction of cerebral ß-amyloidosis by intraperitoneal applications of Aß-containing brain extracts in three Aß-precursor protein (APP) transgenic mouse lines that differ in levels of transgene expression in brain and periphery (APP23 mice, APP23 mice lacking murine APP, and R1.40 mice). Results revealed that beta-amyloidosis induction, which could be blocked with an anti-Aß antibody, was dependent on the amount of inoculated brain extract and on the level of APP/Aß expression in the brain but not in the periphery. The induced Aß deposits in brain occurred in a characteristic pattern consistent with the entry of Aß seeds at multiple brain locations. Intraperitoneally injected Aß could be detected in blood monocytes and some peripheral tissues (liver, spleen) up to 30 d after the injection but escaped histological and biochemical detection thereafter. These results suggest that intraperitoneally inoculated Aß seeds are transported from the periphery to the brain in which corruptive templating of host Aß occurs at multiple sites, most efficiently in regions with high availability of soluble Aß.

Spectral discrimination of cerebral amyloid lesions after peripheral application of luminescent conjugated oligothiophenes.

In vivo imaging of pathological protein aggregates provides essential knowledge of the kinetics and implications of these lesions in the progression of proteopathies, such as Alzheimer disease. Luminescent conjugated oligothiophenes are amyloid-specific ligands that bind and spectrally distinguish different types of amyloid aggregates. Herein, we report that heptamer formyl thiophene acetic acid (hFTAA) passes the blood-brain barrier after systemic administration and specifically binds to extracellular ß-amyloid deposits in the brain parenchyma (Aß plaques) and in the vasculature (cerebral ß-amyloid angiopathy) of ß-amyloid precursor protein transgenic APP23 mice. Moreover, peripheral application of hFTAA also stained intracellular lesions of hyperphosphorylated Tau protein in P301S Tau transgenic mice. Spectral profiling of all three amyloid types was acquired ex vivo using two-photon excitation. hFTAA revealed a distinct shift in its emission spectra when bound to Aß plaques versus Tau lesions. Furthermore, a spectral shift was observed for Aß plaques versus cerebral ß-amyloid angiopathy, indicating that different amyloid types and structural variances of a specific amyloid type can be distinguished. In conclusion, by adding spectral signatures to amyloid lesions, our results pave the way for a new area of in vivo amyloid imaging, allowing in vivo differentiation of amyloid (sub)types and monitoring changes of their structure/composition over time.

Sirtuin-1 sensitive lysine-136 acetylation drives phase separation and pathological aggregation of TDP-43.

Trans-activation response DNA-binding protein of 43 kDa (TDP-43) regulates RNA processing and forms neuropathological aggregates in patients with amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Investigating TDP-43 post-translational modifications, we discovered that K84 acetylation reduced nuclear import whereas K136 acetylation impaired RNA binding and splicing capabilities of TDP-43. Such failure of RNA interaction triggered TDP-43 phase separation mediated by the C-terminal low complexity domain, leading to the formation of insoluble aggregates with pathologically phosphorylated and ubiquitinated TDP-43. Introduction of acetyl-lysine at the identified sites via amber suppression confirmed the results from site-directed mutagenesis. K84-acetylated TDP-43 showed cytoplasmic mislocalization, and the aggregation propensity of K136-acetylated TDP-43 was confirmed. We generated antibodies selective for TDP-43 acetylated at these lysines, and found that sirtuin-1 can potently deacetylate K136-acetylated TDP-43 and reduce its aggregation propensity. Thus, distinct lysine acetylations modulate nuclear import, RNA binding and phase separation of TDP-43, suggesting regulatory mechanisms for TDP-43 pathogenesis.

Experimental evidence for temporal uncoupling of brain Aß deposition and neurodegenerative sequelae.

Brain Aß deposition is a key early event in the pathogenesis of Alzheimer´s disease (AD), but the long presymptomatic phase and poor correlation between Aß deposition and clinical symptoms remain puzzling. To elucidate the dependency of downstream pathologies on Aß, we analyzed the trajectories of cerebral Aß accumulation, Aß seeding activity, and neurofilament light chain (NfL) in the CSF (a biomarker of neurodegeneration) in Aß-precursor protein transgenic mice. We find that Aß deposition increases linearly until it reaches an apparent plateau at a late age, while Aß seeding activity increases more rapidly and reaches a plateau earlier, coinciding with the onset of a robust increase of CSF NfL. Short-term inhibition of Aß generation in amyloid-laden mice reduced Aß deposition and associated glial changes, but failed to reduce Aß seeding activity, and CSF NfL continued to increase although at a slower pace. When short-term or long-term inhibition of Aß generation was started at pre-amyloid stages, CSF NfL did not increase despite some Aß deposition, microglial activation, and robust brain Aß seeding activity. A dissociation of Aß load and CSF NfL trajectories was also found in familial AD, consistent with the view that Aß aggregation is not kinetically coupled to neurotoxicity. Rather, neurodegeneration starts when Aß seeding activity is saturated and before Aß deposition reaches critical (half-maximal) levels, a phenomenon reminiscent of the two pathogenic phases in prion disease.

Prominent microglial inclusions in transgenic mouse models of α-synucleinopathy that are distinct from neuronal lesions.

Alpha-synucleinopathies are a group of progressive neurodegenerative disorders, characterized by intracellular deposits of aggregated α-synuclein (αS). The clinical heterogeneity of these diseases is thought to be attributed to conformers (or strains) of αS but the contribution of inclusions in various cell types is unclear. The aim of the present work was to study αS conformers among different transgenic (TG) mouse models of α-synucleinopathies. To this end, four different TG mouse models were studied (Prnp-h[A53T]αS; Thy1-h[A53T]αS; Thy1-h[A30P]αS; Thy1-mαS) that overexpress human or murine αS and differed in their age-of-symptom onset and subsequent disease progression. Postmortem analysis of end-stage brains revealed robust neuronal αS pathology as evidenced by accumulation of αS serine 129 (p-αS) phosphorylation in the brainstem of all four TG mouse lines. Overall appearance of the pathology was similar and only modest differences were observed among additionally affected brain regions. To study αS conformers in these mice, we used pentameric formyl thiophene acetic acid (pFTAA), a fluorescent dye with amyloid conformation-dependent spectral properties. Unexpectedly, besides the neuronal αS pathology, we also found abundant pFTAA-positive inclusions in microglia of all four TG mouse lines. These microglial inclusions were also positive for Thioflavin S and showed immunoreactivity with antibodies recognizing the N-terminus of αS, but were largely p-αS-negative. In all four lines, spectral pFTAA analysis revealed conformational differences between microglia and neuronal inclusions but not among the different mouse models. Concomitant with neuronal lesions, microglial inclusions were already present at presymptomatic stages and could also be induced by seeded αS aggregation. Although nature and significance of microglial inclusions for human α-synucleinopathies remain to be clarified, the previously overlooked abundance of microglial inclusions in TG mouse models of α-synucleinopathy bears importance for mechanistic and preclinical-translational studies.

Acute targeting of pre-amyloid seeds in transgenic mice reduces Alzheimer-like pathology later in life.

Amyloid-ß (Aß) deposits are a relatively late consequence of Aß aggregation in Alzheimer's disease. When pathogenic Aß seeds begin to form, propagate and spread is not known, nor are they biochemically defined. We tested various antibodies for their ability to neutralize Aß seeds before Aß deposition becomes detectable in Aß precursor protein-transgenic mice. We also characterized the different antibody recognition profiles using immunoprecipitation of size-fractionated, native, mouse and human brain-derived Aß assemblies. At least one antibody, aducanumab, after acute administration at the pre-amyloid stage, led to a significant reduction of Aß deposition and downstream pathologies 6 months later. This demonstrates that therapeutically targetable pathogenic Aß seeds already exist during the lag phase of protein aggregation in the brain. Thus, the preclinical phase of Alzheimer's disease-currently defined as Aß deposition without clinical symptoms-may be a relatively late manifestation of a much earlier pathogenic seed formation and propagation that currently escapes detection in vivo.

Long-Term In Vivo Imaging of Individual Microglial Cells.

Microglia are morphologically dynamic cells, neatly arranged in an interconnected three-dimensional lattice throughout the brain, constantly surveying the parenchyma, and swiftly responding to a variety of external stimuli. Capturing the dynamics of their morphology, reaction to trauma, pathogens, or endogenous stimuli, and studying changes in their network in their physiological environment requires the use of two-photon microscopy, as well as a precise repositioning strategy. Herein, we describe a robust repeatable localization method, coupled with optimized in vivo two-photon microscopy for long-term imaging of single microglia cells in the mouse brain.

Quantitative organization of the excitatory synapses of the primate cerebellar nuclei: further evidence for a specialized architecture underlying the primate cerebellum.

The cerebellar intrinsic connectivity is of remarkable regularity with a similar build repeated many times over. However, several modifications of this basic circuitry occur that can provide important clues to evolutionary adaptations. We have observed differences in the wiring of the cerebellar output structures (the deep cerebellar nuclei, DCN) with higher dendritic length density in the phylogenetically newer DCN. In rats, we showed that an increase in wiring is associated with an increase in the presynaptic vesicular glutamate transporter 1 (vGluT1). In this study, we have extended our analysis to the rhesus monkey and can show similarities and differences between the two species. The similarities confirm a higher density in vGluT1+ boutons in the lateral (LN/dentate) and posterior interpositus nucleus compared to the phylogenetically older DCN. In general, we also observe a lower density of vGluT1 and 2+ boutons in the monkey, which however, yields a similar number of excitatory boutons per neuron in both species. The only exception is the vGlut1+ boutons in the macaque LN/dentate, which showed a significantly lower number of vGluT1+ boutons per neuron. We also detected a higher percentage of co-labelled vGluT1 and 2 boutons in the macaque than we found in the rat. In summary, these results confirm that the hyposcalled dendrites of the monkey LN/dentate also show a lower number of vGluT1+ boutons per neuron. These results provide further support of our model relating the dendritic morphology of the LN/dentate neurons to the morphology of the specially enlarged LN/dentate nucleus in primates.

Long-term adult human brain slice cultures as a model system to study human CNS circuitry and disease.

Most of our knowledge on human CNS circuitry and related disorders originates from model organisms. How well such data translate to the human CNS remains largely to be determined. Human brain slice cultures derived from neurosurgical resections may offer novel avenues to approach this translational gap. We now demonstrate robust preservation of the complex neuronal cytoarchitecture and electrophysiological properties of human pyramidal neurons in long-term brain slice cultures. Further experiments delineate the optimal conditions for efficient viral transduction of cultures, enabling 'high throughput' fluorescence-mediated 3D reconstruction of genetically targeted neurons at comparable quality to state-of-the-art biocytin fillings, and demonstrate feasibility of long term live cell imaging of human cells in vitro. This model system has implications toward a broad spectrum of translational studies, regarding the validation of data obtained in non-human model systems, for therapeutic screening and genetic dissection of human CNS circuitry.

Microglia turnover with aging and in an Alzheimer's model via long-term in vivo single-cell imaging.

To clarify the role of microglia in brain homeostasis and disease, an understanding of their maintenance, proliferation and turnover is essential. The lifespan of brain microglia, however, remains uncertain, and reflects confounding factors in earlier assessments that were largely indirect. We genetically labeled single resident microglia in living mice and then used multiphoton microscopy to monitor these cells over time. Under homeostatic conditions, we found that neocortical resident microglia were long-lived, with a median lifetime of well over 15 months; thus, approximately half of these cells survive the entire mouse lifespan. While proliferation of resident neocortical microglia under homeostatic conditions was low, microglial proliferation in a mouse model of Alzheimer's ß-amyloidosis was increased threefold. The persistence of individual microglia throughout the mouse lifespan provides an explanation for how microglial priming early in life can induce lasting functional changes and how microglial senescence may contribute to age-related neurodegenerative diseases.

Computer-generated ovaries to assist follicle counting experiments.

Precise estimation of the number of follicles in ovaries is of key importance in the field of reproductive biology, both from a developmental point of view, where follicle numbers are determined at specific time points, as well as from a therapeutic perspective, determining the adverse effects of environmental toxins and cancer chemotherapeutics on the reproductive system. The two main factors affecting follicle number estimates are the sampling method and the variation in follicle numbers within animals of the same strain, due to biological variability. This study aims at assessing the effect of these two factors, when estimating ovarian follicle numbers of neonatal mice. We developed computer algorithms, which generate models of neonatal mouse ovaries (simulated ovaries), with characteristics derived from experimental measurements already available in the published literature. The simulated ovaries are used to reproduce in-silico counting experiments based on unbiased stereological techniques; the proposed approach provides the necessary number of ovaries and sampling frequency to be used in the experiments given a specific biological variability and a desirable degree of accuracy. The simulated ovary is a novel, versatile tool which can be used in the planning phase of experiments to estimate the expected number of animals and workload, ensuring appropriate statistical power of the resulting measurements. Moreover, the idea of the simulated ovary can be applied to other organs made up of large numbers of individual functional units.