Long term worsening of amyloid pathology, cerebral function, and cognition after a single inoculation of beta-amyloid seeds with Osaka mutation.

Alzheimer's disease (AD) is characterized by intracerebral deposition of abnormal proteinaceous assemblies made of amyloid-ß (Aß) peptides or tau proteins. These peptides and proteins induce synaptic dysfunctions that are strongly correlated with cognitive decline. Intracerebral infusion of well-defined Aß seeds from non-mutated Aß1-40 or Aß1-42 peptides can increase Aß depositions several months after the infusion. Familial forms of AD are associated with mutations in the amyloid precursor protein (APP) that induce the production of Aß peptides with different structures. The Aß Osaka (Aßosa mutation (E693Δ)) is located within the Aß sequence and thus the Aßosa peptides have different structures and properties as compared to non-mutated Aß1-42 peptides (Aßwt). Here, we wondered if a single exposure to this mutated Aß can worsen AD pathology as well as downstream events including cognition, cerebral connectivity and synaptic health several months after the inoculation. To answer this question we inoculated Aß1-42-bearing Osaka mutation (Aßosa) in the dentate gyrus of APPswe/PS1dE9 mice at the age of two months. Their cognition and cerebral connectivity were analyzed at 4 months post-inoculation by behavioral evaluation and functional MRI. Aß pathology as well as synaptic density were evaluated by histology. The impact of Aßosa peptides on synaptic health was also measured on primary cortical neurons. Remarkably, the intracerebral administration of Aßosa induced cognitive and synaptic impairments as well as a reduction of functional connectivity between different brain regions, 4 months post-inoculation. It increased Aß plaque depositions and increased Aß oligomers. This is the first study showing that a single, sporadic event as Aßosa inoculation can worsen the fate of the pathology and clinical outcome several months after the event. It suggests that a single inoculation of Aß regulates a large cascade of events for a long time.

Development of an AAV-based model of tauopathy targeting retinal ganglion cells and the mouse visual pathway to study the role of microglia in Tau pathology.

Tauopathy is a typical feature of Alzheimer's disease of major importance because it strongly correlates with the severity of cognitive deficits experienced by patients. During the pathology, it follows a characteristic spatiotemporal course which takes its origin in the transentorhinal cortex, and then gradually invades the entire forebrain. To study the mechanisms of tauopathy, and test new therapeutic strategies, it is necessary to set-up relevant and versatile in vivo models allowing to recapitulate tauopathy. With this in mind, we have developed a model of tauopathy by overexpression of the human wild-type Tau protein in retinal ganglion cells in mice (RGCs). This overexpression led to the presence of hyperphosphorylated forms of the protein in the transduced cells as well as to their progressive degeneration. The application of this model to mice deficient in TREM2 (Triggering Receptor Expressed on Myeloid cells-2, an important genetic risk factor for AD) as well as to 15-month-old mice showed that microglia actively participate in the degeneration of RGCs. Surprisingly, although we were able to detect the transgenic Tau protein up to the terminal arborization of RGCs at the level of the superior colliculi, spreading of the transgenic Tau protein to post-synaptic neurons was detected only in aged animals. This suggests that there may be neuron-intrinsic- or microenvironment mediators facilitating this spreading that appear with aging.

Reactive astrocytes promote proteostasis in Huntington's disease through the JAK2-STAT3 pathway.

Huntington's disease is a fatal neurodegenerative disease characterized by striatal neurodegeneration, aggregation of mutant Huntingtin and the presence of reactive astrocytes. Astrocytes are important partners for neurons and engage in a specific reactive response in Huntington's disease that involves morphological, molecular and functional changes. How reactive astrocytes contribute to Huntington's disease is still an open question, especially because their reactive state is poorly reproduced in experimental mouse models. Here, we show that the JAK2-STAT3 pathway, a central cascade controlling astrocyte reactive response, is activated in the putamen of Huntington's disease patients. Selective activation of this cascade in astrocytes through viral gene transfer reduces the number and size of mutant Huntingtin aggregates in neurons and improves neuronal defects in two complementary mouse models of Huntington's disease. It also reduces striatal atrophy and increases glutamate levels, two central clinical outcomes measured by non-invasive magnetic resonance imaging. Moreover, astrocyte-specific transcriptomic analysis shows that activation of the JAK2-STAT3 pathway in astrocytes coordinates a transcriptional program that increases their intrinsic proteolytic capacity, through the lysosomal and ubiquitin-proteasome degradation systems. This pathway also enhances their production and exosomal release of the co-chaperone DNAJB1, which contributes to mutant Huntingtin clearance in neurons. Together, our results show that the JAK2-STAT3 pathway controls a beneficial proteostasis response in reactive astrocytes in Huntington's disease, which involves bi-directional signalling with neurons to reduce mutant Huntingtin aggregation, eventually improving disease outcomes.

giRAff: an automated atlas segmentation tool adapted to single histological slices.

Conventional histology of the brain remains the gold standard in the analysis of animal models. In most biological studies, standard protocols usually involve producing a limited number of histological slices to be analyzed. These slices are often selected into a specific anatomical region of interest or around a specific pathological lesion. Due to the lack of automated solutions to analyze such single slices, neurobiologists perform the segmentation of anatomical regions manually most of the time. Because the task is long, tedious, and operator-dependent, we propose an automated atlas segmentation method called giRAff, which combines rigid and affine registrations and is suitable for conventional histological protocols involving any number of single slices from a given mouse brain. In particular, the method has been tested on several routine experimental protocols involving different anatomical regions of different sizes and for several brains. For a given set of single slices, the method can automatically identify the corresponding slices in the mouse Allen atlas template with good accuracy and segmentations comparable to those of an expert. This versatile and generic method allows the segmentation of any single slice without additional anatomical context in about 1 min. Basically, our proposed giRAff method is an easy-to-use, rapid, and automated atlas segmentation tool compliant with a wide variety of standard histological protocols.

Pathological changes induced by Alzheimer's brain inoculation in amyloid-beta plaque-bearing mice.

Alzheimer's disease (AD) is characterized by intracerebral accumulations of extracellular amyloid-ß (Aß) plaques and intracellular tau pathology that spread in the brain. Three types of tau lesions occur in the form of neuropil threads, neurofibrillary tangles, and neuritic plaques i.e. tau aggregates within neurites surrounding Aß deposits. The cascade of events linking these lesions and synaptic or memory impairments are still debated. Intracerebral infusion of human AD brain extracts in Aß plaque-bearing mice that do not overexpress pathological tau proteins induces tau pathologies following heterotopic seeding of mouse tau protein. There is however little information regarding the downstream events including synaptic or cognitive repercussions of tau pathology induction in these models. In the present study, human AD brain extracts (ADbe) and control-brain extracts (Ctrlbe) were infused into the hippocampus of Aß plaque-bearing APPswe/PS1dE9 mice. Memory, synaptic density, as well as Aß plaque and tau aggregate loads, microgliosis, astrogliosis at the inoculation site and in connected regions (perirhinal/entorhinal cortex) were evaluated 4 and 8 months post-inoculation. ADbe inoculation produced the following effects: (i) memory deficit; (ii) increased Aß plaque deposition in proximity to the inoculation site; (iii) tau pathology induction; (iv) appearance of neuropil threads and neurofibrillary tangles next to the inoculation site with a spreading to connected regions. Neuritic plaque pathology was detected in both ADbe- and Ctrlbe-inoculated animals but ADbe inoculation increased the severity close to and at distance of the inoculation site. (v) Finally, ADbe inoculation reduced synaptic density in the vicinity to the inoculation site and in connected regions as the perirhinal/entorhinal cortex. Synaptic impairments were correlated with increased severity of neuritic plaques but not to other tau lesions or Aß lesions, suggesting that neuritic plaques are a culprit for synaptic loss. Synaptic density was also associated with microglial load.

Multiscale segmentation- and error-guided iterative convolutional neural network for cerebral neuron segmentation in microscopic images.

This article uses microscopy images obtained from diverse anatomical regions of macaque brain for neuron semantic segmentation. The complex structure of brain, the large intra-class staining intensity difference within neuron class, the small inter-class staining intensity difference between neuron and tissue class, and the unbalanced dataset increase the difficulty of neuron semantic segmentation. To address this problem, we propose a multiscale segmentation- and error-guided iterative convolutional neural network (MSEG-iCNN) to improve the semantic segmentation performance in major anatomical regions of the macaque brain. After evaluating microscopic images from 17 anatomical regions, the semantic segmentation performance of neurons is improved by 10.6%, 4.0%, 1.5%, and 1.2% compared with Random Forest, FCN-8s, U-Net, and UNet++, respectively. Especially for neurons with brighter staining intensity in the anatomical regions such as lateral geniculate, globus pallidus and hypothalamus, the performance is improved by 66.1%, 23.9%, 11.2%, and 6.7%, respectively. Experiments show that our proposed method can efficiently segment neurons with a wide range of staining intensities. The semantic segmentation results are of great significance and can be further used for neuron instance segmentation, morphological analysis and disease diagnosis. Cell segmentation plays a critical role in extracting cerebral information, such as cell counting, cell morphometry and distribution analysis. Accurate automated neuron segmentation is challenging due to the complex structure of brain, the large intra-class staining intensity difference within neuron class, the small inter-class staining intensity difference between neuron and tissue class, and the unbalanced dataset. The proposed multiscale segmentation- and error-guided iterative convolutional neural network (MSEG-iCNN) improve the segmentation performance in 17 major anatomical regions of the macaque brain. HIGHLIGHTS: Cell segmentation plays a critical role in extracting cerebral information, such as cell counting, cell morphometry and distribution analysis. Accurate automated neuron segmentation is challenging due to the complex structure of brain, the large intra-class staining intensity difference within neuron class, the small inter-class staining intensity difference between neuron and tissue class, and the unbalanced dataset. The proposed multiscale segmentation- and error-guided iterative convolutional neural network (MSEG-iCNN) improve the segmentation performance in 17 major anatomical regions of the macaque brain.

Transmission of amyloid-beta and tau pathologies is associated with cognitive impairments in a primate.

Amyloid-ß (Aß) pathology transmission has been described in patients following iatrogenic exposure to compounds contaminated with Aß proteins. It can induce cerebral Aß angiopathy resulting in brain hemorrhages and devastating clinical impacts. Iatrogenic transmission of tau pathology is also suspected but not experimentally proven. In both scenarios, lesions were detected several decades after the putatively triggering medico-surgical act. There is however little information regarding the cognitive repercussions in individuals who do not develop cerebral hemorrhages. In the current study, we inoculated the posterior cingulate cortex and underlying corpus callosum of young adult primates (Microcebus murinus) with either Alzheimer's disease or control brain extracts. This led to widespread Aß and tau pathologies in all of the Alzheimer-inoculated animals following a 21-month-long incubation period (n = 12) whereas none of the control brain extract-inoculated animals developed such lesions (n = 6). Aß deposition affected almost all cortical regions. Tau pathology was also detected in Aß-deposit-free regions distant from the inoculation sites (e.g. in the entorhinal cortex), while some regions adjacent, but not connected, to the inoculation sites were spared (e.g. the occipital cortex). Alzheimer-inoculated animals developed cognitive deficits and cerebral atrophy compared to controls. These pathologies were induced using two different batches of Alzheimer brain extracts. This is the first experimental demonstration that tau can be transmitted by human brain extracts inoculations in a primate. We also showed for the first time that the transmission of widespread Aß and tau pathologies can be associated with cognitive decline. Our results thus reinforce the need to organize a systematic monitoring of individuals who underwent procedures associated with a risk of Aß and tau iatrogenic transmission. They also provide support for Alzheimer brain-inoculated primates as relevant models of Alzheimer pathology.

First evidence that emerging pinnatoxin-G, a contaminant of shellfish, reaches the brain and crosses the placental barrier.

Massive proliferation of some toxic marine dinoflagellates is responsible for the occurrence of harmful algal blooms and the contamination of fish and shellfish worldwide. Pinnatoxins (PnTx) (A-H) comprise an emerging phycotoxin family belonging to the cyclic imine toxin group. Interest has been focused on these lipophilic, fast-acting and highly potent toxins because they are widely found in contaminated shellfish, and can represent a risk for seafood consumers. PnTx display a potent antagonist effect on nicotinic acetylcholine receptors (nAChR), and in this study we assessed in vivo the ability of PnTx-G to cross physiological barriers to reach its molecular target. Radiolabeled [3H]-PnTx-G synthesized with good radiochemical purity and yield retained the high affinity of the natural toxin. Oral gavage or intravenous administration to adult rats and digital autoradiographic analyses revealed the biodistribution and toxicokinetics of [3H]-PnTx-G, which is rapidly cleared from blood, and accumulates in the liver and small intestine. The labeling of peripheral and brain adult/embryo rat tissues highlights its ability to cross the intestinal, blood-brain and placental barriers. High-resolution 3D-imaging and in vitro competition studies on rat embryo sections revealed the specificity of [3H]-PnTx-G binding and its selectivity for muscle and neuronal nAChR subtypes (such as α7 subtype). The use of a human perfused cotyledon model and mass spectrometry analyses disclosed that PnTx-G crosses the human placental barrier. The increasing worldwide occurrence of both the dinoflagellate Vulcanodinium rugosum and PnTx-contaminated shellfish, due to climate warming, raises concerns about the potential adverse impact that exposure to pinnatoxins may have for human health.

THY-Tau22 mouse model accumulates more tauopathy at late stage of the disease in response to microglia deactivation through TREM2 deficiency.

The role played by microglia has taken the center of the stage in the etiology of Alzheimer's disease (AD). Several genome-wide association studies carried out on large cohorts of patients have indeed revealed a large number of genetic susceptibility factors corresponding to genes involved in neuroinflammation and expressed specifically by microglia in the brain. Among these genes TREM2, a cell surface receptor expressed by microglia, arouses strong interest because its R47H variant confers a risk of developing AD comparable to the ε4 allele of the APOE gene. Since this discovery, a growing number of studies have therefore examined the role played by TREM2 in the evolution of amyloid plaques and neurofibrillary tangles, the two brain lesions characteristic of AD. Many studies report conflicting results, reflecting the complex nature of microglial activation in AD. Here, we investigated the impact of TREM2 deficiency in the THY-Tau22 transgenic line, a well-characterized model of tauopathy. Our study reports an increase in the severity of tauopathy lesions in mice deficient in TREM2 occurring at an advanced stage of the pathology. This exacerbation of pathology was associated with a reduction in microglial activation indicated by typical morphological features and altered expression of specific markers. However, it was not accompanied by any further changes in memory performance. Our longitudinal study confirms that a defect in microglial TREM2 signaling leads to an increase in neuronal tauopathy occurring only at late stages of the disease.

Neuronal tau species transfer to astrocytes and induce their loss according to tau aggregation state.

Deposits of different abnormal forms of tau in neurons and astrocytes represent key anatomo-pathological features of tauopathies. Although tau protein is highly enriched in neurons and poorly expressed by astrocytes, the origin of astrocytic tau is still elusive. Here, we used innovative gene transfer tools to model tauopathies in adult mouse brains and to investigate the origin of astrocytic tau. We showed in our adeno-associated virus (AAV)-based models and in Thy-Tau22 transgenic mice that astrocytic tau pathology can emerge secondarily to neuronal pathology. By designing an in vivo reporter system, we further demonstrated bidirectional exchanges of tau species between neurons and astrocytes. We then determined the consequences of tau accumulation in astrocytes on their survival in models displaying various status of tau aggregation. Using stereological counting of astrocytes, we report that, as for neurons, soluble tau species are highly toxic to some subpopulations of astrocytes in the hippocampus, whereas the accumulation of tau aggregates does not affect their survival. Thus, astrocytes are not mere bystanders of neuronal pathology. Our results strongly suggest that tau pathology in astrocytes may significantly contribute to clinical symptoms.

Evaluation of automated segmentation algorithms for neurons in macaque cerebral microscopic images.

Accurate cerebral neuron segmentation is required before neuron counting and neuron morphological analysis. Numerous algorithms for neuron segmentation have been published, but they are mainly evaluated using limited subsets from a specific anatomical region, targeting neurons of clear contrast and/or neurons with similar staining intensity. It is thus unclear how these algorithms perform on cerebral neurons in diverse anatomical regions. In this article, we introduce and reliably evaluate existing machine learning algorithms using a data set of microscopy images of macaque brain. This data set highlights various anatomical regions (e.g., cortex, caudate, thalamus, claustrum, putamen, hippocampus, subiculum, lateral geniculate, globus pallidus, etc.), poor contrast, and staining intensity differences of neurons. The evaluation was performed using 10 architectures of six classic machine learning algorithms in terms of typical Recall, Precision, F-score, aggregated Jaccard index (AJI), as well as a performance ranking of algorithms. F-score of most of the algorithms is superior to 0.7. Deep learning algorithms facilitate generally higher F-scores. U-net with suitable layer depth has been evaluated to be excellent classifiers with F-score of 0.846 and 0.837 when performing cross validation. The evaluation and analysis indicate the performance gap among algorithms in various anatomical regions and the strengths and limitations of each algorithm. The comparative result highlights at the same time the importance and difficulty of neuron segmentation and provides clues for future improvement. To the best of our knowledge, this work is the first comprehensive study for neuron segmentation in such large-scale anatomical regions. Neuron segmentation plays a critical role in extracting cerebral information, such as neuron counting and neuron morphological analysis. Accurate automated cerebral neuron segmentation is a challenging task due to different kinds, poor contrast, staining intensity differences, and fuzzy boundaries of neurons. The comprehensive evaluation and analysis of performance among existing machine learning algorithms in diverse anatomical regions allows to make clear of the strengths and limitations of state-of-the-art algorithm. The comprehensive study provides clues for future improvement and creation of automated methods.

Induction of amyloid-ß deposits from serially transmitted, histologically silent, Aß seeds issued from human brains.

In humans, iatrogenic transmission of cerebral amyloid-ß (Aß)-amyloidosis is suspected following inoculation of pituitary-derived hormones or dural grafts presumably contaminated with Aß proteins as well as after cerebral surgeries. Experimentally, intracerebral inoculation of brain homogenate extracts containing misfolded Aß can seed Aß deposition in transgenic mouse models of amyloidosis or in non-human primates. The transmission of cerebral Aß is governed by the host and by the inoculated samples. It is critical to better characterize the propensities of different hosts to develop Aß deposition after contamination by an Aß-positive sample as well as to better assess which biological samples can transmit this lesion. Aß precursor protein (huAPPwt) mice express humanized non-mutated forms of Aß precursor protein and do not spontaneously develop Aß or amyloid deposits. We found that inoculation of Aß-positive brain extracts from Alzheimer patients in these mice leads to a sparse Aß deposition close to the alveus 18 months post-inoculation. However, it does not induce cortical or hippocampal Aß deposition. Secondary inoculation of apparently amyloid deposit-free hippocampal extracts from these huAPPwt mice to APPswe/PS1dE9 mouse models of amyloidosis enhanced Aß deposition in the alveus 9 months post-inoculation. This suggests that Aß seeds issued from human brain samples can persist in furtive forms in brain tissues while maintaining their ability to foster Aß deposition in receptive hosts that overexpress endogenous Aß. This work emphasizes the need for high-level preventive measures, especially in the context of neurosurgery, to prevent the risk of iatrogenic transmission of Aß lesions from samples with sparse amyloid markers.

Complex roles for reactive astrocytes in the triple transgenic mouse model of Alzheimer disease.

In Alzheimer disease (AD), astrocytes undergo complex changes and become reactive. The consequences of this reaction are still unclear. To evaluate the net impact of reactive astrocytes in AD, we developed viral vectors targeting astrocytes that either activate or inhibit the Janus kinase-signal transducer and activator of transcription 3 (JAK2-STAT3) pathway, a central cascade controlling astrocyte reaction. We aimed to evaluate whether reactive astrocytes contribute to tau as well as amyloid pathologies in the hippocampus of 3xTg-AD mice, an AD model that develops tau hyper-phosphorylation and amyloid deposition. JAK2-STAT3 pathway-mediated modulation of reactive astrocytes in 25% of the hippocampus of 3xTg-AD mice did not significantly influence tau phosphorylation or amyloid processing and deposition at early, advanced, and terminal disease stage. Interestingly, inhibition of the JAK2-STAT3 pathway in hippocampal astrocytes did not improve spatial memory in the Y maze but it did reduce anxiety in the elevated plus maze. Our unique approach to specifically manipulate reactive astrocytes in situ show they may impact behavioral outcomes without influencing tau or amyloid pathology.

Impairment of Glycolysis-Derived l-Serine Production in Astrocytes Contributes to Cognitive Deficits in Alzheimer's Disease.

Alteration of brain aerobic glycolysis is often observed early in the course of Alzheimer's disease (AD). Whether and how such metabolic dysregulation contributes to both synaptic plasticity and behavioral deficits in AD is not known. Here, we show that the astrocytic l-serine biosynthesis pathway, which branches from glycolysis, is impaired in young AD mice and in AD patients. l-serine is the precursor of d-serine, a co-agonist of synaptic NMDA receptors (NMDARs) required for synaptic plasticity. Accordingly, AD mice display a lower occupancy of the NMDAR co-agonist site as well as synaptic and behavioral deficits. Similar deficits are observed following inactivation of the l-serine synthetic pathway in hippocampal astrocytes, supporting the key role of astrocytic l-serine. Supplementation with l-serine in the diet prevents both synaptic and behavioral deficits in AD mice. Our findings reveal that astrocytic glycolysis controls cognitive functions and suggest oral l-serine as a ready-to-use therapy for AD.

Assessment of simplified methods for quantification of [18F]-DPA-714 using 3D whole-brain TSPO immunohistochemistry in a non-human primate.

The 18 kDa translocator protein (TSPO) is the main molecular target to image neuroinflammation by positron emission tomography (PET). However, TSPO-PET quantification is complex and none of the kinetic modelling approaches has been validated using a voxel-by-voxel comparison of TSPO-PET data with the actual TSPO levels of expression. Here, we present a single case study of binary classification of in vivo PET data to evaluate the statistical performance of different TSPO-PET quantification methods. To that end, we induced a localized and adjustable increase of TSPO levels in a non-human primate brain through a viral-vector strategy. We then performed a voxel-wise comparison of the different TSPO-PET quantification approaches providing parametric [18F]-DPA-714 PET images, with co-registered in vitro three-dimensional TSPO immunohistochemistry (3D-IHC) data. A data matrix was extracted from each brain hemisphere, containing the TSPO-IHC and TSPO-PET data for each voxel position. Each voxel was then classified as false or true, positive or negative after comparison of the TSPO-PET measure to the reference 3D-IHC method. Finally, receiver operating characteristic curves (ROC) were calculated for each TSPO-PET quantification method. Our results show that standard uptake value ratios using cerebellum as a reference region (SUVCBL) has the most optimal ROC score amongst all non-invasive approaches.

Encephalopathy induced by Alzheimer brain inoculation in a non-human primate.

Alzheimer's disease is characterized by cognitive alterations, cerebral atrophy and neuropathological lesions including neuronal loss, accumulation of misfolded and aggregated ß-amyloid peptides (Aß) and tau proteins. Iatrogenic induction of Aß is suspected in patients exposed to pituitary-derived hormones, dural grafts, or surgical instruments, presumably contaminated with Aß. Induction of Aß and tau lesions has been demonstrated in transgenic mice after contamination with Alzheimer's disease brain homogenates, with very limited functional consequences. Unlike rodents, primates naturally express Aß or tau under normal conditions and attempts to transmit Alzheimer pathology to primates have been made for decades. However, none of earlier studies performed any detailed functional assessments. For the first time we demonstrate long term memory and learning impairments in a non-human primate (Microcebus murinus) following intracerebral injections with Alzheimer human brain extracts. Animals inoculated with Alzheimer brain homogenates displayed progressive cognitive impairments (clinical tests assessing cognitive and motor functions), modifications of neuronal activity (detected by electroencephalography), widespread and progressive cerebral atrophy (in vivo MRI assessing cerebral volume loss using automated voxel-based analysis), neuronal loss in the hippocampus and entorhinal cortex (post mortem stereology). They displayed parenchymal and vascular Aß depositions and tau lesions for some of them, in regions close to the inoculation sites. Although these lesions were sparse, they were never detected in control animals. Tau-positive animals had the lowest performances in a memory task and displayed the greatest neuronal loss. Our study is timely and important as it is the first one to highlight neuronal and clinical dysfunction following inoculation of Alzheimer's disease brain homogenates in a primate. Clinical signs in a chronic disease such as Alzheimer take a long time to be detectable. Documentation of clinical deterioration and/or dysfunction following intracerebral inoculations with Alzheimer human brain extracts could lead to important new insights about Alzheimer initiation processes.

Automated Individualization of Size-Varying and Touching Neurons in Macaque Cerebral Microscopic Images.

In biomedical research, cell analysis is important to assess physiological and pathophysiological information. Virtual microscopy offers the unique possibility to study the compositions of tissues at a cellular scale. However, images acquired at such high spatial resolution are massive, contain complex information, and are therefore difficult to analyze automatically. In this article, we address the problem of individualization of size-varying and touching neurons in optical microscopy two-dimensional (2-D) images. Our approach is based on a series of processing steps that incorporate increasingly more information. (1) After a step of segmentation of neuron class using a Random Forest classifier, a novel min-max filter is used to enhance neurons' centroids and boundaries, enabling the use of region growing process based on a contour-based model to drive it to neuron boundary and achieve individualization of touching neurons. (2) Taking into account size-varying neurons, an adaptive multiscale procedure aiming at individualizing touching neurons is proposed. This protocol was evaluated in 17 major anatomical regions from three NeuN-stained macaque brain sections presenting diverse and comprehensive neuron densities. Qualitative and quantitative analyses demonstrate that the proposed method provides satisfactory results in most regions (e.g., caudate, cortex, subiculum, and putamen) and outperforms a baseline Watershed algorithm. Neuron counts obtained with our method show high correlation with an adapted stereology technique performed by two experts (respectively, 0.983 and 0.975 for the two experts). Neuron diameters obtained with our method ranged between 2 and 28.6 µm, matching values reported in the literature. Further works will aim to evaluate the impact of staining and interindividual variability on our protocol.

Voxel-Based Statistical Analysis of 3D Immunostained Tissue Imaging.

Recently developed techniques to visualize immunostained tissues in 3D and in large samples have expanded the scope of microscopic investigations at the level of the whole brain. Here, we propose to adapt voxel-based statistical analysis to 3D high-resolution images of the immunostained rodent brain. The proposed approach was first validated with a simulation dataset with known cluster locations. Then, it was applied to characterize the effect of ADAM30, a gene involved in the metabolism of the amyloid precursor protein, in a mouse model of Alzheimer's disease. This work introduces voxel-based analysis of 3D immunostained microscopic brain images and, therefore, opens the door to localized whole-brain exploratory investigation of pathological markers and cellular alterations.

Plasma Amyloid Is Associated with White Matter and Subcortical Alterations and Is Modulated by Age and Seasonal Rhythms in Mouse Lemur Primates.

Accumulation of amyloid-ß (Aß) peptides in the brain is a critical early event in the pathogenesis of Alzheimer's disease (AD), the most common age-related neurodegenerative disorder. There is increasing interest in measuring levels of plasma Aß since this could help in diagnosis of brain pathology. However, the value of plasma Aß in such a diagnosis is still controversial and factors modulating its levels are still poorly understood. The mouse lemur (Microcebus murinus) is a primate model of cerebral aging which can also present with amyloid plaques and whose Aß is highly homologous to humans'. In an attempt to characterize this primate model and to evaluate the potential of plasma Aß as a biomarker for brain alterations, we measured plasma Aß40 concentration in 21 animals aged from 5 to 9.5 years. We observed an age-related increase in plasma Aß40 levels. We then evaluated the relationships between plasma Aß40 levels and cerebral atrophy in these mouse lemurs. Voxel-based analysis of cerebral MR images (adjusted for the age/sex/brain size of the animals), showed that low Aß40 levels are associated with atrophy of several white matter and subcortical brain regions. These results suggest that low Aß40 levels in middle-aged/old animals are associated with brain deterioration. One special feature of mouse lemurs is that their metabolic and physiological parameters follow seasonal changes strictly controlled by illumination. We evaluated seasonal-related variations of plasma Aß40 levels and found a strong effect, with higher plasma Aß40 concentrations in winter conditions compared to summer. This question of seasonal modulation of Aß plasma levels should be addressed in clinical studies. We also focused on the amplitude of the difference between plasma Aß40 levels during the two seasons and found that this amplitude increases with age. Possible mechanisms leading to these seasonal changes are discussed.

Potentiating tangle formation reduces acute toxicity of soluble tau species in the rat.

Tauopathies are neurodegenerative diseases characterized by the aggregation of tau protein. These pathologies exhibit a wide variety of clinical and anatomo-pathological presentations, which may result from different pathological mechanisms. Although tau inclusions are a common feature in all these diseases, recent evidence instead implicates small oligomeric aggregates as drivers of tau-induced toxicity. Hence in vivo model systems displaying either soluble or fibrillary forms of wild-type or mutant tau are needed to better identify their respective pathological pathways. Here we used adeno-associated viruses to mediate gene transfer of human tau to the rat brain to develop models of pure tauopathies. Two different constructs were used, each giving rise to a specific phenotype developing in less than 3 months. First, hTAUWT overexpression led to a strong hyperphosphorylation of the protein, which was associated with neurotoxicity in the absence of any significant aggregation. In sharp contrast, its co-expression with the pro-aggregation peptide TauRD-ΔK280 in the hTAUProAggr group strongly promoted its aggregation into Gallyas-positive neurofibrillary tangles, while preserving neuronal survival. Our results support the hypothesis that soluble tau species are key players of tau-induced neurodegeneration.