Structure-Guided Discovery of cis-Hexahydro-pyrido-oxazinones as Reversible, Drug-like Monoacylglycerol Lipase Inhibitors.

Monoacylglycerol lipase (MAGL) is a key enzyme involved in the metabolism of the endogenous signaling ligand 2-arachidonoylglycerol, a neuroprotective endocannabinoid intimately linked to central nervous system (CNS) disorders associated with neuroinflammation. In the quest for novel MAGL inhibitors, a focused screening approach on a Roche library subset provided a reversible benzoxazinone hit exhibiting high ligand efficiency. The subsequent design of the three-dimensional cis-hexahydro-pyrido-oxazinone (cis-HHPO) moiety as benzoxazinone replacement enabled the combination of high MAGL potency with favorable ADME properties. Through enzymatic resolution an efficient synthetic route of the privileged cis-(4R,8S) HHPO headgroup was established, providing access to the highly potent and selective MAGL inhibitor 7o. Candidate molecule 7o matches the target compound profile of CNS drugs as it achieves high CSF exposures after systemic administration in rodents. It engages with the target in the brain and modulates neuroinflammatory processes, thus holding great promise for the treatment of CNS disorders.

Fenebrutinib, a Bruton's tyrosine kinase inhibitor, blocks distinct human microglial signaling pathways.

BACKGROUND: Bruton's tyrosine kinase (BTK) is an intracellular signaling enzyme that regulates B-lymphocyte and myeloid cell functions. Due to its involvement in both innate and adaptive immune compartments, BTK inhibitors have emerged as a therapeutic option in autoimmune disorders such as multiple sclerosis (MS). Brain-penetrant, small-molecule BTK inhibitors may also address compartmentalized neuroinflammation, which is proposed to underlie MS disease progression. BTK is expressed by microglia, which are the resident innate immune cells of the brain; however, the precise roles of microglial BTK and impact of BTK inhibitors on microglial functions are still being elucidated. Research on the effects of BTK inhibitors has been limited to rodent disease models. This is the first study reporting effects in human microglia. METHODS: Here we characterize the pharmacological and functional properties of fenebrutinib, a potent, highly selective, noncovalent, reversible, brain-penetrant BTK inhibitor, in human microglia and complex human brain cell systems, including brain organoids. RESULTS: We find that fenebrutinib blocks the deleterious effects of microglial Fc gamma receptor (FcγR) activation, including cytokine and chemokine release, microglial clustering and neurite damage in diverse human brain cell systems. Gene expression analyses identified pathways linked to inflammation, matrix metalloproteinase production and cholesterol metabolism that were modulated by fenebrutinib treatment. In contrast, fenebrutinib had no significant impact on human microglial pathways linked to Toll-like receptor 4 (TLR4) and NACHT, LRR and PYD domains-containing protein 3 (NLRP3) signaling or myelin phagocytosis. CONCLUSIONS: Our study enhances the understanding of BTK functions in human microglial signaling that are relevant to MS pathogenesis and suggests that fenebrutinib could attenuate detrimental microglial activity associated with FcγR activation in people with MS.

Enhancing Drug Discovery and Development through the Integration of Medicinal Chemistry, Chemical Biology, and Academia-Industry Partnerships: Insights from Roche's Endocannabinoid System Projects.

The endocannabinoid system (ECS) is a critical regulatory network composed of endogenous cannabinoids (eCBs), their synthesizing and degrading enzymes, and associated receptors. It is integral to maintaining homeostasis and orchestrating key functions within the central nervous and immune systems. Given its therapeutic significance, we have launched a series of drug discovery endeavors aimed at ECS targets, including peroxisome proliferator-activated receptors (PPARs), cannabinoid receptors types 1 (CB1R) and 2 (CB2R), and monoacylglycerol lipase (MAGL), addressing a wide array of medical needs. The pursuit of new therapeutic agents has been enhanced by the creation of specialized labeled chemical probes, which aid in target localization, mechanistic studies, assay development, and the establishment of biomarkers for target engagement. By fusing medicinal chemistry with chemical biology in a comprehensive, translational end-to-end drug discovery strategy, we have expedited the development of novel therapeutics. Additionally, this strategy promises to foster highly productive partnerships between industry and academia, as will be illustrated through various examples.

Pharmacological blockade of 2-AG degradation ameliorates clinical, neuroinflammatory and synaptic alterations in experimental autoimmune encephalomyelitis.

The endocannabinoid system (ECS) is critically involved in the pathophysiology of Multiple Sclerosis (MS), a neuroinflammatory and neurodegenerative disease of the central nervous system (CNS). Over the past decade, researchers have extensively studied the neuroprotective and anti-inflammatory effects of the ECS. Inhibiting the degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG) has emerged as a promising strategy to mitigate brain damage in MS. In this study, we investigated the effects of a novel reversible MAGL inhibitor (MAGLi 432) on C57/BL6 female mice with experimental autoimmune encephalomyelitis (EAE), a model of MS. We assessed its implications on motor disability, neuroinflammation, and synaptic dysfunction. Systemic in vivo treatment with MAGLi 432 resulted in a less severe EAE disease, accompanied by increased 2-AG levels and decreased levels of arachidonic acid (AA) and prostaglandins (PGs) in the brain. Additionally, MAGLi 432 reduced both astrogliosis and microgliosis, as evidenced by decreased microglia/macrophage density and a less reactive morphology. Flow cytometry analysis further revealed fewer infiltrating CD45+ and CD3+ cells in the brains of MAGLi 432-treated EAE mice. Finally, MAGLi treatment counteracted the striatal synaptic hyperexcitability promoted by EAE neuroinflammation. In conclusion, MAGL inhibition significantly ameliorated EAE clinical disability and striatal inflammatory synaptopathy through potent anti-inflammatory effects. These findings provide new mechanistic insights into the neuroprotective role of the ECS during neuroinflammation and highlight the therapeutic potential of MAGLi-based drugs in mitigating MS-related inflammatory and neurodegenerative brain damage.

Preclinical Evaluation of the Reversible Monoacylglycerol Lipase PET Tracer (R)-[11C]YH132: Application in Drug Development and Neurodegenerative Diseases.

Monoacylglycerol lipase (MAGL) plays a crucial role in the degradation of 2-arachidonoylglycerol (2-AG), one of the major endocannabinoids in the brain. Inhibiting MAGL could lead to increased levels of 2-AG, which showed beneficial effects on pain management, anxiety, inflammation, and neuroprotection. In the current study, we report the characterization of an enantiomerically pure (R)-[11C]YH132 as a novel MAGL PET tracer. It demonstrates an improved pharmacokinetic profile compared to its racemate. High in vitro MAGL specificity of (R)-[11C]YH132 was confirmed by autoradiography studies using mouse and rat brain sections. In vivo, (R)-[11C]YH132 displayed a high brain penetration, and high specificity and selectivity toward MAGL by dynamic PET imaging using MAGL knockout and wild-type mice. Pretreatment with a MAGL drug candidate revealed a dose-dependent reduction of (R)-[11C]YH132 accumulation in WT mouse brains. This result validates its utility as a PET probe to assist drug development. Moreover, its potential application in neurodegenerative diseases was explored by in vitro autoradiography using brain sections from animal models of Alzheimer's disease and Parkinson's disease.

Identification of (R)-[18F]YH134 for Monoacylglycerol Lipase Neuroimaging and Exploration of Its Use for Central Nervous System and Peripheral Drug Development.

This study aimed to evaluate (R)-[18F]YH134 as a novel PET tracer for imaging monoacylglycerol lipase (MAGL). Considering the ubiquitous expression of MAGL throughout the whole body, the impact of various MAGL inhibitors on (R)-[18F]YH134 brain uptake and its application in brain-periphery crosstalk were explored. Methods: MAGL knockout and wild-type mice were used to evaluate (R)-[18F]YH134 in in vitro autoradiography and PET experiments. To explore the impact of peripheral MAGL occupancy on (R)-[18F]YH134 brain uptake, PET kinetics with an arterial input function were studied in male Wistar rats under baseline and blocking conditions. Results: In in vitro autoradiography, (R)-[18F]YH134 revealed a heterogeneous distribution pattern with high binding to MAGL-rich brain regions in wild-type mouse brain slices, whereas the radioactive signal was negligible in MAGL knockout mouse brain slices. The in vivo brain PET images of (R)-[18F]YH134 in wild-type and MAGL knockout mice demonstrated its high specificity and selectivity in mouse brain. A Logan plot with plasma input function was applied to estimate the distribution volume (V T) of (R)-[18F]YH134. V T was significantly reduced by a brain-penetrant MAGL inhibitor but was unchanged by a peripherally restricted MAGL inhibitor. The MAGL target occupancy in the periphery was estimated using (R)-[18F]YH134 PET imaging data from the brain. Conclusion: (R)-[18F]YH134 is a highly specific and selective PET tracer with favorable kinetic properties for imaging MAGL in rodent brain. Our results showed that blocking of the peripheral target influences brain uptake but not the V T of (R)-[18F]YH134. (R)-[18F]YH134 can be used for estimating the dose of MAGL inhibitor at half-maximal peripheral target occupancy.

A monoacylglycerol lipase inhibitor showing therapeutic efficacy in mice without central side effects or dependence.

Monoacylglycerol lipase (MAGL) regulates endocannabinoid 2-arachidonoylglycerol (2-AG) and eicosanoid signalling. MAGL inhibition provides therapeutic opportunities but clinical potential is limited by central nervous system (CNS)-mediated side effects. Here, we report the discovery of LEI-515, a peripherally restricted, reversible MAGL inhibitor, using high throughput screening and a medicinal chemistry programme. LEI-515 increased 2-AG levels in peripheral organs, but not mouse brain. LEI-515 attenuated liver necrosis, oxidative stress and inflammation in a CCl4-induced acute liver injury model. LEI-515 suppressed chemotherapy-induced neuropathic nociception in mice without inducing cardinal signs of CB1 activation. Antinociceptive efficacy of LEI-515 was blocked by CB2, but not CB1, antagonists. The CB1 antagonist rimonabant precipitated signs of physical dependence in mice treated chronically with a global MAGL inhibitor (JZL184), and an orthosteric cannabinoid agonist (WIN55,212-2), but not with LEI-515. Our data support targeting peripheral MAGL as a promising therapeutic strategy for developing safe and effective anti-inflammatory and analgesic agents.

Multi-parameter optimization: Development of a morpholin-3-one derivative with an improved kinetic profile for imaging monoacylglycerol lipase in the brain.

Monoacylglycerol lipase (MAGL) is a gatekeeper in regulating endocannabinoid signaling and has gained substantial attention as a therapeutic target for neurological disorders. We recently discovered a morpholin-3-one derivative as a novel scaffold for imaging MAGL via positron emission tomography (PET). However, its slow kinetics in vivo hampered the application. In this study, structural optimization was conducted and eleven novel MAGL inhibitors were designed and synthesized. Based on the results from MAGL inhibitory potency, in vitro metabolic stability and surface plasmon resonance assays, we identified compound 7 as a potential MAGL PET tracer candidate. [11C]7 was synthesized via direct 11CO2 fixation method and successfully mapped MAGL distribution patterns on rodent brains in in vitro autoradiography. PET studies in mice using [11C]7 demonstrated its improved kinetic profile compared to the lead structure. Its high specificity in vivo was proved by using MAGL KO mice. Although further studies confirmed that [11C]7 is a P-glycoprotein (P-gp) substrate in mice, its low P-gp efflux ratio on cells transfected with human protein suggests that it should not be an issue for the clinical translation of [11C]7 as a novel reversible MAGL PET tracer in human subjects. Overall, [11C]7 ([11C]RO7284390) showed promising results warranting further clinical evaluation.

A potent and selective inhibitor for the modulation of MAGL activity in the neurovasculature.

Chronic inflammation and blood-brain barrier dysfunction are key pathological hallmarks of neurological disorders such as multiple sclerosis, Alzheimer's disease and Parkinson's disease. Major drivers of these pathologies include pro-inflammatory stimuli such as prostaglandins, which are produced in the central nervous system by the oxidation of arachidonic acid in a reaction catalyzed by the cyclooxygenases COX1 and COX2. Monoacylglycerol lipase hydrolyzes the endocannabinoid signaling lipid 2-arachidonyl glycerol, enhancing local pools of arachidonic acid in the brain and leading to cyclooxygenase-mediated prostaglandin production and neuroinflammation. Monoacylglycerol lipase inhibitors were recently shown to act as effective anti-inflammatory modulators, increasing 2-arachidonyl glycerol levels while reducing levels of arachidonic acid and prostaglandins, including PGE2 and PGD2. In this study, we characterized a novel, highly selective, potent and reversible monoacylglycerol lipase inhibitor (MAGLi 432) in a mouse model of lipopolysaccharide-induced blood-brain barrier permeability and in both human and mouse cells of the neurovascular unit: brain microvascular endothelial cells, pericytes and astrocytes. We confirmed the expression of monoacylglycerol lipase in specific neurovascular unit cells in vitro, with pericytes showing the highest expression level and activity. However, MAGLi 432 did not ameliorate lipopolysaccharide-induced blood-brain barrier permeability in vivo or reduce the production of pro-inflammatory cytokines in the brain. Our data confirm monoacylglycerol lipase expression in mouse and human cells of the neurovascular unit and provide the basis for further cell-specific analysis of MAGLi 432 in the context of blood-brain barrier dysfunction caused by inflammatory insults.

Discovery, synthesis and evaluation of novel reversible monoacylglycerol lipase radioligands bearing a morpholine-3-one scaffold.

Monoacylglycerol lipase (MAGL) is a serine hydrolase that plays an important role in the endocannabinoid degradation in the brain. It has recently emerged as a promising therapeutic target in the treatment of neuroinflammatory and neurodegenerative diseases, such as multiple sclerosis, Alzheimer's disease and Parkinson's disease. Development of MAGL-specific radioligands for non-invasive imaging by positron-emission tomography (PET) would deepen our knowledge on the relevant pathological changes in diseased states and accelerate drug discovery. In this study, we report the selection and synthesis of two morpholine-3-one derivatives as potential reversible MAGL PET tracer candidates based on their multiparameter optimization scores. Both compounds ([11C]1, [11C]2) were radiolabeled by direct [11C]CO2 fixation and the in vitro autoradiographic studies demonstrated their specificity and selectivity towards MAGL. Dynamic PET imaging using MAGL knockout and wild-type mice confirmed the in vivo specificity of [11C]2. Our preliminary results indicate that morpholine-3-one derivative [11C]2 ([11C]RO7279991) binds to MAGL in vivo, and this molecular scaffold could serve as an alternative lead structure to image MAGL in the central nervous system.

Development of High Brain-Penetrant and Reversible Monoacylglycerol Lipase PET Tracers for Neuroimaging.

Monoacylglycerol lipase (MAGL) is one of the key enzymes in the endocannabinoid system. Inhibition of MAGL has been proposed as an attractive approach for the treatment of various diseases. In this study, we designed and successfully synthesized two series of piperazinyl pyrrolidin-2-one derivatives as novel reversible MAGL inhibitors. (R)-[18F]13 was identified through the preliminary evaluation of two carbon-11-labeled racemic structures [11C]11 and [11C]16. In dynamic positron-emission tomography (PET) scans, (R)-[18F]13 showed a heterogeneous distribution and matched the MAGL expression pattern in the mouse brain. High brain uptake and brain-to-blood ratio were achieved by (R)-[18F]13 in comparison with previously reported reversible MAGL PET radiotracers. Target occupancy studies with a therapeutic MAGL inhibitor revealed a dose-dependent reduction of (R)-[18F]13 accumulation in the mouse brain. These findings indicate that (R)-[18F]13 ([18F]YH149) is a highly promising PET probe for visualizing MAGL non-invasively in vivo and holds great potential to support drug development.

The SARS-CoV-2 main protease Mpro causes microvascular brain pathology by cleaving NEMO in brain endothelial cells.

Coronavirus disease 2019 (COVID-19) can damage cerebral small vessels and cause neurological symptoms. Here we describe structural changes in cerebral small vessels of patients with COVID-19 and elucidate potential mechanisms underlying the vascular pathology. In brains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected individuals and animal models, we found an increased number of empty basement membrane tubes, so-called string vessels representing remnants of lost capillaries. We obtained evidence that brain endothelial cells are infected and that the main protease of SARS-CoV-2 (Mpro) cleaves NEMO, the essential modulator of nuclear factor-κB. By ablating NEMO, Mpro induces the death of human brain endothelial cells and the occurrence of string vessels in mice. Deletion of receptor-interacting protein kinase (RIPK) 3, a mediator of regulated cell death, blocks the vessel rarefaction and disruption of the blood-brain barrier due to NEMO ablation. Importantly, a pharmacological inhibitor of RIPK signaling prevented the Mpro-induced microvascular pathology. Our data suggest RIPK as a potential therapeutic target to treat the neuropathology of COVID-19.

Chaperone-mediated autophagy prevents collapse of the neuronal metastable proteome.

Components of the proteostasis network malfunction in aging, and reduced protein quality control in neurons has been proposed to promote neurodegeneration. Here, we investigate the role of chaperone-mediated autophagy (CMA), a selective autophagy shown to degrade neurodegeneration-related proteins, in neuronal proteostasis. Using mouse models with systemic and neuronal-specific CMA blockage, we demonstrate that loss of neuronal CMA leads to altered neuronal function, selective changes in the neuronal metastable proteome, and proteotoxicity, all reminiscent of brain aging. Imposing CMA loss on a mouse model of Alzheimer's disease (AD) has synergistic negative effects on the proteome at risk of aggregation, thus increasing neuronal disease vulnerability and accelerating disease progression. Conversely, chemical enhancement of CMA ameliorates pathology in two different AD experimental mouse models. We conclude that functional CMA is essential for neuronal proteostasis through the maintenance of a subset of the proteome with a higher risk of misfolding than the general proteome.

muscat detects subpopulation-specific state transitions from multi-sample multi-condition single-cell transcriptomics data.

Single-cell RNA sequencing (scRNA-seq) has become an empowering technology to profile the transcriptomes of individual cells on a large scale. Early analyses of differential expression have aimed at identifying differences between subpopulations to identify subpopulation markers. More generally, such methods compare expression levels across sets of cells, thus leading to cross-condition analyses. Given the emergence of replicated multi-condition scRNA-seq datasets, an area of increasing focus is making sample-level inferences, termed here as differential state analysis; however, it is not clear which statistical framework best handles this situation. Here, we surveyed methods to perform cross-condition differential state analyses, including cell-level mixed models and methods based on aggregated pseudobulk data. To evaluate method performance, we developed a flexible simulation that mimics multi-sample scRNA-seq data. We analyzed scRNA-seq data from mouse cortex cells to uncover subpopulation-specific responses to lipopolysaccharide treatment, and provide robust tools for multi-condition analysis within the muscat R package.

Intracellular transport and regulation of transcytosis across the blood-brain barrier.

The blood-brain barrier is a dynamic multicellular interface that regulates the transport of molecules between the blood circulation and the brain parenchyma. Proteins and peptides required for brain homeostasis cross the blood-brain barrier via transcellular transport, but the mechanisms that control this pathway are not well characterized. Here, we highlight recent studies on intracellular transport and transcytosis across the blood-brain barrier. Endothelial cells at the blood-brain barrier possess an intricate endosomal network that allows sorting to diverse cellular destinations. Internalization from the plasma membrane, endosomal sorting, and exocytosis all contribute to the regulation of transcytosis. Transmembrane receptors and blood-borne proteins utilize different pathways and mechanisms for transport across brain endothelial cells. Alterations to intracellular transport in brain endothelial cells during diseases of the central nervous system contribute to blood-brain barrier disruption and disease progression. Harnessing the intracellular sorting mechanisms at the blood-brain barrier can help improve delivery of biotherapeutics to the brain.

High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis.

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. Transcytosis across the BBB regulates the delivery of hormones, metabolites, and therapeutic antibodies to the brain parenchyma. Here, we present a protocol that combines immunofluorescence of free-floating sections with laser scanning confocal microscopy and image analysis to visualize subcellular organelles within endothelial cells at the BBB. Combining this data-set with 3D image analysis software allows for the semi-automated segmentation and quantification of capillary volume and surface area, as well as the number and intensity of intracellular organelles at the BBB. The detection of mouse endogenous immunoglobulin (IgG) within intracellular vesicles and their quantification at the BBB is used to illustrate the method. This protocol can potentially be applied to the investigation of the mechanisms controlling BBB transcytosis of different molecules in vivo.

Sorting Tubules Regulate Blood-Brain Barrier Transcytosis.

Transcytosis across the blood-brain barrier (BBB) regulates key processes of the brain, but the intracellular sorting mechanisms that determine successful receptor-mediated transcytosis in brain endothelial cells (BECs) remain unidentified. Here, we used Transferrin receptor-based Brain Shuttle constructs to investigate intracellular transport in BECs, and we uncovered a pathway for the regulation of receptor-mediated transcytosis. By combining live-cell imaging and mathematical modeling in vitro with super-resolution microscopy of the BBB, we show that intracellular tubules promote transcytosis across the BBB. A monovalent construct (sFab) sorted for transcytosis was localized to intracellular tubules, whereas a bivalent construct (dFab) sorted for degradation formed clusters with impaired transport along tubules. Manipulating tubule biogenesis by overexpressing the small GTPase Rab17 increased dFab transport into tubules and induced its transcytosis in BECs. We propose that sorting tubules regulate transcytosis in BECs and may be a general mechanism for receptor-mediated transport across the BBB.

Region-specific permeability of the blood-brain barrier upon pericyte loss.

The blood-brain barrier (BBB) regulates differing needs of the various brain regions by controlling transport of blood-borne components from the neurovascular circulation into the brain parenchyma. The mechanisms underlying region-specific transport across the BBB are not completely understood. Previous work showed that pericytes are key regulators of BBB function. Here we investigated whether pericytes influence BBB permeability in a region-specific manner by analysing the regional permeability of the BBB in the pdgf-b ret/ret mouse model of pericyte depletion. We show that BBB permeability is heterogeneous in pdgf-b ret/ret mice, being significantly higher in the cortex, striatum and hippocampus compared to the interbrain and midbrain. However, we show that this regional heterogeneity in BBB permeability is not explained by local differences in pericyte coverage. Region-specific differences in permeability were not associated with disruption of tight junctions but may result from changes in transcytosis across brain endothelial cells. Our data show that certain brain regions are able to maintain low BBB permeability despite substantial pericyte loss and suggest that additional, locally-acting mechanisms may contribute to control of transport.

Trafficking of Endogenous Immunoglobulins by Endothelial Cells at the Blood-Brain Barrier.

The Blood-Brain Barrier (BBB) restricts access of large molecules to the brain. The low endocytic activity of brain endothelial cells (BECs) is believed to limit delivery of immunoglobulins (IgG) to the brain parenchyma. Here, we report that endogenous mouse IgG are localized within intracellular vesicles at steady state in BECs in vivo. Using high-resolution quantitative microscopy, we found a fraction of endocytosed IgG in lysosomes. We observed that loss of pericytes (key components of the BBB) in pdgf-b(ret/ret) mice affects the intracellular distribution of endogenous mouse IgG in BECs. In these mice, endogenous IgG was not detected within lysosomes but instead accumulate at the basement membrane and brain parenchyma. Such IgG accumulation could be due to reduced lysosomal clearance and increased sorting to the abluminal membrane of BECs. Our results suggest that, in addition to low uptake from circulation, IgG lysosomal degradation may be a downstream mechanism by which BECs further restrict IgG access to the brain.

Autophagic and lysosomal defects in human tauopathies: analysis of post-mortem brain from patients with familial Alzheimer disease, corticobasal degeneration and progressive supranuclear palsy.

INTRODUCTION: The accumulation of insoluble proteins within neurons and glia cells is a pathological hallmark of several neurodegenerative diseases. Abnormal aggregation of the microtubule-associated protein tau characterizes the neuropathology of tauopathies, such as Alzheimer disease (AD), corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP). An impairment of the lysosomal degradation pathway called macroautophagy, hereafter referred to as autophagy, could contribute to the accumulation of aggregated proteins. The role of autophagy in neurodegeneration has been intensively studied in the context of AD but there are few studies in other tauopathies and it is not known if defects in autophagy is a general feature of tauopathies. In the present study, we analysed autophagic and lysosomal markers in human post-mortem brain samples from patients with early-onset familial AD (FAD) with the APP Swedish mutation (APPswe), CBD and PSP and control individuals. RESULTS: FAD, CBD and PSP patients displayed an increase in LC3-positive vesicles in frontal cortex, indicating an accumulation of autophagic vesicles. Moreover, using double-immunohistochemistry and in situ proximity ligation assay, we observed colocalization of hyperphosphorylated tau with the autophagy marker LC3 in FAD, CBD and PSP patients but not in control individuals. Increased levels of the lysosomal marker LAMP1 was detected in FAD and CBD, and in addition Cathepsin D was diffusely spread in the cytoplasm in all tauopathies suggesting an impaired lysosomal integrity. CONCLUSION: Taken together, our results indicate an accumulation of autophagic and lysosomal markers in human brain tissue from patients with primary tauopathies (CBD and PSP) as well as FAD, suggesting a defect of the autophagosome-lysosome pathway that may contribute to the development of tau pathology.