[Neuropathology of the Neurodegenerative Diseases].

A definite diagnosis of neurodegenerative diseases is required for neuropathological examination during an autopsy. Each neurodegenerative disease has specific vulnerable regions and affected systems (system degeneration), and is typified by an accumulation of abnormal protein with the formation of characteristic morphological aggregates in the nerve and glial cells, called proteinopathy. The most common neurodegenerative diseases are tauopathy, such as progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and Pick's disease (PiD); α-synucleinopathy, including multiple system atrophy (MSA); and TAR DNA-binding protein of 43 kDa (TDP-43) proteinopathy, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). PSP and CBD show characteristic tau-positive astrocytic inclusions known as tufted astrocytes and astrocytic plaques, respectively. PiD shows tau-positive neuronal inclusions termed Pick bodies. MSA is characterized by α-synuclein-positive oligodendroglial inclusions, called glial cytoplasmic inclusions. ALS- and FTLD-TDP show TDP-43-positive neuronal inclusions, such as skein-like and round inclusions. Huntington's disease shows polyglutamine-positive neuronal inclusions, and Creutzfeldt-Jakob disease shows diffuse deposition of granular prions in the neuropil. The atypical proteins in these diseases have abnormal conformational properties. A comprehensive comparison of the clinical findings and neuropathological observations, including neuroanatomy and images acquired during life, is important to improve the sensitivity of clinical diagnosis.

Prominent tauopathy and intracellular ß-amyloid accumulation triggered by genetic deletion of cathepsin D: implications for Alzheimer disease pathogenesis.

BACKGROUND: Cathepsin D (CatD) is a lysosomal protease that degrades both the amyloid-ß protein (Aß) and the microtubule-associated protein, tau, which accumulate pathognomonically in Alzheimer disease (AD), but few studies have examined the role of CatD in the development of Aß pathology and tauopathy in vivo. METHODS: CatD knockout (KO) mice were crossed to human amyloid precursor protein (hAPP) transgenic mice, and amyloid burden was quantified by ELISA and immunohistochemistry (IHC). Tauopathy in CatD-KO mice, as initially suggested by Gallyas silver staining, was further characterized by extensive IHC and biochemical analyses. Controls included human tau transgenic mice (JNPL3) and another mouse model of a disease (Krabbe A) characterized by pronounced lysosomal dysfunction. Additional experiments examined the effects of CatD inhibition on tau catabolism in vitro and in cultured neuroblastoma cells with inducible expression of human tau. RESULTS: Deletion of CatD in hAPP transgenic mice triggers large increases in cerebral Aß, manifesting as intense, exclusively intracellular aggregates; extracellular Aß deposition, by contrast, is neither triggered by CatD deletion, nor affected in older, haploinsufficient mice. Unexpectedly, CatD-KO mice were found to develop prominent tauopathy by just ∼ 3 weeks of age, accumulating sarkosyl-insoluble, hyperphosphorylated tau exceeding the pathology present in aged JNPL3 mice. CatD-KO mice exhibit pronounced perinuclear Gallyas silver staining reminiscent of mature neurofibrillary tangles in human AD, together with widespread phospho-tau immunoreactivity. Striking increases in sarkosyl-insoluble phospho-tau (∼ 1250%) are present in CatD-KO mice but notably absent from Krabbe A mice collected at an identical antemortem interval. In vitro and in cultured cells, we show that tau catabolism is slowed by blockade of CatD proteolytic activity, including via competitive inhibition by Aß42. CONCLUSIONS: Our findings support a major role for CatD in the proteostasis of both Aß and tau in vivo. To our knowledge, the CatD-KO mouse line is the only model to develop detectable Aß accumulation and profound tauopathy in the absence of overexpression of hAPP or human tau with disease-associated mutations. Given that tauopathy emerges from disruption of CatD, which can itself be potently inhibited by Aß42, our findings suggest that impaired CatD activity may represent a key mechanism linking amyloid accumulation and tauopathy in AD.

Precision proteoform design for 4R tau isoform selective templated aggregation.

Prion-like spread of disease-specific tau conformers is a hallmark of all tauopathies. A 19-residue probe peptide containing a P301L mutation and spanning the R2/R3 splice junction of tau folds and stacks into seeding-competent fibrils and induces aggregation of 4R, but not 3R tau. These tau peptide fibrils propagate aggregated intracellular tau over multiple generations, have a high ß-sheet content, a colocalized lipid signal, and adopt a well-defined U-shaped fold found in 4R tauopathy brain-derived fibrils. Fully atomistic replica exchange molecular dynamics (MD) simulations were used to compute the free energy landscapes of the conformational ensemble of the peptide monomers. These identified an aggregation-prohibiting ß-hairpin structure and an aggregation-competent U-fold unique to 4R tauopathy fibrils. Guided by MD simulations, we identified that the N-terminal-flanking residues to PHF6, which slightly vary between 4R and 3R isoforms, modulate seeding. Strikingly, when a single amino acid switch at position 305 replaced the serine of 4R tau with a lysine from the corresponding position in the first repeat of 3R tau, the seeding induced by the 19-residue peptide was markedly reduced. Conversely, a 4R tau mimic with three repeats, prepared by replacing those amino acids in the first repeat with those amino acids uniquely present in the second repeat, recovered aggregation when exposed to the 19-residue peptide. These peptide fibrils function as partial prions to recruit naive 4R tau-ten times the length of the peptide-and serve as a critical template for 4R tauopathy propagation. These results hint at opportunities for tau isoform-specific therapeutic interventions.

Mouse serum albumin induces neuronal apoptosis and tauopathies.

The elderly frequently present impaired blood-brain barrier which is closely associated with various neurodegenerative diseases. However, how the albumin, the most abundant protein in the plasma, leaking through the disrupted BBB, contributes to the neuropathology remains poorly understood. We here demonstrated that mouse serum albumin-activated microglia induced astrocytes to A1 phenotype to remarkably increase levels of Elovl1, an astrocytic synthase for very long-chain saturated fatty acids, significantly promoting VLSFAs secretion and causing neuronal lippoapoptosis through endoplasmic reticulum stress response pathway. Moreover, MSA-activated microglia triggered remarkable tau phosphorylation at multiple sites through NLRP3 inflammasome pathway. Intracerebroventricular injection of MSA into the brains of C57BL/6J mice to a similar concentration as in patient brains induced neuronal apoptosis, neuroinflammation, increased tau phosphorylation, and decreased the spatial learning and memory abilities, while Elovl1 knockdown significantly prevented the deleterious effect of MSA. Overall, our study here revealed that MSA induced tau phosphorylation and neuron apoptosis based on MSA-activated microglia and astrocytes, respectively, showing the critical roles of MSA in initiating the occurrence of tauopathies and cognitive decline, and providing potential therapeutic targets for MSA-induced neuropathology in multiple neurodegenerative disorders.

Overlaps and divergences between tauopathies and synucleinopathies: a duet of neurodegeneration.

Proteinopathy, defined as the abnormal accumulation of proteins that eventually leads to cell death, is one of the most significant pathological features of neurodegenerative diseases. Tauopathies, represented by Alzheimer's disease (AD), and synucleinopathies, represented by Parkinson's disease (PD), show similarities in multiple aspects. AD manifests extrapyramidal symptoms while dementia is also a major sign of advanced PD. We and other researchers have sequentially shown the cross-seeding phenomenon of α-synuclein (α-syn) and tau, reinforcing pathologies between synucleinopathies and tauopathies. The highly overlapping clinical and pathological features imply shared pathogenic mechanisms between the two groups of disease. The diagnostic and therapeutic strategies seemingly appropriate for one distinct neurodegenerative disease may also apply to a broader spectrum. Therefore, a clear understanding of the overlaps and divergences between tauopathy and synucleinopathy is critical for unraveling the nature of the complicated associations among neurodegenerative diseases. In this review, we discuss the shared and diverse characteristics of tauopathies and synucleinopathies from aspects of genetic causes, clinical manifestations, pathological progression and potential common therapeutic approaches targeting the pathology, in the aim to provide a timely update for setting the scheme of disease classification and provide novel insights into the therapeutic development for neurodegenerative diseases.

The Chronic Effects of a Single Low-Intensity Blast Exposure on Phosphoproteome Networks and Cognitive Function Influenced by Mutant Tau Overexpression.

Blast-induced neurotrauma (BINT) is a pressing concern for veterans and civilians exposed to explosive devices. Affected personnel may have increased risk for long-term cognitive decline and developing tauopathies including Alzheimer's disease-related disorders (ADRD) or frontal-temporal dementia (FTD). The goal of this study was to identify the effect of BINT on molecular networks and their modulation by mutant tau in transgenic (Tg) mice overexpressing the human tau P301L mutation (rTg4510) linked to FTD or non-carriers. The primary focus was on the phosphoproteome because of the prominent role of hyperphosphorylation in neurological disorders. Discrimination learning was assessed following injury in the subsequent 6 weeks, using the automated home-cage monitoring CognitionWall platform. At 40 days post injury, label-free phosphoproteomics was used to evaluate molecular networks in the frontal cortex of mice. Utilizing a weighted peptide co-expression network analysis (WpCNA) approach, we identified phosphopeptide networks tied to associative learning and mossy-fiber pathways and those which predicted learning outcomes. Phosphorylation levels in these networks were inversely related to learning and linked to synaptic dysfunction, cognitive decline, and dementia including Atp6v1a and Itsn1. Low-intensity blast (LIB) selectively increased pSer262tau in rTg4510, a site implicated in initiating tauopathy. Additionally, individual and group level analyses identified the Arhgap33 phosphopeptide as an indicator of BINT-induced cognitive impairment predominantly in rTg4510 mice. This study unveils novel interactions between ADRD genetic susceptibility, BINT, and cognitive decline, thus identifying dysregulated pathways as targets in potential precision-medicine focused therapeutics to alleviate the disease burden among those affected by BINT.

Mathematical topology and geometry-based classification of tauopathies.

Neurodegenerative diseases, like Alzheimer's, are associated with the presence of neurofibrillary lesions formed by tau protein filaments in the cerebral cortex. While it is known that different morphologies of tau filaments characterize different neurodegenerative diseases, there are few metrics of global and local structure complexity that enable to quantify their structural diversity rigorously. In this manuscript, we employ for the first time mathematical topology and geometry to classify neurodegenerative diseases by using cryo-electron microscopy structures of tau filaments that are available in the Protein Data Bank. By employing mathematical topology metrics (Gauss linking integral, writhe and second Vassiliev measure) we achieve a consistent, but more refined classification of tauopathies, than what was previously observed through visual inspection. Our results reveal a hierarchy of classification from global to local topology and geometry characteristics. In particular, we find that tauopathies can be classified with respect to the handedness of their global conformations and the handedness of the relative orientations of their repeats. Progressive supranuclear palsy is identified as an outlier, with a more complex structure than the rest, reflected by a small, but observable knotoid structure (a diagrammatic structure representing non-trivial topology). This topological characteristic can be attributed to a pattern in the beginning of the R3 repeat that is present in all tauopathies but at different extent. Moreover, by comparing single filament to paired filament structures within tauopathies we find a consistent change in the side-chain orientations with respect to the alpha carbon atoms at the area of interaction.

Transgenic Dendra2::tau expression allows in vivo monitoring of tau proteostasis in Caenorhabditis elegans.

Protein homeostasis is perturbed in aging-related neurodegenerative diseases called tauopathies, which are pathologically characterized by aggregation of the microtubule-associated protein tau (encoded by the human MAPT gene). Transgenic Caenorhabditis elegans serve as a powerful model organism to study tauopathy disease mechanisms, but moderating transgenic expression level has proven problematic. To study neuronal tau proteostasis, we generated a suite of transgenic strains expressing low, medium or high levels of Dendra2::tau fusion proteins by comparing integrated multicopy transgene arrays with single-copy safe-harbor locus strains generated by recombinase-mediated cassette exchange. Multicopy Dendra2::tau strains exhibited expression level-dependent neuronal dysfunction that was modifiable by known genetic suppressors or an enhancer of tauopathy. Single-copy Dendra2::tau strains lacked distinguishable phenotypes on their own but enabled detection of enhancer-driven neuronal dysfunction. We used multicopy Dendra2::tau strains in optical pulse-chase experiments measuring tau turnover in vivo and found that Dendra2::tau turned over faster than the relatively stable Dendra2. Furthermore, Dendra2::tau turnover was dependent on the protein expression level and independent of co-expression with human TDP-43 (officially known as TARDBP), an aggregating protein interacting with pathological tau. We present Dendra2::tau transgenic C. elegans as a novel tool for investigating molecular mechanisms of tau proteostasis.

Distinct and Convergent Alterations of Entorhinal Cortical Circuits in Two Mouse Models for Alzheimer's Disease and Related Disorders.

Background: The impairment of neural circuits controlling cognitive processes has been implicated in the pathophysiology of Alzheimer's disease and related disorders (ADRD). However, it is largely unclear what circuits are specifically changed in ADRD, particularly at the early stage. Objective: Our goal of this study is to reveal the functional changes in the circuit of entorhinal cortex (EC), an interface between neocortex and hippocampus, in AD. Methods: Electrophysiological, optogenetic and chemogenetic approaches were used to examine and manipulate entorhinal cortical circuits in amyloid-ß familial AD model (5×FAD) and tauopathy model (P301S Tau). Results: We found that, compared to wild-type mice, electrical stimulation of EC induced markedly smaller responses in subiculum (hippocampal output) of 5×FAD mice (6-month-old), suggesting that synaptic communication in the EC to subiculum circuit is specifically blocked in this AD model. In addition, optogenetic stimulation of glutamatergic terminals from prefrontal cortex (PFC) induced smaller responses in EC of 5×FAD and P301S Tau mice (6-month-old), suggesting that synaptic communication in the PFC to EC pathway is compromised in both ADRD models. Chemogenetic activation of PFC to EC pathway did not affect the bursting activity of EC neurons in 5×FAD mice, but partially restored the diminished EC neuronal activity in P301S Tau mice. Conclusions: These data suggest that 5×FAD mice has a specific impairment of short-range hippocampal gateway (EC to subiculum), which may be caused by amyloid-ß deposits; while two ADRD models have a common impairment of long-range cortical to hippocampal circuit (PFC to EC), which may be caused by microtubule/tau-based transport deficits. These circuit deficits provide a pathophysiological basis for unique and common impairments of various cognitive processes in ADRD conditions.

In vitro evaluation of [3H]PI-2620 and structural derivatives in non-Alzheimer's tauopathies.

Alzheimer's disease (AD) and non-AD tauopathies such as chronic traumatic encephalopathy (CTE), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD) are characterized by the abnormal aggregation of three-repeat (3R) and/or four-repeat (4R) tau isoforms. Several tau-PET tracers have been applied for human imaging of AD and non-AD tauopathies including [18F]PI-2620. Our objective is to evaluate [3H]PI-2620 and two promising structural derivatives, [3H]PI-2014 and [3H]F-4, using in vitro saturation assays and competitive binding assays against new chemical entities based on this scaffold in human AD tissues for comparison with PSP, CBD and CTE tissues. Thin section autoradiography was employed to assess specific binding and distribution of [3H]PI-2620 and [3H]F-4 in fresh-frozen human post-mortem AD, PSP, CBD and CTE tissues. Immunohistochemistry was performed for phospho-tau (AT8) and 4R-tau (RD4). Homogenate filtration binding assays were performed for saturation analysis and competitive binding studies against [3H]PI-2620. All compounds bound with high affinity in AD tissue. In PSP tissue [3H]PI-2620 demonstrated the highest affinity (5.3 nM) and in CBD tissue [3H]F-4 bound with the highest affinity (9.4 nM). Over 40 fluorinated derivatives based on PI-2620 and F-4 were screened in AD and PSP tissue. Notably, compound 2 was the most potent derivative in PSP tissue (Ki = 7.3 nM). By autoradiography, [3H]PI-2620 and [3H]F-4 demonstrated positive signals similar in intensity in AD, PSP and CTE tissues that were displaced by homologous blockade. Binding of both radiotracers aligned with immunostaining for 4R-tau. This work demonstrates that [3H]PI-2620 and [3H]F-4 show promise for imaging 4R-tau aggregates in non-AD tauopathies. PI-2620 continues to serve as a structural scaffold for PET radiotracers with higher affinity for non-AD tau over AD tau.

Assays for the Screening and Characterization of Tau Aggregation Inhibitors.

Aggregation of tau protein is a pathological hallmark of Alzheimer's disease and other neurodegenerative tauopathies. Inhibition of tau aggregation may provide a method for treatment of these disorders. Methods to identify tau aggregation inhibitors (TAIs) in vitro are useful and here we describe assays for TAIs using purified recombinant tau protein fragments in a cell-free immunoassay format and in a stably transfected cell model to create a more physiological environment.

Quantification of Methylation and Phosphorylation Stoichiometry.

Tauopathies including Alzheimer's disease (AD) are neurodegenerative disorders accompanied by the conversion of functional forms of the microtubule associated protein Tau into non-functional aggregates. A variety of post-translational modifications (PTMs) on Tau precede or accompany the conversion, placing them in position to modulate Tau function as well as its propensity to aggregate. Although Tau PTMs can be characterized by their sites of modification, their total stoichiometry when summed over all sites also is an important metric of their potential impact on function. Here we provide a protocol for rapidly producing recombinant Tau with enzyme-specific PTMs at high stoichiometry in vitro and demonstrate its utility in the context of hyperphosphorylation. Additionally, protocols for estimating phosphorylation and methylation stoichiometry on Tau proteins isolated from any source are presented. Together these methods support experimentation on Tau PTM function over a wide range of experimental conditions.

Western Blot of Tau Protein from Mouse Brains Extracts: How to Avoid Signal Artifacts.

Tau is a microtubule-associated protein enriched in the axonal compartment. Its most well-known function is to bind and stabilize microtubules. In Alzheimer's disease and other neurodegenerative diseases known as tauopathies, tau undergoes several abnormal post-translational modifications including hyperphosphorylation, conformational changes, oligomerization, and aggregation. Numerous mouse models of tauopathies have been developed, and Western blotting remains an invaluable tool in studying tau protein physiological and pathological changes in these models. However, many of the antibodies that have been developed to analyze tau post-translational modifications are mouse monoclonal, which are at risk of producing artifactual signals in Western blotting procedures. This risk does not arise due to their lack of specificity, but rather because the secondary antibodies used to detect them will also react with the heavy chain of endogenous mouse immunoglobulins (Igs), leading to a non-specific signal at the same molecular weight as tau protein (around 50 kDa). Here, we present the use of anti-light-chain secondary antibodies as a simple and efficient technique to prevent non-specific Ig signals around 50 kDa. We demonstrate the efficacy of this method by either eliminating or identifying artifactual signals when using monoclonal antibodies directed at non-phosphorylated epitopes (T49, Tau3R, Tau4R), phosphorylated epitopes (MC6, AT180, CP13), or an abnormal tau conformation (MC1), in wild-type (WT) mice with tau hyperphosphorylation (hypothermic), transgenic mice overexpressing human tau (hTau mice), and tau knockout (TKO) mice.

The O-GlcNAc Modification of Recombinant Tau Protein and Characterization of the O-GlcNAc Pattern for Functional Study.

The neuronal microtubule-associated tau protein is characterized in vivo by a large number of post-translational modifications along the entire primary sequence that modulates its function. The primary modification of tau is phosphorylation of serine/threonine or tyrosine residues that is involved in the regulation of microtubule binding and polymerization. In neurodegenerative disorders referred to as tauopathies including Alzheimer's disease, tau is abnormally hyperphosphorylated and forms fibrillar inclusions in neurons progressing throughout different brain area during the course of the disease. The O-ß-linked N-acetylglucosamine (O-GlcNAc) is another reversible post-translational modification of serine/threonine residues that is installed and removed by the unique O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA), respectively. This modification was described as a potential modulator of tau phosphorylation and functions in the physiopathology. Moreover, reducing protein O-GlcNAc levels in the brain upon treatment of tauopathy mouse models with an OGA inhibitor reveals a beneficial effect on tau pathology and neurodegeneration. However, whether the role of tau O-GlcNAcylation is responsible of the protective effect against tau toxicity remains to be determined. The production of O-GlcNAc modified recombinant tau protein is a valuable tool for the investigations of the impact of O-GlcNAcylation on tau functions, modulation of interactions with partners and crosstalk with other post-translational modifications, including but not restricted to phosphorylation. We describe here the in vitro O-GlcNAcylation of tau with recombinant OGT for which we provide an expression and purification protocol. The use of the O-GlcNAc tau protein in functional studies requires the analytical characterization of the O-GlcNAc pattern. Here, we describe a method for the O-GlcNAc modification of tau protein with recombinant OGT and the analytical characterization of the resulting O-GlcNAc pattern by a combination of methods for the overall characterization of tau O-GlcNAcylation by chemoenzymatic labeling and mass spectrometry, as well as the quantitative, site-specific pattern by NMR spectroscopy.

Methods for Biochemical Isolation of Insoluble Tau in Rodent Models of Tauopathies.

The intracellular accumulation of microtubule-associated protein tau is a characteristic feature of tauopathies, a group of neurodegenerative diseases including Alzheimer's disease. Formation of insoluble tau aggregates is initiated by the abnormal hyperphosphorylation and oligomerization of tau. Over the past decades, multiple transgenic rodent models mimicking tauopathies have been develop, showcasing this neuropathological hallmark. The biochemical analysis of insoluble tau in these models has served as a valuable tool to understand the progression of tau-related pathology. In this chapter, we provide a comprehensive review of the two primary methods for isolating insoluble tau, namely, sarkosyl and formic acid extraction (and their variants), which are employed for biochemical analysis in transgenic mouse models of tauopathy. We also analyze the strengths and limitations of these methods.

Studying Microtubule Dynamics in Human Neurons: Two-Dimensional Microtubule Tracing and Kymographs in iPSC- and SH-SY5Y-Derived Neurons for Tau Research.

The study of microtubule (MT) dynamics is essential for the understanding of cellular transport, cell polarity, axon formation, and other neurodevelopmental mechanisms. All these processes rely on the constant transition between assembly and disassembly of tubulin polymers to/from MTs, known as dynamic instability. This process is well-regulated, among others, by phosphorylation of microtubule-associated proteins (MAP), including the Tau protein. Protein kinases, in particular the microtubule affinity regulating kinase (MARK), regulate the MT-Tau interaction, inducing Tau dissociation by phosphorylation. Phosphorylated Tau dissociates from microtubules forming insoluble aggregates known as neurofibrillary tangles. These accumulations of hyperphosphorylated Tau in the neurons disrupt the physiological MT-based transport machinery within the cell and can potentially lead to the development of neurodegenerative disorders, such as Alzheimer's disease (AD) and related tauopathies. Further investigations on the MT cytoskeleton dynamics are essential as they may elucidate pathomechanisms of neurodegenerative diseases - particularly tauopathies - as well as fundamental neurodevelopmental processes.The study of the dynamic assembly and disassembly of the MT network requires live-cell imaging rather than conventional immunocytochemistry based on fixed samples. To investigate MT dynamics, we perform live-cell imaging of neurons transfected with a fluorescently tagged version of the microtubule plus-end tracking protein (+TIP) EB3. This protein associates with the growing ends of MTs and thus visualizes MT growth in real time. Our imaging analysis protocol allows the determination of quantity, orientation, and velocity of MT growth in the soma and neurites of transfected neurons, using ImageJ-based tracking software and kymographs. Furthermore, functional effects of Tau and MARK kinases on the MT cytoskeleton can be assessed by overexpression or downregulation experiments of the respective protein prior to the live imaging assay. We use two different human neuronal cell models, naive and differentiated SH-SY5Y neuroblastoma cells, and neurons derived from induced pluripotent stem cells (iPSCs), both of which have shown success as models to study Tau-related pathologies.This protocol describes an optimized method for analysis of microtubule dynamics using fluorescent tagged EB3 protein as microtubule plus end marker. In this chapter, we outline the process of neuronal transfection, live-cell imaging, and necessary time-lapse image analysis based on ImageJ in two human-derived neuronal systems, which are suitable for the analysis of Tau trafficking and sorting studies.

Differentiating SH-SY5Y Cells into Polarized Human Neurons for Studying Endogenous and Exogenous Tau Trafficking: Four Protocols to Obtain Neurons with Noradrenergic, Dopaminergic, and Cholinergic Properties.

Pathological alterations of the neuronal Tau protein are characteristic for many neurodegenerative diseases, called tauopathies. To investigate the underlying mechanisms of tauopathies, human neuronal cell models are required to study Tau physiology and pathology in vitro. Primary rodent neurons are an often used model for studying Tau, but rodent Tau differs in sequence, splicing, and aggregation propensity, and rodent neuronal physiology cannot be compared to humans. Human-induced pluripotent stem cell (hiPSC)-derived neurons are expensive and time-consuming. Therefore, the human neuroblastoma SH-SY5Y cell line is a commonly used cell model in neuroscience as it combines convenient handling and low costs with the advantages of human-derived cells. Since naïve SH-SY5Y cells show little similarity to human neurons and almost no Tau expression, differentiation is necessary to obtain human-like neurons for studying Tau protein-related aspects of health and disease. As they express in principle all six Tau isoforms seen in the human brain, differentiated SH-SY5Y-derived neurons are suitable for investigating the human microtubule-associated protein Tau and, for example, its sorting and trafficking. Here, we describe and discuss a general cultivation procedure as well as four differentiation methods to obtain SH-SY5Y-derived neurons resembling noradrenergic, dopaminergic, and cholinergic properties, based on the treatment with retinoic acid (RA), brain-derived neurotrophic factor (BDNF), and 12-O-tetrade canoylphorbol-13-acetate (TPA). TPA and RA-/TPA-based protocols achieve differentiation efficiencies of 40-50% after 9 days of treatment. The highest differentiation efficiency (~75%) is accomplished by a combination of RA and BDNF; treatment only with RA is the most time-efficient method as ~50% differentiated cells can be obtained already after 7 days.

Isolation and Detection of Pathological Tau Species in a Tauopathy Mouse Model.

Tau protein in Alzheimer's disease (AD) and tauopathies becomes insoluble due to hyperphosphorylation, conformational alterations, and aggregation. To analyze insoluble tau and pathological tau species, this study employs a methodology that utilizes wild-type and transgenic tau mice (P310S Tau) tissue extraction using 1% Sarkosyl or N-Lauroylsarcosine sodium salt and the radio immunoprecipitation assay (RIPA) buffer. However, the commonly used methods to study the insoluble tau fraction using detergents like Sarkosyl and RIPA require a large amount of homogenate, which can pose challenges when dealing with small tissue samples. Additionally, the study employs immunohistochemistry to visualize and quantify the pathological tau species in the brain tissue of transgenic mice, aiming to identify and analyze pathological tau species such as hyperphosphorylated tau to further our understanding of tauopathies such as Alzheimer's disease.

Cholesterol 25-hydroxylase mediates neuroinflammation and neurodegeneration in a mouse model of tauopathy.

Alzheimer's disease (AD) is characterized by amyloid plaques and neurofibrillary tangles, in addition to neuroinflammation and changes in brain lipid metabolism. 25-Hydroxycholesterol (25-HC), a known modulator of both inflammation and lipid metabolism, is produced by cholesterol 25-hydroxylase encoded by Ch25h expressed as a "disease-associated microglia" signature gene. However, whether Ch25h influences tau-mediated neuroinflammation and neurodegeneration is unknown. Here, we show that in the absence of Ch25h and the resultant reduction in 25-HC, there is strikingly reduced age-dependent neurodegeneration and neuroinflammation in the hippocampus and entorhinal/piriform cortex of PS19 mice, which express the P301S mutant human tau transgene. Transcriptomic analyses of bulk hippocampal tissue and single nuclei revealed that Ch25h deficiency in PS19 mice strongly suppressed proinflammatory signaling in microglia. Our results suggest a key role for Ch25h/25-HC in potentiating proinflammatory signaling to promote tau-mediated neurodegeneration. Ch25h may represent a novel therapeutic target for primary tauopathies, AD, and other neuroinflammatory diseases.

EB1 Increases the Dynamics of Tau Droplets and Inhibits Tau Aggregation: Implications in Tauopathies.

EB1, a microtubule plus end-tracking protein (+TIP), regulates microtubule dynamics. Recent evidence indicates cross-talk between EB proteins and tau, a microtubule-associated neuronal protein that is important for the growth and stability of microtubules. We investigated the interaction between tau and EB1 and the effect of binding of EB1 on tau function and aggregation. EB1 colocalized with tau in SH-SY5Y cells and coimmunoprecipitated with tau. Further, purified EB1 impaired the ability of adult tau to induce tubulin polymerization in vitro. EB1 bound to tau with a dissociation constant of 2.5 ± 0.7 µM. EB1 reduced heparin-induced tau aggregation with a half-maximal inhibitory concentration of 4.3 ± 0.2 µM, and increased the dynamics of tau in phase-separated droplets. The fluorescence recovery rate in tau droplets increased from 0.02 ± 0.01 to 0.07 ± 0.03 s-1, while the half-time of recovery decreased from 44.5 ± 14 to 13.5 ± 6 s in the presence of 8 µM EB1, suggesting a delay in the transition of tau from the soluble to aggregated form in tau liquid-liquid phase separation. EB1 decreased the rate of aggregation and increased the critical concentration of tau aggregation. Dynamic light scattering, atomic force microscopy, dot blot assays, and SDS-PAGE analysis showed that EB1 inhibited the formation of oligomers and higher-order aggregates of tau. The data suggest a novel role for EB1 as a regulator of tau function and aggregation, and the findings indicated the role of the EB family proteins in neuronal function and neurodegeneration.