Human Tau Research Articles

Tau “islands” affects axonal transport in vitro and in vivo

AbstractBackgroundTau is a protein abundantly expressed in neurons where it modulates the stability of axonal microtubules, thus contributing to the regulation of axonal transport of several organelles. Tau aggregates in a group of neurodegenerative diseases named tauopathies, which include frontotemporal dementia (FTD) and Alzheimer’s disease. Recent in vitro work has uncovered the existence of tau “islands”, a microtubule‐bound multimeric state of tau, which is distinct from pathological aggregates. However, whether these structures exist in intact neurons remains unclear.MethodWe investigated both cultured mouse neurons and mice in vivo, using immunoistochemistry, live imaging and two photon microscopy.Resultswe found that human tau showed regions of higher density along axons, reminiscent of tau islands. FTD‐linked mutations, known to increase pathological phosphorylation and aggregation of tau, induces larger islands, an effect that is reversed by inhibition of p38 MAPK, known to phosphorylate tau at multiple sites. Functionally, axonal transport of BDNF‐containing secretory granules is affected by FTD‐linked mutant tau as assessed both in vitro and in vivo by using a new assay based on two‐photon microscopy. Interestingly, this impairment occurred very early on, before overt tau aggregation. Inhibition of p38 MAPK was able to partially rescue the defects in axonal transport both in vitro and in vivo.ConclusionOur data suggests that tau island size regulates axonal transport, an effect dependent on tau phosphorylation. Inefficient organelles transport may have severe consequences on the activity and plasticity of neuronal circuits. The evidence that reducing tau phosphorylation by inhibiting p38 MAPK potentiated axonal transport points towards inhibition of p38 MAPK as a promising therapeutic strategy in tauopathies.

Dysregulation of memory‐induced protein synthesis in tauopathies

AbstractBackgroundProtein synthesis is a vital biological process, important for many neuronal and cognitive processes such as synaptic plasticity and the formation, updating, and extinction of long‐term memories. Recent studies have identified dysregulated translation as a molecular hallmark of many neurodegenerative diseases, including tauopathies such as Alzheimer’s disease (AD), frontotemporal dementia (FTD), and corticobasal degeneration (CBD) (Evans et al., EMBO J, 2019; Evans et al., Acta Neuropathoc Comms, 2021; Elder et al., Commun Biol. 2021). This dysregulation of protein synthesis is thought to be driven in part by pathogenic alterations to tau, a neuronally enriched microtubule binding protein.MethodHere, we have utilized a series of de novo proteomic analyses, including in vivo non‐canonical amino acid (NCAA) tagging, surface sensing of translation‐based ribosome speed of elongation (SunRiSE), and polysome profiling, to explore both in vitro and in vivo how mutant human tau alters translation in a variety of tauopathies models, including the PS19 mouse strain, human neurons from a FTD patient‐derived inducible pluripotent stem cells (iPSCs) line, as well as in transfected HEK293 cells.ResultUsing these techniques, we have found that both P301S and V337M mutant human tau can severely impair protein synthesis, even when protein degradation is inhibited. Our data also suggests that the dysregulated translation is caused, at least in part, by impaired elongation, with SunRiSE assays showing that P301S and V337M mutant human tau both slow elongation rates. We also have demonstrated that FTD‐mutant tau impairs protein synthesis in CA1 hippocampal neurons in PS19 mice and are determining whether learning‐induced increases in hippocampal protein synthesis following contextual fear conditioning are impaired in these mice.ConclusionTogether, our results demonstrate that FTD‐mutant tau can severely impact several important components of the cellular translational machinery. Furthermore, our results also contribute to the growing evidence that impairments in protein synthesis are a feature of neurodegenerative diseases. This work was supported by the Alzheimer’s Association (H.T.E), Leon Levy foundation (H.T.E.), the Rainwater foundation (H.T.E.) and NIH grant NS121786 (E.K.).

Investigating neuronal senescence marker, p19INK4d, in mouse and human brains

AbstractBackgroundThe cellular senescence stress response allows damaged, toxic cells to survive. Clearing senescent cells is a promising therapeutic approach and has moved to clinical trials for Alzheimer’s disease and related dementias (ADRD). We previously identified a link between the cell cycle inhibitor CDKN2D/p19INK4d and neuronal senescence in human postmortem brains with various levels of AD neuropathology. We hypothesized that senescence could be induced by overexpressing this gene in mouse neurons in vivo.MethodAdeno‐associated virus 9 (AAV9) with the neuron specific human Synapsin I (hSyn1) promoter was used to deliver experimental Cdkn2d‐GFP, or control GFP gene expression. Precise delivery of 0.5µl of 1013 IU virus into four neuronal populations per mouse, bilateral hippocampal CA1 and cortical layer 5 neurons was accomplished with the assistance of a stereotaxic robot. Male and female wild type, human tau transgenic and mouse tau knockout mice were used (12‐13 months‐old, n = 47). Mice were euthanized at either two weeks or eight weeks post‐surgery to determine the short‐ and long‐term effects of Cdkn2d expression. GeoMx digital spatial profiler was utilized to quantify protein expression between Cdkn2d infected and uninfected neighboring neurons. ROI selection is displayed in Figure 1.ResultHistological analyses confirmed p19INK4d protein expression in AAV9‐infected cells. GeoMx DSP analyses performed on hippocampus, cortex, and thalamus revealed a protein expression pattern consistent with senescence (i.e., resistance to apoptosis), in Cdkn2d‐expressing neurons of the hippocampus and cortex compared to the un‐infected thalamus (Figure 2). Moreover, proteins associated with apoptosis were higher in the thalamus compared to the cortex and hippocampus.ConclusionsThe data indicates that AAV9‐hSyn1‐Cdkn2d‐GFP is an appropriate method to increase p19INK4d expression in mouse neurons in vivo. Over‐expressing p19INK4d caused neurons to acquire a protein expression signature consistent with senescence. The concomitant expression of apoptosis proteins in the thalamus leads me to hypothesize that the senescent cells in the cortex may cause cell death in the thalamus through the corticothalamic tract. Further studies with increased sample size are needed to confirm the results and continue exploring the effects of Cdkn2d on neuronal senescence, and how tau protein may influence this stress response.

TREM2 protects against complement‐mediated synaptic loss via binding to C1q in neurodegeneration

AbstractBackgroundThe triggering receptor expressed on myeloid cells 2 (TREM2) is recognized as the strongest immune‐specific genetic risk factor for Alzheimer’s disease (AD), since its heterozygous R47H variant confers a 3‐4‐fold increased risk of AD. It has been widely acknowledged that TREM2 plays versatile roles in regulating microglial functions by interacting with a variety of ligands.MethodWe performed affinity‐purification coupled to mass spectrometry with the TREM2‐Fc fusion construct to identify new binding partners for TREM2. The binding of TREM2 to new partners was further confirmed by a solid‐phase binding assay, surface plasmon resonance assay, dot blot binding assay and proximity ligation assay. Next, the functional effects of TREM2 binding to new partners were determined both in vitro and in vivo. We further performed serial truncations to determine the amino acid sequence in TREM2 responsible for binding to new partners. Finally, the functional effects of TREM2 peptide binding to new partners were examined in both the physiological and pathological contexts.ResultHere we show that C1q protein, the initiator of the classical complement pathway, is a binding partner for TREM2. TREM2 specifically attenuated the activation of classical complement cascade via high‐affinity binding to C1q in vitro. The formation of TREM2‐C1q complexes was also detected in human AD brain. Remarkably, the increased density of TREM2‐C1q complexes was associated with lower deposition of C3 but higher density of PSD95, pinpointing to a protective role of TREM2 against complement activity in AD brain. In mice expressing mutant human tau, Trem2 haploinsufficiency increased complement‐mediated microglial engulfment of synapses and accelerated synaptic loss with negligible effects on microglial density and tau pathology. We further identified a 41 amino acid sequence in TREM2 responsible for binding to C1q. In vivo administration of this short peptide reduced synapse removal by microglia and rescued synapse density in both the amyloidosis and tauopathy mouse models of AD.ConclusionTogether, our results demonstrate a critical role for microglial TREM2 in restricting complement‐mediated synaptic elimination in neurodegeneration, providing new mechanistic insights into the protective roles of TREM2 against AD pathogenesis.

Basic Science and Pathogenesis - Part 2.

Traumatic Brain Injury (TBI) is a known risk factor for developing a neurodegenerative disease later in life, including Alzheimer's Disease (AD) and other forms of tauopathy such as chronic traumatic encephalopathy (CTE) and frontal-temporal dementia (FTD). Although several studies highlight the relationship between the severity and frequency of a TBI and the development of these tauopathies, the mechanism behind this association remains unknown. Proper meningeal lymphatic function is required for clearance of waste products and protein aggregates such as amyloid beta and tau from the brain parenchyma. While post-TBI inflammation is beneficial for clearing debris and promoting tissue repair, prolonged glial activation and decreased meningeal lymphatic drainage have been linked to worsened cognitive function. A mild TBI (mTBI) was delivered to three-month-old young adult mice overexpressing P301S mutant human tau (PS19) through the skull on the right inferior temporal lobe of the brain. Sham and TBI injured mice were tested on behavioral tests at 5 months of age. At 7 months of age, brains were examined for tau pathology and neuroinflammation using confocal microscopy. Four months after mTBI, the brains of TBI PS19 mice exhibit increased levels of microglia and astrocytes, and increased P-S202/T-205-Tau in the cortex and thalamus of the ipsilateral hemisphere compared to the contralateral side and to Sham PS19 controls. These findings correlate with our behavior study showing increased hyperactivity, risk-taking behavior, and impaired short term working memory. PS19 TBI mice also present larger ventricles than their uninjured controls suggesting a potential loss of white matter in the cortex and/or hippocampus. P-S202/T-205-Tau orginated from the cortex near the injury site can aggregate and promote the formation of more toxic tau that spreads to deeper regions of the cortex and in the thalamus primarily through the thalamocortical circuit. Also, microglia and astrocyte activation and production of inflammatory mediators has been associated with synapse pruning and engulfment of damaged neurons expressing 'eat me signals' leading to neurodegeneration.

P301L‐Tau Expression Induces Skeletal Muscle Abnormalities in Mouse Model of Tauopathy

AbstractBackgroundPatients suffering from Alzheimer’s disease (AD) and related tauopathies often report signs of muscle weakness. The pathological accumulation of insoluble tau that forms neurofibrillary tangles (NFTs) is a hallmark of AD, yet it is unclear whether pathological tau impacts skeletal muscle. Proper muscle function is necessary for movement and consequently impacts quality of life in tauopathy patients, while concurrently contributing to the physiological maintenance of whole‐body endocrine and metabolic homeostasis. Skeletal muscle crosstalks with other organ systems; therefore, disruption of skeletal muscle health impacts whole‐body processes, such as the formation of pathological tau. The objective of this study was to determine the links between tauopathy and muscle function.MethodI performed intracerebroventricular injections with adeno‐associated viral (AAV) vector encoding P301L human tau (N = 4 females, 7 males) or a control plasmid (empty or a yellow fluorescent protein construct) (N = 5 females, 6 males). At 3 months, I measured grip strength. I harvested the brain, soleus, and tibialis anterior (TA) muscles at 15 months, and measured muscle mechanics concurrently in the soleus and extensor digitorum longus (EDL) muscles. I biochemically detected soluble tau and visualized TA structure immunohistochemically.ResultsGrip strength was reduced at 3 months in the P301L tau‐expressing mice of both sexes. The intrinsic force production of the soleus, but not the EDL, was reduced in P301L tau‐expressing males at 15 months. At this same time point, soluble tau accumulated in the cortex but not the soleus. Aggregation of nuclei were found histologically in the TA of P301L tau expressing mice but not in controls.ConclusionChanges in grip strength preceded insoluble tau accumulation and cognitive decline typical of this model. Reductions in the soleus force production indicate that P301L tau overexpression in the brain impacts intrinsic muscle strength, and occurs independently of innervation. The nuclear clumps detected in the TA of P301L tau‐expressing mice suggest that AAV‐P301L‐tau injections in the brain promote an inflammatory response in muscle. Together these data suggest that pathological tau species that are expressed in the brain reach peripheral tissues and exert deleterious consequences such as skeletal muscle abnormalities.

Inhibition of EV biogenesis reduces tau propagation in Alzheimer’s Disease mouse models

AbstractBackgroundMounting evidence correlates the propagation of hyperphosphorylated tau (pTau) along synaptically connected networks in the brain with progressive cognitive decline in Alzheimer’s Disease (AD). Recent findings have highlighted extracellular vesicle (EV)s in enabling transcellular transmission of pathological tau and identified the partial inhibition of EV biogenesis via small‐molecule inhibitors of nSMase2 as a potential therapeutic avenue. However, there are no suitable compounds for clinical development so far.MethodThrough high‐throughput screening, we identified PDDC, a highly selective and potent nSMase2 inhibitor with excellent brain penetration and oral bioavailability. To evaluate PDDC’s effect on tau propagation in vivo, we chronically administered PDDC‐containing chow to PS19 transgenic mice and measured brain ceramides, nSMase2 activity, tau levels, glial activation, hippocampal neuronal cell layer thickness, and mossy fiber synaptophysin staining. In addition, neuronally‐derived EVs from plasma were isolated and characterized. To directly monitor the effect of PDDC on tau propagation, we developed a murine model where an AAV encoding for P301L/S320F double mutant human tau was stereotaxically‐injected unilaterally into the hippocampus and transfer to the contralateral dentate gyrus (DG) was monitored ± PDDC treatment.ResultPS19 mice exhibited robust elevation of multiple ceramide species and nSMase2 enzymatic activity in the brain, both of which were normalized by PDDC treatment. PS19 mice treated with PDDC had significantly reduced hippocampal total tau and Thr181 pTau, reduced glial activation, protected synapses, and improved pyramidal and granule cell layer thickness. Plasma NEVs of treated mice were fewer in number and had lower p181‐Tau levels than the untreated group; FCA confirmed the decrease of NEVs carrying p262‐Tau at the single EV level. Similarly, the AAV‐hTau‐seeded mice treated with PDDC had reduced tau staining intensity in the contralateral DG.ConclusionData in two AD models using PDDC provides strong preclinical support for using nSMase2 inhibition as a therapeutic strategy to slow tau propagation in AD.

Extracellular Tau Oligomers Damage the Axon Initial Segment by an Endogenous Tau‐Dependent Mechanism

AbstractBackgroundNeuronal polarity and synaptic connectivity are compromised in Alzheimer’s disease (AD) and other tauopathies. The axon initial segment (AIS) is a key structure for regulating polarity and functions of neurons. It occupies the first 20‐60 µm of the axon, comprises a diffusion barrier that segregates axon‐enriched from somatodendritic‐enriched molecules, and has a high concentration of voltage‐gated ion channels that generate action potentials. Extracellular amyloid‐β oligomers compromise AIS integrity. However, effects on the AIS of toxic tau species, including extracellular oligomers (xcTauOs) that spread tau pathology from neuron to neuron by a prion‐like process, whereas unknown. Therefore, we wanted to test the hypothesis that AIS structure is sensitive to xcTauOs.MethodPrimary cortical neurons derived from either wild type (WT), or tau knockout (KO) mice were exposed to xcTauOs or vehicle. Quantitative western blotting and immunofluorescence microscopy with an antibody against the AIS‐enriched protein TRIM46 was used to monitor effects on the AIS. The same methods were also used to compare TRIM46 and two other AIS proteins, ankyrin‐G and neurofascin‐186 in human hippocampal tissue obtained from AD and age‐matched non‐AD donors.ResultIn cultured WT, but not TKO neurons, xcTauOs cause a trend toward AIS shortening and reduce the concentration of the resident AIS protein, TRIM46, without affecting total TRIM46 levels. Lentiviral‐driven human tau expression in tau KO neurons rescues TRIM46 sensitivity to xcTauOs. In human AD hippocampus, AIS length and TRIM46 concentration within the AIS are reduced in neurons containing neurofibrillary tangles (NFTs), without affecting the overall protein levels of multiple resident AIS proteins.ConclusionThese collective findings demonstrate that in cultured neurons, xcTauOs cause partial AIS damage by a mechanism dependent on intracellular tau, thereby raising the possibility that AIS reduction in AD is caused by xcTauOs working in concert with endogenous neuronal tau.

Outbreak of tauopathy in the olfactory system of PS19 mice: an early marker for Alzheimer’s disease diagnosis?

AbstractBackgroundClinical data indicate that impairment of olfactory function is an early indicator of neurodegenerative diseases, including Alzheimer’s disease (AD)(Attems et al., 2014). Our hypothesis is that typical AD lesions could appear in olfactory neurons from the olfactory epithelium (OE). The olfactory epithelium can be easily collected in human individuals (e.g., by nasal brushing), which provides an important tool to investigate the pathology in patients. Here, we address the question of the occurrence of tauopathy in the OE of transgenic mice and its correlation with pathology in the central nervous system (CNS). We use Tau P301S mice (PS19 mice), a tauopathy mouse model. These mice express human tau protein (1N4R) carrying the P301S mutation. They develop neurofibrillary tangle‐like inclusions in the cortex and hippocampus, leading to progressive neurodegeneration at eight months (Yoshiyama et al., 2007).MethodMouse samples were collected from heterozygous PS19 and wild‐type (WT) mice. Immunohistochemical staining (DAB) was used to demonstrate the presence of hyperphosphorylated human tau protein (hTau). We investigated regions of the olfactory system to correlate with data obtained in CNS regions (e.g., hippocampal formation, entorhinal cortex). Mouse heads decalcification was also implemented to preserve the olfactory epithelium structure. Western blots were performed to monitor tau levels and tau phosphorylation pattern.ResultThe hyperphosphorylated human tau protein (Ser202, Thr205) is only detected in PS19 mice sections while the endogenous murine tau protein is expressed both in WT and PS19 mice. The localization and expression of these proteins vary according to the region (OE or CNS subregions), the age (3, 6 or 9 months), and the genotype of the mice (WT or heterozygous PS19). It also seems that tau lesions may spread between brain regions that are directly connected, notably through the olfactory pathway.ConclusionIn PS19 mice, a pathological phosphorylation pattern peaked in the OE prior to vulnerable CNS regions or olfactory bulbs. We hypothesize that tau lesions appearing early in the OE may spread in the olfactory bulb and finally reach the entorhinal cortex and piriform cortex, highlighting the significance of the olfactory system investigation as an early diagnosis tool.

Salivary tau as a probable diagnostic method for Alzheimer’s disease

AbstractBackgroundBiomarkers can be detected in blood levels and, more recently, the feasibility of identifying TAU protein in salivary samples was verified.MethodThis is a case‐control study carried out in patients diagnosed with probable AD and cognitively healthy and non‐AD patients who had research centers in the cities of Ribeirão Preto, Jaboticabal, Louveira and São Paulo, Brazil. For the quantification of t‐Tau by the ELISA method in the saliva of research participants, we used the Kit MBS022635 Human Tau Protein ELISA. And for the quantification of phosphorylated TAU (pTAU), the Kit E‐EL‐H5314 Human pMAPT pTAU (microtubule‐associated phosphorylated tau protein) ELISA Kit was usedResultTotal TAU protein concentrations showed no evidence for differences in concentrations in the three groups of patients, with and without Alzheimer’s disease. However, the phosphorylated TAU protein brings a very interesting finding, as it showed variations between young and elderly patients with and without the disease.ConclusionTotal TAU protein does not seem to be a likely biomarker to aid in the diagnosis of the disease. However, our results show that phosphorylated TAU was elevated in the elderly and, in the elderly with AD, it was even higher.

Modulation of cannabinoid receptor‐2 alters brain clearance of human tau

AbstractBackgroundMultiple studies have demonstrated a strong neuroinflammatory response in the presence of Tau pathology, which is strongly associated with the clinical symptoms and cognitive decline found in Alzheimer’s disease (AD). The correlation between AD progression and tau pathologies, rather than Ab accumulation, suggests that targeting pathological tau may be a more effective therapeutic approach. Microglia have been implicated in tauopathies as they may contribute to tau phosphorylation and aggregation and the uptake and spread of seed‐competent tau to healthy neurons due to dysfunctional degradation. Cannabinoid type 2 receptors (CB2) are highly expressed in immune cells and upregulated in activated microglia under conditions of neurologic disease, such as AD. The pharmacological modulation of CB2 have demonstrated a reduction in inflammation and plaque deposition, suggesting a role of CB2 on the immune system to influence AD‐related pathologies and phenotypes. However, there are limited findings on the CB2 regulation of tau accumulation.MethodWe have generated organotypic brain slice cultures from CB2‐EGFP/f/f mice and transduced with rAAV‐P301L/S320F‐human tau, which previously has shown to accumulate total tau and develop insoluble tau species. Slices were treated with CB2‐selective compounds and evaluated for tau pathology and microglial phenotypes using real‐time imaging and biochemical analyses.ResultPrevious investigation in our lab of a synucleinopathy animal model revealed the capacity of CB2 signaling to alter the function of immune cells and reduce accumulation of phosphorylated human alpha‐synuclein. Furthermore, we have determined that treatment with a CB2 inverse agonist will increase phagocytosis of pHrodo E. coli bioparticles in microglia BV2 cultures stimulated by LPS. Our preliminary work in brain slices over‐expressing mutant human tau show a trend toward reduced ratio of phosphorylated tau (Ser202/Thr205) to total tau in the soluble protein fraction (p = 0.0549) in slices treated with CB2 inverse agonist SR144528 compared to vehicle. We are repeating the experiment and performing further evaluations in organotypic brain slice cultures from CB2‐KO mice.ConclusionThese studies will advance our understanding of the role of brain CB2 in pathologic tau removal and potentially highlight CB2 as a therapeutic target against tauopathies such as AD.

A polygenic risk score (PRS) predicts cognitive decline in the APOE3 population in an early Alzheimer’s disease clinical trial cohort

AbstractBackgroundAlzheimer’s disease (AD) is one of the most common forms of dementia globally and is defined by accumulation of extracellular amyloid plaques and intracellular tau‐containing neurofibrillary tangles in neuronal cells. These pathological changes occur more than a decade prior to symptom onset. Given that disease modifying therapies are geared towards asymptomatic/early AD patients, identification of asymptomatic individuals at risk of progression is key. Genome‐Wide Association Studies(GWAS) for AD have implicated many single nucleotide polymorphisms (SNPs) associated with disease risk. Whereas a minority of early onset AD cases are driven by mutations in genes, sporadic AD cases have complex genetic architecture and are highly heritable. Polygenic Risk Scores (PRS), which aggregate genome‐wide risk loci, have been shown to predict AD onset with AUCs of 75‐84%.MethodsThe PRS approach used in this study is built from a sum of weighted contributions of over 100,000s SNPs associated with disease. We utilized PRS (genoSCORE™) to predict cognitive function decline in a cohort of 280 AD patients in a Ph2 clinical trial for Tilavonemab, a monoclonal antibody targeting the N‐terminus of human tau. Additionally, genoTOR™ was calculated to identify pathways most impacted by the genetic variants enriched in the cohort.ResultsGiven that tilavonemab did not alter Clinical Dementia Rating‐Sum of Boxes(CDR‐SB), we tested differences in cognitive decline between high PRS and low PRS individuals defined using a threshold of 0.6 in the entire cohort and found no difference. Upon stratification by APOE status, higher PRS(> = 0.6) predicted an increase in CDR‐SB of an average of 1.0 at week 72 (p<0.1) and week 96 (p<0.05) compared to participants with lower PRS(<0.6) within the APOE3 population. Additional analyses of the genoTOR™ signaling pathways are ongoing.ConclusionsThe results of these analyses demonstrate that genoSCORE™ may be helpful to enrich for APOE3 individuals most susceptible to cognitive decline over time. Since the majority of genetic risk studies have focused on an APOE4 population, this method provides a novel tool to enrich for individuals at high risk for cognitive decline over time in a less studied population in which there is difficulty with prediction of disease progression.

Investigation of tau post‐translational modification based on T217 and T231 in human plasma as new biomarkers

AbstractBackgroundPost‐translational modification (PTM) of human tau protein is of high importance as an indicator for predicting the progress of Alzheimer’s disease (AD). Particularly, phosphorylation and O‐glycosylation on tau have been observed at relatively high levels in Alzheimer’s disease (AD) patients and healthy controls, respectively [1]. Recently, a study reported that an increase in the level of phosphorylation at T217 and T231 was observed in the initial stages of the preclinical Alzheimer’s continuum [2]. Reference: [1] C. Alquezar et al. Frontiers in Neurology, 11 (2021) 595532. [2] Marc Suárez‐Calvet et al. EMBO Molecular Medicine, 12 (2020) e12921.MethodTo investigate the PTM of tau protein in human blood plasma, an electrochemical sensor with 1 µm gap between working and counter electrodes was performed and obtained Taumeter indicating the ratio of phosphorylation and O‐glycosylation on the surface of tau protein, where antibodies for phosphorylated T217 and T231 were used [3]. To explore the clinical association, standardized uptake value ratio (SUVR) of amyloid‐positron emission tomography (PET), mini‐mental state examination (MMSE), and clinical dementia rating scale sum of boxes (CDR‐SB) were also investigated and compared to Taumeters. Reference: [3] Yoon et al. Alzheimer’s & Dementia, 18 (2022) e061465.ResultA total of 43 participants (normal control (NC, n = 15), mild cognitive impairment (MCI, n = 14), and AD (n = 14) were investigated. Taumeters for T217 (p = 0.00423, AUC = 0.82667) and T231 (p = 0.00034, AUC = 0.86222) were obtained and analyzed through one‐way analysis of variance (ANOVA) between NC and AD groups (Figure 2). In regression analysis, Taumeter for T217 (r = 0.23824, p = 0.12394) was more correlated with amyloid‐PET SUVR than Taumeter for T231 (r = 0.28072, p = 0.06824) (Figures 3a‐b). Taumeter for T217 (r = ‐0.51246, p = 0.00044) was also more correlated with MMSE score than Taumeter for T231 (r = ‐0.07186, p = 0.64701) (Figures 3c‐d). Taumeter for T217 (r = 0.57937, p<0.0001) was more correlated with the CDR‐SB score than Taumeter for T231 (r = 0.16254, p = 0.29768) (Figures 3e‐f).ConclusionIn this study, we investigated the correlation between Taumeters for T217 and T231, amyloid‐PET SUVR, MMSE score, and CDR‐SB score. As a result, Taumeter for T217 indicates more strong correlation with analytical results of amyloid‐PET SUVR, MMSE score, and CDR‐SB score than Taumter for T231.

Effect of Low Concentration of Nitroxides on SH-SY5Y Cells Transfected with the Tau Protein

Nitroxides, stable synthetic free radicals, are promising antioxidants, showing many beneficial effects both at the cellular level and in animal studies. However, the cells are usually treated with high millimolar concentrations of nitroxides which are not relevant to the concentrations that could be attained in vivo. This paper aimed to examine the effects of low (≤10 μM) concentrations of three nitroxides, 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO), 4-hydroxy-TEMPO (TEMPOL) and 4-amino-TEMPO (TEMPAMINE), in pure chemical systems and on SH-SY5Y cells transfected with the human tau protein (TAU cells), a model of chronic cellular oxidative stress, and transfected with the empty plasmid (EP cells). All nitroxides were active in antioxidant-activity tests except for the 2,2′-azinobis-(3-ethylbenzthiazolin-6-sulfonate) radical (ABTS•) decolorization assay and reduced Fe3+, inhibited autoxidation of adrenalin and pyrogallol and oxidation of dihydrorhodamine123 by 3-morpholino-sydnonimine SIN-1. TEMPO protected against fluorescein bleaching from hypochlorite, but TEMPAMINE enhanced the bleaching. Nitroxides showed no cytotoxicity and were reduced by the cells to non-paramagnetic derivatives. They decreased the level of reactive oxygen species, depleted glutathione, and increased mitochondrial-membrane potential in both types of cells, and increased lipid peroxidation in TAU cells. These results demonstrate that even at low micromolar concentrations nitroxides can affect the cellular redox equilibrium and other biochemical parameters.

DLK-MAPK Signaling Coupled with DNA Damage Promotes Intrinsic Neurotoxicity Associated with Non-Mutated Tau

Alzheimer’s disease (AD) is the most prevalent form of neurodegeneration. Despite the well-established link between tau aggregation and clinical progression, the major pathways driven by this protein to intrinsically damage neurons are incompletely understood. To model AD-relevant neurodegeneration driven by tau, we overexpressed non-mutated human tau in primary mouse neurons and observed substantial axonal degeneration and cell death, a process accompanied by activated caspase 3. Mechanistically, we detected deformation of the nuclear envelope and increased DNA damage response in tau-expressing neurons. Gene profiling analysis further revealed significant alterations in the mitogen-activated protein kinase (MAPK) pathway; moreover, inhibitors of dual leucine zipper kinase (DLK) and c-Jun N-terminal kinase (JNK) were effective in alleviating wild-type human tau-induced neurodegeneration. In contrast, mutant P301L human tau was less toxic to neurons, despite causing comparable DNA damage. Axonal DLK activation induced by wild-type tau potentiated the impact of DNA damage response, resulting in overt neurotoxicity. In summary, we have established a cellular tauopathy model highly relevant to AD and identified a functional synergy between the DLK-MAPK axis and DNA damage response in the neuronal degenerative process.

The Discovery of Putative Small Molecules via Ligand-based Pharmacophore Modelling Targeting Human Tau Protein for an Effective Treatment of Parkinson’s Disease

Introduction: The human tau protein is a key protein involved in various neurodegenerative disease (NDs) including Parkinson’s disease (PD). The protein has high tendency to aggregate into oligomers, subsequently generating insoluble mass in the brain. Symptoms of PD include tremor, bradykinesia, rigidity, and postural instability. Currently drugs for PD treatment are only symptom-targeted while effective therapeutic treatment remains a challenge. The objective of this study is to identify novel promising anti-PD drugs using computational techniques. Method: ligand-based (LB) receptor modelling was conducted using LigandScout, validated and subjected to Glide XP docking, virtual screening, ADMET, and molecular dynamics predictions. Results: The adopted LB modelling generated pharmacophoric features of 5 hydrogen bond donors, 1 aromatic rings, and 7 hydrogen bond acceptors. The validation result indicated GH score of 0.73 and EF of 36.30 as validation protocols, probing it to be an ideal model. Using 3D query of the modelling a total of 192 compounds were retrieved from interbioscreen database containing 70,436 natural compounds. Interestingly, ligands 1, 2, 3, 4 and 5 orderly indicated higher binding affinities to the receptor with Glide XP docking of -7.451, -7.368, -7.101, -6.878, and -6.789 compared to a clinical drug Anle138b with -4.552 kcal/mol respectively. Furthermore, molecular dynamics and pkCSM pharmacokinetics demonstrated ligands 1, 2, & 4 having better stability and low toxicity profiles compared to the reference. Conclusion: In summary, the study pave way for discovery of small molecules that could be recommended as adjuvant /single candidate as ant-PD candidates upon further translational study.

A novel transgenic mouse line with hippocampus-dominant and inducible expression of truncated human tau

BackgroundIntraneuronal accumulation of hyperphosphorylated tau is a defining hallmark of Alzheimer’s disease (AD). However, mouse models imitating AD-exclusive neuronal tau pathologies are lacking.MethodsWe generated a new tet-on transgenic mouse model expressing truncated human tau N1-368 (termed hTau368), a tau fragment increased in the brains of AD patients and aged mouse brains. Doxycycline (dox) was administered in drinking water to induce hTau368 expression. Immunostaining and Western blotting were performed to measure the tau level. RNA sequencing was performed to evaluate gene expression, and several behavioral tests were conducted to evaluate mouse cognitive functions, emotion and locomotion.ResultsDox treatment for 1–2 months at a young age induced overt and reversible human tau accumulation in the brains of hTau368 transgenic mice, predominantly in the hippocampus. Meanwhile, the transgenic mice exhibited AD-like high level of tau phosphorylation, glial activation, loss of mature neurons, impaired hippocampal neurogenesis, synaptic degeneration and cognitive deficits.ConclusionsThis study developed a well-characterized and easy-to-use tool for the investigations and drug development for AD and other tauopathies.

A cell-penetrating peptide derived from SARS-CoV-2 protein Orf9b allosterically inhibits MARK4 activity and mitigates tau toxicity

Abnormal activation of microtubule affinity-regulating kinase 4 (MARK4) and its phosphorylation of the microtubule-associated protein tau are believed to play a role in the pathogenesis of Alzheimer's disease, and MARK4 inhibition can be a strategy to develop disease-modifying therapy. Here we report the development of a membrane-permeable peptide that inhibits MARK4 activity in an allosteric manner. The SARS-CoV-2-derived protein Orf9b inhibited MARK4-mediated tau phosphorylation in primary neurons and Drosophila. Orf9b inhibited MARK4 activity in an allosteric manner and did not inhibit the activity of MARK2, which is another MARK family member and is closely related to MARK4. Co-expression of Orf9b in the fly retina expressing human tau and MARK4 suppressed phosphorylation of tau at the microtubule-binding repeats and tau-induced neurodegeneration. We identified the minimal sequence of Orf9b required to suppress MARK4 activity and fused it to a cell-permeable sequence (TAT-Orf9b10-18_78–95). Extracellular supplementation of TAT-Orf9b10-18_78–95 inhibited MARK4 activity in primary neurons, and feeding TAT-Orf9b10-18_78–95 to a fly model of tauopathy lowered phospho-tau levels and suppressed neurodegeneration. These results suggest that TAT-Orf9b10-18_78–95 is a unique class of MARK4 inhibitor and can be used to modify tau toxicity.

Reevaluating tau reduction as a therapeutic approach for tauopathies: Insights and perspectives.

Tau, one of the most abundant microtubule-associated protein in neurons plays a role in regulating microtubule dynamics in axons, as well as shaping the overall morphology of the axon. Recent studies challenge the traditional view of tau as a microtubule stabilizer and shed new light on the complexity of its role in regulating various properties of the microtubule. While reducing tau levels shows therapeutic promise for early tauopathies, efficacy wanes in later stages due to resilient toxic tau aggregates and neurofibrillary tangles. Notably, tauopathies involve factors beyond toxic tau alone, necessitating a broader therapeutic approach. Overexpression of human tau in mouse models, although useful for answering some questions, may not accurately reflect disease mechanisms in patients with tauopathies. Furthermore, the interplay between tau and MAP6, another microtubule-associated protein, adds complexity to tau's regulation of microtubule dynamics. Tau promotes the formation and elongation of labile microtubule domains, vital for cellular processes, while MAP6 stabilizes microtubules. A delicate balance between these proteins is important for neuronal function. Therefore, tau reduction therapies require a comprehensive understanding of disease progression, considering functional tau loss, toxic aggregates, and microtubule dynamics. Stage-dependent application and potential unintended consequences must be carefully evaluated. Restoring microtubule dynamics in late-stage tauopathies may necessitate alternative strategies. This knowledge is valuable for developing effective and safe treatments for tauopathies.

Apathy-like behaviour in tau mouse models of Alzheimer’s disease and frontotemporal dementia

Apathy is the most common behavioural and psychological symptom in Alzheimer’s disease (AD) and other neurodegenerative diseases including frontotemporal dementia (FTD) and Parkinson’s disease (PD). In patients, apathy can include symptoms of loss of motivation, initiative, and interest, listlessness, and indifference, flattening of emotions, absence of drive and passion. Researchers have later refined this to a reduction in goal direct behaviours. In animals, specific symptoms of apathy-like behaviour have been modelled including goal directed or nest-building behaviour which are seen as indicative of proxies for motivation and daily activities. In the present study a nest-building protocol was established using four different inbred mouse strains (CD1, BALB/c, C57Bl/6J, C3H) before assessing AD and FTD tau transgenic mice of Line 1 (L1) and Line 66 (L66) in this paradigm. Female mice aged 5 – 6 months were assessed in the home cage over a period of 7 days with nest-building behaviour scored by three independent experimenters at intervals of 1-, 2- and 7-days post nestlet introduction. Inbred mouse strains displayed different levels of nesting behaviour. BALB/c mice were more proficient than CD1 and C3H mice, while all strains displayed similar nest-building behaviour by day 7. In the tau mouse models, L66 presented with impaired nesting compared to wild-type on days 1 and 2 (not day 7), whereas L1 performed like wild-type on all days. Anhedonia measured in a sucrose preference test was only observed in L66. Anhedonia and low nesting scores in L66 mice are indicative of apathy-like phenotypes. Differences evident between the L1 and L66 tau transgenic mouse models are likely due to the different human tau species expressed in these mice.