4R Tau Research Articles

Microscopy assessment of a fluorescence [18F] flortaucipir analog (T726) shows neuropathological overlap with 3R and 4R tau lesions.

[18F] flortaucipir (FTP) binding to paired helical filament (PHF) tau in Alzheimer's disease (AD) is well accepted. Binding to 3R and 4R tau in frontotemporal lobar degeneration (FTLD) is controversial. We aimed to investigate whether an FTP fluorescent analog (T726) can help shed light on this controversy. We assessed T726 binding to amyloid beta (Aβ) and different tau isoforms in nine subjects (one control, three with Alzheimer's disease [AD], and five with FTLD) with different 3R and 4R tauopathies using fluorescence confocal microscopy. T726 did not colocalize with Aβ but showed significant co-localization with PHF tau in AD. We also observed some, albeit limited, co-localization of T726 with 3R and 4R tau lesions in FTLD. This study's findings support FTP binding to some 3R and 4R tau lesions in FTLD. Further studies are needed to understand the biology of why FTP binds some but not all FTLD tau lesions. Flortaucipir analog (T726) showed significant co-localization with paired helical filament (PHF) tau in Alzheimer's disease (AD). Colocalization between T726 with 3R and 4R tau lesions was observed in frontotemporal lobar degeneration (FTLD). Not all 4R tau lesions bind to T726 across different FTLD brain regions.

MAPT haplotype-associated transcriptomic changes in progressive supranuclear palsy

Progressive supranuclear palsy (PSP) is a neurodegenerative movement and cognitive disorder characterized by abnormal accumulation of the microtubule-associated protein tau in the brain. Biochemically, inclusions in PSP are enriched for tau proteoforms with four microtubule-binding domain repeats (4R), an isoform that arises from alternative tau pre-mRNA splicing. While preferential aggregation and reduced degradation of 4R tau protein is thought to play a role in inclusion formation and toxicity, an alternative hypothesis is that altered expression of tau mRNA isoforms plays a causal role. This stems from the observation that PSP is associated with common variation in the tau gene (MAPT) at the 17q21.31 locus which contains low copy number repeats flanking a large recurrent genomic inversion. The complex genomic structural changes at the locus give rise to two dominant haplotypes, termed H1 and H2, that have the potential to markedly influence gene expression. Here, we explored haplotype-dependent differences in gene expression using a bulk RNA-seq dataset derived from human post-mortem brain tissue from PSP (n = 84) and controls (n = 77) using a rigorous computational pipeline, including alternative pre-mRNA splicing. We found 3579 differentially expressed genes in the temporal cortex and 10,011 in the cerebellum. We also found 7214 differential splicing events in the temporal cortex and 18,802 in the cerebellum. In the cerebellum, total tau mRNA levels and the proportion of transcripts encoding 4R tau were significantly increased in PSP compared to controls. In the temporal cortex, the proportion of reads that expressed 4R tau was increased in cases compared to controls. 4R tau mRNA levels were significantly associated with the H1 haplotype in the temporal cortex. Further, we observed a marked haplotype-dependent difference in KANSL1 expression that was strongly associated with H1 in both brain regions. These findings support the hypothesis that sporadic PSP is associated with haplotype-dependent increases in 4R tau mRNA that might play a causal role in this disorder.

Astrocyte tau deposition in progressive supranuclear palsy is associated with dysregulation of MAPT transcription

Progressive supranuclear palsy (PSP) is a neurodegenerative disease characterized by 4R tau deposition in neurons as well as in astrocytes and oligodendrocytes. While astrocytic tau deposits are rarely observed in normal aging (so-called aging-related tau astrogliopathy, ARTAG) and Alzheimer’s disease (AD), astrocytic tau in the form of tufted astrocytes is a pathognomonic hallmark of PSP. Classical biochemical experiments emphasized tau synthesis in neurons in the central nervous system, suggesting that astrocytic tau inclusions might be derived from uptake of extracellular neuronal-derived tau. However, recent single-nucleus RNAseq experiments highlight the fact that MAPT, the gene encoding tau, is also expressed by astrocytes, albeit in lower amounts. We, therefore, revisited the question of whether astrocyte-driven expression of tau might contribute to astrocytic tau aggregates in PSP by performing fluorescent in situ hybridization/immunohistochemical co-localization in human postmortem brain specimens from individuals with PSP and AD with ARTAG as well as normal controls. We find that, in PSP but not in AD, tau-immunoreactive astrocytes have higher levels of MAPT mRNA compared to astrocytes that do not have tau aggregates. These results suggest that astrocytic responses in PSP are unique to this tauopathy and support the possibility that fundamental changes in PSP astrocyte-endogenous mRNA biology contribute to increased synthesis of tau protein and underlies the formation of the astrocytic tau deposits characteristic of PSP.

G272V and P301L Mutations Induce Isoform Specific Tau Mislocalization to Dendritic Spines and Synaptic Dysfunctions in Cellular Models of 3R and 4R Tau Frontotemporal Dementia.

Tau pathologies are detected in the brains of some of the most common neurodegenerative diseases including Alzheimer's disease (AD), Lewy body dementia (LBD), chronic traumatic encephalopathy (CTE), and frontotemporal dementia (FTD). Tau proteins are expressed in six isoforms with either three or four microtubule-binding repeats (3R tau or 4R tau) due to alternative RNA splicing. AD, LBD, and CTE brains contain pathological deposits of both 3R and 4R tau. FTD patients can exhibit either 4R tau pathologies in most cases or 3R tau pathologies less commonly in Pick's disease, which is a subfamily of FTD. Here, we report the isoform-specific roles of tau in FTD. The P301L mutation, linked to familial 4R tau FTD, induces mislocalization of 4R tau to dendritic spines in primary hippocampal cultures that were prepared from neonatal rat pups of both sexes. Contrastingly, the G272V mutation, linked to familial Pick's disease, induces phosphorylation-dependent mislocalization of 3R tau but not 4R tau proteins to dendritic spines. The overexpression of G272V 3R tau but not 4R tau proteins leads to the reduction of dendritic spine density and suppression of mEPSCs in 5-week-old primary rat hippocampal cultures. The decrease in mEPSC amplitude caused by G272V 3R tau is dynamin-dependent whereas that caused by P301L 4R tau is dynamin-independent, indicating that the two tau isoforms activate different signaling pathways responsible for excitatory synaptic dysfunction. Our 3R and 4R tau studies here will shed new light on diverse mechanisms underlying FTD, AD, LBD, and CTE.

Subcortical tau is linked to hypoperfusion in connected cortical regions in 4-repeat tauopathies.

Four-repeat (4R) tauopathies are neurodegenerative diseases characterized by cerebral accumulation of 4R tau pathology. The most prominent 4R tauopathies are progressive supranuclear palsy (PSP) and corticobasal degeneration characterized by subcortical tau accumulation and cortical neuronal dysfunction, as shown by PET-assessed hypoperfusion and glucose hypometabolism. Yet, there is a spatial mismatch between subcortical tau deposition patterns and cortical neuronal dysfunction, and it is unclear how these two pathological brain changes are interrelated. Here, we hypothesized that subcortical tau pathology induces remote neuronal dysfunction in functionally connected cortical regions to test a pathophysiological model that mechanistically links subcortical tau accumulation to cortical neuronal dysfunction in 4R tauopathies. We included 51 Aβ-negative patients with clinically diagnosed PSP variants (n = 26) or corticobasal syndrome (n = 25) who underwent structural MRI and 18F-PI-2620 tau-PET. 18F-PI-2620 tau-PET was recorded using a dynamic one-stop-shop acquisition protocol to determine an early 0.5-2.5 min post tracer-injection perfusion window for assessing cortical neuronal dysfunction, as well as a 20-40 min post tracer-injection window to determine 4R-tau load. Perfusion-PET (i.e. early window) was assessed in 200 cortical regions, and tau-PET was assessed in 32 subcortical regions of established functional brain atlases. We determined tau epicentres as subcortical regions with the highest 18F-PI-2620 tau-PET signal and assessed the connectivity of tau epicentres to cortical regions of interest using a resting-state functional MRI-based functional connectivity template derived from 69 healthy elderly controls from the ADNI cohort. Using linear regression, we assessed whether: (i) higher subcortical tau-PET was associated with reduced cortical perfusion; and (ii) cortical perfusion reductions were observed preferentially in regions closely connected to subcortical tau epicentres. As hypothesized, higher subcortical tau-PET was associated with overall lower cortical perfusion, which remained consistent when controlling for cortical tau-PET. Using group-average and subject-level PET data, we found that the seed-based connectivity pattern of subcortical tau epicentres aligned with cortical perfusion patterns, where cortical regions that were more closely connected to the tau epicentre showed lower perfusion. Together, subcortical tau-accumulation is associated with remote perfusion reductions indicative of neuronal dysfunction in functionally connected cortical regions in 4R-tauopathies. This suggests that subcortical tau pathology may induce cortical dysfunction, which may contribute to clinical disease manifestation and clinical heterogeneity.

Pick's Disease Presenting as Tremulous Parkinsonism with Limited Levodopa Response-A Rare Cause of Corticobasal Syndrome.

Corticobasal syndrome is a clinical diagnosis and common pathological causes are corticobasal degeneration, progressive supranuclear palsy and Alzheimer's disease. We would like to highlight a rare but important differential of corticobasal syndrome. A 78-year-old female had a 4-year history of predominantly right-hand rest tremor, worsening of handwriting but no change in cognition. The clinical examination showed right upper limb postural and kinetic tremor, mild wrist rigidity and reduced amplitude of right-sided finger tapping. She was initially diagnosed as idiopathic Parkinson's disease. Five years after onset of symptoms, she demonstrated bilateral myoclonic jerks and right upper limb dystonic posturing. She could not copy movements with the right hand. The magnetic resonance imaging (MRI) revealed disproportionate atrophy in the parietal lobes bilaterally. The clinical diagnosis was changed to probable corticobasal syndrome. She passed away 11 years from onset of symptoms at the age of 85 years. She underwent a post-mortem. The anterior and posterior frontal cortex, anterior cingulate, temporal neocortex, hippocampus and amygdaloid complex demonstrated considerable tau-related pathology consisting of a dense background of neuropil threads, and rounded, paranuclear neuronal inclusions consistent with Pick bodies. The immunostaining for three microtubule binding domain repeats (3R) tau performed on sections from the frontal and temporal lobes, basal ganglia and midbrain highlighted several inclusions whilst no 4R tau was observed. She was finally diagnosed with Pick's disease. Pick's disease can rarely present with clinical features of corticobasal syndrome.

Plasma extracellular vesicle tau and TDP-43 as diagnostic biomarkers in FTD and ALS

Minimally invasive biomarkers are urgently needed to detect molecular pathology in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Here, we show that plasma extracellular vesicles (EVs) contain quantifiable amounts of TDP-43 and full-length tau, which allow the quantification of 3-repeat (3R) and 4-repeat (4R) tau isoforms. Plasma EV TDP-43 levels and EV 3R/4R tau ratios were determined in a cohort of 704 patients, including 37 genetically and 31 neuropathologically proven cases. Diagnostic groups comprised patients with TDP-43 proteinopathy ALS, 4R tauopathy progressive supranuclear palsy, behavior variant FTD (bvFTD) as a group with either tau or TDP-43 pathology, and healthy controls. EV tau ratios were low in progressive supranuclear palsy and high in bvFTD with tau pathology. EV TDP-43 levels were high in ALS and in bvFTD with TDP-43 pathology. Both markers discriminated between the diagnostic groups with area under the curve values >0.9, and between TDP-43 and tau pathology in bvFTD. Both markers strongly correlated with neurodegeneration, and clinical and neuropsychological markers of disease severity. Findings were replicated in an independent validation cohort of 292 patients including 34 genetically confirmed cases. Taken together, the combination of EV TDP-43 levels and EV 3R/4R tau ratios may aid the molecular diagnosis of FTD, FTD spectrum disorders and ALS, providing a potential biomarker to monitor disease progression and target engagement in clinical trials.

Differentiation and characterization of healthy versus pathological tau using chemical exchange saturation transfer.

Neurofibrillary tangles of tau constitute one of the key biological hallmarks of Alzheimer's disease. Currently, the assessment of regional tau accumulation requires intravenous administration of radioactive tracers for PET imaging. A noninvasive MRI-based solution would have significant clinical implications. Herein, we utilized an MRI technique known as chemical exchange saturation transfer (CEST) to determine the imaging signature of tau in both its monomeric and pathologic fibrillated conformations. Three sets of purified recombinant full-length (4R) tau protein were prepared for collection of CEST spectra using a 9.4 T NMR spectrometer at varying temperatures (25, 37, and 42°C) and RF intensities (0.7, 1.0, 1.5, and 2.2 μT). Monomeric and fibrillated tau were readily distinguished based on their Z-spectrum profiles. Fibrillated tau demonstrated a less prominent peak at 3.5 ppm with additional peaks near 0.5 and 1.5 ppm. No significant differences were identified between fibrillated tau prepared using heparin versus seed-competent tau. In conclusion, monomeric and fibrillated tau can be readily detected and distinguished based on their CEST-derived Z-spectra, pointing to the potential utility of CEST-MRI as a noninvasive biomarker of regional pathologic tau accumulation in the brain. Further testing and validation in vitro and in vivo will be necessary before this can be applied clinically.

Human iPSC 4R tauopathy model uncovers modifiers of tau propagation

Tauopathies are age-associated neurodegenerative diseases whose mechanistic underpinnings remain elusive, partially due to a lack of appropriate human models. Here, we engineered human induced pluripotent stem cell (hiPSC)-derived neuronal lines to express 4R Tau and 4R Tau carrying the P301S MAPT mutation when differentiated into neurons. 4R-P301S neurons display progressive Tau inclusions upon seeding with Tau fibrils and recapitulate features of tauopathy phenotypes including shared transcriptomic signatures, autophagic body accumulation, and reduced neuronal activity. A CRISPRi screen of genes associated with Tau pathobiology identified over 500 genetic modifiers of seeding-induced Tau propagation, including retromer VPS29 and genes in the UFMylation cascade. In progressive supranuclear palsy (PSP) and Alzheimer's Disease (AD) brains, the UFMylation cascade is altered in neurofibrillary-tangle-bearing neurons. Inhibiting the UFMylation cascade invitro and invivo suppressed seeding-induced Tau propagation. This model provides a robust platform to identify novel therapeutic strategies for 4R tauopathy.

MAPT Mutations V337M and N297K Alter Organelle Trafficking in Frontotemporal Dementia Patient-Specific Motor Neurons.

Frontotemporal dementia (FTD) is a neurodegenerative disease characterized by the progressive loss of neurons mainly in the frontal and temporal lobes of the brain. Mutations (e.g., V337M, N297K) in the microtubule-associated protein TAU (MAPT) are responsible 5-20% of familial FTD cases and have been associated with defects in organelle trafficking that plays a critical role in the proper function of cells, including transport of essential molecules and degradation of waste products. Due to the critical role of TAU mutations in microtubule stabilization and organelle transportation, it is of great interest to study these molecular mechanisms to develop effective therapeutic strategies. Therefore, herein, we analyzed mitochondrial and lysosomal trafficking in disease-specific spinal motor neurons by using live cell imaging in undirected (uncompartmentalized) and directed (compartmentalized) cell culture systems. While V337M neurons only expressed 3R TAU, the N297K mutant neurons expressed both 3R and 4R TAU. Axonal trafficking was affected differentially in V337M and N297 MAPT mutated neurons. These findings suggest that the MAPT mutations V337M and N297K impaired axon physiology differentially, which highlights the need for mutation- and/or 3R/4R TAU-specific therapeutic approaches.

Lateral geniculate body is spared of tau pathology in Pick disease.

The pathobiology of tau is of great importance for understanding the mechanisms of neurodegeneration in aging and age-associated disorders such as Alzheimer disease (AD) and frontotemporal dementias. It is critical to identify neuronal populations and brain regions that are vulnerable or resistant to tau pathological changes. Pick disease (PiD) is a three-repeat (3R) tauopathy that belongs to the group of frontotemporal lobar degenerations. The neuropathologic changes of PiD are characterized by globular tau-positive neuronal intracytoplasmic inclusions, called Pick bodies, in the granule cells of the dentate gyrus and frontal and temporal neocortices, and ballooned neurons, named Pick neurons, in the neocortex. In the present study, we examined 13 autopsy-confirmed cases of PiD. Using immunohistochemistry for phospho-tau (AT8) and 3R tau isoform, all PiD cases demonstrated extensive lesions involving the hippocampus and neocortex. However, the lateral geniculate body (LGB) is spared of significant tau lesions in contrast to the neighboring hippocampus and other thalamic nuclei. Only 1 PiD case (7.7%) had tau-positive neurons, and 4 cases had tau-positive neurites (31%) in the LGB. By contrast, the LGB does consistently harbor tau lesions in other tauopathies including progressive supranuclear palsy, corticobasal degeneration, and AD.

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.

A nonhuman primate model with Alzheimer’s disease-like pathology induced by hippocampal overexpression of human tau

BackgroundAlzheimer’s disease (AD) is one of the most burdening diseases of the century with no disease-modifying treatment at this time. Nonhuman primates (NHPs) share genetic, anatomical, and physiological similarities with humans, making them ideal model animals for investigating the pathogenesis of AD and potential therapies. However, the use of NHPs in AD research has been hindered by the paucity of AD monkey models due to their long generation time, ethical considerations, and technical challenges in genetically modifying monkeys.MethodsHere, we developed an AD-like NHP model by overexpressing human tau in the bilateral hippocampi of adult rhesus macaque monkeys. We evaluated the pathological features of these monkeys with immunostaining, Nissl staining, cerebrospinal fluid (CSF) analysis, magnetic resonance imaging (MRI), positron emission tomography (PET), and behavioural tests.ResultsWe demonstrated that after hippocampal overexpression of tau protein, these monkeys displayed multiple pathological features of AD, including 3-repeat (3R)/4-repeat (4R) tau accumulation, tau hyperphosphorylation, tau propagation, neuronal loss, hippocampal atrophy, neuroinflammation, Aβ clearance deficits, blood vessel damage, and cognitive decline. More interestingly, the accumulation of both 3R and 4R tau is specific to NHPs but not found in adult rodents.ConclusionsThis work establishes a tau-induced AD-like NHP model with many key pathological and behavioural features of AD. In addition, our model may potentially become one of the AD NHP models adopted by researchers worldwide since it can be generated within 2 ~ 3 months through a single injection of AAVs into the monkey brains. Hence, our model NHPs may facilitate mechanistic studies and therapeutic treatments for AD.

The enigma of depression in corticobasal degeneration, a frequent but poorly understood co-morbidity.

Depression is one of the most frequent neuropsychiatric symptoms in corticobasal degeneration (CBD), a rare, sporadic, and late-onset progressive neurodegenerative disorder of unknown etiology. It is clinically characterized by a levodopa-poorly responsible akinetic-rigid syndrome, apraxia, limb dystonia, cognitive, mood, behavioral, and language disorders. This 4-repeat (4R) tauopathy is morphologically featured by asymmetric frontoparietal atrophy, neuronal loss, and gliosis in cortex and subcortex including substantia nigra, ballooned/achromatic neurons with filamentous 4R tau aggregates in cortex and striatum, widespread thread-like structures, pathognomonic "astroglial plaques", "tufted astrocytes", and numerous "coiled bodies" (in astrocytes and oligodendroglia) in cerebral white matter. CBD is non-specific, as pathologically proven cases include several clinical phenotypes. Pubmed and Google Scholar were systematically analyzed until October 2023, with focus on the prevalence, clinical manifestation, neuroimaging data, and treatment options of depression in CBD. Its prevalence is about 30-40% which is more frequent than in most other atypical parkinsonian syndromes. Depression usually does not correlate with motor and other clinical parameters, suggesting different pathophysiological mechanisms. Asymmetric atrophy and hypometabolism of frontoparietal cortical areas are associated with disruption of fronto-subcortical circuits, nigrostriatal dopaminergic, and cholinergic deficiency. Since no specific neuroimaging, neuropathological, or biomarker studies of depression in CBD are available, its pathobiological mechanisms and pathogenesis are poorly understood. Antidepressive therapy may be useful, but is often poorly tolerated. Depression in CBD, like in other parkinsonian syndromes, may be related to multi-regional patterns of cerebral disturbances and complex pathogenic mechanisms that deserve further elucidation as a basis for early diagnosis and adequate treatment to improve the quality of life in this fatal disease.

Native PLGA nanoparticles attenuate Aβ-seed induced tau aggregation under in vitro conditions: potential implication in Alzheimer's disease pathology.

Evidence suggests that beta-amyloid (Aβ)-induced phosphorylation/aggregation of tau protein plays a critical role in the degeneration of neurons and development of Alzheimer's disease (AD), the most common cause of dementia affecting the elderly population. Many studies have pursued a variety of small molecules, including nanoparticles conjugated with drugs to interfere with Aβ and/or tau aggregation/toxicity as an effective strategy for AD treatment. We reported earlier that FDA approved PLGA nanoparticles without any drug can attenuate Aβ aggregation/toxicity in cellular/animal models of AD. In this study, we evaluated the effects of native PLGA on Aβ seed-induced aggregation of tau protein using a variety of biophysical, structural and spectroscopic approaches. Our results show that Aβ1-42 seeds enhanced aggregation of tau protein in the presence and absence of heparin and the effect was attenuated by native PLGA nanoparticles. Interestingly, PLGA inhibited aggregation of both 4R and 3R tau isoforms involved in the formation of neurofibrillary tangles in AD brains. Furthermore, Aβ seed-induced tau aggregation in the presence of arachidonic acid was suppressed by native PLGA. Collectively, our results suggest that native PLGA nanoparticles can inhibit the Aβ seed-induced aggregation of different tau protein isoforms highlighting their therapeutic implication in the treatment of AD.

1,4-Diurea- and 1,4-Dithiourea-Substituted Aromatic Derivatives Selectively Inhibit α-Synuclein Oligomer Formation In Vitro.

Parkinson's disease (PD) is the second most common neurodegenerative disease, affecting the elderly population worldwide. In PD, the misfolding of α-synuclein (α-syn) results in the formation of inclusions referred to as Lewy bodies (LB) in midbrain neurons of the substantia nigra and other specific brain localizations, which is associated with neurodegeneration. There are no approved strategies to reduce the formation of LB in the neurons of patients with PD. Our drug discovery program focuses on the synthesis of urea and thiourea compounds coupled with aminoindole moieties to abrogate α-syn aggregation and to slow down the progression of PD. We synthesized several urea and thiourea analogues with a central 1,4-phenyl diurea/thiourea linkage and evaluated their effectiveness in reducing α-syn aggregation with a special focus on the selective inhibition of oligomer formation among other proteins. We utilized biophysical methods such as thioflavin T (ThT) fluorescence assays, transmission electron microscopy (TEM), photoinduced cross-linking of unmodified proteins (PICUP), as well as M17D intracellular inclusion cell-based assays to evaluate the antiaggregation properties and cellular protection of our best compounds. Our results identified compound 1 as the best compound in reducing α-syn fibril formation via ThT assays. The antioligomer formation of compound 1 was subsequently superseded by compound 2. Both compounds selectively curtailed the oligomer formation of α-syn but not tau 4R isoforms (0N4R, 2N4R) or p-tau (isoform 1N4R). Compounds 1 and 2 failed to abrogate tau 0N3R fibril formation by ThT and atomic force microscopy. Compound 2 was best at reducing the formation of recombinant α-syn fibrils by TEM. In contrast to compound 2, compound 1 reduced the formation of α-syn inclusions in M17D neuroblastoma cells in a dose-dependent manner. Compound 1 may provide molecular scaffolds for the optimization of symmetric molecules for its α-syn antiaggregation activity with potential therapeutic applications and development of small molecules in PD.

Physiological and pathological 3R and 4R tau expression in marmoset brain

AbstractBackgroundCommon marmosets (Callithrix jacchus) have recently emerged as an important model for studying aging and neurodegenerative disorders, including Alzheimer’s disease (AD). Amyloid‐β deposits in the cerebral cortex of marmosets present naturally with aging, which has now been widely reported. However, the role of tau in the marmoset brain has been understudied. The purpose of the present studies was to comprehensively examine 3R and 4R tau isoform expression in the marmoset brain.MethodsThe mRNA expression in the marmoset brain was confirmed by RT‐PCR. The total mRNA was isolated from frozen tissue of various age groups and reverse transcribed to cDNA, which was used for RT‐PCR. The sequence of each fragment was confirmed by the Sanger sequencing method. The tau protein expression in Sarkosyl soluble and insoluble fractions was examined by western blot using tau‐specific antibodies, and expression levels of 3R/4R were quantified by Dot blot. Synaptic tau expression was analyzed from crude synaptosome extractions of the frontal cortex. Pathological tau aggregates in cryo‐slices of a 10‐year‐old marmoset brain were visualized by immunohistochemistry.Results3R and 4R tau were expressed in the marmoset brain, with 3R tau predominately expressed in neonatal brains and 4R tau in adult/aged brains. 3R and 4R tau were also localized in synaptic regions of the frontal cortex. The Sarkosyl soluble tau was phosphorylated on T181, T217, and T231 sites with evidence of oligomers. Insoluble tau was phosphorylated on S396/S404 and S202/S205 sites and contained high molecular weight aggregates comprising 3R and 4R tau. Moreover, a 10‐year‐old marmoset brain case study demonstrated a paired helical filament structure in the hippocampus and intracellular 3R and 4R tau accumulation.ConclusionsOur results reveal physiological and pathological 3R/4R tau distributions in the frontal and temporal cortex of the marmoset brain, confirming the presence of 3R tau and AD‐like pathological tau in aging marmosets. These data support the utility of common marmosets as appropriate models for studying AD and neurodegenerative disorders.

Phosphorylation differentially regulates tau isoforms

AbstractBackgroundSix major tau isoforms are alternatively spliced in an age‐dependent manner: 3R tau (3‐repeat domain) is expressed in development, while both 3R and 4R (4‐repeat domain) tau isoforms are expressed in adult brain. While FTDP‐17‐causing point mutations in tau show isoform‐dependent effects on aggregation and microtubule stabilization, there are not tau mutations linked to Alzheimer’s disease. Meanwhile, effects of phosphorylation on different tau isoforms remain largely unexplored.MethodWe use a combination of recombinant proteins, mouse aging studies and iPSC‐derived neurons to test for isoform‐dependent effects of tau phosphorylation on its function and dysfunction.ResultPhosphorylation at certain sites selectively diminishes microtubule polymerization activity by the fetal tau isoform while a comparable adult isoform was largely unaffected. Conversely, phosphorylation at these same sites accelerated in vitro aggregation of an adult isoform compared to the fetal isoform.ConclusionPhosphorylation to tau has isoform‐dependent effects on its function and dysfunction, thus emphasizing the importance of distinguishing pathological and regulatory phospho‐tau species in future therapeutic efforts.

PRX005, a novel anti‐MTBR tau monoclonal antibody: results from a first‐in‐human double‐blind, placebo‐controlled, single ascending dose phase 1 study

AbstractBackgroundMultiple lines of evidence indicate that tau protein spreads throughout the brain via synaptically connected pathways and that this propagation could be mediated by tau “seeds” containing the microtubule binding region (MTBR) of tau. In addition, cerebrospinal fluid (CSF) MTBR‐tau levels correlate with tau tangles and clinical stages of Alzheimer’s disease. To block the spread of tau pathology, we developed PRX005, a humanized monoclonal tri‐epitopic antibody that binds with high affinity to the R1, R2, and R3 repeats in the MTBR region of tau, and targets both 3R and 4R tau isoforms.MethodHealthy volunteers were randomly assigned to three dose cohorts and randomized in a 3:1 manner to receive either PRX005 or placebo. Primary endpoint was safety and tolerability with up to 2 months of follow‐up. Secondary endpoints included PRX005 pharmacokinetic parameters in plasma and CSF penetrance.ResultA total of 19 healthy volunteer participants received study drug or placebo. PRX005 was well tolerated and had a pharmacokinetic profile typical of IgG1 antibodies, with dose‐proportional increase in plasma concentration and central nervous system (CNS) penetrance. No persistent drug‐induced anti‐drug antibodies were observed following treatment with PRX005. Additional results from this Phase 1 will be presented.ConclusionIn this first‐in‐human, single ascending dose study, PRX005 was well tolerated and achieved CNS concentrations for pharmacological targeting of MTBR‐tau. Our results support the progression of the clinical development of PRX005 in an ongoing, multiple ascending dose study in patients with Alzheimer’s disease.

Posterior Primary Progressive Prosopagnosia: Imaging and Neuropathology

AbstractBackgroundProsopagnosia is common with right temporal lesions, often in the fusiform gyrus when caused by vascular pathology. When caused by frontotemporal dementia, the lesion typically begins in the temporal tip. We are reporting advanced imaging, including tau and inflammation, as well as neuropathology in a patient with pathology centered at the posterior extent of the fusiform gyrus.MethodA 69‐year‐old man with apperceptive prosopagnosia and a negative amyloid positron emission tomogram (PET) underwent MRI and PET using 11C‐PBR28, an inflammation tracer, and 18F‐flortaucipir. V T values for 11C‐PBR28 were calculated with the Logan plot and a metabolite‐corrected arterial input function. For 18F‐flortaucipir, the SUV ratio over the cerebellar gray matter was calculated for t = 80‐100 min. A neuropathological examination was conducted using standard procedures as well as phospho‐tau histochemistry.ResultAtrophy and 18F‐flortaucipir uptake involved a similar region at the posterior extent of the right fusiform gyrus and, minimally, a symmetrical region of the left fusiform gyrus and an area in right premotor cortex. Involving the same regions, the 11C‐PBR28 signal was however more extensive than atrophy or 18F‐flortaucipir signal. At autopsy, the patient had corticobasal degeneration (CBD), with severe 4R tau involvement of temporo‐occipital, inferior parietal, and frontal cortices, as well as limbic regions and subcortical nuclei. The location of CBD pathology in this patient was highly atypical, as shown by comparing the neuropathological findings with the findings in two typical CBD patients.ConclusionAlthough prosopagnosia from ischemic and other lesions in the right posterior temporo‐occipital region is common, primary progressive prosopagnosia in the absence of amyloid has been described mostly with involvement of the right anterior temporal region. As exemplified by our patient, it can rarely happen with posterior temporal involvement and in this case the pathology was not TDP‐43 type C, as present in most patients with anterior temporal involvement, but corticobasal degeneration, a 4R tauopathy.