Phospho-tau Levels Research Articles

Association of Early β-Amyloid Accumulation and Neuroinflammation Measured With [11C]PBR28 in Elderly Individuals Without Dementia.

ObjectiveTo examine whether early β–amyloid (Aβ) accumulation and metabolic risk factors are associated with neuroinflammation in elderly individuals without dementia.MethodsWe examined 54 volunteers (mean age 70.0 years, 56% women, 51% APOE ɛ4 carriers) with the translocator protein (TSPO) tracer [11C]PBR28 to assess neuroinflammation and with [11C] Pittsburgh compound B (PiB) to assess cerebral Aβ accumulation. [11C]PBR28 and [11C]PiB standardized uptake value ratios (SUVRs) were quantified in 6 regions of interests by using the cerebellar cortex as a pseudo-reference and reference region, respectively. Fasting venous glucose, insulin, and high-sensitivity C-reactive protein (hs-CRP) values were determined. Homeostatic model assessment of insulin resistance (HOMA-IR) was calculated. A subset of individuals (n = 11) underwent CSF sampling, and Aβ40, Aβ42, total tau, phospho-tau, soluble TREM2, and YKL-40 levels were measured.ResultsAmong the whole study group, no significant association was found between [11C]PiB and [11C]PBR28 SUVR composite scores (slope 0.02, p = 0.30). However, higher [11C]PiB binding was associated with higher [11C]PBR28 binding among amyloid-negative ([11C]PiB composite score ≤1.5) (TSPO genotype–, age- and sex-adjusted slope 0.26, p = 0.008) but not among amyloid-positive (slope −0.004, p = 0.88) participants. Higher CSF soluble TREM2 (rs = 0.72, p = 0.01) and YKL-40 (rs = 0.63, p = 0.04) concentrations were associated with a higher [11C]PBR28 composite score. Higher body mass index, HOMA-IR, and hs-CRP were associated with higher [11C]PBR28 binding in brain regions where Aβ accumulation is first detected in Alzheimer disease.ConclusionsWhile there was no association between amyloid and neuroinflammation in the overall study group, neuroinflammation was associated with amyloid among the subgroup at early stages of amyloid pathology.

Oxidative Stress in Alzheimer's Disease: In Vitro Therapeutic Effect of Amniotic Fluid Stem Cells Extracellular Vesicles.

Alzheimer's disease (AD) is characterized by abnormal protein aggregation, deposition of extracellular β-amyloid proteins (Aβ), besides an increase of oxidative stress. Amniotic fluid stem cells (AFSCs) should have a therapeutic potential for neurodegenerative disorders, mainly through a paracrine effect mediated by extracellular vesicles (EV). Here, we examined the effect of EV derived from human AFSCs (AFSC-EV) on the disease phenotypes in an AD neuron primary culture. We observed a positive effect of AFSC-EV on neuron morphology, viability, and Aβ and phospho-Tau levels. This could be due to the apoptotic and autophagic pathway modulation derived from the decrease in oxidative stress. Indeed, reactive oxygen species (ROS) were reduced, while GSH levels were enhanced. This modulation could be ascribed to the presence of ROS regulating enzymes, such as SOD1 present into the AFSC-EV themselves. This study describes the ROS-modulating effects of extracellular vesicles alone, apart from their deriving stem cell, in an AD in vitro model, proposing AFSC-EV as a therapeutic tool to stop the progression of AD.

Enhancing microtubule stabilization rescues cognitive deficits and ameliorates pathological phenotype in an amyloidogenic Alzheimer\u2019s disease model

In Alzheimer’s disease (AD), and other tauopathies, microtubule destabilization compromises axonal and synaptic integrity contributing to neurodegeneration. These diseases are characterized by the intracellular accumulation of hyperphosphorylated tau leading to neurofibrillary pathology. AD brains also accumulate amyloid-beta (Aβ) deposits. However, the effect of microtubule stabilizing agents on Aβ pathology has not been assessed so far. Here we have evaluated the impact of the brain-penetrant microtubule-stabilizing agent Epothilone D (EpoD) in an amyloidogenic model of AD. Three-month-old APP/PS1 mice, before the pathology onset, were weekly injected with EpoD for 3 months. Treated mice showed significant decrease in the phospho-tau levels and, more interesting, in the intracellular and extracellular hippocampal Aβ accumulation, including the soluble oligomeric forms. Moreover, a significant cognitive improvement and amelioration of the synaptic and neuritic pathology was found. Remarkably, EpoD exerted a neuroprotective effect on SOM-interneurons, a highly AD-vulnerable GABAergic subpopulation. Therefore, our results suggested that EpoD improved microtubule dynamics and axonal transport in an AD-like context, reducing tau and Aβ levels and promoting neuronal and cognitive protection. These results underline the existence of a crosstalk between cytoskeleton pathology and the two major AD protein lesions. Therefore, microtubule stabilizers could be considered therapeutic agents to slow the progression of both tau and Aβ pathology.

Probing the therapeutic potential of TRPC6 for Alzheimer's disease in live neurons from patient-specific iPSCs.

The induced pluripotent stem cells (iPSCs) offer an unprecedented opportunity to model and study Alzheimer’s disease (AD) under patient-specific genetic background. The lower expression of transient receptor potential canonical 6 (TRPC6) was associated with AD patients, which might be involved in AD pathogenesis. However, the role of TRPC6 that played in AD process still needs more investigation in patient-relevant neurons. In this study, the iPSCs were generated from peripheral blood cells of sporadic AD patients and efficiently differentiated into mature cortical neurons. These sporadic AD-bearing neurons displayed higher levels of AD pathological markers Aβ and phospho-tau, but lower levels of TRPC6, than those of control neurons. Treatment of AD neurons with TRPC6 protein fragment or agonist inhibited the elevation of Aβ and phospho-tau. Our results in live AD neurons manifest that the compromised expression of TRPC6 substantially contributed to Aβ pathology of sporadic AD, suggesting that targeting TRPC6 could help to develop novel therapeutic strategies for the treatments of AD.

Intermittent Calorie Restriction Ameliorates Behavioral Changes and Tau Hyperphosphorylation in Chronic Immobilization Stress in Rats.

Background: Chronic stress has been linked to mood and anxiety disorders and alteration of cognitive functions, such as memory. Intermittent calorie restriction (ICR) is a repeated mild stress that enhances the cell ability to combat more severe stress. Aim: Examination of the effects of ICR on rat behaviour and Tau hyperphosphorylation in chronic immobilization stress (CIS) rat model. Methods: 32 rats were divided into 4 equal groups: control, ICR (subjected to ICR protocol), CIS (subjected to CIS protocol), and CIS+ICR (subjected to both CIS and ICR protocols). After performing behavioural (open field and Barnes maze) tests, serum level of corticosterone, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were determined, and hippocampal tissue level of phosphorylated Tau (phospho-Tau), noradrenaline, and serotonin were measured. Results: We found that, ICR significantly altered the stress-induced anxiety-like behavior and memory disturbances (P<0.001). Rats exposed to both ICR and CIS protocols had significantly lower levels of phospho-Tau, corticosterone, and TNF-α, and enhanced levels of noradrenaline and serotonin than those subjected to CIS alone (P<0.01). Conclusion: our results suggest that ICR protects against chronic stress-induced anxiety-like behaviour, memory disturbance and Tau hyperphosphorylation, possibly through inhibition of hypothalamic pituitary adrenal (HPA) axis, anti-inflammatory effects, and enhancement of noradrenaline and serotonin hormones.

Aβ1-42 and Tau as Potential Biomarkers for Diagnosis and Prognosis of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease, but its definitive diagnosis delays around 12 months. Although the research is highly active in the biomarker field, the absence of specific biomarkers for diagnosis contributes to this long delay. Another strategy of biomarker identification based on less specific but sensitive molecules may be of interest in clinical practice. For example, markers related to other neurodegenerative diseases such as Alzheimer’s disease (AD) could be fully explored. Here, we compared baseline levels of amyloidβ1-42 (Aβ1-42), total Tau, and phosphorylated-Tau (phospho-Tau) protein in the cerebrospinal fluid (CSF) of ALS patients to controls and correlated it with clinical parameters of ALS progression collected over 12 months. We observed increased levels of Aβ1-42 (controls: 992.9 ± 358.3 ng/L; ALS: 1277.0 ± 296.6 ng/L; p < 0.0001) and increased Aβ1-42/phospho-Tau ratio and Innotest Amyloid Tau Index (IATI) (both p < 0.0001). IATI and the phospho-Tau/total Tau ratio correlated positively with ALSFRS-R and weight at baseline. Multivariate analysis revealed that baseline ALSFRS-R was associated with Aβ1-42 and phospho-Tau/total Tau ratio (p = 0.0109 and p = 0.0013, respectively). Total Tau and phospho-Tau levels correlated negatively with ALSFRS-R variation at months 6 and 9, respectively (p = 0.02 and p = 0.04, respectively). Phospho-Tau/total Tau ratio correlated positively with ALSFRS-R variation at month 9 (p = 0.04). CSF levels of Aβ1-42 could be used as a complementary tool to ALS diagnosis, and total Tau and phospho-Tau levels may help establishing the prognosis of ALS. Further studies merit exploring the pathophysiological mechanisms associated with these markers. Despite their lack of specificity, phospho-Tau/total Tau and Aβ1-42 should be combined to other biological and clinical markers in order to improve ALS management.

A microfiber scaffold-based 3D in vitro human neuronal culture model of Alzheimer's disease.

Increasing evidence indicates superiority of three-dimensional (3D) in vitro cell culture systems over conventional two-dimensional (2D) monolayer cultures in mimicking native in vivo microenvironments. Tissue-engineered 3D culture models combined with stem cell technologies have advanced Alzheimer's disease (AD) pathogenesis studies. However, existing 3D neuronal models of AD overexpress mutant genes or have heterogeneities in composition, biological properties and cell differentiation stages. Here, we encapsulate patient induced pluripotent stem cell (iPSC) derived neural progenitor cells (NPC) in poly(lactic-co-glycolic acid) (PLGA) microtopographic scaffolds fabricated via wet electrospinning to develop a novel 3D culture model of AD. First, we enhanced cellular infiltration and distribution inside the scaffold by optimizing various process parameters such as fiber diameter, pore size, porosity and hydrophilicity. Next, we compared key neural stem cell features including viability, proliferation and differentiation in 3D culture with 2D monolayer controls. The 3D microfibrous substrate reduces cell proliferation and significantly accelerates neuronal differentiation within seven days of culture. Furthermore, 3D culture spontaneously enhanced pathogenic amyloid-beta 42 (Aβ42) and phospho-tau levels in differentiated neurons carrying familial AD (FAD) mutations, compared with age-matched healthy controls. Overall, our tunable scaffold-based 3D neuronal culture platform serves as a suitable in vitro model that robustly recapitulates and accelerates the pathogenic characteristics of FAD-iPSC derived neurons.

Melatonin Reduces GSK3β-Mediated Tau Phosphorylation, Enhances Nrf2 Nuclear Translocation and Anti-Inflammation.

Alzheimer’s disease is a neuropathological condition with abnormal accumulation of extracellular Amyloid-β plaques and intracellular neurofibrillary tangles of Microtubule-associated protein Tau (Tau) in the brain. In pathological conditions, Tau undergoes post-translational modifications such as hyperphosphorylation by the activity of cellular kinases, which eventually leads to protein aggregation in neurons. Melatonin is a neuro-hormone that is mainly secreted from the pineal gland and functions to modulate the cellular kinases. In our study, we have checked the neuroprotective function of Melatonin by MTT and LDH assay, where Melatonin inhibited the Tau aggregates-mediated cytotoxicity and membrane leakage in Neuro2A cells. The potency of Melatonin has also been studied for the quenching of intracellular reactive oxygen species level by DCFDA assay and caspase 3 activity. Melatonin was shown to reduce the GSK3β mRNA and subsequent protein level as well as the phospho-Tau level (pThr181 and pThr212-pSer214) in okadaic acid-induced Neuro2A cells, as observed by western blot and immunofluorescence assay. Further, Melatonin has increased the cellular Nrf2 level and its nuclear translocation as an oxidative stress response in Tauopathy. The Melatonin was found to induce pro- and anti-inflammatory cytokines levels in N9 microglia. The mRNA level of cellular kinases such as as-GSK3β, MAPK were also studied by qRT-PCR assay in Tau-exposed N9 and Neuro2A cells. The immunomodulatory effect of Melatonin was evident as it induced IL-10 and TGF-β cytokine levels and activated MAP3K level in Tau-exposed microglia and neurons, respectively. Melatonin also downregulated the mRNA level of pro-inflammatory markers, IL-1β and Cyclooxygenase-2 in N9 microglia. Together, these findings suggest that Melatonin remediated the cytokine profile of Tau-exposed microglia, reduced Tau hyperphosphorylation by downregulating GSK3β level, and alleviated oxidative stress via Nrf2 nuclear translocation.

Pre-analytical protocol for measuring Alzheimer's disease biomarkers in fresh CSF.

IntroductionWe aimed to establish a standardized, routine‐use pre‐analytical protocol for measuring Alzheimer's disease (AD) biomarkers in cerebrospinal fluid (CSF).MethodsThe effect of pre‐analytical factors (sample collection/handling/storage/transportation) on biomarker levels was assessed using freshly collected CSF. Tube type/sterilization was assessed using previously frozen samples. A low‐bind false‐bottom tube (FBT, Sarstedt) was used for all experiments, except tube types/sterilization experiments. Biomarkers were measured using Elecsys CSF assays.ResultsAmyloid beta (Aβ)1‐42 levels varied by tube type, using a low‐bind FBT reduced variation. Aβ1‐42 levels were higher with no mixing versus roller/inversion mixing. Aβ1‐42 levels were lower with horizontal versus upright transportation; this was resolved by maximal tube filling and storage at 2°C to 8°C. Aβ1‐40 levels were less strongly affected. Phospho‐tau and total‐tau levels were largely unaffected.DiscussionWe propose an easy‐to‐use, standardized, routine‐use pre‐analytical protocol, using low‐bind FBTs, for measuring AD CSF biomarkers in clinical practice.

Overexpressing Low-Density Lipoprotein Receptor Reduces Tau-Associated Neurodegeneration Via ApoE-Dependent and Independent Mechanisms

APOE is the strongest genetic risk factor for late-onset Alzheimer’s disease. ApoE exacerbates tau-associated neurodegeneration by driving microglial activation. However, how apoE regulates microglial activation and whether targeting apoE is therapeutically beneficial in tauopathy is unclear. Here we show that overexpressing a low-density lipoprotein receptor (LDLR) transgene in P301S tau transgenic mice markedly reduces brain apoE and ameliorates tau pathology and neurodegeneration. ApoE specifically interacts with a high-molecular-weight tau species, and highly correlates with phospho-tau and insoluble tau levels. Microglial expression of the LDLR transgene reduces intracellular apoE and is associated with less microglial activation. snRNA-seq analysis of apoE-deficient or LDLR-overexpressing brains reveals that apoE deficiency drives microglial catabolism and increases the oligodendrocyte progenitor cell population. LDLR overexpression shares overlapping mechanisms, but uniquely upregulates microglial expression of specific ion channels and neurotransmitter receptors in tauopathy. A subset of disease-associated astrocytes with both neuroprotective and neurotoxic gene signatures is also identified.

Dementia with lewy bodies: GBA1 mutations are associated with cerebrospinal fluid alpha-synuclein profile.

Patients with dementia with Lewy bodies reveal a variable pathology including alpha-synuclein, amyloid-beta, and Tau. Mutations in GBA1 are specifically associated with synucleinopathies. PD patients with GBA1 mutations show reduced CSF levels of total alpha-synuclein. Whether GBA1 mutations are associated with a CSF alpha-synuclein profile in dementia with Lewy bodies. Screening of the GBA1 gene and single-nucleotide polymorphisms in SNCA rs356220, APOE rs429358, and MAPT rs1052587 as well as CSF levels of total alpha-synuclein, amyloid-beta1-42 , total-Tau, phospho-Tau, and neurofilament light chain were assessed in 100 dementia with Lewy bodies and 39 controls cross-sectionally. Severity of GBA1 mutations was associated with a younger age at onset and higher prevalence of rapid eye movement sleep behavior disorder. CSF levels of total alpha-synuclein were lowest in DLBGBA_pathogenic compared to DLBGBA_mild and DLBGBA_wildtype . Similar to PD, pathogenic GBA1 mutations seem to be associated with CSF alpha-synuclein profiles in dementia with Lewy bodies. That might be useful for patient stratification for specific alpha-synuclein-lowering compounds. © 2019 International Parkinson and Movement Disorder Society.

Choroid Plexus Acts as Gatekeeper for TREM2, Abnormal Accumulation of ApoE, and Fibrillary Tau in Alzheimer's Disease and in Down Syndrome Dementia.

Background:Genetic factors that influence Alzheimer’s disease (AD) risk include mutations in TREM2 and allelic variants of Apolipoprotein E, influencing AD pathology in the general population and in Down syndrome (DS). Evidence shows that dysfunction of the choroid plexus may compromise the blood-cerebrospinal fluid (CSF) barrier, altering secretary, transport and immune function that can affect AD pathology.Objective:To investigate the genotype and phenotype of DS individuals in relation to choroid plexus damage and blood-CSF barrier leakage to identify markers that could facilitate early diagnosis of AD in DS.Methods:To assess allele frequency and haplotype associations ApoE, Tau, TREM2, and HLA-DR were analyzed by SNP analysis in DS participants (n = 47) and controls (n = 50). The corresponding plasma protein levels were measured by ELISA. Postmortem brains from DS, AD, and age-matched controls were analyzed by immunohistochemistry.Results:Haplotype analysis showed that individuals with Tau H1/H1 and ApoE ɛ4 genotypes were more prevalent among DS participants with an earlier diagnosis of dementia (17%) compared to H1/H2 haplotypes (6%). Plasma TREM2 levels decreased whereas phospho-tau levels increased with age in DS. In AD and DS brain, insoluble tau and ApoE were found to accumulate in the choroid plexus.Conclusion:Accumulation of tau and ApoE in the choroid plexus may increase the oligomerization rate of Aβ42 and impair tau trafficking, leading to AD pathology. We have identified a high-risk haplotype: ApoE ɛ4, Tau/H1, and TREM2/T, that manifests age-related changes potentially opening a window for treatment many years prior to the manifestation of the AD dementia.

Distinct Microglial Responses in Two Transgenic Murine Models of TAU Pathology.

Microglial cells are crucial players in the pathological process of neurodegenerative diseases, such as Alzheimer’s disease (AD). Microglial response in AD has been principally studied in relation to amyloid-beta pathology but, comparatively, little is known about inflammatory processes associated to tau pathology. In the hippocampus of AD patients, where tau pathology is more prominent than amyloid-beta pathology, a microglial degenerative process has been reported. In this work, we have directly compared the microglial response in two different transgenic tau mouse models: ThyTau22 and P301S. Surprisingly, these two models showed important differences in the microglial profile and tau pathology. Where ThyTau22 hippocampus manifested mild microglial activation, P301S mice exhibited a strong microglial response in parallel with high phospho-tau accumulation. This differential phospho-tau expression could account for the different microglial response in these two tau strains. However, soluble (S1) fractions from ThyTau22 hippocampus presented relatively high content of soluble phospho-tau (AT8-positive) and were highly toxic for microglial cells in vitro, whereas the correspondent S1 fractions from P301S mice displayed low soluble phospho-tau levels and were not toxic for microglial cells. Therefore, not only the expression levels but the aggregation of phospho-tau should differ between both models. In fact, most of tau forms in the P301S mice were aggregated and, in consequence, forming insoluble tau species. We conclude that different factors as tau mutations, accumulation, phosphorylation, and/or aggregation could account for the distinct microglial responses observed in these two tau models. For this reason, deciphering the molecular nature of toxic tau species for microglial cells might be a promising therapeutic approach in order to restore the deficient immunological protection observed in AD hippocampus.

Characterization of a 3xTg-AD mouse model of Alzheimer's disease with the senescence accelerated mouse prone 8 (SAMP8) background.

No model fully recapitulates the neuropathology of Alzheimer's disease (AD). Although the triple-transgenic mouse model of AD (3xTg-AD) expresses Aβ plaques and tau-laden neurofibrillary tangles, as well as synaptic and behavioral deficits, it does not display frank neuronal loss. Because old age is the most important risk factor in AD, senescence-related interactions might be lacking to truly establish an AD-like environment. To investigate this hypothesis, we bred the 3xTg-AD mouse with the senescence-accelerated mouse prone 8 (SAMP8), a model of accelerated aging. We generated four groups of heterozygous mice with either the SAMP8 or SAMR1 (senescence-resistant-1) genotype, along with either the 3xTg-AD or non-transgenic (NonTg) genotype. Despite no differences among groups in total latency to escape the Barnes maze, a greater number of errors were noticed before entering the target hole in 19-month-old P8/3xTg-AD mice at day 5, compared to other groups. Postmortem analyses revealed increased cortical levels of phospho-tau (Thr231) in female P8/3xTg-AD mice (+277% vs. R1/3xTg-AD mice), without other tau-related changes. Female P8/3xTg-AD mice exhibited higher cortical soluble Aβ40 and Aβ42 concentrations (Aβ40, +85%; Aβ42, +35% vs. R1/3xTg-AD), whereas insoluble forms remained unchanged. Higher Aβ42 load coincided with increased astroglial activation in female P8/3xTg-AD mice, as measured with glial fibrillary acidic protein (GFAP) (+57% vs. R1/3xTg-AD mice). To probe neuronal degeneration, concentrations of neuronal nuclei (NeuN) were measured, but no differences were detected between groups. Altogether, the SAMP8 genotype had deleterious effects on spatial memory and exerted female-specific aggravation of AD neuropathology without overt neurodegeneration in 3xTg-AD mice.

Aging African green monkeys manifest transcriptional, pathological, and cognitive hallmarks of human Alzheimer's disease

While many preclinical models of Alzheimer's disease (AD) have been reported, none fully recapitulate the disease. In an effort to identify an appropriate preclinical disease model, we characterized age-related changes in 2 higher order species, the African green monkey (AGM) and the rhesus macaque. Gene expression profiles in the dorsolateral prefrontal cortex and the visual cortex showed age-related changes in AGMs that are strikingly reminiscent of AD, whereas aged rhesus were most similar to healthy elderly humans. Biochemically, age-related changes in AGM cerebrospinal fluid levels of tau, phospho-tau, and amyloid beta were consistent with AD. Histologically, aged AGMs displayed pathological hallmarks of the disease, plaques, and 2 AGMs showed evidence of neurofibrillary tangle-like structures. We hypothesized and confirmed that AGMs have age-related cognitive deficits via a prefrontal cortex-dependent cognition test, and that symptomatic treatments that improve cognition in AD patients show efficacy in AGMs. These data suggest that the AGM could represent a novel and improved translational model to assist in the development of therapeutics for AD.

ApoE4-associated phospholipid dysregulation contributes to development of Tau hyper-phosphorylation after traumatic brain injury

The apolipoprotein E4 (ApoE4) genotype combines with traumatic brain injury (TBI) to increase the risk of developing Alzheimer’s Disease (AD). However, the underlying mechanism(s) is not well-understood. We found that after exposure to repetitive blast-induced TBI, phosphoinositol biphosphate (PIP2) levels in hippocampal regions of young ApoE3 mice were elevated and associated with reduction in expression of a PIP2 degrading enzyme, synaptojanin 1 (synj1). In contrast, hippocampal PIP2 levels in ApoE4 mice did not increase after blast TBI. Following blast TBI, phospho-Tau (pTau) levels were unchanged in ApoE3 mice, whereas in ApoE4 mice, levels of pTau were significantly increased. To determine the causal relationship between changes in pTau and PIP2/synj1 levels after TBI, we tested if down-regulation of synj1 prevented blast-induced Tau hyper-phosphorylation. Knockdown of synj1 decreased pTau levels in vitro, and abolished blast-induced elevation of pTau in vivo. Blast TBI increased glycogen synthase kinase (GSK)-3β activities in ApoE4 mice, and synj1 knockdown inhibited GSK3β phosphorylation of Tau. Together, these data suggest that ApoE proteins regulate brain phospholipid homeostasis in response to TBI and that the ApoE4 isoform is dysfunctional in this process. Down-regulation of synj1 rescues blast-induced phospholipid dysregulation and prevents development of Tau hyper-phosphorylation in ApoE4 carriers.

Aging Does Not Affect Axon Initial Segment Structure and Somatic Localization of Tau Protein in Hippocampal Neurons of Fischer 344 Rats.

Little is known about the specific contributions of aging to the neuron dysfunction and death in Alzheimer’s disease (AD). AD is characterized by the pathological accumulation of abnormal tau (a microtubule-associated protein), and the mislocalization of tau from the axon to the somatodendritic compartment is thought to play an important role in disease pathogenesis. The axon initial segment (AIS) is thought to play a role in the selective localization of tau in the axonal compartment. Thus, disruption in the AIS barrier may allow tau to diffuse freely back into the somatodendritic compartment and potentially lead to neurotoxicity. Here, we analyzed AISs using stereological methods and protein immunoblotting, and the localization of tau was assessed with immunofluorescence optical density measurements and protein immunoblotting. None of the outcome measurements assessed, including AIS structure, AIS protein levels, the distribution of tau in neurons of the hippocampus (HP), and total tau or phospho-tau protein levels were different in young, middle-, and old-age Fischer 344 rats. The outcome measurements assessed, including AIS structure, AIS protein levels, the distribution of tau in neurons of the HP, and total tau or phospho-tau protein levels were not different in young, middle-, and old-age Fischer 344 rats, with the exception of a small reduction in AIS volume and diameter in the CA2 region of aged animals. These data suggest that aging largely has no effect on these properties of the AIS or tau distribution, and thus, may not contribute directly to tau mislocalization.

MUTAGENESIS OF THE GENERAL AMYLOID INTERACTION MOTIF (GAIM) REVEALS A STRUCTURE-ACTIVITY RELATIONSHIP FOR MISFOLDED BETA-AMYLOID AND TAU AGGREGATES

The general amyloid interaction motif (GAIM) derived from the M13 phage tip protein g3p binds a wide variety of amyloid aggregates in a conformation-dependent manner (Krishnan 2014). Dimeric GAIM-Ig-fusions robustly bind and remodel Aβ42 amyloid aggregates and inhibit tau aggregate propagation in primary neuronal cells. In transgenic models of AD and tauopathy, GAIM-fusion treatment reduces Aβ plaque load, phospho-tau levels and improves cognition (Levenson 2016). In this study, we explored the mechanism of GAIM-mediated remodeling of amyloid aggregates by mutagenesis. Using this data, we designed a next generation Ig-fusion, NPT189, which in addition to showing improved binding potency to multiple aggregates, has reduced potential for immunogenicity after removal of potential T-cell epitopes. Data obtained from H/D exchange NMR studies, GAIM-peptide-based inhibition assays, TolA binding, and computational modeling was used for targeted mutagenesis of the GAIM scaffold. Stable, high expressing variants were then screened for binding to Aβ42 and Tau-K18 fibers using SPR and binding ELISA assays. High binding variants were further screened for Aβ42 fiber remodeling as well as tau and α-synuclein transmission inhibition activities. An independent mutagenesis study was carried out to sequentially eliminate four potential T-cell epitopes of GAIM. Analysis of binding and remodeling data collected from GAIM variants illuminated a novel mechanism for GAIM activity. Amyloid binding is mediated by residues facing the interdomain groove of N1 and N2 domains of GAIM. Binding specificity depends on the stability of both domains and the melting temperature for domain separation. Over-stabilization of N1 or N2 domains leads to reduced binding activity, while destabilization of the domains leads to increased non-specific binding. We hypothesize progressive binding and rearrangement of GAIM β-strands enables GAIM to remodel amyloid aggregates into non-fibrous and non-pathogenic aggregates. GAIM-Ig-fusions represent a novel class of therapeutics to treat protein misfolding disorders. Systemic administration of these molecules slows down the accumulation of amyloid plaque and intracellular tau tangles and provides cognitive benefits in transgenic animals.

THE ROLE OF COMPLEMENT C3 AND C3AR RECEPTOR IN TAU PATHOLOGY IN ALZHEIMER’S DISEASE

Besides the pathological hallmarks of β-amyloid (Aβ) plaques and neurofibrillary tangles (NFTs), increasing evidence suggests that neuroinflammation plays a significant role in AD pathogenesis. The complement pathway is a key regulator of innate immunity. Recent findings from our laboratory showed that the central complement component C3 is upregulated in astrocytes of AD patients and mouse models. Moreover, elevated astroglial C3 can modulate Aβ dynamics and pathology and reduce dendritic morphology and synaptic function through microglial and neuronal C3a receptor (C3aR), respectively. Giving the importance of the C3-C3aR axis in the CNS, we are interested in understanding whether the complement pathway also impacts the NFT pathology. We analyzed the expression patterns of complement pathway genes in AD, MCI and non-cognitive impaired brain samples obtained from the Religious Orders Study (ROS) and Rush Memory and Aging Project (MAP) cohorts and in the PS19 mouse model of tauopathy before or after the onset of NFT pathology. Next, we studied the functional effects of the C3-C3aR pathway on reactive gliosis and NFT pathology by genetically deleting C3aR or overexpressing neuronal C3aR in PS19 animals. Finally, we utilized RNA sequencing and reverse-phase proteomics (RPPA) to identify C3aR downstream signaling pathways that promote such effects. Analysis of RNA sequencing datasets from human brains revealed a strong upregulation of complement pathway genes that is highly correlated with Braak staging and cognitive decline. Prominent complement upregulation was also observed in PS19 mice in response to NFT pathology. AAV-driven neuronal overexpression of C3aR exacerbates tau/NFT pathology in PS19 animals. Conversely, knocking out C3aR in PS19 mice results in significant reduction of inflammation, phospho-tau levels and associated pathology and improved behavioral deficits of PS19 animals. Mechanistically, we identify the Jak-Stat3 pathway as a potent regulator of reactive gliosis and tau pathology in PS19 mice downstream of C3aR.

OBJECT AND SCENE MEMORY ARE DIFFERENTIALLY ASSOCIATED WITH CSF MARKERS OF ALZHEIMER’S DISEASE AND MRI VOLUMETRY

Alzheimer's Disease (AD) is characterized by progressive tau-pathology in perirhinal and entorhinal cortices (ErC), hippocampus, parahippocampal cortex and the retrosplenial/posterior cingulate region (RsC/PCC). The latter also shows early amyloid plaque accumulation. While these regions form a network for complex objects-in-scene memories, there is a segregation of object memory into a perirhinal-entorhinal and scene-layout memory into a RsC/PCC-parahippocampal-entorhinal pathway. Here we investigated whether complex objects-in-scene memory, as opposed to isolated object or scene-layout memory is correlated with CSF levels of Aß42 and phospho-tau and MR volume and thickness measures. 204 participants (cognitively healthy, subjective memory complaints, mild cognitive impairment or early AD) of the DELCODE study of the DZNE performed a complex scene recognition memory task. CSF samples were available from 80 participants. 78 participants also performed a second task on mnemonic discrimination of objects (in the absence of scenes) and scenes (in the absence of objects). Of these 28 CSF samples were available. MR thickness measures were obtained from T1 and T2-weighted images using Freesurfer 6. Objects-in-scene recognition memory correlated with right and left PCC volume (p=0.005; p=0.047), right and left hippocampal volume (p=0.002; p=0.001), Aß42, phospho-tau and their ratio (p=0.002; p=0.002; p=0.000). Isolated object and scene discrimination correlated with left and right hippocampal volume (Scene: p=0.039; p=0.006; Object: p=0.011; p=0.001); scene memory correlated with right and object memory with both left and right ErC thickness (Scene: p=0.027; Object: p=0.024; p=0.031). A scene-minus-object difference score correlated with Aß42 levels indicating that decreasing levels of Aß42 were associated with selective decline of scene discrimination (p=0.034). When considering only healthy older adults, object discrimination relatively selectively correlated with right and left hippocampal volume (n=57; rp=0.022; p=0.005), left entorhinal thickness (p=0.042) and with phospho-tau levels (n=19; p=0.052). These data suggest that complex object-in-scene recognition captures variance caused by both tau and amyloid pathology. A domain-specific breakdown, however, indicates that tau-related variance tends to be captured by object discrimination whereas amyloid pathology is captured by scene discrimination performance. The findings are compatible with the possibility that object and scene discrimination track different stages of disease progression in AD.