Familial Alzheimer's Disease Research Articles

Loss of tubulin tyrosine ligase, a novel feature of sporadic and familial AD, is an enhancer of tau phosphorylation

AbstractBackgroundWe have recently shown that loss of tubulin tyrosine ligase (TTL) and subsequent disruption of a‐tubulin retyrosination is a feature of AD and a promoter of synaptic loss by inhibiting microtubule entries into dendritic spines. TTL+/‐ mice have cognitive deficits, altered LTP, synapse loss and increased levels of detyrosinated and D2 tubulin, two tubulin modifications that accumulate on non‐dynamic microtubules. We also found that low levels of phospho‐tau correlated with higher levels of detyrosinated and D2 tubulin in pyramidal neurons residing in the anterior hippocampal formation of human AD brains, raising the question of whether improper microtubule longevity drives early stages of tau hyperphosphorylation in AD. Here, we demonstrate that loss of TTL enhances both AMPK activation and tau phosphorylation in cultured rat hippocampal and human cortical neurons, and present preliminary evidence on the mechanisms by which this regulation may occur and contribute to TTL‐loss dependent synaptic dysfunction.MethodHuman cortical neurons derived from iPSC lines in which the London familial APP mutation V717I was knocked into one allele of the IMR90 control using CRISPR/Cas9, were used as a cellular model of familial AD. Lentiviral infection with shRNA against TTL was used to silence TTL expression. Microtubule dynamics were measured by confocal time‐lapse microscopy in neurons expressing EB3‐EGFP, a protein that tracks the growing plus ends of microtubules. Immunoblotting was performed to measure both unmodified and modified levels of TLL, tubulins, AMPK subunits, total tau, p262 and AT8 phospho‐tau.ResultIn the APP neurons, we found that a reduction in TTL levels correlated with an increase in p262/AT8 phospho‐tau variants and phospho‐AMPK, the active state of one of the kinases that phosphorylates serine 262, a key residue in the binding of tau to microtubules. Knockdown of TTL was sufficient to reduce microtubule dynamics and increase both active AMPK levels and phospho‐tau.ConclusionOur results indicate that loss of TTL alone reduces dynamic microtubules, triggers tau kinase activation, and results in hyperphosphorylated tau. Together with our previously published data, these findings suggest that reduced levels of TTL act as one of the early drivers of tau hyperphosphorylation in AD.

Assessing amyloid plaque burden in aging marmosets and genetically engineered PSEN1 mutation carriers using 11C‐PiB PET

AbstractBackgroundThere is an urgent need to develop novel animal models to investigate the underlying cellular and molecular root causes of the pathogenesis and progression of AD. Our group has worked to establish the common marmoset as the first primate‐specific model of AD to reveal the earliest cellular and molecular events of AD processes and allow charting of AD progression from its inception. For this, we established a colony of outbred marmosets and used genetic engineering techniques to generate marmosets carrying the PSEN1‐C410Y and PSEN1‐A426P mutations that cause early‐onset, familial AD. The present study aims to use PET imaging to access and track the progression of Ab plaque burden non‐invasively and longitudinally in our marmoset models of AD.MethodMarmosets anesthetized with isoflurane delivered via a facemask were placed in a preclinical PET/CT scanner customized with marmoset‐specific beds. Respiratory rate, heart rate, arterial O2 saturation, and rectal temperature were monitored and maintained at normal physiological values. Our PET imaging protocol follows the Alzheimer’s Disease Neuroimaging Initiative (ADNI). The amyloid‐binding 11C‐Pittsburgh Compound B (11C‐PiB) was delivered intravenously in doses varying from 18.5 – 185 MBq (0.5 – 5 mCi). A 90‐min dynamic acquisition was performed immediately following the radiotracer injection, followed by a 3D CT scan acquired at 200 µm isotropic resolution. After the PET/CT neuroimaging scan, the marmosets were recovered from anesthesia and returned to their home cages.ResultWe observed positive 11C‐PiB binding and retention in the brains of aging outbred marmosets and in marmosets harboring PSEN1 mutations. Significant 11C‐PiB retention was observed in the cortex and white matter tracts. The areas with the highest 11C‐PiB retention were the lateral frontal cortex, anterior cingulate cortex, and temporal lobe regions.ConclusionThis study demonstrates using 11C‐PiB to track Ab plaque burden in the brains of aging marmosets and marmosets harboring PSEN1 mutations. As observed in human patients of AD, the amount of 11C‐PiB bound (measured in SUV and SUVR units) varied from animal to animal, and we are currently performing group analysis to investigate the dependence of 11C‐PiB retention on age and sex. This research was funded by NIH/NIA grants R24AG073190 and U19AG074866.

Assessing amyloid plaque burden in aging marmosets and genetically engineered PSEN1 mutation carriers using 11C‐PiB PET

AbstractBackgroundThere is an urgent need to develop novel animal models to investigate the underlying cellular and molecular root causes of the pathogenesis and progression of AD. Our group has worked to establish the common marmoset as the first primate‐specific model of AD to reveal the earliest cellular and molecular events of AD processes and allow charting of AD progression from its inception. For this, we established a colony of outbred marmosets and used genetic engineering techniques to generate marmosets carrying the PSEN1‐C410Y and PSEN1‐A426P mutations that cause early‐onset, familial AD. The present study aims to use PET imaging to access and track the progression of Ab plaque burden non‐invasively and longitudinally in our marmoset models of AD.MethodMarmosets anesthetized with isoflurane delivered via a facemask were placed in a preclinical PET/CT scanner customized with marmoset‐specific beds. Respiratory rate, heart rate, arterial O2 saturation, and rectal temperature were monitored and maintained at normal physiological values. Our PET imaging protocol follows the Alzheimer’s Disease Neuroimaging Initiative (ADNI). The amyloid‐binding 11C‐Pittsburgh Compound B (11C‐PiB) was delivered intravenously in doses varying from 18.5 – 185 MBq (0.5 – 5 mCi). A 90‐min dynamic acquisition was performed immediately following the radiotracer injection, followed by a 3D CT scan acquired at 200 µm isotropic resolution. After the PET/CT neuroimaging scan, the marmosets were recovered from anesthesia and returned to their home cages.ResultWe observed positive 11C‐PiB binding and retention in the brains of aging outbred marmosets and in marmosets harboring PSEN1 mutations. Significant 11C‐PiB retention was observed in the cortex and white matter tracts. The areas with the highest 11C‐PiB retention were the lateral frontal cortex, anterior cingulate cortex, and temporal lobe regions.ConclusionThis study demonstrates using 11C‐PiB to track Ab plaque burden in the brains of aging marmosets and marmosets harboring PSEN1 mutations. As observed in human patients of AD, the amount of 11C‐PiB bound (measured in SUV and SUVR units) varied from animal to animal, and we are currently performing group analysis to investigate the dependence of 11C‐PiB retention on age and sex.This research was funded by NIH/NIA grants R24AG073190 and U19AG074866.

Model‐EyeD: Harnessing the potential of the eye as a biomarker for Alzheimer’s disease and related dementias

AbstractBackgroundDisease‐modifying treatments for Alzheimer’s disease and related dementias (ADRD) will be most effective early in the disease process. Clinical use of these therapies will require practical, widely accessible biomarkers. Plasma‐based protein biomarkers hold promise for identifying core AD pathology, but this complex disease likely requires other markers to identify people at highest risk of progression to dementia. Studies support eye exams as a synergistic strategy since retinal imaging would be cost effective, but significant hurdles remain. The timing and subtypes of retinal biomarkers have not yet been determined and the identification of ADRD‐specific biomarkers independent of common ocular diseases is challenging. Here, we introduce a new program, Model‐EyeD, to evaluate the eye as an accessible biomarker in the mouse model and human subjects.MethodFor familial AD and mixed dementia, we have backcrossed the APPSAA knock‐in allele (humanized Abeta with Swedish, Artic, Austrian, mutations) from C57BL/6J (B6) to genetically diverse NZO/HILtJ (prone to obesity and diabetes), and WSB/EiJ (susceptible to cerebrovascular deficits) strains. To study late‐onset AD (LOAD), we are using strains created by the IU/JAX/PITT MODEL‐AD Center, including the common 677C>T variant in methylene tetrahydrofolate reductase gene (Mthfr677C>T ), a cardiovascular and cerebrovascular risk factor. A battery of ocular assays (pattern electroretinograms, fluorescence angiography, optical coherence tomography) are performed on four to 24 months old male and female mice. Results are being compared to MRI, PET/CT, cognition and blood biomarkers.ResultUnlike previous transgenic amyloid models, B6.APPSAA and WSB.APPSAA mice show no retinal amyloid deposition or retinal vascular deficits at 12 months. NZO (in the absence of amyloid deposition) show retinopathy lesions (cotton wool spots, vascular leakage), suggesting NZO.APPSAA are ideal to study interactions between metabolic syndrome retinopathy and mixed dementia. Young B6.Mthfr677C>T mice show cerebral and retinal vascular deficits. Aging studies are ongoing. Results will be confirmed in human subjects with clinical and biomarker‐confirmed AD and mixed AD/small vessel vascular disease.ConclusionOur extensive collection of genetically diverse mouse models, coupled with our alignment to data from human subjects, show promise in developing eye‐based protocols to identify early stages of different clinical phenotypes of ADRD in humans.

Impaired Microtubule Dynamics Are a Driver of Tau Phosphorylation

AbstractBackgroundThe microtubule cytoskeleton is both stable and dynamic, and both states are necessary for normal neuronal function. Stable microtubules support long‐term structures like dendrites and axons, while dynamic microtubules promote synapse maintenance and are mostly composed of tyrosinated a‐tubulin, a condition that affects microtubule associated protein binding and motor protein recruitment. We have recently shown that human iPSC neurons carrying the familial AD APP London V717I mutation have impaired microtubule dynamics and significant increases in tubulin post‐translational modification (PTMs) associated with microtubule longevity. These changes result from decreased levels of the enzyme tubulin tyrosine ligase (TTL), which refreshes detyrosinated tubulin to its original, tyrosinated state. In human AD brains, TTL levels also decrease, while long‐lived microtubule PTMs increase. Here, we report that by compromising normal microtubule dynamics or by reducing TTL expression alone we can drive tau phosphorylation in rodent hippocampal neurons and explore the mechanisms by which this regulation occurs.MethodHuman cortical neurons derived from iPSC lines in which the London familial APP mutation V717I was knocked into one allele of the IMR90 control using CRISPR/Cas9, were used as a cellular model of familial AD. Inhibition of microtubule dynamics was performed with taxol or through lentiviral knockdown of TTL. Microtubule dynamics were measured by confocal time‐lapse microscopy in neurons expressing EB3‐EGFP, a protein that tracks the growing plus ends of microtubules. Immunoblotting was performed to measure protein levels.ResultWe found that in addition to increased levels of p262 tau, APP London neurons possess higher levels of active AMPK, a kinase that can phosphorylate serine 262 on tau and an important regulator of tau pathology in vitro and in vivo. We also demonstrate that decreasing microtubule dynamics with taxol or TTL silencing alone is sufficient to promote tau phosphorylation and that inhibition of AMPK or knockdown of its regulatory subunit can prevent it.ConclusionThese findings demonstrate that reduced levels of TTL and loss of dynamic microtubules drive AMPK‐mediated tau phosphorylation and suggest that TTL‐dependent loss of dynamic microtubules may contribute to tau pathology in AD.

Refinement of Neurofilament light Dynamics in CSF and Blood for familial Alzheimer’s Disease

AbstractBackgroundDisease‐modifying therapies for Alzheimer’s Disease (AD) are likely most beneficial when initiated in the pre‐symptomatic phase. To track success of such interventions fluid biomarkers became instrumental, with neurofilament light chain (NfL) showing particular promise. We previously reported that serum NfL increases in pre‐symptomatic phases of familial (autosomal‐dominantly inherited) AD (fAD) and that within‐person rate‐of‐change in NfL is an earlier predictor of fAD symptom onset compared to cross‐sectional levels. The aim of the study at hand was to extend these initial findings in a larger cohort.MethodParticipants enrolled in the Dominantly Inherited Alzheimer Network (DIAN) with matched cross‐sectional and longitudinal cerebrospinal fluid (CSF; n = 962) as well as plasma (n = 1294) samples were used. NfL measurements have been performed on the SIMOA platform using commercially available assay‐kits. To investigate the influence of physiological factors independent from AD, we first analyzed whether age and body mass index (BMI) could account for observed variation in inter‐individual CSF and plasma NfL levels considering non‐carrier family‐members (NC) as a healthy control group. Next, utilizing estimated years to symptom onset (EYO) and previously published methods (Preische et al., 2019), we determined the point in disease course when baseline and longitudinal CSF and plasma NfL concentrations started to increase in mutation carriers (MC) relative to NC, after adjusting for age and BMI.ResultOur results reveal a tight correlation of CSF and blood NfL values within MC as well as NC (figure 1A‐B). However, within NC, after correction for age and BMI, some unexplained variability remained (figure 1C). Further, a decrease of the NfL plasma/CSF ratio over age was found after correcting for BMI (figure 1D). The discrimination of MC from NC was equally good for plasma compared to CSF, being possible on the group level as early as around ‐15 to almost ‐20 EYO depending on the modelling (figure 2).ConclusionOur results support plasma equivalently to CSF NfL as a clinically useful biomarker to longitudinally track neurodegeneration in fAD. The systemic factors influencing physiological NfL values in blood should be investigated in future studies to further improve interpretation of this biomarker.

Deficits in mitochondrial function and glucose metabolism seen in sporadic and familial Alzheimer’s disease derived Astrocytes are ameliorated by increasing hexokinase 1

AbstractBackgroundAstrocytes have multiple roles including providing neurons with metabolic substrates and maintaining neurotransmitters at neuronal synapses. Astrocyte glucose metabolism plays a key role in learning and memory with astrocytic glycogen a key substrate supporting memory encoding. The homeostatic role the astrocyte provides for neurons leads to metabolic demands, meaning that abnormalities in the function of astrocyte mitochondria and glycolysis could affect this relationship. Changes to cellular metabolism are seen early in Alzheimer’s disease (AD). Understanding cellular metabolism changes in AD astrocytes could be exploited as a new biomarker or synergistic therapeutic agent when combined with anti‐amyloid treatments in AD.MethodsIn this project, we characterised mitochondrial and glycolytic dysfunction in astrocytes derived from patients with sporadic (n = 6) and familial (PSEN1, n = 3) AD, and associated controls (n = 9). Astrocytes were derived using direct reprogramming technology. Astrocyte metabolic outputs; ATP, and extracellular lactate levels were measured using luminescent and fluorescent protocols. Mitochondrial respiration and glycolytic function were measured using a Seahorse XF Analyzer. Hexokinase deficits identified where corrected by transfecting astrocytes with an adenovirus viral vector containing the hexokinase 1 gene.ResultsIn sAD astrocytes a 20% reduction (p = 0.05) and in fAD a 48% (p<0.01) reduction in total cellular ATP was seen. A 44% reduction (p<0.05), and 80% reduction in Mitochondrial spare capacity was seen in sAD and fAD respectively. Reactive oxygen species (ROS) where increased in both AD astrocyte types (p = 0.05). Mitochondrial complex I and II was significantly increased in sAD (p<0.05) but not in fAD. Astrocyte glycolytic reserve and extracellular lactate was significantly reduced when compared to controls in both sAD and fAD (p<0.05). We identified that astrocytes had a deficit in the glycolytic pathway enzyme hexokinase, and that correcting this deficit restored total cellular ATP levels, extracellular lactate and reduced ROS in sAD but not fAD astrocytes.ConclusionAD astrocytes have abnormalities in functional capacity of mitochondria and the process of glycolysis. These deficits are likely to impede metabolic support for neurons. These functional deficits can be improved by correcting hexokinase deficits. This suggests that hexokinase 1 deficiency could potentially be exploited as a new therapeutic target for AD.

Adult‐ and childhood onset dementias: related causes and related solutions?

AbstractBackgroundThe endo‐lysosomal pathway (ELP) of cells is central to their importation of nutrients and for recycling of cellular components. Much genetic evidence supports the importance of dysfunction of the ELP in Alzheimer’s disease (AD). However, AD is a complex disorder and how AD‐associated genetic variation affects ELP function is unclear. Sanfilippo syndrome childhood dementia is a consequence of extreme ELP dysfunction, where inherited recessive mutations result in the ELP’s inability to properly catabolise a particular polysaccharide. Unlike AD, Sanfilippo is genetically simple. All the causative genes have been identified and the biochemical consequences of their mutations are understood. Animal models of Sanfilippo are considered excellent models of the human disease. Using zebrafish as a model organism, we are dissecting shared disease mechanisms between these two dementia subtypes.MethodZebrafish models of both (early‐onset familial) AD and Sanfilippo syndrome were generated using CRISPR‐Cas9‐based approaches. The brain transcriptomes (RNA‐seq) and metabolomes (metabolomics/fluxomics) of both models are compared within single zebrafish families. Spatial working memory in both models is assessed using a Y‐maze.ResultPreliminary brain transcriptome analyses have identified changes‐in‐common to both oxidative phosphorylation and ribosome gene expression in both AD and Sanfilippo model zebrafish. Metabolomics and behavioural analyses of these models are ongoing.ConclusionNeurodegeneration in Sanfilippo syndrome and Alzheimer’s disease may share common mechanisms that are amenable to common therapeutic approaches.

Subclinical mesial temporal lobe activity in Alzheimer’s Disease

AbstractBackgroundAnimal and human studies of Alzheimer’s disease (AD) demonstrated that aberrant network activity with neuronal hyperexcitability plays a role in disease pathogenesis and cognitive functioning. Suppressing network hyperexcitability with levetiracetam improved cognitive functioning in familial AD mice and executive functioning in patients with AD and epileptiform activity. Manifestations of network hyperexcitability are unsensitive biomarkers since they are mostly subclinical and not visible on scalp EEG. (Lam et al. 2019, Vossel et al. 2021, Sanchez et al. 2012). Our purpose was to identify subclinical epileptiform activity in two patients with mild cognitive impairment (MCI) and one patient with moderate AD, with a suspicion of network hyperexcitability based on either spikes on 24‐h scalp EEG or a history suspect for seizures without epileptiform discharges on scalp EEG.MethodThree patients were implanted with bilateral foramen ovale (FO) electrodes to obtain more direct measures of mesial temporal lobe epileptiform activity. Scalp EEG according to the 10‐20 system and polysomnography were simultaneously recorded in all subjects for three to five consecutive days. Polysomnography was scored according to the AASM scoring rules. Persyst 14 software was used for automatic spike detection.ResultInvasive recordings showed interictal epileptic activity in all three patients, which was occasional during wakefulness and abundant during sleep. In one patient two nocturnal unilateral mesiotemporal seizures, characterized by arousal, were recorded during N1 sleep, while at the same time no epileptiform activity was visible on scalp EEG.ConclusionIn patients with AD subclinical mesial temporal epileptiform activity can be present with a normal scalp EEG. Epileptic activity is predominantly found during sleep. Our findings suggest an important role for network hyperexcitability in AD disease pathogenesis.

Proteomic characterization of small vessel pathology in PSEN1 E280A familial Alzheimer’s disease

AbstractBackgroundVascular pathology is a main contributor to Alzheimer’s disease (AD) progression and severity. We have previously characterized vascular pathology in the PSEN1E280A Colombian kindred. In postmortem analyses we identified that the occipital cortex (OC) is the most affected region by small vessel disease (SVD), including other cortical areas and basal ganglia. Interestingly, frontal cortex (FC) is spared from tau pathology in protected PSEN1E280A cases, while the OC is not. Here we study the proteome of isolated vessels from frontal and occipital cortices to identify differences in molecular pathways.MethodApproximately 400 mg of FC and OC of postmortem brain tissue from PSEN1E280A familial AD (FAD, n = 10), sporadic AD (SAD, n = 10) and controls (HC, n = 5) were homogenized, centrifuged at 2000 g / 5 minutes before using gradient centrifugation at 10000 g / 15 minutes with dextran to receive the microvessel containing pellet which was retrieved and resuspended in lysis buffer for tandem mass tag (TMT) proteomics. Proteomics spectral data were matched to peptide sequences in the human UniProt protein database using the MaxQuant software package. The MaxQuant output files were analysed by MSstatsTMT using R software for statistical analysis. clusterProfiler package was used to perform Gene Ontology enrichment analyses.ResultMicrovessels isolated from AD OC showed 1597 significantly dysregulated proteins when compared to controls, while FC showed 523. FAD showed the majority of upregulated proteins in FC (181 > 88 in SAD), and OC (428 > 84). This pattern was similar in downregulated proteins in the OC (391>171), but not in the FC (106<156) (Fig. 1A). The comparison between FAD cases and controls was the only one that identified a variety of biological processes (BP) during Protein set enrichment analysis. Notably, FAD FC showed upregulation of BP associated to vessel proliferation (Fig. 1B), while the OC showed downregulation of BP associated to immune system and mRNA regulation (Fig. 1C).ConclusionWe have identified the OC as a critical region of FAD pathology. Proteomic analysis of isolated microvessels from this region point towards a more deleterious profile while the FC microvessel proteome indicates an earlier stage of SVD pathology.

Functional and structural changes of cholinergic basal forebrain in asymptomatic PSEN1 E280A mutation carriers compared with non‐mutation carriers from the API ADAD Colombia cohort

AbstractBackgroundImaging studies showed early atrophy of the cholinergic basal forebrain in prodromal sporadic Alzheimer’s disease (AD) and alterations of the posterior basal forebrain functional connectivity in amyloid positive, but cognitively normal people. Similar investigations in familial cases of AD are still lacking. Here we investigated whether basal forebrain volume, metabolism, and posterior connectivity were already reduced in preclinical stages of autosomal dominant AD.MethodWe analyzed baseline data from 242 cognitively unimpaired subjects (69% PSEN1 E280A mutation carriers; mean age = 38 years; 62% female; mean education = 9 years) enrolled in the API ADAD Colombia Trial including available MRI, FDG‐PET and cognitive measures. We determined volume and metabolism of the basal forebrain, and of the hippocampus as a comparison region. In addition, we created connectivity maps from resting state fMRI data using anterior and posterior BF seed regions. We carried out Bayesian ANCOVA to determine evidence in favor or against an effect of carriers’ status on volumes, metabolism, functional connectivity and cognitive scores.ResultWe found inconclusive to moderate evidence in favor of no effect of carrier status on basal forebrain (BF10 = 0.884) and hippocampal volumes (BF10 = 0.964) or metabolism (BF10 = 0.187) (see Figure) in preclinical (CDR = 0) and prodromal (CDR = 0.5) subjects. However, we found extreme evidence that delayed memory was decreased already in preclinical mutation carriers (BF10 = 4066.73). Yet, we found no conclusive evidence that this subtle memory impairment was associated with basal forebrain (BF10 = 0.582) or hippocampal (BF10 = 0.906) atrophy.ConclusionOur findings indicate absence of basal forebrain and hippocampus atrophy or metabolic impairment in preclinical mutation carriers. Interestingly, memory impairment was already present in preclinical mutation carriers. This is consistent with results of the Dominantly Inherited Alzheimer Network (DIAN) cohort showing early cognitive impairment in mutation carriers, but no significant group differences in hippocampal volume until later stages of the disease. These previous and our findings may suggest that different brain regions and networks are affected in preclinical stages of familial AD than in preclinical sporadic AD, which should be investigated in future studies.

Seizures exacerbate tau spread in the 5XFAD mouse model

AbstractBackgroundCentral to the progression of Alzheimer’s disease (AD) is the spread of pathological tau along neuronal connections. This aspect of pathological progression can be recapitulated in WT mice by intracerebral injection of human AD brain derived tau lysate (AD‐tau). AD‐tau spread is enhanced in the amyloidosis mouse model, five times familial AD (5XFAD), suggesting that tau propagation is facilitated by β‐amyloid (Aβ) plaques. Once assumed to simply be a biproduct of Aβ‐induced hyperexcitability, a bidirectional relationship between seizures and AD progression has been established. Our lab has demonstrated worsened tau and Aβ pathology and cognitive deficits in AD patients with seizure history and in seizure‐kindled 5XFAD mice. Thus, we hypothesized that seizures would exacerbate the spread of AD‐tau in the 5XFAD mouse model and that 5XFAD mice would show increased seizure susceptibility and related neuronal activity.MethodTo investigate seizure‐pathology interactions, we crossed the 5XFAD mouse model with targeted recombination in active populations (TRAP; 5X‐TRAP) mice to permanently label seizure‐activated neurons. We injected AD‐tau unilaterally into the hippocampus and overlying cortex (1 µg/site) at 3 months of age in 5X‐TRAP and WT‐TRAP littermates. Seizures were induced with pentylenetetrazol (PTZ) kindling 2‐3 weeks following surgery and seizure‐activated neurons were labeled (tdTomato) on the final day of kindling via 4‐hydroxytamoxifen administration (50 mg/kg). Mice were euthanized three months post AD‐tau injection and immunohistochemistry for phospho‐tau (AT8) was performed.ResultWe found that kindled 5X‐TRAP mice show increased pathological tau (% AT8 coverage) in the dentate gyrus (DG) and CA1 contralateral to injection site compared to non‐kindled mice (p<0.05). We also found a trend towards elevated AT8 in the ipsilateral DG across seizure kindled groups (p = 0.083) and that 5X‐TRAP mice showed increased AT8 in the ipsilateral CA1 compared to WT‐TRAP (p<0.05). Further, we found a trend towards increased seizure susceptibility (0.059) in 5X‐TRAP mice and significantly increased seizure activated neurons (tdTomato+) in the DG of kindled 5X‐TRAP mice compared to WT‐TRAP (p<0.05).ConclusionOverall, our data further indicate a bidirectional relationship between seizures and AD. We also demonstrate that seizures facilitate the spread of pathological tau, thus suggesting that targeting seizure activity may slow AD progression.

Transient CSF1R Inhibition in the Early Stage of AD Retains Homeostatic Microglia with Reduced Inflammasomes and Increased autophagy in the 5XFAD Model

AbstractBackgroundChronic neuroinflammation is one of the most conspicuous features of Alzheimer’s disease (AD). While microglia play a vital role in phagocytosing amyloid beta (Aβ) and restraining plaque extension, disease‐associated microglia are known to perpetuate neuroinflammation. Previous studies have shown that ∼80% microglia elimination through a month‐long colony‐stimulating factor 1 receptor (CSF1R) inhibition in the advanced stage of AD in 5X Familial AD (5XFAD) mice does not reduce Aβ levels or plaques but restrains synapse loss and neurodegeneration. On the other hand, prolonged CSF1R inhibition has been shown to prevent parenchymal plaque development. However, the immediate effects of transient CSF1R inhibition in the early stage of AD on residual microglial morphology and their metabolic fitness are unknown.MethodWe investigated the impact of transient CSF1R inhibition in three‐month‐old 5XFAD mice, a stage at which neuroinflammation has commenced, but Aβ plaques were minimal. Microglia from the hippocampus and the somatosensory cortex (SSC) of 5XFAD mice receiving a diet containing either the CSF1R inhibitor PLX5622 or a standard diet for ten days were examined for morphology, markers of NOD‐, LRR‐, and pyrin domain‐containing protein 3 (NLRP3) inflammasome complex, autophagy, and mechanistic target of rapamycin (mTOR) signaling.ResultTen days of CSF1R inhibition resulted in 67‐70% depletion of microglia in the hippocampus and SSC. The residual microglia displayed a noninflammatory phenotype, typified by highly branched and ramified processes, reduced incidence of NLRP3 inflammasome complexes, and increased autophagy. Interestingly, Aβ plaque load was reduced, and mTOR signaling was unaltered in microglia but reduced in neurons. However, transient CSF1R inhibition did not impact astrocyte hypertrophy or neurogenesis in the hippocampus.ConclusionThe results showed that transient CSF1R inhibition in the early stage of AD promotes the retention of homeostatic microglia with reduced inflammasomes and better metabolic fitness but with no changes in mTOR signaling, which can restrain the perpetuation of neuroinflammation and improve phagocytosis of Aβ by microglia.

Protective variants in Alzheimer’s Disease from healthy elderly APOE‐ε4 participants

AbstractBackgroundPresence of the APOE‐ε4 allele is the most consistently confirmed genetic risk factor for late‐onset Alzheimer’s disease (AD) in white, non‐Hispanic populations, but the associated risks observed in African Americans and Hispanics are somewhat lower. The objective of this study was to identify protective variants that could modify or reduce the effect of APOE‐ε4 on AD risk.MethodsWe analyzed WGS data in over 3500 individuals from 600 multiplex AD families of non‐Hispanic White (NHW) and Caribbean Hispanic (CH) ancestry to identify protective variants in AD. APOE ε4 homozygosity was found in 48 healthy individuals over the age of 70, and APOE ε4 heterozygosity was found in 155 healthy individuals over the age of 80 were present in families with two of more affected samples. Protective variants were found segregating in the APOE ε4 homozygote and heterozygote individuals. We required that these variants be rare (MAF<0.1%) in GnomAD and prioritized exon coding variants that were putatively damaging to the resulting protein product. Novel or rare coding mutations segregating in ε4 unaffected subjects were tested for the effects on the age of onset of symptoms.ResultsWe identified 632 putatively protective variants that were present in at least 1% of APOE ε4 carriers and were absent in those with other APOE genotypes. 67 protective variants were present in both NHW and CH families. Pathway analysis of these segregating variants in APOE ε4 carriers are enriched in pathways involved in biological pathways and molecular functions such “actin binding”, “microtubule binding”, “extracellular matrix structural constituent”. These results suggest that protective variants promote activity of the extracellular matrix and cytoskeletal proteins leading to enhanced clearing of Aβ peptides. Variants in NBEAL1 delayed the age of onset in AD by 8.33 years respectively.ConclusionsWGS in CH and NHW families multiply affected by AD identified some healthy elderly, non‐demented APOE ε4 carriers with rare coding variants associated with delayed age of onset. Characterizing the functional role of these protective variants in AD will facilitate the creation of tractable models for investigation of disease mechanisms and potential therapies.

Can escapees of familial AD help identify protective factors? A case report of an extremely resilient PSEN1 carrier

AbstractBackgroundCarriers of mutations in PSEN1, PSEN2 or APP develop early‐onset Alzheimer’s dementia around a certain age depending on the specific mutation. However, some individuals “escape their fate”, meaning they remain asymptomatic far beyond their expected age at onset (EAO). We call these individuals “escapees”. A well‐known example is the resilient PSEN1 carrier that led to the identification of the protective APOEε3‐Christchurch mutation (Arboleda‐Velasquez et al., 2019). Here we present an extremely resilient PSEN1‐P264L mutation carrier where the cause of resilience has not yet been identified.MethodCarriers of PSEN1‐P264L have an EAO of 47 (SD = 4.3 years; Ryman et al., 2014). Our patient remains asymptomatic at age 58, which less than 0.6% of mutation carriers are expected to reach unaffected. We characterized this individual over the course of eight years with neuropsychological tests (n = 9), MRI scans (n = 5), amyloid (n = 3, [18F]florbetapir) and tau (n = 3, [18F]flortaucipir) PET scans and cerebrospinal fluid (CSF; n = 1). Genetic analysis of APOE and polygenic risk score (PRS) is described.ResultNeuropsychological test scores were within normal range at baseline and no significant cognitive decline was observed from age 50 to 58. On five consecutive MRI scans, no abnormalities or evidence of neurodegeneration was observed with visual read. At age 50, the patient was amyloid‐positive and tau‐positive in CSF (table 1). The patient was also amyloid‐positive on their first PET at age 51 (fig. 1). Widespread amyloid‐PET binding was observed, which increased over time. On tau‐PET, the patient was negative at age 51 and 53 (fig. 1), but positive at age 56, with an atypical binding pattern that shows high occipital load with relative sparing of the temporal and parietal cortex. This tau pattern and progression are remarkably similar to that seen in the escapee with the APOEε3‐Christchurch mutation. The APOE status of the patient is APOE ε3ε3 and the patient had an average PRS (+0.2SD from population average). Whole genome sequencing is ongoing.ConclusionWe describe an “escapee” of a PSEN1 mutation in which we have not yet identified the protective factor(s). Escapees represent rare but potent resilience phenotypes that should be studied systematically.

Characterization of brain metabolism, volume, and vasculature of the 5xFAD mouse model of Alzheimer’s Disease across lifespan.

AbstractBackgroundThe MODEL‐AD Consortium seeks to develop the next generation of Alzheimer’s Disease models based on human data. A popular model of familial AD is the 5xFAD mouse, characterized by early amyloid‐β deposition and cognitive decrements. Despite numerous studies, the 5xFAD mouse has not been comprehensively phenotyped for vascular and metabolic features over its lifespan.MethodMale and female 5xFAD and WT littermates underwent in vivo 18F‐FDG‐PET imaging at 4, 6, and 12 months of age to evaluate regional glucose metabolism. A separate cohort of mice (4, 8, 12 months) underwent “vessel painting” that labels all cerebral vessels with a fluorescent dye. “Vessel painted” brains were analyzed for vascular characteristics such as vessel density, junction density, vessel length, network complexity, number of collaterals and vessel diameter.ResultsOur analyses revealed that vessel length, vessel and junction densities increased from 4 to 12 months on the cortical surface in both 5xFAD and WT mice. The number of collateral vessels between the middle cerebral artery (MCA) and the anterior and posterior cerebral arteries decreased with age but their diameters were significantly increased only in 5xFAD mice. MCA average vessel length was decreased in 5xFAD mice compared to WT. Analysis of 18F‐FDG cortical uptake found significant interactions between WT and 5xFAD mice spanning 4‐12 months of age in retrosplenial, somatosensory and visual cortices. Broadly, 5xFAD males had increased 18F‐FDG uptake at 12 months of age compared to WT mice. In most cortical regions, female 5xFAD mice had reduced FDG uptake compared to WT across the lifespan. In males these metabolic increases coincided with decreased vessel characteristics.ConclusionThe 5xFAD mouse exhibits AD‐like cognitive deficits with age that are associated with increasing amyloid‐β deposition. No significant differences were found in cortical vascular features although males and females exhibited opposite effects in 18F‐FDG uptake. The MCA supplies blood to large portions of the motor cortex and increased vessel lengths and decreased collaterals along with higher metabolic rates in 5xFAD mice may be related to increasing behavioral deficits via metabolic insufficiency or other mechanisms.

Characterization of brain metabolism, volume, and vasculature of the 5xFAD mouse model of Alzheimer’s Disease across lifespan

AbstractBackgroundThe MODEL‐AD Consortium seeks to develop the next generation of Alzheimer’s Disease models based on human data. A popular model of familial AD is the 5xFAD mouse, characterized by early amyloid‐ß deposition and cognitive decrements. Despite numerous studies, the 5xFAD mouse has not been comprehensively phenotyped for vascular and metabolic features over its lifespan.MethodMale and female 5xFAD and WT littermates underwent in vivo 18F‐FDG‐PET imaging at 4, 6, and 12 months of age to evaluate regional glucose metabolism. A separate cohort of mice (4, 8, 12 months) underwent “vessel painting” that labels all cerebral vessels with a fluorescent dye. “Vessel painted” brains were analyzed for vascular characteristics such as vessel density, junction density, vessel length, network complexity, number of collaterals and vessel diameter.ResultOur analyses revealed that vessel length, vessel and junction densities increased from 4 to 12 months on the cortical surface in both 5xFAD and WT mice. The number of collateral vessels between the middle cerebral artery (MCA) and the anterior and posterior cerebral arteries decreased with age but their diameters were significantly increased only in 5xFAD mice. MCA average vessel length was decreased in 5xFAD mice compared to WT. Analysis of 18F‐FDG cortical uptake found significant interactions between WT and 5xFAD mice spanning 4‐12 months of age in retrosplenial, somatosensory and visual cortices. Broadly, 5xFAD males had increased 18F‐FDG uptake at 12 months of age compared to WT mice. In most cortical regions, female 5xFAD mice had reduced FDG uptake compared to WT across the lifespan. In males these metabolic increases coincided with decreased vessel characteristics.ConclusionThe 5xFAD mouse exhibits AD‐like cognitive deficits with age that are associated with increasing amyloid‐ß deposition. No significant differences were found in cortical vascular features although males and females exhibited opposite effects in 18F‐FDG uptake. The MCA supplies blood to large portions of the motor cortex and increased vessel lengths and decreased collaterals along with higher metabolic rates in 5xFAD mice may be related to increasing behavioral deficits via metabolic insufficiency or other mechanisms.

Inhibition of microfold cells ameliorates early pathological phenotypes by modulating microglial functions in Alzheimer’s disease mouse model

BackgroundThe gut microbiota has recently attracted attention as a pathogenic factor in Alzheimer’s disease (AD). Microfold (M) cells, which play a crucial role in the gut immune response against external antigens, are also exploited for the entry of pathogenic bacteria and proteins into the body. However, whether changes in M cells can affect the gut environments and consequently change brain pathologies in AD remains unknown.MethodsFive familial AD (5xFAD) and 5xFAD-derived fecal microbiota transplanted (5xFAD-FMT) naïve mice were used to investigate the changes of M cells in the AD environment. Next, to establish the effect of M cell depletion on AD environments, 5xFAD mice and Spib knockout mice were bred, and behavioral and histological analyses were performed when M cell-depleted 5xFAD mice were six or nine months of age.ResultsIn this study, we found that M cell numbers were increased in the colons of 5xFAD and 5xFAD-FMT mice compared to those of wild-type (WT) and WT-FMT mice. Moreover, the level of total bacteria infiltrating the colons increased in the AD-mimicked mice. The levels of M cell-related genes and that of infiltrating bacteria showed a significant correlation. The genetic inhibition of M cells (Spib knockout) in 5xFAD mice changed the composition of the gut microbiota, along with decreasing proinflammatory cytokine levels in the colons. M cell depletion ameliorated AD symptoms including amyloid-β accumulation, microglial dysfunction, neuroinflammation, and memory impairment. Similarly, 5xFAD-FMT did not induce AD-like pathologies, such as memory impairment and excessive neuroinflammation in Spib−/− mice.ConclusionTherefore, our findings provide evidence that the inhibiting M cells can prevent AD progression, with therapeutic implications.

Lipophorin receptors genetically modulate neurodegeneration caused by reduction of Psn expression in the aging Drosophila brain.

Mutations in the Presenilin (PSEN) genes are the most common cause of early-onset familial Alzheimer's disease (FAD). Studies in cell culture, in vitro biochemical systems, and knockin mice showed that PSEN mutations are loss-of-function mutations, impairing γ-secretase activity. Mouse genetic analysis highlighted the importance of Presenilin (PS) in learning and memory, synaptic plasticity and neurotransmitter release, and neuronal survival, and Drosophila studies further demonstrated an evolutionarily conserved role of PS in neuronal survival during aging. However, molecular pathways that interact with PS in neuronal survival remain unclear. To identify genetic modifiers that modulate PS-dependent neuronal survival, we developed a new DrosophilaPsn model that exhibits age-dependent neurodegeneration and increases of apoptosis. Following a bioinformatic analysis, we tested top ranked candidate genes by selective knockdown (KD) of each gene in neurons using two independent RNAi lines in Psn KD models. Interestingly, 4 of the 9 genes enhancing neurodegeneration in Psn KD flies are involved in lipid transport and metabolism. Specifically, neuron-specific KD of lipophorin receptors, lpr1 and lpr2, dramatically worsens neurodegeneration in Psn KD flies, and overexpression of lpr1 or lpr2 does not alleviate Psn KD-induced neurodegeneration. Furthermore, lpr1 or lpr2 KD alone also leads to neurodegeneration, increased apoptosis, climbing defects, and shortened lifespan. Lastly, heterozygotic deletions of lpr1 and lpr2 or homozygotic deletions of lpr1 or lpr2 similarly lead to age-dependent neurodegeneration and further exacerbate neurodegeneration in Psn KD flies. These findings show that LpRs modulate Psn-dependent neuronal survival and are critically important for neuronal integrity in the aging brain.

Exploratory Study of Gastrointestinal Redox Biomarkers in the Presymptomatic and Symptomatic Tg2576 Mouse Model of Familial Alzheimer's Disease: Phenotypic Correlates and Effects of Chronic Oral d-Galactose.

The gut might play an important role in the etiopathogenesis of Alzheimer's disease (AD) as gastrointestinal alterations often precede the development of neuropathological changes in the brain and correlate with disease progression in animal models. The gut has an immense capacity to generate free radicals whose role in the etiopathogenesis of AD is well-known; however, it remains to be clarified whether gastrointestinal redox homeostasis is associated with the development of AD. The aim was to (i) examine gastrointestinal redox homeostasis in the presymptomatic and symptomatic Tg2576 mouse model of AD; (ii) investigate the effects of oral d-galactose previously shown to alleviate cognitive deficits and metabolic changes in animal models of AD and reduce gastrointestinal oxidative stress; and (iii) investigate the association between gastrointestinal redox biomarkers and behavioral alterations in Tg2576 mice. In the presymptomatic stage, Tg2576 mice displayed an increased gastrointestinal electrophilic tone, characterized by higher lipid peroxidation and elevated Mn/Fe-SOD activity. In the symptomatic stage, these alterations are rectified, but the total antioxidant capacity is decreased. Chronic oral d-galactose increased the antioxidant capacity and reduced lipid peroxidation in the Tg2576 but had the opposite effects in the wild-type animals. The total antioxidant capacity of the gastrointestinal tract was associated with greater spatial memory. Gut redox homeostasis might be involved in the development and progression of AD pathophysiology and should be further explored in this context.