AD Brain Research Articles

Perturbed glycogen metabolism is an Alzheimer’s disease therapeutic target

AbstractBackgroundThree clinical hallmarks of AD are amyloid beta (Aβ) plaques, neurofibrillary tangles, and glucose hypometabolism. Multiple agents can decrease Aβ and tau levels, such as the recently FDA‐approved Aducanumab. However, improvements in patient cognition are disappointing and this disappointment is potentially due to a failure to normalize cerebral glucose hypometabolism.Clinical AD symptom severity strongly correlates with the degree of glucose hypometabolism. In the brain, one route of glucose metabolism is the biosynthesis of glycogen, a branched molecule that functions as an energy cache in times of energetic stress. Importantly, glycogen is critical for neuronal function and memory formation, and defective glycogen metabolism is an underlying driver of several neurological glycogen storage disorders (GSDs). An excess glycogen phenotype was reported in AD brains in the early 1980s, but to date, little is known about its involvement in AD symptoms.MethodIn this study, we first report that AD brains have an excess glycogen phenotype and that AD glycogen correlates with disease severity in human patient samples. Furthermore, the human excess AD glycogen is phenocopied in multiple AD mouse models and the accumulation of glycogen in these mice precedes cognitive deficits. Based on rigorous and striking data, we propose glycogen as a novel target for AD therapy that warrants additional preclinical development.ResultWe developed the enzyme therapy VAL‐0417 that combines a glycogen‐degrading amylase fused to an antibody fragment that facilitates intracellular delivery of recombinant enzymes. VAL‐0417 ablates excess glycogen and rescues disease phenotypes in a fatal GSD that causes childhood dementia called Lafora disease (LD). LD mice share several phenotypes with AD mouse models, including neurotoxic glycogen accumulation. Excitingly, VAL‐0417 also degrades AD glycogen both in vitro from human patient samples and in vivo in the 5xFAD AD mouse model. Furthermore, VAL‐0417 significantly reduces Ab plaque burden in 5xFAD mice and improves hippocampal long‐term potentiation.ConclusionThese studies suggest that targeting AD glycogen with VAL‐0417, either alone or in combination with other therapies targeting Aβ and/or tau, is an exciting and novel preclinical therapeutic target.

Interferon regulatory factor 5 modulates microglia response to tau by interfering with phagocytosis

AbstractBackgroundThe role of the immune system in responding to neurodegenerative pathology is well established. Microglia are at the center of such response, conveying on one end a positive protective effect, but on the other end, as with senescence, contributing to the hyperinflammation that is a core feature of neurodegeneration. We have identified the transcription factor interferon regulatory factor 5 (IRF5) as a mediator of microglia activation in response to pathological tau, described its specific activation by human‐derived PHF tau and its over‐representation in AD brains. We are now proposing that IRF5 can control the phagocytic activity of microglia, hence modulating their tau‐directed activity.MethodWe utilized age‐ and sex‐matched Irf5+/+ (WT) and Irf5− / − littermate mice for the analysis of ex vivo phagocytic activity of primary microglia, at steady‐state and triggered by tau. We examined microglia morphology and numbers under these conditions and with ageing. Last, we injected PHF tau intracranially to WT and Irf5− / − mice, monitoring tau accumulation and IRF5 response.Result1) Irf5− / − microglia have higher baseline phagocytic activity compared to WT microglia, which may be modulated by tau. 2) Microglia numbers are reduced in ageing Irf5− / − 3) PHF tau‐injected Irf5− / − mice show significant soluble tau accumulation, but a different pattern of oligomeric and insoluble tau accumulation with age. 4) In WT mice, the PHF tau‐injected hemisphere showed significantly higher levels of IRF5 compared to the contralateral, confirming a specific response of IRF5 to tau.ConclusionTau is a specific activator of microglia and of the IRF5 pathway, which can modulate microglia response to pathology. Ageing is an active variable in the process, affecting the pattern of microglia response to tau, differentially in WT and Irf5− / − mice. Modulating the IRF5 pathway may be a viable therapeutic approach in tauopathies.

Multiple patterns of structural brain change trajectories in AD: MRI deep learning verification with enhanced sequential pattern analysis

AbstractBackgroundOur recently proposed interpretable Ensemble 3DCNN deep learning (DL) technique has allowed the derivation of a P‐score based on brain structural magnetic resonance imaging (sMRI) modelling to predict patterned neurodegeneration in AD (https://doi.org/10.1002/advs.202204717). Sequential association rules with a support rate greater than 18% have been used to define a neurodegenerative progression pattern. To more prudently test the meaningfulness of the patterns found, we further calculated the lift values of the various sequential patterns underlying the suggested longitudinal sMRI changes.MethodThe lift value of an association rule is defined as the rule’s confidence rate divided by the support rate of the rule consequent, i.e., the support rate of the rule divided by the product of the support values of the rule consequent and the rule antecedent. For example, with {L.NAC}→{L.NAC, R.NAC} (https://doi.org/10.1002/advs.202204717), among the 167 AD subjects employed for analyzing neurodegenerative progression patterns, the number (N) of the subjects with at least an sMRI image containing both L.NAC and R.NAC as neurodegenerative regions at all time points examined was firstly calculated. Secondly, among the above‐mentioned 167 AD subjects, the number (M) of the subjects with at least an sMRI image containing only L.NAC as neurodegenerative regions at all time points examined was counted and the time point (P) corresponding to the selected (earlier/earliest) sMRI image was saved. Thirdly, among the above selected M subjects, the number (K) of the subjects with at least an sMRI image containing both L.NAC and R.NAC as neurodegenerative regions at all time points latter than the P was computed. Thus, lift value = (K/M)/(N/167).ResultThe lift values of the fifty sequential association rules reported in the recent paper (https://doi.org/10.1002/advs.202204717) ranged between 1.27 and 2.59(i.e., all > 1; Table 1), further confirming the longitudinal sMRI change patterns in AD.ConclusionThis study analyzing the lift values of the sequential association rules further verified the multiple patterns of longitudinal sMRI trajectories in AD brains, as reported recently. The research demonstrated featured and heterogeneous whole‐brain structural degeneration with AD progression.

Efficacy of the murine version of E2814 in a validated AD brain seed‐injection model in hTau mice

AbstractBackgroundAs Alzheimer’s disease progresses, pathological tau spreads through the brain via synaptically connected pathways. Tau proteins containing the microtubule binding region (MTBR) may be essential to form extracellular ‘seeds’ that initiate and propagate pathology. To simulate this process preclinically, we have further characterised an AD brain‐derived seed‐injection in vivo model using hTau mice that overexpress all six isoforms of human wild‐type tau (Andorfer et al., 2003). In further follow up studies, mice were treated with the 7G6‐IgG2a antibody, which is the murine counterpart of E2814, an anti‐tau antibody currently in clinical trials that recognises the MTBR (Roberts et al., 2020).MethodIn a validation study, insoluble fraction or whole homogenate from human AD brain (‘seed’) was directly injected into the left hippocampus of 6‐month‐old hTau mice or their tau null littermates. At different times after seed injection, the generation of insoluble tau was quantified in selected brain regions (hippocampus and cortex) by western blotting following tissue fractionation with detergents.In a subsequent therapeutic intervention study, animals were treated peripherally with a control mouse IgG2a antibody or 7G6‐IgG2a at different doses (3, 10, and 40 mg/kg). Levels of insoluble tau in the hippocampus and cortex were quantified as described above. Terminal CSF was also collected to measure target engagement of antibody. Additionally, immunohistochemistry using the AT8 antibody that detects pathological forms of phospho‐tau proteins was performed on brains from a limited number of animals receiving the highest dose of control IgG2a or 7G6‐IgG2a.ResultAD brain insoluble fraction was superior to homogenate when used as a seed in the hTau mouse.Peripheral treatment of seeded hTau mice with the 7G6‐IgG2a antibody (40 mg/kg) resulted in a significant reduction of insoluble tau in multiple brain regions. Antibody target engagement also increased in the CSF from treated animals in a dose‐dependent manner.ConclusionAD seed‐injection in hTau mice has been further validated and can be used in therapeutic intervention studies. The murine version of E2814, 7G6‐IgG2a, reduced pathological tau seeding and spreading in this model. The nonclinical data obtained here further supports E2814 being tested in clinical trials.

Targeting ceramides in 5XFAD mice reduces amyloid plaque deposition and gliosis

AbstractBackgroundAlzheimer’s Disease and other neurodegenerative disorders have been linked to metabolic disorders associated with aberrant lipid deposition. Sphingolipids, such as ceramides, are among the most deleterious lipids that accumulate in the AD brain. Increased ceramides have been measured in post‐mortem AD brain samples and are abundant in Aβ‐plaques. As biomarkers, ceramides in the plasma and cerebrospinal fluid correlate with AD risk. The potential of ceramide‐lowering strategies to treat or prevent AD has been underexplored. We hypothesize that ceramides are causal contributors to AD pathologies and amenable to therapeutic targeting to improve AD outcomes.MethodTo address ceramides in AD, we have targeted serine palmitoyl‐transferase (SPT), the first enzyme and rate‐limiting step in ceramide de novo synthesis, with the drug myriocin in 5XFAD mice on a regular chow diet and an obesogenic high‐fat diet. We have also generated inducible genetic models to study loss‐of‐ceramides (overexpression of the ceramide‐degrading enzyme Asah1) or gain‐of‐ceramides (overexpression of the SPT complex), specifically in neurons in the 5XFAD model. We used immunocytochemistry to assess Aβ plaque numbers and size and both astrocyte and microglial activation and numbers. Cognitive behavior was measured using a Barnes maze.Result5XFAD mice treated with myriocin for 17 weeks had a reduction of hippocampal ceramides. As expected, control vehicle‐treated 5XFAD mice showed a significant memory deficit during behavioral testing. Myriocin‐treated mice had significantly improved memory of the Barnes maze. Treated mice had fewer and smaller Aβ plaques and had a reduction in both astrocyte numbers and arborization in the cortex and hippocampus. Microglia were reduced in the hippocampus of treated mice. In 5XFAD mice with neuron‐specific Asah1 overexpression, Aβ plaque size and number remained unchanged compared to a 5XFAD control. However, Gfap+ astrocytes and Iba1+ microglia/monocyte activation and migration were eliminated, resulting in plaques with no associated glia and glial morphology like non‐5XFAD mice.ConclusionDecreasing ceramide production by myriocin treatment in 5XFAD mice reduced Aβ plaque number and size and reduced gliosis in the cortex and hippocampus. Reducing ceramides in neurons results in the abrogation of gliosis throughout the brain of a 5XFAD mouse.

Alternative splicing and characterisation of the tauopathy‐induced gene Growth Arrest DNA Damage Induced 45 alpha

AbstractBackgroundThe Gadd45 family are a set of 3 genes classically induced by DNA damage. They are also induced by wider stresses such as axonal injury, ischemia and degenerative diseases like ALS. Increased Gadd45 protein staining is observed in AD brains, however their association with pathogenic tau is unknown. The Gadd45 genes undergo alternative splicing to generate 8 functionally distinct proteins, whose significance in neuronal systems are poorly understood. We aim to investigate if the Gadd45 family are upregulated in tauopathies and whether this is accompanied by changes to mRNA splicing of these genes, with a view to understand whether these differentially affect cell health.MethodTransgenic mice (rTg4510) over‐expressing and FTD‐associated mutant tau (TauP301L) were used to investigate tau‐associated upregulation of the Gadd45 genes. Subsequently, we analyse which splice variants are present in primary cortical neurons and express plasmid constructs of these variants to investigate their sub‐cellular localisation and function, using apoptotic cell death as a read‐out.ResultGadd45α, but not β or γ members of the same family, is upregulated in response to pathological tau. Untreated cortical neurones express a single isoform of the Gadd45α gene, however upon cellular stress 2 additional isoforms are induced. The identified variants show distinct sub‐cellular distribution, including changes to nuclear localisation and the development of punctate staining. Furthermore, these differentially affect apoptosis.ConclusionGadd45α is selectively upregulated by tau pathology. The gene undergoes alternative splicing in murine neurones in response to cellular stress, and these variants show evidence for distinct functions. We will next investigate the association of these variants with pathological tau.We thank the Alzheimer’s Society for funding this work.

A preliminary in vitro study on Adult Neural Stem cells from 3xTg mice

AbstractBackgroundIn AD, abnormalities in brain wiring and variations of the brain energetic metabolism are considered a key pathological framework of cognitive decline (Heininger, 2000). Neural stem cells (NSCs) can contribute to brain regeneration and, their transplantation improves the behaviour of neurodegenerative mice models (De Gioia et.al., 2021) although, the molecular mechanisms involved are still unidentified. Our preliminary study aims to explore the potential differences between NSCs‐derived neurons from 6‐months‐old healthy and AD mice in terms of neuronal network morphology. Furthermore, the study wants to provide a tool to investigate whether cell energy metabolism is modified, considering that bioenergetic failure can influence the regenerative potential of adult AD brain, an issue that is still ill defined.MethodNSCs were isolated from subventricular zone of healthy (H) and 3xTg (AD) mice. NSCs selection was based on their ability to proliferate and to form 3D floating neurospheres. Isolated cells were amplified and characterised for their expression of stem‐related markers. Subsequently, neuron and astrocyte differentiation protocols were set up. Morphological analyses were performed on H and AD NSC‐derived neurons and the expressions of glutamate transporters and sodium/calcium exchangers (NCXs) were investigated.ResultH and AD NSCs were strongly positive for Nestin and Sox2 stem markers and a faint expression of NeuN and Gfap, markers related to neuron and astrocyte mature phenotype respectively, was detected. These data confirmed the purity of the cell cultures as well as the reliability of the protocols. Morphological investigations highlighted a different distribution of the neurite network between NSCs‐derived neurons isolated from H and AD mice. Furthermore, H and AD mature cells expressed glutamate transporters essential not only for cellular transmission but also for energy production by their interplay with NCXs.ConclusionPreliminary data suggest that the pathological features of adult neurons from AD brain can be traced back to “stem level” and continue during differentiation. The different morphological organization of neurite network observed in NSCs‐derive AD neurons could be involved in the alteration of the impulse transmission and plasticity. Further studies will be necessary to better clarify the consequence of dysregulated glutamate transporters as well the role of astrocytes.

Structural and functional Analysis of novel 3‐Nitrotyrosine Aβ‐specific Antibodies

AbstractBackgroundPosttranslational modified (PTM) amyloid peptides are increasingly within the spotlight of Alzheimer’s drug research. Some of these modifications, such as the formation of pyroglutamate or isoaspartate, are associated with the deposition and increased toxicity of amyloid peptides. The role of nitration at Tyr10 of Aβ and its abundance in AD brain is less well understood. How 3NY10‐Aβ is formed is controversial, as well as the influence on aggregation behaviour. Hence, we here aimed at generation and characterization of monoclonal antibodies specific for 3NY10‐Aβ as research tool and potential therapeutic molecules.MethodMonoclonal antibodies were developed by immunizing mice with 3NY10‐Aβ(6‐14) and derivation of corresponding hybridoma cell lines. Characterisation of the antibodies was achieved by a variety of biochemical and kinetic analyses: isothermal titration calorimetry (ITC), dot blot analysis and ThioflavinT aggregation studies. X‐ray crystallography was used for structure determination. We used immunohistochemical (IHC) staining of brain tissue from 5xFAD mice and enzyme linked immune sorbent assay (ELISA) to assess the formation of 3NY10‐Aβ in transgenic mice.ResultTwo highly affine 3NY10‐Aβ antibodies 4C3 and 3B3 have been generated, both binding 3NY10‐Aβ(1‐16) in the nanomolar range (KD4C3 = 11 nM, KD3B3 = 88,5 nM) as determined by ITC. The specificity of the antibodies was confirmed by dot blot analysis, using several post‐translationally modified Aβ‐peptides. Aggregation studies of 3NY10‐Aβ(1‐40/42) peptides in presence of 3B3 or 4C3 showed an influence of fibril formation, as could be verified by means of transmission electron microscopy. Co‐crystallisation of 4C3 Fab‐fragment and 3NY10‐Aβ(1‐16) at a resolution of 2.5 Å revealed a binding of 3NY10. To investigate the role of 3NY10‐Aβ in AD, IHC‐staining of brain from mice with AD‐like pathology (5xFAD mice) was performed. The data support presence of 3NY10‐Aβ within amyloid deposits, although staining appeared weak. Likewise, a Sandwich‐ELISA of homogenized murine brain tissue displayed no significant proportion of 3NY10‐modified Aβ in relation to the total Aβ(1‐40/42).ConclusionIt has been demonstrated that the unique monoclonal 3NY10‐Aβ specific antibodies 4C3 and 3B3 are a suitable tool for investigating the role of 3NY10‐Aβ in AD. Future research aims to quantify the level of 3NY10‐Aβ in human brain tissue with AD‐pathology.

Distinct immune signatures associated with sex and genotype in the 12m novel hAPP hAPOE mouse risk model of Alzheimer’s disease

AbstractBackgroundUnderstanding the biology of model systems is critically important in the identification of biomarkers to delay or prevent Alzheimer’s disease. Previous findings in our group demonstrate sex differences in immune transcripts in the AD brain and in the humanized apolipoprotein E (APOE) mouse risk model. With this study, we extend these discoveries to further investigate the effect of APOE genotype using a novel risk mouse model with humanized amyloid precursor protein (APP) and humanized‐APOE transgenes (Jackson Laboratory). We test the hypothesis that during midlife aging, hAPP hAPOE ε4/4 genotype, and female sex will show increased risk profiles for Aβ 42:40 ratio and immunophenotypes associated with Alzheimer’s disease.MethodTo identify midlife (12 months; human correlate 44 years) immunophenotypes that may contribute to AD 3 different risk genotypes were studied: hAPP (6 male /6 female), hAPP hAPOE ε3/3 (16 male/16 female), and hAPP hAPOE ε4/4 (16 male /16 female). The left hemisphere of the brain was collected and prepared for multi‐color flow cytometry. Microglial reactivity (MHCII, CD68), phagocytosis (pHrodo), oxidative stress (CellRox), and the presence of lymphocytes (CD3, CD4, CD8, CD19) were determined. Peripheral blood was collected and assessed for monocytes and lymphocytes while plasma was isolated and ELISA was used to detect the presence of Aβ 40‐42, and levels of pro‐inflammatory cytokines. Female animals were monitored daily by lavage to determine midlife endocrine status.ResulthAPP hAPOE ε4/4 animals demonstrated accelerated endocrine aging compared to hAPP hAPOE ε3/3 animals. Plasma beta‐amyloid 42:40 ratio, and levels of IL‐1B, and IL‐6 showed a significant interaction between sex and hAPP hAPOE genotype (p<0.05, p<0.05, p<0.05). Plasma levels of IL‐5 and CXCL1 trended toward genotype significance with p = 0.0544 and p = 0.0556, respectively. Percentage of MHCII+ activated microglia was significantly impacted by sex (p = 0.0005) and by genotype (p<0.0001).ConclusionThe interaction between hAPP and hAPOE genotype, in coordination with biological sex significantly impacts immunophenotypes of the CNS and periphery during midlife aging. Precision interventions targeting sex and genotype‐dependent prodromal immune biomarkers may serve as an opportunity to support homeostatic immune milieu or to modulate harmful inflammatory signaling and reduce risk of AD or delay AD onset.

Neuroprotective effect of Tropisetron in Streptozotocin induced animal model of Alzheimer’s disease

AbstractBackgroundAlzheimer’s disease is a neurodegenerative pathology having genetic and non‐genetic forms. Non‐genetic forms of AD dominate over the genetic condition and is mainly sporadic. However, both forms affect cognitive functions. Although exact mechanisms are not fully understood, amyloid β deposition & tau tangle formation are considered central mechanisms to cause neurodegenerative changes in the AD brain. On the other hand, cognitive deterioration began way before the accumulation of amyloid or tau tangle formation. Cognitive functions, including learning and memory, have been majorly controlled/regulated by the cholinergic system. Here in the present study, we have explored the neuroprotective effect of tropisetron in streptozotocin‐induced animal model of Alzheimer’s disease. During the last two decades, the involvement of other neurotransmitter systems has been explored, and the serotonergic system has been demonstrated to play an essential role in memory and learning. The serotonergic system is the most extensive monoaminergic system in the brain, and alterations in serotonergic signals have been linked with Aβ and tau tangle formation. Moreover, serotonin acting through its receptor antagonist has been reported beneficial in various experimental AD models.MethodIn the present study animals were infused with STZ alternatively on 1st and 3rd day. From 15th day animals were treated with tropisteron with two subsequent doses (2.2.5mg/kg) and (5mg/kg). On 28th day animals were sacrificed. Further animals were tested for behavioral, biochemical and histopathological parameters.ResultTropisetron significantly attenuated oxidative damage and restored the reduced levels of anti‐oxidant enzymes. ICV‐STZ treatment had shown significant neuronal damage in hippocampal and cortical regions of AD brain in histopathological examination. Moreover, hippocampal neurons were observed to be irregular in morphology with dark pyknotic nuclei. This confirms the neurodegeneration in ICV‐STZ animals. Target drug tropisetron resulted in significant neuroprotection against ICV‐STZ.ConclusionThe observed outcomes of the present study suggest the neuroprotective effect of tropisteron in STZ induced animal model of Alzheimer’s disease.It would be safe to conclude that tropisetron may improve the behavioral outcomes by attenuating oxidative burden & its neuroprotective potential as seen in our histological assessment.

The AI‐based AlphaFold 2 algorithm predicts an open structure for tauC3

AbstractBackgroundTauC3 is an especially noxious C‐terminal tau fragment produced by caspase‐3 cleavage at Asp421. Two decades of accumulating evidence support its importance in the pathogenesis of AD and other tauopathies, notably FTD and PSP. TauC3 promotes aggregation and filament formation, recruits full length tau (FLT) to incipient fibrils, and despite low concentration in AD brain tissue relative to total tau, has been reported to account for substantially all the seeding activity in AD brain extracts. TauC3 has also been shown to slow fast axonal transport (FAT), with deleterious consequences. Although tau’s largely disordered structure prevents analysis by X‐ray crystallography, FRET analysis suggested the existence of a hairpin conformation in which the N‐terminus folds over the C‐terminus close to the MTBR. In this model, Ser432 is the point of closest contact with Pro301. Opening of the hairpin has been hypothesized to 1) make FLT prone to aggregation by exposing the MTBR, and 2) impair FAT by overexposing the phosphatase activating domain near the N‐terminus. We postulated that tauC3 cannot form the hairpin because it lacks Ser432 and thus has an “open” conformation. This would explain tauC3’s substantially increased pathogenicity compared to FLT. The AI‐based tool AlphaFold 2 (AF2) is being used to predict the conformation of many proteins with great accuracy. Applying it to FLT and tauC3 could provide valuable data about the hairpin and whether caspase cleavage alters it.MethodWe tested AF2’s ability to predict the conformation of 4‐repeat FLT and tauC3 from FASTA sequences, aligning the models produced with the MTBR.ResultAF2 modeling revealed a dramatically different conformation for tauC3 compared to FLT, specifically, an open conformation with the MTBR and N‐terminus far apart (Figure). Interestingly, it also revealed that the entire C‐terminus region of FLT starting at Gln423 is composed of a single α‐helix.ConclusionAF2 predictions support the existence of the hairpin conformation for tau and underscore the critical importance of residue 432 in maintaining it. The extended C‐terminus α‐helix may have functional implications. TauC3’s open conformation is consistent with its slowing of FAT and its ability to seed aggregation even at low concentrations.

Amygdala volume as neuroimaging marker of limbic‐predominant age‐related TDP‐43 encephalopathy (LATE) in neurodegenerative disease: a post‐mortem MRI and pathology study

AbstractBackgroundLimbic‐predominant age‐related TDP‐43 encephalopathy (LATE) is characterised by hyper‐phosphorylated TAR DNA‐binding protein 43 (TDP‐43) aggregates within the amygdala and (para)hippocampal regions. LATE neuropathological change can occur in patients with neurodegenerative disorders including Alzheimer’s (AD) and Parkinson’s disease (PD), as well as in healthy controls. LATE is associated with lower cognitive performance in AD patients. No neuroimaging marker for LATE is currently available, but identification would be of importance for accurate disease prognosis and as potential exclusion criteria for e.g. AD clinical trials. The current study aims to assess whether MRI‐measured amygdala and hippocampal volume can differentiate LATE status in neuropathological‐confirmed AD and PD brain donors.MethodPost‐mortem in‐situ 3DT1 3T‐MRI data were collected for 15 cases (9 AD and 6 PD) with post‐mortem confirmed LATE neuropathological change, as well as 30 non‐LATE (18 AD and 12 PD) donors. Each donor with LATE was matched with two non‐LATE donors on age, sex and neuropathological (i.e. Thal and Braak) staging. Neuropathological assessment was done on tissue of the right hemisphere. Amygdala volume, including amygdala subfield volumes, as well as hippocampal volume, were calculated using Freesurfer (version 7.1.1) and corrected for total intracranial volume. Group differences were assessed with univariate analyses using post‐mortem delay as covariate, subfield analysis were Bonferroni corrected.ResultBrain donors with LATE neuropathological change showed significantly lower right amygdala (p = .002) and right hippocampal (p = .007) volumes (figure 1). Amygdala subfield volumes were available for 13 LATE and 27 non‐LATE cases. When investigating the amygdala subfields, we found significant LATE compared to non‐LATE differences within the anterior amygdaloid area (p = 0.005) and the lateral nucleus (p = 0.002) (figure 2). In AD (n = 27), LATE cases showed significantly lower right amygdala (p = .023) and right hippocampal (p = .003) volumes compared to non‐LATE cases. In PD (n = 18), only the volume of the right amygdala was significantly lower in LATE cases compared to non‐LATE cases (p = .043) (figure 3).ConclusionMRI‐measured amygdala volume, especially of the anterior and lateral nucleus, can differentiate LATE neuropathological change, and may therefore serve as a promising neuroimaging biomarker in neurodegenerative disease.

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.

The role of the blood‐brain barrier in brain insulin signaling

AbstractBackgroundDefects in central response to insulin is suspected to play a role in AD. However, to act on the brain, pancreas‐secreted insulin must interact first with the blood‐brain barrier (BBB). The insulin receptor (INSR) consists in 2 insulin‐binding extracellular α chains, either short (INSRα‐A) or long (INSRα‐B) isoforms, as well as 2 intracellular β‐chains (INSRβ) containing auto‐phosphorylation sites. The aim of the present study was to better understand the role of INSR located on the BBB in AD.MethodWe used microvessel‐enriched brain samples from individuals classified as Controls, MCI or AD and from 3xTg‐AD mice from different ages. In situ cerebral perfusion was used to directly assess the interaction between circulating insulin and the BBB.ResultsWe first show that INSR in human and mouse brains are concentrated in blood microvessels, with INSRα‐B being almost absent from the parenchymal fractions. Lower levels of INSRα‐B were observed in microvessel extracts from the parietal cortex of AD persons, associated with cognitive decline. The resulting shift toward a higher INSRα‐A:INSRα‐B ratio is consistent with insulin resistance situated on the BBB. Intracarotid infusion of insulin induced a rapid phosphorylation of INSRβ retrieved from cerebrovascular extracts, but not from parenchymal extracts of the mouse brain. In the 3xTg‐AD mouse model of AD neuropathology, lower levels of cerebrovascular INSRα‐B were observed whereas activation of INSRβ was blunted upon intracarotid administration of insulin. Finally, we provide some insights on a possible association between BACE1 activation and the reduction of cerebrovascular INSR.ConclusionOverall, data gathered from postmortem AD brains and an animal model of AD neuropathology suggest that brain insulin resistance in AD may occur at the level of INSR located on microvessels. These results also indicate that INSR and its downstream signaling within endothelial cells of the BBB are systemically accessible drug targets in AD.

The effect of prion protein expression on amyloid‐beta pathology

AbstractBackgroundWe have developed multiple knock‐in mouse models that express humanised Aβ (AppNL‐F) with varying levels of PrPC expression (AppNL‐F, AppNL‐F_Tg20, AppNL‐F_PrPKO, AppNL‐F_hTauKI and AppNL‐F_hTauKI_PrPKO) to investigate the role of PrPC in knock‐in mice before and after inoculation with human AD brain homogenates.MethodUninoculated knock‐in mice with varying levels of PrPC were characterised using biochemical and histological techniques. We went on to characterise AppNL‐F, AppNL‐F_Tg20, AppNL‐F_PrPKO, AppNL‐F_hTauKI and AppNL‐F_hTauKI_PrPKO mice after inoculation with human brain homogenates.ResultAn age‐dependent increase in Aβ42 levels in all animals at 10 months old was observed compared to their 6 month old counterparts. We saw an accumulation of activated astrocytes around the Aβ plaques with higher levels of astrogliosis seen in the AppNL‐F_Tg20 animals at all ages compared to AppNL‐F, AppNL‐F_PrPKO, AppNL‐F_hTauKI and AppNL‐F_hTauKI_PrPKO mice. Synaptic protein fractions extracted from AppNL‐F_Tg20 mice inoculated human AD brain homogenate showed a significant increase in Fyn and Pyk2 protein phosphorylation when compared to vehicle and control human brain homogenate inoculated AppNL‐F_Tg20 mice. This effect was statistically significant for both proteins at 8 months post‐inoculation (mpi) and was further exacerbated at 12 mpi. The results at 8 and 12mpi were independent of guanidine hydrochloride‐soluble Aβ42 levels and Aβ plaque coverage in the mouse brains. We went on to find significant differences in tau hyperphosphorylation and Aβ plaque coverage between inoculated AppNL‐F_hTauKI and AppNL‐F_hTauKI_PrPKO mice which was independent of guanidine hydrochloride‐soluble Aβ42 levels.ConclusionThe level of PrPC expression in two knock‐in mouse models inoculated with human brain homogenate was shown to have an effect on synaptic protein phosphorylation at 8 and 12mpi. This was independent of Aβ42 levels and Aβ plaque coverage in the mouse brains.

Targeting hyperactive antiviral response to treat tauopathy

AbstractBackgroundCyclic GMP‐AMP synthase (cGAS, Cgas), the major antiviral DNA sensor, binds double‐stranded DNA (dsDNA) and catalyzes cyclic GMP‐AMP (cGAMP) formation, which activates stimulator of interferon genes (STING). Activation of cGAS‐STING has been implicated in several neurodegenerative conditions. The role of cGAS in tauopathy remains unknown.MethodWe investigated cGAS‐STING‐IFN activation in microglia of tauopathy mice and human AD brains. We crossed cGAS knockout mice with P301S tauopathy mice and performed behavioral, immunohistochemistry, electrophysiology and single‐nuclei (sn) RNA‐seq.Resultwe demonstrated potent protective effects of Cgas genetic deletion against synaptic and cognitive deficits associated with induction of transcriptional network of MEF2C, a gene implicated in cognitive resilience against amyloid and tau pathology. SnRNA‐seq analyses of human AD brains revealed dysregulation of microglial IFN‐I genes and neuronal MEF2C transcriptional network.ConclusionOur current study linked a hyperactive cGAS‐IFN antiviral response with diminished MEF2C‐associated cognitive resilience. In diseased conditions, cGAS hyperactivation promoted microglial IFN‐I response, reduced neuronal MEF2C transactivation, and rendered loss of cognitive resilience. cGAS ablation diminished microglial IFN‐I response and enhanced neuronal MEF2C transcription network and associated cognitive resilience against tau pathology. Pharmacological inhibition of cGAS with TDI‐6570 enhanced MEF2C target genes and restored synaptic integrity and memory, supporting the therapeutic potential of targeting cGAS‐MEF2C axis to improve resilience against AD and related neurological disorders.

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.

Distinct immune signatures associated with sex and genotype in the 15m novel hAPP+hAPOE mouse risk model of Alzheimer’s disease

AbstractBackgroundAlzheimer’s disease research continues to struggle with the translatability of animal models to human patients. Exploring whole systems is integral to early AD biomarker discovery in the pursuit of therapeutics to delay or prevent AD. Previous finds in our lab indicate sex differences in immune transcription levels in human AD brain and in the humanized apolipoprotein E (APOE) mouse model. Herein we investigated the impact of the APOE genotype in a novel mouse model, humanized amyloid precursor protein (APP), and humanized APOE transgenes (Jackson Laboratory).MethodsTo identify immunophenotypes at late‐midlife (15 months: late 40s – early 50s human correlative) a novel hAPP+hAPOE mouse model was utilized: hAPP (5M:5F), hAPP+hAPOE ε3/3 (16M:16F), & hAPP+hAPOE ε4/4 (16M:16F). The brain was hemisected. The left side was processed for multi‐color flow cytometry analysis. Peripheral blood was taken via cardiac puncture. The plasma was isolated and ELISA was used to detect levels of Aβ 40‐42 and pro‐inflammatory cytokines. PBMCs were isolated from the peripheral blood for multi‐color flow cytometry analysis. Microglia (CD45, CD11b), microglia reactivity (MHCII, CD68), phagocytosis (pHrodo), and oxidative stress (CellRox) levels were determined in the brain. Lymphocytes (CD3, CD4, CD8, CD19), lymphocyte activity (CD69), and oxidative stress (CellRox) levels were determined in both the brain and peripheral blood. Midlife endocrine status was determined for the female mice by daily lavage.ResultshAPP+hAPOE ε4/4 animals exhibited accelerated endocrine aging evidence by increased proportion of irregular cycling mice, compared to hAPP hAPOE ε3/3 animals. Microglial activation marker by MHCII was significantly impacted by sex (p<0.0001) and less so by genotype (p = 0.0551). Microglial function measured by pHrodo was impacted by sex (p<0.0001) with hAPP+hAPOE ε3/3 males exhibiting greater phagocytosis in MHCII+ microglia relative to their female counterpart.ConclusionThe interaction between the genotypes APP and APOE in combination with sex has a significant impact on immune phenotypes in the late‐midlife brain. These indications offer unique insights for future targeted therapeutics. Our findings support further investigation into neuroimmune activation in mid‐life to delay or prevent AD.

LATE-NC in Alzheimer's disease: Molecular aspects and synergies.

Alzheimer's disease (AD) is classically characterized by senile plaques and neurofibrillary tangles (NFTs). However, multiple copathologies can be observed in the AD brain and contribute to the development of cognitive decline. Limbic-predominant age-related TDP-43 encephalopathy neuropathological changes (LATE-NC) accumulates in the majority of AD cases and leads to more severe cognitive decline compared with AD pathology alone. In this review, we focus on the synergistic relationship between LATE-NC and tau in AD, highlighting the aggravating role of TDP-43 aggregates on tau pathogenesis and its impact on the clinical picture and therapeutic strategies. Additionally, we discuss to what extent the molecular patterns of LATE-NC in AD differ from frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP) neuropathological changes. Thus, we highlight the importance of tau and TDP-43 synergies for subtyping AD patients, which may respond differently to therapeutic interventions depending on the presence of comorbid LATE-NC.

Intensity-dependent gamma electrical stimulation regulates microglial activation, reduces beta-amyloid load, and facilitates memory in a mouse model of Alzheimer’s disease

BackgroundGamma sensory stimulation may reduce AD-specific pathology. Yet, the efficacy of alternating electrical current stimulation in animal models of AD is unknown, and prior research has not addressed intensity-dependent effects.MethodsThe intensity-dependent effect of gamma electrical stimulation (GES) with a sinusoidal alternating current at 40 Hz on Aβ clearance and microglia modulation were assessed in 5xFAD mouse hippocampus and cortex, as well as the behavioral performance of the animals with the Morris Water Maze.ResultsOne hour of epidural GES delivered over a month significantly (1) reduced Aβ load in the AD brain, (2) increased microglia cell counts, decreased cell body size, increased length of cellular processes of the Iba1 + cells, and (3) improved behavioral performance (learning & memory). All these effects were most pronounced when a higher stimulation current was applied.ConclusionThe efficacy of GES on the reduction of AD pathology and the intensity-dependent feature provide guidance for the development of this promising therapeutic approach.